A plant lactobacillus YYS-J1 that can degrade nitrite and its application

Abstract

This invention belongs to the field of microbial technology, specifically relating to a *Lactobacillus plantarum* YYS-J1 strain capable of degrading nitrite and its applications. The *Lactobacillus plantarum* YYS-J1 provided by this invention possesses strong acid and bile salt resistance, exhibiting good tolerance in simulated gastric and intestinal fluids. It has a highly efficient function in degrading nitrite and trimethylamine; when applied to the preparation of fermented foods, it can significantly reduce nitrite and trimethylamine content, improving product safety and added value, laying the foundation for large-scale production of green and healthy traditional fermented foods. Its highly efficient function in degrading trimethylamine allows it to be used as a preservative for aquatic products. Similarly, when applied to probiotic products, it can eliminate nitrite, trimethylamine, and their compounds from the body.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a plant lactobacillus YYS-J1 that can degrade nitrite and its applications. Background Technology

[0002] Nitrites are commonly found in leafy green vegetables. Under high temperatures or during incomplete pickling, they can easily form nitrosamines, which are harmful to human health and can cause cancer and birth defects. Nitrites are also commonly used in cured meat products, where they readily react with amine compounds such as trimethylamine to form carcinogenic N-nitrosamines. Furthermore, nitrosamines can cross the placenta and enter the fetus, causing birth defects.

[0003] Therefore, the issue of nitrite residue in fermented products is a matter of great public concern and urgently needs to be addressed. To this end, many countries have introduced relevant standards and laws to prevent excessive nitrite residues in food. my country's GB2760-2014 "Standard for the Use of Food Additives" stipulates that the maximum amount of nitrite added in meat processing shall not exceed 150 mg / kg, and the residue shall not exceed 30 mg / kg; the residue in pickled vegetables shall not exceed 20 mg / kg.

[0004] Currently, methods for reducing nitrite levels include physical degradation, chemical degradation, and biodegradation. The most common biodegradation method is lactic acid bacteria degradation, which offers advantages such as high efficiency and safety.

[0005] Li Lu et al. used *Lactobacillus fermentum* G9 to ferment kimchi and found that *Lactobacillus fermentum* can effectively reduce the accumulation of nitrite and biogenic amines (including at least one of tryptamine, β-phenylethylamine, tyramine, histamine, putrescine, spermine, and spermidine) during the fermentation process. The nitrite degradation rate approached 100%, and the degradation time was effectively shortened from 9 days in traditional fermentation to 5 days. Zhang Jun et al. screened a *Lactobacillus plantarum* G11 strain with the advantage of rapid and efficient nitrite degradation, achieving a nitrite degradation rate of 89.86% within 24 hours.

[0006] Furthermore, trimethylamine is a low-boiling-point, nitrogen-containing small molecule compound that is readily absorbed by intestinal epithelial cells. Once absorbed, trimethylamine enters the bloodstream and exerts cytotoxic effects on cardiomyocytes, and is considered a risk marker for cardiovascular disease. Simultaneously, trimethylamine enters the liver via the bloodstream and is oxidized to form trimethylamine oxide. Trimethylamine oxide induces atherosclerosis by promoting inflammatory responses, affecting cholesterol metabolism and oxidative stress, and participating in thrombus formation. Clinical studies have shown that high levels of trimethylamine oxide in the blood are closely related to the occurrence and development of cardiovascular disease.

[0007] Furthermore, trimethylamine oxide is also a natural component of fish. When fish die, trimethylamine oxide is converted into trimethylamine and other amines and aldehydes with putrefactive properties through the action of microorganisms and their own enzymes. Therefore, reducing trimethylamine from food sources is of great significance.

[0008] Current methods for preserving fresh fish mainly include chemical and physical methods. Chemical preservatives are detrimental to human health, while low-temperature preservation methods have adverse effects on the taste and nutritional value of fish. Tuo Yanfeng et al. used Lactobacillus sakei B2-4 for biological preservation of fish and meat, and their research found that this bacterium has a preservative effect on fish.

[0009] Currently, the reported strains can only degrade either nitrite or trimethylamine; there are no reports of strains that can degrade both nitrite and trimethylamine simultaneously. Summary of the Invention

[0010] To address the shortcomings of the prior art mentioned in the background section, this invention provides a *Lactobacillus plantarum* YYS-J1 strain capable of efficiently degrading nitrite and trimethylamine, the technical solution of which is as follows:

[0011] The *Lactobacillus plantarum* YYS-J1 provided by this invention has the following Latin scientific name: Lactiplantibacillus plantarum It was deposited on July 28, 2023, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 28056.

[0012] The plant lactobacillus YYS-J1 was isolated from oil residue in an oil mill in Yiting County, Yunnan Province. Gram-positive and catalase-negative strains were selected and identified as plant lactobacillus by physiological and biochemical experiments and molecular biology, and named plant lactobacillus YYS-J1.

[0013] This strain has the following characteristics:

[0014] (1) It has the ability to degrade nitrite;

[0015] (2) It has the ability to degrade trimethylamine;

[0016] (3) Acid and bile salt resistance.

[0017] The present invention also provides the application of Lactobacillus plantarum YYS-J1 as a preservative in the preservation of aquatic products, as described above.

[0018] The present invention also provides the application of Lactobacillus plantarum YYS-J1 as described above as a starter in the preparation of fermented foods.

[0019] The present invention also provides a fermentation agent whose components include *Lactobacillus plantarum* YYS-J1 as described above.

