Lactobacillus rhamnosus and application thereof

Abstract

This invention discloses a strain of *Lactobacillus rhamnosus* H7 and its applications, belonging to the field of food microbiology technology. This invention provides a *Lactobacillus rhamnosus* H7 isolated from fermented meat, deposited at the Guangdong Provincial Microbial Culture Collection Center. This strain has the function of degrading nitrite. When inoculated into dried sausage, the residual nitrite and nitrosamine levels in the dried sausage are reduced, and the color of the meat product is significantly improved. When inoculated into fermented mandarin fish, the residual nitrite and nitrosamine levels in the fermented mandarin fish are also reduced. Therefore, inoculating fermented meat products with *Lactobacillus rhamnosus* H7 not only helps improve the safety of fermented meat products but also enhances their quality. This strain has good application prospects as a starter culture for fermented meat products.

Description

Technical Field

[0001] This invention relates to a strain of Lactobacillus rhamnosus H7 and its application in fermented foods, belonging to the field of food microbiology technology. Background Technology

[0002] Fish products such as fermented mandarin fish are often cured with coarse salt. The nitrates in this salt can be reduced by microorganisms to form nitrites. Furthermore, nitrites are often used as an important additive in fermented sausages, serving to protect color, inhibit bacteria, and impart a unique flavor to fermented meat products. Although nitrites gradually decrease during fermentation, some residues remain in the final product. These residual nitrites can lead to methemoglobinemia in the body. In addition, nitrites pose a potential carcinogenic risk. As a precursor, they can react with amines to form N-nitrosamines, which are extremely harmful to human health. However, due to the widespread use of coarse salt in fish product production and the lack of a complete alternative for the color-protecting and antibacterial functions of nitrites in fermented sausages, minimizing nitrite residues in fermented meat products is a crucial issue for improving the safety of fermented foods.

[0003] As consumers' demands for product quality increase, improving the quality of fermented meat products while reducing nitrite is becoming increasingly important. Studies have shown that some lactic acid bacteria can reduce nitrite under anaerobic conditions, and adding them to fermented meat products such as sausages and fish can help reduce nitrite residues. For example, Sadeghi et al. reported that applying *Lactobacillus rhamnosus* to fermented trout sausages significantly reduced nitrite residues. Furthermore, adding lactic acid bacteria to meat products also improves the quality of fermented meat products. For instance, Tukel et al. found that inoculating with *Lactobacillus rhamnosus* improved the color of salami. Research on adding lactic acid bacteria to reduce nitrite residues while simultaneously improving the color of meat products has also been reported. For example, Liang Xiaobo et al. discovered a strain of *Staphylococcus equi* that can not only degrade nitrite but also improve the color of meat products. However, no research has yet found a lactic acid bacterium that, when added to fermented sausages, can simultaneously degrade nitrite residues, improve the color of meat products, and effectively degrade nitrosamines; nor has it been found to simultaneously degrade both nitrites and nitrosamines when added to fermented fish products such as stinky mandarin fish. Therefore, this invention aims to screen for a lactic acid bacterium that, through single-strain inoculation of fermented sausages and fermented fish, can simultaneously reduce residual nitrites and nitrosamines and improve the color of meat products. Summary of the Invention

[0004] In view of the shortcomings of the prior art, the purpose of this invention is to screen out a strain of Lactobacillus rhamnosus H7 and apply it to fermented foods. It can not only reduce nitrite and nitrosamine residues, but also significantly improve the color of meat products.

[0005] The first technical solution provided by this invention is a strain of Lactobacillus rhamnosus H7, which was deposited on May 20, 2024 at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No. 64655, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province.

[0006] The second technical solution provided by the present invention is a microbial preparation containing *Lactobacillus rhamnosus* H7 as described in the first technical solution.

[0007] In some embodiments, the microbial preparation may be in solid or liquid form, including but not limited to lyophilized powder, fermentation broth, etc.

[0008] In some embodiments, the amount of *Lactobacillus rhamnosus* H7 added to the microbial preparation is not less than 1 × 10⁻⁶. 6 CFU / g or 1×10 6 CFU / mL.

[0009] Furthermore, in the microbial preparation, the amount of *Lactobacillus rhamnosus* H7 added is not less than 1×10⁻⁶. 7 CFU / g or 1×10 7 CFU / mL.

[0010] The third technical solution provided by the present invention is a fermentation agent containing *Lactobacillus rhamnosus* H7 as described in the first technical solution or the microbial preparation as described in the second technical solution, and with the addition of other additives or microbial preparations.

[0011] In some embodiments, the amount of *Lactobacillus rhamnosus* H7 added to the starter culture is not less than 1 × 10⁻⁶. 6 CFU / g or 1×10 6 CFU / mL.

[0012] Furthermore, the amount of *Lactobacillus rhamnosus* H7 added to the fermentation agent is not less than 1 × 10⁻⁶. 7 CFU / g or 1×10 7 CFU / mL.

[0013] In some embodiments, the preparation method of Lactobacillus rhamnosus H7 fermentation antagonist is as follows: Select strains stored at -80℃, culture them on MRS solid medium at 30-37℃ for 16-24 hours, extract single colonies and inoculate them into MRS liquid medium, culture them at 37℃ and 200-250 rpm for 16-24 hours, inoculate them into MRS liquid medium at a 1% inoculation rate in shake flasks for expansion culture, wash the obtained Lactobacillus rhamnosus H7 with sterile physiological saline twice, collect the bacterial cells by centrifugation again, suspend them in an appropriate amount of sterile water, and prepare 1×10⁻⁶ fermentation antagonists. 6 -1×10 8 CFU / mL bacterial suspension.