[0020] In one embodiment, the fermenting agent is a vegetable fermenting agent or a meat product fermenting agent; the components of the fermenting agent include *Lactobacillus plantarum* YYS-J1 (e.g., added in the form of freeze-dried powder) and other fermentation aids (e.g., other *Lactobacillus plantarum* powders, maltodextrin, etc.).

[0021] The present invention also provides a freeze-dried powder, the components of which include *Lactobacillus plantarum* YYS-J1 as described above.

[0022] In one embodiment, the number of viable bacteria in the freeze-dried powder is 3000-8000 billion CFU / g.

[0023] The present invention also provides a microbial agent, wherein the components of the microbial agent comprise the freeze-dried powder as described above, and / or, the components of the microbial agent comprise *Lactobacillus plantarum* YYS-J1 as described above.

[0024] In one embodiment, the microbial agent further includes other ingredients, which are existing ingredients suitable for microbial agents, such as at least one of prebiotics, fillers, acidulants, solvents, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, lubricants, wetting agents, stabilizers, flow aids, flavoring agents, preservatives, coating materials, fragrances, anti-adhesion agents, binding agents, thickeners, and inclusion agents.

[0025] This invention also provides a method for preparing freeze-dried powder, which includes the following steps:

[0026] The *Lactobacillus plantarum* YYS-J1 strain described above was fermented until the viable count in the fermentation broth was ≥3 × 10⁻⁶. 9 When the concentration of cfu / mL is reached, fermentation is stopped; *Lactobacillus plantarum* YYS-J1 cells are collected from the fermentation broth, and the *Lactobacillus plantarum* YYS-J1 cells are mixed with a freeze-drying protectant and then subjected to emulsification and embedding treatment to obtain an emulsion; the emulsion is freeze-dried, pulverized, and *Lactobacillus plantarum* YYS-J1 freeze-dried powder is obtained.

[0027] The *Lactobacillus plantarum* YYS-J1 provided by this invention has the following beneficial effects:

[0028] The *Lactobacillus plantarum* YYS-J1 provided by this invention has strong acid and bile salt resistance and good tolerance in artificial gastric and intestinal fluids. It has the function of efficiently degrading nitrite and trimethylamine. When applied to the preparation of fermented foods, it can significantly reduce nitrite and trimethylamine content, improve product safety and added value, and lay the foundation for large-scale production of green and healthy traditional fermented foods. Its efficient trimethylamine degradation function allows it to be used as a preservative for aquatic products. Similarly, when applied to probiotic products, it can remove nitrite, trimethylamine, and their compounds from the body. Attached Figure Description

[0029] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0030] Figure 1 This is a standard curve of sodium nitrite used in Example 3;

[0031] Figure 2 This is a graph showing the change in nitrite content during the fermentation of Chinese cabbage by *Lactobacillus plantarum* YYS-J1 in Example 5.

[0032] Figure 3 This is a graph showing the pH changes during the fermentation of Chinese cabbage by *Lactobacillus plantarum* YYS-J1 in Example 5.

[0033] Figure 4 This is a graph showing the change in total acid content during the fermentation of Chinese cabbage by *Lactobacillus plantarum* YYS-J1 in Example 5.

[0034] Figure 5 This is a graph showing the pH changes during the fermentation of meat using *Lactobacillus plantarum* YYS-J1 in Example 6.

[0035] Figure 6 The graph shows the change in nitrite content during the fermentation of meat by Lactobacillus plantarum YYS-J1 in Example 6.

[0036] Figure 7 The graph shows the change in TVB-N content during storage of sausage fermented with Lactobacillus plantarum YYS-J1 in Example 6.

[0037] Figure 8 The graph shows the change in trimethylamine content during storage of sausage fermented with Lactobacillus plantarum YYS-J1 in Example 6.

[0038] Figure 9 The graph shows the effect of soaking in *Lactobacillus plantarum* YYS-J1 suspension on the TVB-N content of tilapia in Example 7.

[0039] Figure 10 This is a graph showing the effect of soaking in *Lactobacillus plantarum* YYS-J1 bacterial suspension on the trimethylamine content of tilapia in Example 7.

[0040] Figure 11 This is a process flow diagram of fermenting sauerkraut in Example 5;

[0041] Figure 12 This is a process flow diagram of the fermented meat product in Example 6. Detailed Implementation

[0042] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0043] Unless otherwise specified, the percentage sign "%" in the implementation plan refers to the mass percentage; the percentage of solution "% (m / v)" refers to the number of grams of solute contained in 100 mL of solution; "V / V" refers to the volume percentage.

[0044] This invention provides a *Lactobacillus plantarum* YYS-J1:

[0045] Lactobacillus plantarum ( Lactiplantibacillus plantarum YYS-J1 was deposited on July 28, 2023, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 28056.

[0046] Source: The plant lactobacillus YYS-J1 was isolated from oil residue in an oil mill in Yiting County, Yunnan Province. Gram-positive and catalase-negative strains were selected and identified as plant lactobacillus by physiological and biochemical experiments and molecular biology, and named plant lactobacillus YYS-J1.

[0047] Colony morphology: milky white, raised surface, round, with neat edges, smooth and moist surface.

[0048] Functions: It has strong resistance to acid and bile salts and is well tolerated in artificial gastric and intestinal fluids; it has a high efficiency in degrading trimethylamine and nitrite.

[0049] This invention also provides an operational example of a method for preparing freeze-dried products:

[0050] Lactobacillus plantarum YYS-J1 was fermented and cultured until the viable count in the fermentation broth was ≥3×10⁻⁶. 9 When the concentration of cfu / mL is reached, fermentation is stopped; *Lactobacillus plantarum* YYS-J1 cells are collected from the fermentation broth, and the *Lactobacillus plantarum* YYS-J1 cells are mixed with a freeze-drying protectant and then subjected to emulsification and embedding treatment to obtain an emulsion; the emulsion is freeze-dried, pulverized, and *Lactobacillus plantarum* YYS-J1 freeze-dried powder is obtained.

[0051] Specifically, the preparation steps include the following:

[0052] 1) Preparation of seed culture of Lactobacillus plantarum YYS-J1;

[0053] 2) Seed culture expansion;

[0054] 3) Seed culture fermentation to obtain fermentation broth;

[0055] 4) Centrifuge the fermentation broth to obtain bacterial sludge;

[0056] 5) After mixing the bacterial sludge with the freeze-drying protectant, emulsify and embed it to obtain an emulsion;

[0057] 6) The emulsion is freeze-dried and pulverized to obtain freeze-dried powder of Lactobacillus plantarum YYS-J1.

[0058] The specific practical examples of the above steps are as follows:

[0059] Step 1) Preparation of seed culture: Inoculate Lactobacillus plantarum YYS-J1 into a test tube containing seed culture medium for activation to obtain primary seed culture;

[0060] Preferably, the activation time is 18-24 hours; the activation temperature is 32-37°C; and the activation is performed in anaerobic conditions.

[0061] Step 2) Expanding the culture:

[0062] Step 2.1) The primary seed culture is inoculated into an Erlenmeyer flask containing seed culture medium and activated under anaerobic conditions to obtain the secondary seed culture;

[0063] Preferably, the primary seed culture is inoculated into the seed culture medium and activated under anaerobic conditions for 16–24 h; the inoculation amount of the primary seed culture into the seed culture medium is preferably 3–5% (volume percentage); and the anaerobic culture temperature of the primary seed culture is preferably 32–37 °C.

[0064] Step 2.2) Inoculate the secondary seed culture into the seed culture medium for fermentation to obtain the tertiary seed culture;

[0065] Preferably, the anaerobic fermentation time of the secondary seed liquid is 15-16 hours, the fermentation temperature is 35-40℃, and the inoculum amount is 3%-5% (volume percentage).

[0066] Step 3) Seed culture fermentation to obtain fermentation broth:

[0067] The tertiary seed culture was inoculated into a fermentation medium for fermentation (in a seed tank) to obtain the fermentation broth;

[0068] The preferred anaerobic fermentation time is 8–10 h, the preferred fermentation temperature is 35–40 °C, the preferred inoculum amount is 3–5% (volume percentage), the preferred seed tank pressure is 0.03–0.05 MPa, the preferred stirring speed is 50–100 r / min, the preferred fermentation pH is 5.0–6.0, and the alkali supplementation solution is 10–30% NaOH solution.

[0069] Step 4) Centrifuge the fermentation broth to obtain bacterial sludge;

[0070] Preferably, the centrifugation conditions for the fermentation broth are a rotation speed of 5000-8000 r / min and a centrifugation time of 10-20 min.

[0071] Step 5) Mix the bacterial sludge with the freeze-drying protectant and then emulsify and embed it to obtain an emulsion;

[0072] Preferably, the emulsification and embedding time is 15–50 min, and the emulsification and embedding temperature is preferably 10–25°C, more preferably 15–20°C. The mass ratio of bacterial cells (bacterial sludge) to freeze-drying protectant is preferably 1:(0.5–1.0); the freeze-drying protectant comprises 5–20% trehalose, 5–10% skim milk powder, 0–5% monosodium glutamate, 0–5% glycerol, and the balance being water.

[0073] Step 6) After obtaining the emulsion, the emulsion is pre-frozen, vacuum freeze-dried, and pulverized to obtain plant lactobacillus YYS-J1 freeze-dried powder;

[0074] The preferred temperature for pre-freezing is -45 to -40°C, and the preferred time is 6 to 10 hours; the preferred temperature for vacuum freezing is -45 to -30°C, and the preferred time is 30 to 40 hours; the preferred vacuum degree for vacuum freeze drying is 3 to 5 Pa.

[0075] Preferably, the seed culture medium used in the above steps comprises, by mass percentage: 1-3% glucose, 1-3% peptone, 0.5-3% yeast extract, 0.8-1.2% beef extract, 0.2-1% anhydrous sodium acetate, 0.02-0.08% magnesium sulfate, 0.02-0.08% manganese sulfate, 0.08-0.15% Tween 80, and the balance being water, with a pH of 6.2-7.0;

[0076] The fermentation medium used was: 1.5-2.5% glucose, 2-2.5% yeast peptone, 1-3% yeast extract, 0.4-0.8% anhydrous sodium acetate, 0.04-0.06% magnesium sulfate, 0.03-0.05% manganese sulfate, 0.1-0.15% Tween 80 and the balance being water.

[0077] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0078] Example 1: Isolation and identification of Lactobacillus plantarum YYS-J1

[0079] Aseptic sampling was performed on oil residue using the plate coating method. 5g of oil residue sample was placed in a sterile homogenizing bag, and 45mL of 0.85% physiological saline was added and homogenized thoroughly to obtain the sample. 100 μL of the sample was then serially diluted 10-fold. -2 10 -3 10 -4 100 μL of the sample was spread on MRS solid medium containing 2.5% CaCO3 and incubated upside down at 37°C for 24 h.

[0080] Select colonies with good growth and large calcium dissolution zones, and repeatedly isolate and purify them using the streak plate method until a single colony is obtained. Name the isolated strain YYS-J1, add glycerol and preserve it in a -80℃ culture chamber.