[0014] The fourth technical solution provided by this invention is the application of Lactobacillus rhamnosus H7 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 foods.

[0015] In some embodiments, the fermented food includes fermented sausages, fermented fish, etc.

[0016] Furthermore, the fermented sausage is an air-dried sausage; the fermented fish includes stinky mandarin fish.

[0017] The fifth technical solution provided by the present invention is the role of Lactobacillus rhamnosus H7 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.

[0018] The sixth technical solution provided by the present invention is a method for reducing nitrite and / or nitrosamines in fermented foods. The method involves introducing the Lactobacillus rhamnosus H7 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 food.

[0019] In some embodiments, the concentration of *Lactobacillus rhamnosus* H7 in the fermentation system is 1 × 10⁻⁶. 6 ~1×10 8 CFU / g.

[0020] In some embodiments, the fermented meat product includes fermented sausage, fermented fish, etc. Further, the fermented sausage is a dried sausage; the fermented fish is stinky mandarin fish.

[0021] In some embodiments, the preparation of the fermented sausage includes the following steps:

[0022] S1. Freshly slaughtered lean pork and fat are minced in a meat grinder with a 1.5cm aperture sieve plate and mixed at a lean-to-fat ratio of 1:9 to 3:7.

[0023] S2. Based on 100% of the total weight of lean and fatty meat, add 1.5-2.5 wt% salt, 0.2%-0.5% wt% monosodium glutamate, 0.5%-1.5 wt% baijiu (Chinese white liquor), 1.0-4.0% wt% white sugar, 1.0-1.5 wt% soy sauce, 0.5-1.5% wt% chili powder, and 0.015 wt% sodium nitrite. Inoculate with Lactobacillus rhamnosus H7 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. After stirring evenly, marinate at room temperature for 30 minutes and then stuff into sausages.

[0024] S3. Ferment the sausages stuffed in S2 at 10-15℃ for 10-12 days to obtain air-dried sausages.

[0025] In some embodiments, the preparation of the fermented fish meat includes the following steps:

[0026] S1. Clean the fresh mandarin fish by removing its internal organs, scales, and gills;

[0027] 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 the weight of the drinking water as 100%. Inoculate with Lactobacillus rhamnosus H7 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.

[0028] 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.

[0029] In some embodiments, the preparation of the fermented fish meat includes the following steps:

[0030] S1. Remove the internal organs, scales, and gills from the fresh mandarin fish, drain it, and weigh it.

[0031] 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 rhamnosus H7 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;

[0032] 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.

[0033] Compared with existing methods, the present invention has the following advantages:

[0034] This invention provides a strain of Lactobacillus rhamnosus H7, which was isolated from fermented meat and has a strong ability to degrade nitrite. When this strain is inoculated into fermented foods, the residual amount of nitrosamines is significantly reduced, the residual amount of nitrites is significantly reduced, and the color of meat products is significantly improved.

[0035] Biological Preservation Materials

[0036] The Lactobacillus rhamnosus H7 strain provided by this invention is classified as Lacticaseibacillus rhamnosus. It was deposited on May 20, 2024, at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No. 64655. The deposit address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, Guangdong Provincial Center for Microbial Culture Collection. Attached Figure Description

[0037] Figure 1 Photograph of MRS solid plate culture of Lactobacillus rhamnosus H7;

[0038] Figure 2 The image shows the results of the screening test for the nitrite degradation ability of lactic acid bacteria. Detailed Implementation

[0039] 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.

[0040] Test method:

[0041] 1. Nitrite residue was determined by spectrophotometry according to GB 5009.33-2016 "National Food Safety Standard - Determination of Nitrite and Nitrate in Food". The specific method is as follows:

[0042] (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.

[0043] (2) Nitrite determination: Pipette 40.0 mL of the above filtrate into a 50 mL stoppered colorimetric tube, and 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 (equivalent to 0.0 μg, 1.0 μg, 2.0 μg, 3.0 μg, 4.0 μg, 5.0 μg, 7.5 μg, 10.0 μg, and 12.5 μg sodium nitrite) into 50 mL stoppered colorimetric tubes. Add 2 mL of 4 g / L p-aminobenzenesulfonic acid solution to both the standard tube and the sample tube, mix well, and let stand for 3-5 minutes. Then add 1 mL of 2 g / L naphthylethylenediamine hydrochloride solution to each tube, add water to the mark, mix well, and let stand for 15 minutes. 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.

[0044] 2. The method for determining nitrosamine residue is as follows:

[0045] (1) Sample pretreatment: Weigh 10g of homogenized sample, add 10uL of NDPA-d14 internal standard (concentration 2μg / L) and 15mL of acetonitrile, vortex mix, and freeze at -20℃ for 30 minutes. Add 4g of magnesium sulfate and 1g of sodium chloride, shake for 30 seconds, and centrifuge at 8000rpm / min for 10 minutes at 4℃. Take 6mL of supernatant and add it to a centrifuge tube containing 50mg PSA, 150mg C18E and 900mg anhydrous sodium sulfate. Vortex thoroughly and centrifuge at 8000rpm / min for 10 minutes. Take 5mL of supernatant, blow nitrogen to 1mL, filter through a 0.22um organic filter membrane, and prepare for analysis.