[0081] The MRS liquid culture medium formula is as follows: 10.0 g beef extract, 20.0 g glucose, 10.0 g tryptone, 5.0 g yeast extract, 1.0 mL Tween 80, 2.0 g dipotassium hydrogen phosphate, 2.0 g ammonium citrate, 5.0 g anhydrous sodium acetate, 0.5 g magnesium sulfate, 0.25 g manganese sulfate monohydrate, 1.0 L deionized water, pH 6.5 (adding 1.5% agar makes it MRS solid culture medium).

[0082] The screened and purified strain YYS-J1 was subjected to Gram staining and catalase tests, and its physiological and biochemical indicators were measured. The results were compared with those in Bergey's Manual of Systematic Bacteriology, 8th Edition, for preliminary identification of the species. The tests showed that the screened strain YYS-J1 stained purple with a Gram stain, indicating a positive result. It was rod-shaped, catalase-negative, and did not form spores.

[0083] DNA was extracted from YYS-J1 according to the instructions of the bacterial DNA extraction kit and amplified by PCR. The amplified product was sent to Guangzhou Qingke Biotechnology Co., Ltd. for sequencing. The strain YYS-J1 was identified as *Lactobacillus plantarum*. The phylogenetic tree is shown below. Figure 1 As shown, its gene sequence is as follows:

[0084]

[0085] Example 2: Preparation of Lyophilized Powder of Lactobacillus plantarum YYS-J1

[0086] The culture medium and lyophilization protectant used in this embodiment are as follows:

[0087] The seed culture medium, by weight percentage, consists of: 3% glucose, 2% peptone, 1.5% yeast extract, 1% beef extract, 0.5% anhydrous sodium acetate, 0.05% magnesium sulfate, 0.06% manganese sulfate, 0.1% Tween 80, and the balance being water, with a pH of 6.8.

[0088] The fermentation medium, by weight percentage, consists of 2.3% glucose, 2.5% yeast peptone, 2.2% yeast extract, 0.6% anhydrous sodium acetate, 0.05% magnesium sulfate, 0.03% manganese sulfate, 0.1% Tween 80, and the balance being water.

[0089] The freeze-drying protectant comprises, by weight percentage, 10% trehalose, 8% skim milk powder, 1% monosodium glutamate, 0.5% glycerol and the balance being water.

[0090] The preparation of freeze-dried powder includes the following steps:

[0091] 1) Inoculate Lactobacillus plantarum YYS-J1 into a test tube containing seed culture medium for activation, and anaerobic culture in a constant temperature incubator at 37℃ for 20h to obtain the first-grade seed liquid.

[0092] 2) Inoculate the primary seed culture into an Erlenmeyer flask containing seed culture medium at a volume of 3% (volume percentage), and anaerobic culture in a constant temperature incubator at 37℃ for 16 hours to obtain the secondary seed culture.

[0093] 3) Inoculate the secondary seed culture into an Erlenmeyer flask containing seed culture medium at a volume of 5% (volume percentage), and anaerobic culture in a constant temperature incubator at 37℃ for 16 hours to obtain the tertiary seed culture.

[0094] 4) Inoculate the tertiary seed culture into a fermenter containing fermentation medium at a volume percentage of 5%. The preferred pressure in the seed tank is 0.04 MPa, the preferred stirring speed is 50 r / min, the pH for alkali supplementation fermentation is 5.5, and the culture is anaerobic at 37°C for 9 h.

[0095] 5) The viable count of the fermentation broth is ≥5×10⁻⁶ 9 When the cfu / mL level is reached, stop fermentation and collect the cells by centrifugation (6000 r / min, 15 min).

[0096] 6) The bacterial cells and lyophilization protectant were emulsified and encapsulated at a mass ratio of 1:0.8 at 20°C for 15 minutes.

[0097] 7) Pre-freeze the emulsion at -40℃ for 6 hours, then freeze-dry it under vacuum at -30℃ for 32 hours at a vacuum degree of 3 Pa. Pulverize to obtain *Lactobacillus plantarum* YYS-J1 freeze-dried powder, with a viable count of 4.6 × 10⁻⁶. 11 cfu / g.

[0098] Example 3: Study on the acid and bile salt resistance characteristics of Lactobacillus plantarum YYS-J1

[0099] 1. Study on acid and bile salt resistance

[0100] The *Lactobacillus plantarum* YYS-J1 lyophilized powder obtained in Example 2 was inoculated at a rate of 1% (w / v) into MRS liquid medium (pH 6.0, pH 2.0, and bile salt concentrations of 0% (w / v) and 0.4% (the MRS liquid medium was the same as in Example 1) at 37°C for 3 hours. Viable cell counts were performed using the plate count method. The results are shown in Tables 1 and 2.

[0101] Table 1. Study on acid resistance of Lactobacillus plantarum YYS-J1

[0102]

[0103] As shown in Table 1, under pH 2.0 and pH 6.0 conditions, Lactobacillus plantarum YYS-J1 still had a sufficient number of viable bacteria that could pass through the gastric environment after 3 hours.

[0104] Table 2. Study on bile salt tolerance of Lactobacillus plantarum YYS-J1

[0105]

[0106] It can be seen that at a bile salt concentration of 0.4%, Bacillus plantarum YYS-J1 still has a sufficient number of viable bacteria after 3 hours, indicating that Bacillus plantarum YYS-J1 has good bile salt tolerance.

[0107] 2. Tolerance in artificial gastric and intestinal fluids

[0108] A certain amount of pepsin was dissolved in a PBS buffer solution at pH 3.0 and adjusted to a final concentration of 3 g / L to obtain artificial gastric fluid. A certain amount of trypsin was dissolved in a PBS buffer solution at pH 8.0 and adjusted to a final concentration of 1 g / L to obtain artificial intestinal fluid. Both artificial gastric and artificial intestinal fluids were filtered through a 0.22 μm filter membrane under sterile conditions.