[0046] (2) Gas chromatography parameters: Helium was used as the carrier gas, and the flow rate was constant at 1.0 mL / min. The temperature program was set as follows: the initial temperature was 40 °C, held for 0.5 min; then the temperature was increased to 115 °C at a rate of 6 °C / min, held for 1 min; then the temperature was increased to 150 °C at a rate of 15 °C / min, held for 0.5 min; finally, the temperature was increased to 180 °C at a rate of 2 °C / min, held for 2 min. The solvent delay time was 8.5 min.

[0047] (3) Mass spectrometry parameters: Electron ionization (EI) was used for ionization; the ion source temperature was set to 240℃; the injection port temperature was set to 240℃; the quadrupole temperature was set to 150℃; the interface temperature was set to 240℃; the detection mode was set to multiple reaction monitoring (MRM); and the gain factor was set to 1.

[0048] 3. The colorimetric determination method is as follows: The color of dried sausage was determined using an UltraScan Pro colorimeter. The instrument was set to reflective mode and calibrated using a white standard plate. The b* / a* value was used as the evaluation index of the color of dried sausage. Each sample was measured in parallel 6 times and the average value was calculated.

[0049] Raw materials used in the examples:

[0050] 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.

[0051] 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.

[0052] 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.

[0053] YPD solid medium: peptone 10.0 g / L, glucose 20.0 g / L, yeast extract 5.0 g / L, agar 14.0 g / L.

[0054] YPD liquid medium: peptone 20.0 g / L, glucose 20.0 g / L, yeast extract 10.0 g / L, pH 6.5 ± 0.2.

[0055] In the following comparative examples, *Lactobacillus plantarum* CICC 22220, CICC 20242, CICC 20264, CICC 22205, CICC 20038, CICC 20363, CICC20659; *Lactobacillus rhamnosus* CICC 6163, CICC 20255, CICC20053, CICC 22151, CICC 6137, CICC 6161; *Acetobacter pastoris* CICC 20001, CICC 7006; *Lactobacillus acidophilus* CICC 20244, CICC 20248, CICC 6005, CICC 25190, CICC 22162, CICC 20272; *Lactobacillus curvaturei* CICC 25172, CICC 25190, CICC 25190; and *Streptococcus thermophilus* CICC... 20370, *Saccharomyces cerevisiae* CICC 1012, *Staphylococcus equi* CICC 10435, and *Staphylococcus xylose* CICC 21145 were all purchased from the China Industrial Microbial Culture Collection Center; *Lactobacillus bulgaricus* ATCC 11057, *Staphylococcus equi* ATCC 43958, *Staphylococcus xylose* ATCC 29971, *Lactobacillus rhamnosus* ATCC 53193, *Staphylococcus aureus* ATCC 51136 and ATCC 51137, and *Lactobacillus paracasei* ATCC 334 were purchased from Jitai Biotechnology; *Saccharomyces cerevisiae* CGMCC 2.148 and 2.1364, *Lactobacillus paracasei* CGMCC 1.12731, and *Acetobacter pasteurellosis* CGMCC 1.41 were purchased from the Guangdong Provincial Microbial Culture Collection Center; *Lactobacillus paracasei* SICC 1.1375 was purchased from Ningbo Taisto Biotechnology Co., Ltd., and *Bifidobacterium lactis* BB12 was purchased from Chr. Hansen.

[0056] Example 1: Isolation, screening and identification of strain Lactobacillus rhamnosus H7.

[0057] (1) Isolation of the strain: The strain was isolated from fermented meat products.

[0058] (2) Screening method for nitrite degradation capacity:

[0059] 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 (equivalent to 0.0 μg, 1.0 μg, 2.0 μg, 3.0 μg, 4.0 μg, 5.0 μg, 7.5 μg, 10.0 μg, and 12.5 μg sodium nitrite), respectively, into 15 mL centrifuge tubes. Add 2 mL of 4 g / L p-aminobenzenesulfonic acid solution to the tubes, mix well, and let stand for 3-5 min. Then add 1 mL of 2 g / L naphthylethylenediamine hydrochloride solution, add water to a final volume of 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.

[0060] (1) Nitrite degradation capacity determination: Frozen lactic acid bacteria were cultured on MRS solid medium at 37℃ for 1 day. Single colonies were picked from the plates and inoculated into MRS liquid medium and cultured 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, and 1 mL of the supernatant was collected into 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:

[0061]

[0062] In the formula, the units for the initial nitrite content and the nitrite content after cultivation are both μg / mL.

[0063] Appendix Figure 2 The results of the nitrite degradation rate determination for the strains are shown in Table 1. The strain numbers and corresponding strain names are listed in Table 1. The determination showed that strain H7 had a nitrite degradation rate of 95.78%, the highest among all screened strains.

[0064] Table 1: Number and Name of Strains Selected for Nitrite Degradation Capacity

[0065]

[0066]

[0067]

[0068] (3) Identification of strain H7: The genome of strain H7 was extracted for 16S rDNA identification. The genome extraction method was carried out according to the glass bead method in the "Concise Guide to Molecular Biology Experiments".

[0069] The PCR reaction system (50 μL) consisted of: 25 μL 2×PCR Mix, 2 μL Forward Primer, 2 μL Reverse Primer, 2 μL genomic template, and sterile water to a final volume of 50 μL. Primer sequences: Forward Primer (27F): AGAGTTTGATCMTGGCTCAG; Reverse Primer (1492R): GGTTACCTTGTTACGACTT. The PCR program was as follows: 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; 4℃ indefinitely. After PCR, the PCR products were verified by electrophoresis and then sent for sequencing.

[0070] The 16S rDNA sequence of strain H7 is shown in SEQ ID NO:1.

[0071] The sequencing results were analyzed by Blasten, which showed that the bacterium had the highest homology with Lacticaseibacillus rhamnosus, with a similarity of 99.93%, and the identification result was Lacticaseibacillus rhamnosus.

[0072] This strain grew well in MRS solid medium, and after culturing at 37°C for 24 hours, as... Figure 1 As shown, the Lactobacillus rhamnosus H7 formed on MRS solid medium appears as round, white, raised, smooth, moist single colonies with neat edges.

[0073] Based on morphological and 16S rDNA identification, the strain was named Lacticaseibacillus rhamnosus H7 and deposited on May 20, 2024, at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC No. 64655). The deposit address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province.

[0074] Example 2: Lactobacillus rhamnosus H7 at 3×10 6 CFU / g inoculated into dried sausage

[0075] 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.

[0076] S2: Preparation of bacterial culture:

[0077] First, bacterial cells were prepared as follows: *Lactobacillus rhamnosus* H7 strains stored at -80℃ were revived using MRS solid medium at 37℃ for 24 hours. Single colonies of the revived strain were then transferred to MRS liquid medium and cultured at 37℃, 200 rpm for 16 hours on a shaker. A 1% inoculum was then added to the MRS liquid medium and cultured at 37℃, 200 rpm for 16 hours on a shaker. The bacterial cells were collected by centrifugation, washed twice with sterile physiological saline, and collected again by centrifugation. The cells were then resuspended in a suitable amount of sterile water and adjusted to a pH of 10. 6 CFU / mL, yielding Lactobacillus rhamnosus H7 bacterial suspension.

[0078] 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, 0.015 wt% sodium nitrite, and Lactobacillus rhamnosus H7 bacterial culture; wherein the Lactobacillus rhamnosus H7 bacterial culture is prepared at a concentration of 3 × 10⁻⁶. 6 Inoculate the meat with CFU / g, mix well, marinate at room temperature for 30 minutes, and then stuff into sausages.

[0079] S4: Fermentation: The sausages obtained in S3 are fermented at 15°C for 12 days to make air-dried sausages, which are named Example 2.

[0080] Example 3: Lactobacillus rhamnosus H7 at 6×10 7 CFU / g inoculated into dried sausage

[0081] S1: Same as Example 2S1.

[0082] S2: Preparation of bacterial culture:

[0083] Lactobacillus rhamnosus H7 cells were prepared according to the specific steps in Example 2S2, and suspended in an appropriate amount of sterile water and adjusted to 10. 7 CFU / mL, yielding Lactobacillus rhamnosus H7 bacterial suspension.

[0084] S3: Specific ingredient ratios are shown in Example 2S3; wherein the *Lactobacillus rhamnosus* H7 bacterial culture is prepared at a concentration of 6 × 10⁻⁶. 7 Inoculate the meat with CFU / g, mix well, marinate at room temperature for 30 minutes, and then stuff into sausages.

[0085] S4: Same as Example 2S4.

[0086] Example 4: Lactobacillus rhamnosus H7 at 4×10 8 CFU / g inoculated into dried sausage

[0087] S1: Same as Example 2S1.

[0088] S2: Preparation of bacterial culture:

[0089] Lactobacillus rhamnosus H7 cells were prepared according to the specific steps in Example 2S2, and suspended in an appropriate amount of sterile water and adjusted to 10. 8 CFU / mL, yielding Lactobacillus rhamnosus H7 bacterial suspension.

[0090] S3: Specific ingredient ratios are shown in Example 2S3; wherein the *Lactobacillus rhamnosus* H7 bacterial culture is prepared at a concentration of 4×10⁻⁶. 8 Inoculate the meat with CFU / g, mix well, marinate at room temperature for 30 minutes, and then stuff into sausages.

[0091] S4: Same as Example 2S4.

[0092] Example 5: Lactobacillus rhamnosus H7 at 2×10 7 CFU / mL was inoculated into wet-cured stinky mandarin fish.

[0093] S1: Remove the internal organs, scales, and gills from fresh mandarin fish.

[0094] S2: Preparation of bacterial culture

[0095] First, bacterial cells were prepared as follows: *Lactobacillus rhamnosus* H7 strains stored at -80℃ were revived using MRS solid medium at 37℃ for 24 hours. Single colonies of the revived strain were then transferred to MRS liquid medium and cultured at 37℃, 200 rpm for 16 hours on a shaker. A 1% inoculum was then added to the MRS liquid medium and cultured at 37℃, 200 rpm for 16 hours on a shaker. The bacterial cells were collected by centrifugation, washed twice with sterile physiological saline, and collected again by centrifugation. The cells were then resuspended in a suitable amount of sterile water and adjusted to a pH of 10. 8 CFU / mL, yielding Lactobacillus rhamnosus H7 bacterial suspension.

[0096] 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 rhamnosus H7 bacterial culture, based on the weight of the drinking water as 100%. The concentration of Lactobacillus rhamnosus H7 in the fermentation broth is 2×10⁻⁶. 7 CFU / mL.

[0097] 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 5.

[0098] Example 6: Lactobacillus rhamnosus H7 at 4×108 CFU / mL was inoculated into wet-cured stinky mandarin fish.

[0099] S1: Remove the internal organs, scales, and gills from fresh mandarin fish.

[0100] S2: Preparation of bacterial culture

[0101] Bacterial cells were prepared according to the steps in Example 5S2, and suspended in an appropriate amount of sterile water and adjusted to 10. 9 CFU / mL, yielding Lactobacillus rhamnosus H7 bacterial suspension.