[0109] The *Lactobacillus plantarum* YYS-J1 lyophilized powder obtained in Example 2 was inoculated at a 1% (w / v) in MRS liquid medium (consistent with Example 1) and artificial gastric fluid, and cultured at 37°C for 3 hours. Then, the fermentation broth from the artificial gastric fluid was inoculated into artificial intestinal fluid at a 10% inoculation rate and cultured at 37°C for 3 hours. Viable bacteria were then counted using the plate count method. The results are shown in Table 3.

[0110] Table 3. Tolerance of *Lactobacillus plantarum* YYS-J1 in simulated gastric and intestinal fluids.

[0111]

[0112] It can be seen that even after passing through artificial gastric fluid and then artificial intestinal fluid, a sufficient number of live bacteria remain in the YYS-J1 bacteria. This indicates that Lactobacillus plantarum YYS-J1 has good tolerance and can successfully reach the intestines to colonize and exert its effects.

[0113] Example 3: Salt and nitrate tolerance and nitrite degradation of Lactobacillus plantarum YYS-J1

[0114] 1. Salt and nitrate resistance

[0115] The *Lactobacillus plantarum* YYS-J1 seed culture was inoculated into MRS liquid medium and cultured for 24 h. Then, at a 3% inoculation rate, it was inoculated into MRS liquid medium containing 0%, 2%, 4%, and 6% NaCl, as well as MRS liquid medium containing 50 mg / kg, 100 mg / kg, and 150 mg / kg NaNO2, and cultured anaerobicly at 37°C for 24 h. After the culture was completed, the test tubes were removed, shaken well, and the values ​​were measured at 600 nm using a spectrophotometer. The growth of the lactic acid bacteria was determined based on the principle of turbidimetry. Detailed test results are shown in Tables 4-5 (the MRS liquid medium used was the same as in Example 1).

[0116] Table 4 Salt tolerance of Lactobacillus plantarum YYS-J1

[0117]

[0118] Table 5. Nitrate resistance of Lactobacillus plantarum YYS-J1

[0119]

[0120] As shown in Tables 4 and 5, *Lactobacillus plantarum* YYS-J1 can tolerate 6% NaCl and 150 mg / kg NaNO2 with a very high survival rate. Traditional fermented sauerkraut commonly uses 6% NaCl, and traditional fermented meat uses 100 mg / kg nitrite, indicating that *Lactobacillus plantarum* YYS-J1 can be used in traditional fermented foods, including fermented meat and fermented sauerkraut.

[0121] 2. Sodium nitrite degradation ability

[0122] The *Lactobacillus plantarum* strain YYS-J1 was inoculated into MRS liquid medium and activated three times. Then, 3% (v / v) of the inoculum was inoculated into MRS liquid medium containing 100 mg / kg NaNO2 and cultured anaerobicly at 37°C for 24 h. The residual NaNO2 content in the culture medium was determined according to the naphthylethylenediamine hydrochloride method in the national standard GB 5009.33—2016 "Determination of Nitrite and Nitrate in Food". The results are detailed in Table 6. The standard curve established for the determination of sodium nitrite residue is shown in Table 6. Figure 1 As shown (the MRS liquid culture medium used in this example is the same as that in Example 1).

[0123] Table 6. Sodium nitrite degradation rate of Lactobacillus plantarum YYS-J1

[0124]

[0125] As shown in Table 6, the degradation rate of NaNO2 by strain Lactobacillus plantarum YYS-J1 in NaNO2-containing medium is as high as 99.06±0.89%. Its application in traditional fermented foods is beneficial for the efficient removal of nitrites and provides safer and more reliable traditional fermented foods.

[0126] Example 4: Degradation of trimethylamine by Lactobacillus plantarum YYS-J1

[0127] The activated Lactobacillus plantarum YYS-J1 was inoculated at a 3% (v / v) in MRS liquid medium containing 100 mg / kg trimethylamine (as in Example 1) and incubated at 37°C for 24 h.

[0128] Weigh approximately 10 g (accurate to 0.001 g) of the prepared sample into a 50 mL plastic centrifuge tube, add 20 mL of 5% trichloroacetic acid solution, homogenize for 1 min, centrifuge at 4000 r / min for 5 min, add a small amount of defatted cotton to a glass funnel, filter the supernatant into a 50 mL volumetric flask, and repeat the above extraction process twice with 15 mL and 10 mL of 5% trichloroacetic acid solution respectively. Combine the filtrates and dilute to 50 mL with 5% trichloroacetic acid solution. Determine the trimethylamine residue using the first method of headspace gas chromatography-mass spectrometry (HGC-MS) according to GB5009.179—2016 "National Food Safety Standard - Determination of Trimethylamine Content in Food". The results are detailed in Table 7.

[0129] Table 7. Trimethylamine degradation rate of Lactobacillus plantarum YYS-J1

[0130]

[0131] As shown in Table 7, Lactobacillus plantarum YYS-J1 has a good degradation effect on trimethylamine and has broad application prospects.

[0132] Example 5: Application of Lactobacillus plantarum YYS-J1 in fermented sauerkraut

[0133] 1. Process flow as follows Figure 11 As shown;

[0134] 2. Recipe: Chinese cabbage, 3% salt, 3% sugar, 0.5% cooking wine (the percentages refer to the weight ratio of each ingredient to the Chinese cabbage).