[0102] S3: Prepare the fermentation broth according to the steps described in Example 5S3; the concentration of Lactobacillus rhamnosus H7 in the fermentation broth is 4×10⁻⁶. 8 CFU / mL.

[0103] S4: Same as Example 5S4.

[0104] Example 7: Lactobacillus rhamnosus H7 at 2×10 7 CFU / mL was inoculated into dried and salted mandarin fish.

[0105] S1: Remove the internal organs, scales, and gills from the fresh mandarin fish, drain it, and weigh it.

[0106] S2: Same as Example 5S2.

[0107] 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 2×10⁻⁶. 7 CFU / g.

[0108] 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 7.

[0109] Example 8: Lactobacillus rhamnosus H7 at 4×10 8 CFU / mL was inoculated into dried and salted mandarin fish.

[0110] S1: Remove the internal organs, scales, and gills from the fresh mandarin fish, drain it, and weigh it.

[0111] S2: The preparation of bacterial culture is the same as in Example 6S2.

[0112] 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 × 10⁻⁶.8 CFU / g.

[0113] 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 8.

[0114] Comparative Example 1: Naturally Fermented and Air-Dried Sausage

[0115] S1: Same as Example 2S1.

[0116] 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 0.015wt% sodium nitrite. Mix well and marinate at room temperature for 30 minutes before stuffing into sausages.

[0117] S3. Fermentation: The sausages obtained in S2 are fermented at 15℃ for 12 days to make air-dried sausages, which are named Comparative Example 1.

[0118] Comparative Example 2: Wet-process naturally fermented stinky mandarin fish

[0119] S1. Clean the fresh mandarin fish by removing its internal organs, scales, and gills.

[0120] 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%.

[0121] 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 2.

[0122] Comparative Example 3: Dry-fermented stinky mandarin fish

[0123] S1: Remove the internal organs, scales, and gills from the fresh mandarin fish, drain it, and weigh it.

[0124] 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.

[0125] 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 3.

[0126] Comparative Example 4: The fermentation broth of the compound microbial agent was mixed in equal volume proportions with *Lactobacillus plantarum* CICC 22220, *Lactobacillus paracasei* ATCC 334, *Lactobacillus rhamnosus* CICC 6163, *Lactobacillus acidophilus* CICC 6005, and *Acetobacter pasteurellum* CICC 20001 to form a compound solid microbial agent, which was then inoculated into air-dried sausages.

[0127] S1: Same as Example 2S1.

[0128] S2: Preparation of bacterial culture:

[0129] First, bacterial cells were prepared as follows: *Lactobacillus plantarum* CICC 22220, *Lactobacillus paracasei* ATCC 334, *Lactobacillus rhamnosus* CICC 6163, *Lactobacillus acidophilus* CICC 6005, and *Acetobacter pasteurellosis* CICC 20001, stored at -80℃, were revived using MRS solid medium at 37℃ for 24 hours. Single colonies of the revived cells were picked and transferred to MRS liquid medium and cultured at 37℃, 200 rpm for 16 hours on a shaker. Then, a 1% inoculum was added to the MRS liquid medium and cultured at 37℃, 200 rpm for 16 hours on a shaker. The bacterial cells were collected by centrifugation, washed twice with sterile physiological saline, centrifuged again, and resuspended in an appropriate amount of sterile water to a final concentration of 10⁻⁶. 6 CFU / mL, yielding bacterial suspensions of Lactobacillus plantarum CICC 22220, Lactobacillus paracasei ATCC 334, Lactobacillus rhamnosus CICC 6163, Lactobacillus acidophilus CICC 6005, and Acetobacter pasteurellium CICC 20001.

[0130] S3: Fermentation broth preparation: *Lactobacillus plantarum* CICC 22220, *Lactobacillus paracasei* ATCC 334, *Lactobacillus rhamnosus* CICC 6163, *Lactobacillus acidophilus* CICC 6005, and *Acetobacter pasteurellium* CICC 20001 were mixed in equal volume ratios to prepare the fermentation broth, with a final concentration of 10. 6 CFU / mL.

[0131] S4: Based on 100% of the 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, and 0.015 wt% sodium nitrite, along with the mixed fermentation liquid described in S3; wherein the mixed fermentation liquid described in S3 is prepared at a concentration of 3 × 10⁻⁶... 6 After inoculating the meat with CFU / g, mix the above ingredients evenly, marinate at room temperature for 30 minutes, and then stuff the sausages.

[0132] S5: Fermentation: The sausages obtained in S4 are fermented at 15℃ for 12 days to make air-dried sausages, which are named Comparative Example 4.

[0133] Comparative Example 5: Equal volumes of the fermentation broth containing *Lactobacillus plantarum* CICC 20242, *Lactobacillus paracasei* CGMCC 1.12731, *Lactobacillus rhamnosus* CICC 20255, *Lactobacillus acidophilus* CICC 20244, and *Acetobacter pasteurellum* CICC 7006 were mixed and fermented to form a compound solid microbial agent, which was then inoculated into air-dried sausages.

[0134] Except for the fermentation broth being a mixture of Lactobacillus plantarum CICC 20242, Lactobacillus paracasei CGMCC 1.12731, Lactobacillus rhamnosus CICC 20255, Lactobacillus acidophilus CICC 20244, and Acetobacter pasteurellum CICC 7006, the specific implementation methods were the same as those in Comparative Example 4.

[0135] Comparative Example 6: The fermentation broth of the compound microbial agent was mixed in equal proportions with Lactobacillus plantarum CICC 20264, Lactobacillus paracasei SICC 1.1375, Lactobacillus rhamnosus ATCC 53193, Lactobacillus acidophilus CICC 20248, and Acetobacter pasteurellum CGMCC 1.41 to form a compound solid microbial agent, which was then inoculated into air-dried sausages.

[0136] Except for the fermentation broth being a composite strain of Lactobacillus plantarum CICC 20264, Lactobacillus paracasei SICC 1.1375, Lactobacillus rhamnosus ATCC 53193, Lactobacillus acidophilus CICC 20248, and Acetobacter pasteurellum CGMCC 1.41, the specific implementation methods were the same as those in Comparative Example 4.

[0137] Comparative Example 7: A compound probiotic containing *Lactobacillus plantarum* CICC 22205, *Lactobacillus acidophilus* CICC 6005, *Lactobacillus rhamnosus* CICC 6163, and *Lactobacillus curvatureii* CICC 25172 was added to air-dried intestines.

[0138] S1: Same as Example 2S1

[0139] S2: Preparation of bacterial culture:

[0140] Bacterial suspensions of *Lactobacillus plantarum* CICC 22205, *Lactobacillus acidophilus* CICC 6005, *Lactobacillus rhamnosus* CICC 6163, and *Lactobacillus curvatureii* CICC 25172, stored at -80℃, were prepared according to the steps in S2 of Comparative Example 4.

[0141] S3: A compound fermentation broth was prepared by mixing *Lactobacillus plantarum* CICC 22205, *Lactobacillus acidophilus* CICC 6005, *Lactobacillus rhamnosus* CICC 6163, and *Lactobacillus curvatureii* CICC 25172 bacterial cultures in a ratio of 5:5:1:1. The final concentration of the fermentation broth was 10. 6 CFU / mL.

[0142] S4: Prepared according to the formula described in S4 of Comparative Example 4, wherein the mixed fermentation broth is the composite fermentation broth described in S3.

[0143] S5: Fermentation: The sausages obtained in S4 are fermented at 15℃ for 12 days to make air-dried sausages, which are named Comparative Example 7.

[0144] Comparative Example 8: A compound probiotic containing *Lactobacillus plantarum* CICC 20038, *Lactobacillus acidophilus* CICC 25190, *Lactobacillus rhamnosus* CICC 20053, and *Lactobacillus curvature* CICC 25190 was added to air-dried intestines.

[0145] Except for the fermentation broth being a composite strain of Lactobacillus plantarum CICC 20038, Lactobacillus acidophilus CICC 25190, Lactobacillus rhamnosus CICC 20053, and Lactobacillus curvature CICC 25190, the other specific implementation methods are the same as those in Comparative Example 7.

[0146] Comparative Example 9: A compound probiotic containing *Lactobacillus plantarum* CICC 20363, *Lactobacillus acidophilus* CICC 22162, *Lactobacillus rhamnosus* CICC 6161, and *Lactobacillus curvatureii* CICC 25190 was added to air-dried intestines.

[0147] Except for the fermentation broth being a composite strain of Lactobacillus plantarum CICC 20363, Lactobacillus acidophilus CICC 22162, Lactobacillus rhamnosus CICC 6161, and Lactobacillus curvature CICC 25190, the other specific implementation methods are the same as those in Comparative Example 7.

[0148] Comparative Example 10: Lactobacillus rhamnosus CICC 22151 was inoculated into dried intestines.

[0149] Except for the use of Lactobacillus rhamnosus CICC 22151 as the disinfectant solution, the specific implementation method is the same as in Example 2.

[0150] Comparative Example 11: Lactobacillus rhamnosus CICC 6137 was inoculated into dried intestines

[0151] Except for the use of Lactobacillus rhamnosus CICC 6137 as the disinfectant solution, the specific implementation method is the same as in Example 2.

[0152] Comparative Example 12: Lactobacillus rhamnosus CICC 20053 was inoculated into dried intestines.

[0153] Except for the use of Lactobacillus rhamnosus CICC 20053 as the sterilization solution, the specific implementation method is the same as in Example 2.

[0154] Comparative Example 13: Bifidobacterium lactis BB12 was inoculated into dried intestines.

[0155] Except for the use of Bifidobacterium lactis BB12 as the disinfectant solution, the specific implementation method is the same as in Example 2.

[0156] Comparative Example 14: Lactobacillus rhamnosus CICC 22151, Lactobacillus acidophilus CICC 20272, Lactobacillus bulgaricus ATCC 11057, Streptococcus thermophilus CICC 20370, and Saccharomyces cerevisiae CICC 1012 were inoculated into air-dried intestines.

[0157] S1: Same as Example 2S1.

[0158] S2: Preparation of bacterial culture:

[0159] First, bacterial cells were prepared. The bacterial cells of *Lactobacillus rhamnosus* CICC 22151, *Lactobacillus acidophilus* CICC 20272, *Lactobacillus bulgaricus* ATCC 11057, and *Streptococcus thermophilus* CICC 20370 were prepared as follows: *Lactobacillus rhamnosus* CICC 22151, *Lactobacillus acidophilus* CICC 20272, *Lactobacillus bulgaricus* ATCC 11057, and *Streptococcus thermophilus* CICC 20370, stored at -80℃, were revived using MRS solid medium at 37℃ for 24 hours. Single colonies of the revived bacteria were then transferred to MRS liquid medium and cultured at 37℃ and 200 rpm for 16 hours. Finally, a 1% inoculum was added to the MRS liquid medium and cultured at 37℃ and 200 rpm for 16 hours. Collect bacterial cells by centrifugation, wash the bacterial cells twice with sterile physiological saline, collect the bacterial cells by centrifugation again, suspend the cells in an appropriate amount of sterile water and adjust to 10⁻⁶. 6 CFU / ml yielded bacterial suspensions of Lactobacillus rhamnosus CICC 22151, Lactobacillus acidophilus CICC20272, Lactobacillus bulgaricus ATCC 11057, and Streptococcus thermophilus CICC 20370.