[0135] 3. Experimental Groups:

[0136] Natural fermentation group (aseptic addition);

[0137] Inoculate with *Lactobacillus plantarum* YYS-J1 (inoculum size: 10). 7 CFU / g);

[0138] 4. Making fermented sauerkraut

[0139] Select vegetables without insect holes and of good quality according to the process flow, wash them clean, cut them, and dry them. Add salt, sugar, cooking wine, and fermentation agent powder according to steps 2 and 3, seal the container, and ferment at room temperature for 7 days.

[0140] 5. Indicator Measurement

[0141] The pH value, total acid (calculated as lactic acid), and nitrite content were measured daily until the fermentation ended after 7 days.

[0142] Experimental results:

[0143] like Figure 2 , Figure 3 , Figure 4 As shown ( Figure 2-4 The horizontal axis represents the number of days. After inoculation with the starter culture, the rate of pH decrease increased significantly. After 3 days, the pH value tended to stabilize, and the final pH was significantly lower than that of the naturally fermented group. The total acidity after inoculation with the starter culture was significantly lower than that of the naturally fermented group (pH value was lower than that of the naturally fermented group), providing a better-flavored and safer fermented sauerkraut product.

[0144] After 7 days of fermentation, the sodium nitrite content in the naturally fermented group was 22.12 mg / kg, which is still higher than the national standard of 20 mg / kg, posing a serious food safety hazard. After inoculation with Lactobacillus plantarum YYS-J1, the sodium nitrite degradation rate was significantly improved, with a degradation rate of over 98% after 3 days. The residual amount of sodium nitrite was very low, close to 0, which meets the national standard.

[0145] This indicates that Lactobacillus plantarum YYS-J1, when used as a starter culture for sauerkraut and other traditional fermented foods, can significantly increase the fermentation speed, shorten the fermentation time to 3 days, improve the flavor, effectively remove nitrites, and enhance product safety.

[0146] Example 6: Application of Lactobacillus plantarum YYS-J1 in fermented meat products

[0147] 1.1 For detailed process flow, please refer to [link / reference]. Figure 12 :

[0148] 1.2 Fermented Sausage Recipe

[0149] Raw materials and ingredients: 75% lean meat, 25% fat meat. Other seasonings by weight of meat: 3.5% salt, 0.5% sucrose, 0.5% glucose, 0.01% NaNO2, 0.05% vitamin C, 5% ice water, 1% cooking wine, 0.6% chili powder, and 0.5% Sichuan peppercorn powder.

[0150] 1.3 Experimental Grouping:

[0151] Natural fermentation group (aseptic addition);

[0152] Inoculate with *Lactobacillus plantarum* YYS-J1 (fermentation agent combination, inoculation amount of 10). 7 CFU / g);

[0153] 1.4 Making Fermented Sausage

[0154] Select fresh pork (lean to fat ratio 1:3), add seasonings and marinate at 4℃ for 12 hours, add fermentation agent according to 1.3, stuff into sausages, and ferment at 30℃ for 24 hours.

[0155] 1.5 Index Measurement

[0156] (1) Determination of pH and nitrite content

[0157] The pH of the fermented meat was measured every 6 hours until 24 hours, and the nitrite content was measured after fermentation was completed.

[0158] The pH determination method is as follows: Weigh 5g of sample, crush it in a mortar, add 50g of distilled water, and measure the pH value. The nitrite determination method refers to the naphthylethylenediamine hydrochloride method for determining the residual NaNO2 content in the national standard GB 5009.33—2016 "Determination of Nitrite and Nitrate in Food".

[0159] (2) Determination of trimethylamine and volatile basic nitrogen content

[0160] The fermented sausages were air-dried at room temperature and hung in a cool, well-ventilated place. The content of trimethylamine and volatile basic nitrogen in the fermented sausages was measured every 15 days.

[0161] Trimethylamine determination: Remove sausage casings, take approximately 100g, mince using a meat grinder or finely chop with a knife, and mix well. Weigh approximately 10g (accurate to 0.001g) of the prepared sample into a 50mL plastic centrifuge tube, add 20mL of 5% trichloroacetic acid solution, homogenize for 1min, centrifuge at 4000r / min for 5min, add a small amount of defatted cotton to a glass funnel, filter the supernatant into a 50mL volumetric flask, repeat the above extraction process twice with 15mL and 10mL of 5% trichloroacetic acid solution respectively, combine the filtrates, and dilute to 50mL with 5% trichloroacetic acid solution. Determine the trimethylamine residue using the first method of headspace gas chromatography-mass spectrometry (HGC-MS) according to GB5009.179—2016 "National Food Safety Standard - Determination of Trimethylamine Content in Food".

[0162] Volatile basic nitrogen (TVB-N) determination: The determination was performed using a semi-micro distillation method. After removing the sausage casing, 10.00 g of the minced sausage was weighed and placed in a beaker. Distilled water was added to a final volume of 100 mL. After thorough stirring and standing for 30 minutes, the mixture was filtered. 5 mL of the filtrate was mixed with 5 mL of a 10 g / L magnesium oxide suspension and distilled in the reaction chamber of a distiller. The distilled liquid was titrated with 0.01 mol / L standard hydrochloric acid solution. The TVB-N value was calculated based on the amount of hydrochloric acid consumed. The TVB-N content in the fermented sausage was measured every 7 days. Currently, a TVB-N value exceeding 30 mg / 100 g is generally considered sufficient to determine that the meat product has spoiled.