[0160] The preparation method for Saccharomyces cerevisiae CICC 1012 cells was as follows: Saccharomyces cerevisiae CICC 1012 stored at -80℃ was picked and revived using YPD solid medium at 30℃ for 24 hours. Single colonies of the revived cells were picked and transferred to YPD liquid medium and cultured at 30℃, 200 rpm for 16 hours on a shaker. Then, 1% of the culture was inoculated into YPD liquid medium and cultured at 30℃, 200 rpm for 16 hours on a shaker. The cells were collected by centrifugation, washed twice with sterile physiological saline, centrifuged again, and resuspended in an appropriate amount of sterile water to a final concentration of 10⁻⁶. 6 CFU / mL yielded a culture of Saccharomyces cerevisiae CICC 1012.

[0161] S3: A compound fermentation broth was prepared by mixing *Lactobacillus rhamnosus* CICC 22151, *Lactobacillus acidophilus* CICC 20272, *Lactobacillus bulgaricus* ATCC 11057, *Streptococcus thermophilus* CICC 20370, and *Saccharomyces cerevisiae* CICC 1012 bacterial cultures in a ratio of 5:4:3:2:3. The final concentration of the fermentation broth was 10. 6 CFU / mL.

[0162] S4: Prepared according to the formula described in S4 of Comparative Example 3, wherein the mixed fermentation broth is the composite fermentation broth described in S3.

[0163] S5: Fermentation: The sausages obtained in S4 are fermented at 15℃ for 12 days to make air-dried sausages, which are named Comparative Example 14.

[0164] Comparative Example 15: Staphylococcus equi ATCC 43958 was added and inoculated into dried intestines.

[0165] Except for the use of Staphylococcus equi ATCC 43958 as the disinfectant solution, the specific implementation method is the same as in Example 2.

[0166] Comparative Example 16: Staphylococcus equi CICC 10435 was added and inoculated into dried intestines.

[0167] Except for the use of Staphylococcus aureus CICC 10435 as the disinfectant solution, the specific implementation method is the same as in Example 2.

[0168] Comparative Example 17: Fish fermentation Staphylococcus aureus ATCC 51136 was added and inoculated into dried sausage.

[0169] Except for the use of fish fermentation Staphylococcus aureus ATCC 51136 as the sterilization solution, the other specific implementation methods are the same as in Example 2.

[0170] Comparative Example 18: Fish fermentation Staphylococcus aureus ATCC 51137 was added and inoculated into dried intestines.

[0171] Except for the use of fish fermentation Staphylococcus aureus ATCC 51137 as the sterilization solution, the other specific implementation methods are the same as in Example 2.

[0172] Comparative Example 19: Lactobacillus plantarum CICC 20038 was added and inoculated into air-dried intestines.

[0173] Except for the use of Lactobacillus plantarum CICC 20038 as the sterilization solution, the specific implementation method is the same as in Example 2.

[0174] Comparative Example 20: Lactobacillus plantarum CICC 20242 was added and inoculated into air-dried intestines.

[0175] Except for the use of Lactobacillus plantarum CICC 20242 as the sterilization solution, the other specific implementation methods are the same as in Example 2.

[0176] Comparative Example 21: Lactobacillus plantarum CICC 20659 was added and inoculated into air-dried intestines.

[0177] Except for the use of Lactobacillus plantarum CICC 20659 as the sterilization solution, the specific implementation method is the same as in Example 2.

[0178] Comparative Example 22: Lactobacillus plantarum CICC 22220, Saccharomyces cerevisiae CGMCC 2.1364, and Staphylococcus xylose ATCC29971 were inoculated into wet-cured stinky mandarin fish in equal proportions.

[0179] S1: Remove the internal organs, scales, and gills from fresh mandarin fish.

[0180] S2: Preparation of bacterial culture

[0181] First, bacterial cells were prepared as follows: *Lactobacillus plantarum* CICC 22220 and *Staphylococcus xylose* ATCC 29971, stored at -80℃, were revived on MRS solid medium at 37℃ for 24 hours. Single colonies of the revived cells were then transferred to MRS liquid medium and cultured at 37℃, 200 rpm for 16 hours on a shaker. A 1% inoculum was then added to the MRS liquid medium and cultured at 37℃, 200 rpm for 16 hours on a shaker. The bacterial cells were collected by centrifugation, washed twice with sterile physiological saline, and collected again by centrifugation. The cells were then resuspended in a suitable amount of sterile water and adjusted to a pH of 10. 8 CFU / mL, yielding bacterial suspensions of Lactobacillus plantarum CICC 22220 and Staphylococcus xylose ATCC 29971.