[0163] (3) Test results:

[0164] like Figure 5 , Figure 6 As shown ( Figure 5 The horizontal axis represents time (in hours). After 24 hours of fermentation, the naturally fermented group showed a very slow fermentation rate (the rate of pH decrease reflects the fermentation speed; a slow pH decrease indicates a slow fermentation rate). Adding a starter culture significantly increased the fermentation speed, greatly reducing the fermentation time for meat products. After 24 hours of fermentation, the residual nitrite content in the naturally fermented group was as high as 41.35 mg / kg, exceeding the national standard of 30 mg / kg, posing a significant safety hazard. After fermentation with the starter culture, the sodium nitrite content significantly decreased, meeting national standards, with a sodium nitrite removal rate exceeding 90%.

[0165] like Figure 7 , Figure 8 As shown ( Figure 7-8 The horizontal axis represents time (in days). After 60 days of storage, the TVB-N and trimethylamine contents of the *Lactobacillus plantarum* YYS-J1 fermented sausage were significantly lower in the experimental group compared with the naturally fermented group. This indicates that fermentation with *Lactobacillus plantarum* YYS-J1 can extend the storage time of the sausage and increase the safety of the product.

[0166] Example 7: Application of Lactobacillus plantarum YYS-J1 in the preservation of aquatic products

[0167] 1. Experimental materials

[0168] Fresh tilapia, purchased from the market.

[0169] 2. Experimental Methods

[0170] 1% of the lyophilized *Lactobacillus plantarum* YYS-J1 powder obtained in Example 2 was added to 0.85% physiological saline and vortexed until homogeneous to obtain a bacterial suspension. Fresh aquatic products were cut into pieces approximately 15g each. The experimental group was soaked in the homogenized bacterial suspension for 30 minutes, while the control group was soaked in 0.85% physiological saline for 30 minutes. After drying, the pieces were wrapped in plastic wrap, placed in resealable bags, and stored at 4°C. The trimethylamine content and volatile basic nitrogen content were measured at 1, 3, 5, and 7 days.

[0171] Trimethylamine determination: Remove fish scales and skin, take about 100g of muscle, mince it using a meat grinder or finely chop it with a knife, and mix well. Weigh about 10g (accurate to 0.001g) of the prepared sample into a 50mL plastic centrifuge tube, add 20mL of 5% trichloroacetic acid solution, homogenize for 1min, centrifuge at 4000r / min for 5min, add a little defatted cotton to a glass funnel, filter the supernatant into a 50mL volumetric flask, repeat the above extraction process twice with 15mL and 10mL of 5% trichloroacetic acid solution respectively, combine the filtrates and make up to 50mL with 5% trichloroacetic acid solution. Determine the trimethylamine residue using the first method of headspace gas chromatography-mass spectrometry in GB5009.179—2016 "National Food Safety Standard - Determination of Trimethylamine Content in Food".

[0172] Determination of volatile basic nitrogen: The determination was performed using a semi-micro distillation method. Weigh 10.00 g of the chopped substance and place it in a beaker. Add distilled water to a final volume of 100 mL, stir evenly, let stand for 30 min, and then filter. Take 5 mL of the filtrate and mix it with 5 mL of 10 g / L magnesium oxide suspension in the reaction chamber of a distiller for distillation. Titrate the distilled liquid with 0.01 mol / L standard hydrochloric acid solution. Calculate the volatile basic nitrogen value based on the amount of hydrochloric acid consumed.

[0173] like Figure 9 , Figure 10 As shown ( Figure 9-10 The horizontal axis represents time (in days). After soaking in a suspension of Lactobacillus plantarum YYS-J1, the TVB-N and trimethylamine contents of tilapia were significantly reduced compared with the control group, indicating that Lactobacillus plantarum YYS-J1 helps preserve aquatic and seafood products.

[0174] Example 8: Preparation of Vegetable Fermentation Agent

[0175] Mix 10-20 grams of *Lactobacillus plantarum* YYS-J1 freeze-dried powder, 10-20 grams of *Lactobacillus plantarum* BXM2 powder, and 60-80 grams of maltodextrin obtained in Example 2, package them, and you will get a fermentation agent product capable of fermenting 1 ton of vegetables. Wash and cut commercially available vegetables, add salt, sucrose, cooking wine, and the fermentation agent, seal and ferment at room temperature for 3 days to obtain a healthy fermented vegetable product with a rich sour aroma, crisp texture, and low nitrite content.

[0176] Example 9: Preparation of meat product fermentation agent

[0177] Mix 20-40 grams of *Lactobacillus plantarum* YYS-J1 freeze-dried powder obtained in Example 2 with 60-80 grams of maltodextrin, and package to obtain a fermentation agent product capable of fermenting 1 ton of meat products. Mince commercially available frozen pork, add salt, sucrose, cooking wine, etc., marinate for 12 hours, then add the fermentation agent and ferment at 37℃ for 24 hours to obtain healthy fermented meat products with low nitrite content that meet national standards.

[0178] It should be noted that when using *Lactobacillus plantarum* YYS-J1 freeze-dried powder or other forms of *Lactobacillus plantarum* YYS-J1 as a starter culture in food fermentation, it can be used in combination with other fermentation strains and starter culture adjuvants. Other fermentation strains and starter culture adjuvants can be existing strains, adjuvants and additives, including but not limited to maltodextrin and *Lactobacillus plantarum* BXM2 powder as described in the examples.

[0179] Example 10 Preparation of Seafood Preservative

[0180] Package 10-20 grams of the freeze-dried Lactobacillus plantarum YYS-J1 powder obtained in Example 2 to obtain a preservative that can preserve 1 ton of seafood and fish products. Clean commercially available fish by removing the internal organs, add one packet of the preservative product, water, and an appropriate amount of salt, soak for 30 minutes, dry, and vacuum package to extend the shelf life of seafood and aquatic products.