[0182] The preparation method for Saccharomyces cerevisiae CGMCC 2.1364 cells was as follows: Saccharomyces cerevisiae CGMCC 2.1364 stored at -80℃ was picked and revived using YPD solid medium at 30℃ for 24 hours. Single colonies of the revived cells were picked and transferred to YPD liquid medium and cultured at 30℃, 200 rpm on a shaker for 16 hours. Then, a 1% inoculum was added to YPD liquid medium and cultured at 30℃, 200 rpm on a shaker for 16 hours. The cells were collected by centrifugation, washed twice with sterile physiological saline, centrifuged again, and resuspended in an appropriate amount of sterile water to a final concentration of 10⁻⁶. 8 CFU / mL yielded a culture of Saccharomyces cerevisiae CGMCC 2.1364.

[0183] 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 2×10⁻⁶ solution. 7 CFU / mL.

[0184] 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 22.

[0185] Comparative Example 23: Lactobacillus plantarum CICC 20242, Saccharomyces cerevisiae CGMCC 2.148, and Staphylococcus xylose CICC21145 were inoculated into wet-cured stinky mandarin fish in equal proportions.

[0186] Except for the fermentation broth being a composite strain of Lactobacillus plantarum CICC 20242, Saccharomyces cerevisiae CGMCC 2.148, and Staphylococcus xylose CICC21145, the other specific implementation methods are the same as those in Comparative Example 22.

[0187] Comparative Example 24: 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.

[0188] S1: Remove the internal organs, scales, and gills from fresh mandarin fish.

[0189] S2: Preparation of Compound Bacterial Solution

[0190] 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. 8 CFU / mL bacterial culture

[0191] 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... 8 CFU / mL bacterial culture

[0192] Lactobacillus plantarum CICC 22220, Saccharomyces cerevisiae CGMCC 2.1364, and Staphylococcus xylose ATCC 29971 bacterial cultures were mixed in equal volume ratios to prepare 10 8 A compound bacterial solution with CFU / mL.

[0193] 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 2×10 7 CFU / g.

[0194] S4: 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. The product is named Comparative Example 24.

[0195] Comparative Example 25: 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.

[0196] S1: Remove the internal organs, scales, and gills from fresh mandarin fish.

[0197] S2: Preparation of bacterial culture

[0198] 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.

[0199] The preparation method of Saccharomyces cerevisiae CGMCC 2.1364 bacterial culture was the same as that of Comparative Example 22S2.

[0200] Lactobacillus plantarum CICC 20242, Saccharomyces cerevisiae CGMCC 2.148, and Staphylococcus xylose ATCC 29971 bacterial cultures were mixed in equal volume ratios to prepare 10 8 A compound bacterial solution with CFU / mL.

[0201] S3, S4: Same as comparative example 24S3, S4.

[0202] Test case

[0203] The residual amounts of nitrite, nitrosamines, and color of the dried sausages from the examples and comparative examples were measured, and the results are shown in Table 2. Table 2 shows that among all the experimental groups of dried sausages, Examples 2-4 had the lowest residual amounts of nitrite, and the total residual amount of nitrosamines was reduced by 46%-71% compared to Comparative Example 1. Compared to Comparative Example 1, the b* / a* value of inoculated with *Lactobacillus rhamnosus* H7 was lower, and the color was more vibrant. Therefore, inoculating dried sausages with *Lactobacillus rhamnosus* H7 can not only reduce the residues of nitrite and nitrosamines but also improve the color of the dried sausages. In all the examples of fermented mandarin fish, nitrite was undetectable, and NDMA, NYPR, and NMorPH were significantly reduced compared to the comparative example, while other comparative examples of fermented mandarin fish did not achieve such effects. This indicates that inoculation with *Lactobacillus rhamnosus* H7 can simultaneously degrade nitrite and nitrosamines in fermented mandarin fish.

[0204] Table 2. Residual amounts and color values ​​(b* / a*) of nitrite (mg / kg), nitrosamines (ug / kg)

[0205]

[0206] In summary, this application uses *Lactobacillus rhamnosus* H7 as a starter culture, which can significantly reduce residual nitrites and nitrosamines in fermented meat products, thereby improving their safety. Furthermore, *Lactobacillus rhamnosus* H7 can significantly enhance the color of fermented meat products, improving product quality.

[0207] 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 rhamnosus ( Lacticaseibacillus rhamnosus H7, characterized in that, It was deposited at the Guangdong Provincial Center for Microbial Culture Collection on May 20, 2024, with accession number GDMCC No. 64655.

2. A microbial preparation, characterized in that, Contains Lactobacillus rhamnosus H7 as described in claim 1.

3. The microbial preparation according to claim 2, characterized in that, In the microbial preparation, the amount of *Lactobacillus rhamnosus* H7 added is not less than 1×10⁻⁶. 6 CFU / g or 1×10 6 CFU / mL.

4. A fermenting agent, characterized in that, The fermenting agent contains *Lactobacillus rhamnosus* H7 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 rhamnosus H7 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 foods.

6. The application according to claim 5, characterized in that, The fermented food includes fermented sausage and fermented fish; optionally, the fermented sausage is air-dried sausage; and the fermented fish is stinky mandarin fish.

7. The use of Lactobacillus rhamnosus H7 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 foods, characterized in that, The method involves introducing the Lactobacillus rhamnosus H7 of claim 1, or the microbial preparation of any one of claims 2 to 3, or the starter culture of claim 4 into the fermentation system of the fermented food.

9. The method according to claim 8, characterized in that, In the fermentation system, the concentration of *Lactobacillus rhamnosus* H7 was 1 × 10⁻⁶. 6 ~1×10 8 CFU / g.

10. The method according to claim 9, characterized in that, The fermented food includes fermented sausage and fermented fish; optionally, the fermented sausage is air-dried sausage; and the fermented fish is stinky mandarin fish.

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