[0181] Example 11: Application of Lactobacillus plantarum YYS-J1 freeze-dried powder in the preparation of solid beverages

[0182] By mixing 2% of the freeze-dried Lactobacillus plantarum YYS-J1 powder obtained in Example 2, 35% of resistant dextrin, 24% of erythritol, 20% of fructooligosaccharides, and 19% of galactooligosaccharides (by mass), a solid beverage capable of removing nitrite and trimethylamine can be obtained.

[0183] It should be noted that when adding *Lactobacillus plantarum* YYS-J1 freeze-dried powder or other forms of *Lactobacillus plantarum* YYS-J1 to food, the ingredients can be existing food additives and excipients, including but not limited to the resistant dextrin described in the examples.

[0184] Based on the results of the above embodiments, the *Lactobacillus plantarum* YYS-J1 provided by the present invention has the following properties and effects:

[0185] 1. It has strong resistance to acid and bile salts, good tolerance in artificial gastric and intestinal fluids, and excellent salt and nitrate resistance.

[0186] 2. It can efficiently degrade nitrite and trimethylamine:

[0187] 2.1 The study investigated the traditional fermentation process of this product. Compared with the control group, it significantly reduced the nitrite content, shortened the fermentation cycle, enhanced the product flavor, improved product safety and added value, laying the foundation for large-scale production of green and healthy traditional fermented foods.

[0188] In the examples, YYS-J1 bacteria were used to ferment sauerkraut, shortening the fermentation cycle to 3 days and increasing the total acidity and flavor of the fermentation. YYS-J1 bacteria were also used to ferment meat products, significantly reducing nitrite and trimethylamine content and effectively preventing the production of carcinogens such as nitrosamines.

[0189] 2.2 The study investigated its effect on the preservation of aquatic products. Compared with the control group, it can significantly reduce the level of trimethylamine, remove the fishy smell of aquatic products, reduce the level of volatile basic nitrogen, and extend the shelf life of aquatic products, laying the foundation for the application of lactic acid bacteria in the preservation of aquatic and seafood products.

[0190] It should be noted that:

[0191] (1) Application of strains:

[0192] In the example embodiment, the *Lactobacillus plantarum* YYS-J1 is applied to a solid beverage based on the functional characteristics of YYS-J1 in degrading trimethylamine. According to the above design concept and mechanism of action, it can also be other food types suitable for and designed for human consumption, including but not limited to solid beverages, liquid beverages, etc.

[0193] Example illustration: Lactobacillus plantarum YYS-J1 can be used as a fermentation agent in sauerkraut fermentation and meat product fermentation, and can also be used as a preservative to preserve fish. According to the above design concept, this strain can be applied to the fermentation of various vegetables or meat products, and can be applied to the preservation of various aquatic products, including but not limited to the example illustration.

[0194] (2) Definition:

[0195] The term "food" as used herein is used in a broad sense, including human food and drink. In some embodiments, the food product is suitable for and designed for human consumption. This application can be used to prepare solid dosage forms such as powders, tablets, and gels, and also to disperse in liquids to prepare liquid dosage forms, including but not limited to the embodiments described herein.

[0196] As used in this article, "aquatic products" refers to all aquatic animal and plant products and their processed products produced by marine and freshwater fisheries. Seafood refers to a wide range of products from the ocean that are edible or usable.

[0197] "OD" is an abbreviation for optical density, also known as absorbance. The energy difference before and after light passes through a analyte is the energy absorbed by the analyte. At a specific wavelength, there is a quantitative relationship between the concentration of the same analyte and the absorbed energy, which can be used to determine the concentration of the analyte. "ODx" is the optical density value measured when the wavelength is set to x nm. It is a standard indicator for tracking the density of microorganisms in liquid cultures, usually used to indicate bacterial cell density. The method for measuring "OD" values ​​is existing technology, and its principles and methods will not be elaborated here.

[0198] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

[0199] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A plant lactobacillus that can degrade nitrite ( Lactiplantibacillus plantarum YYS-J1, characterized in that, Its accession number is CGMCC No. 28056.

2. The use of *Lactobacillus plantarum* YYS-J1 as described in claim 1 in the preparation of products for degrading nitrite.

3. The use of *Lactobacillus plantarum* YYS-J1 as described in claim 1 in the preparation of products for degrading trimethylamine.

4. The application of Lactobacillus plantarum YYS-J1 as a preservative in the preservation of aquatic products as described in claim 1.

5. The application of *Lactobacillus plantarum* YYS-J1 as a starter culture in the preparation of fermented foods, as described in claim 1.

6. A fermenting agent, characterized in that: Its components include *Lactobacillus plantarum* YYS-J1 as described in claim 1.

7. A freeze-dried powder, characterized in that: Its components include *Lactobacillus plantarum* YYS-J1 as described in claim 1.

8. A microbial agent, characterized in that: The microbial agent comprises the freeze-dried powder as described in claim 7, or the microbial agent comprises *Lactobacillus plantarum* YYS-J1 as described in claim 1.

9. A method for preparing a freeze-dried powder, characterized in that: Includes the following steps: Lactobacillus plantarum YYS-J1 was fermented and cultured until the viable count in the fermentation broth was ≥3×10⁻⁶. 9 Stop fermentation when cfu / mL; Collect *Lactobacillus plantarum* YYS-J1 cells from the fermentation broth, mix the *Lactobacillus plantarum* YYS-J1 cells with a freeze-drying protectant, and then perform emulsification and encapsulation treatment to obtain an emulsion. The emulsion was freeze-dried and pulverized to obtain freeze-dried powder of Lactobacillus plantarum YYS-J1; The *Lactobacillus plantarum* YYS-J1 mentioned herein is the *Lactobacillus plantarum* YYS-J1 as described in claim 1.

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