Staphylococcus carnosus strain lYN3 with drug resistance and nitrite reduction and application thereof

Abstract

The application provides a drug-resistant and nitrite-reducing Staphylococcus carnosus LYN3 and an application thereof, and belongs to the technical field of microorganisms. The strain is preserved in the Guangdong Microbial Culture Collection Center, and the preservation number is GDMCC No: 68349. The strain can resist 10% high salt, resist gastric acid and intestinal bile salt, has excellent intestinal colonization ability, has good self-aggregation and cell surface hydrophobicity, and has DPPH, ABTS and hydroxyl radical scavenging antioxidant activity. The strain can efficiently degrade nitrite in food, and the 24h nitrite degradation rate is as high as 94.44%. The strain can be widely applied to the preparation of fermented food and pickled food, can reduce the nitrite content of finished products, can improve the antioxidant capacity of products, and has good development and application prospect.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a drug-resistant, nitrite-lowering Staphylococcus aureus LYN3 and its applications. Background Technology

[0002] Fermented and pickled foods are traditional Chinese specialties. Products such as chili sauce, cured meat, and pickled vegetables are widely loved by consumers for their unique flavors. However, the production process of these products commonly suffers from the industry-wide problem of excessive nitrite levels. Raw vegetables and meats naturally contain endogenous nitrates. During the fermentation and pickling process, nitrate reductase produced by bacteria reduces nitrates to nitrites. Nitrites not only react with amines in food to form potent carcinogenic nitrosamines, but also irritate the human digestive tract and cause blood oxygen transport disorders. Long-term intake poses significant health risks. Therefore, how to effectively reduce the nitrite content in fermented and pickled foods is a critical issue that urgently needs to be addressed in the food processing industry.

[0003] Existing methods for degrading nitrite mainly fall into three categories: physical, chemical, and microbial. Physical degradation relies on high temperatures and ultraviolet irradiation, which easily damages the flavor, color, and nutritional components of food, resulting in high processing costs and limited degradation efficiency. Chemical methods often involve adding exogenous antioxidants such as vitamin C and tea polyphenols, which can reduce nitrite, but these exogenous additives alter the original flavor of the product. Furthermore, there are issues such as limits on additive usage and consumer resistance to chemical additives. In contrast, microbial degradation utilizes the bacterial strain's own metabolic enzyme system to decompose nitrite, offering advantages such as safety, naturalness, and preservation of food flavor, making it the mainstream direction for current industry research and development.

[0004] Currently, most strains used to reduce nitrite in fermented foods are lactic acid bacteria. However, traditional lactic acid bacteria have significant limitations: First, pickling and fermentation systems generally contain 4% to 10% high concentrations of salt, and most lactic acid bacteria have weak salt tolerance. In high-salt environments, live bacteria die rapidly, and their nitrite degradation activity decreases significantly. Second, after being orally administered to the human body, strains need to tolerate the low pH of gastric acid and the high bile salt environment of the intestine before they can colonize and exert their probiotic effects. Conventional lactic acid bacteria have poor acid and bile salt tolerance, resulting in low survival rates in the gastrointestinal tract. Third, fermented foods are prone to oxidative browning during storage, and traditional lactic acid bacteria lack sufficient antioxidant capacity to simultaneously address the problem of product oxidation and deterioration. Fourth, food processing and the human intestinal environment present various antibiotic pressures. Most existing nitrite-reducing strains are sensitive to antibiotics. If antibiotic residues are present during processing or in the intestines, the strains will quickly become inactive, limiting their application scenarios.

[0005] Staphylococcus carnosus is a naturally occurring, safe symbiotic microorganism found in meat products and fermented sauces. Current research confirms its role in flavor compound formation, but existing isolated strains still have limitations: most Staphylococcus carnosus strains have low nitrite degradation efficiency, with a 24-hour degradation rate of less than 70%; they have poor survival rates under high salt and artificial gastric / intestinal stress, making them difficult to adapt to high-salt curing systems and unable to stably colonize the human gut; they also generally lack antioxidant activity to scavenge free radicals, making it difficult to simultaneously meet the dual needs of nitrite reduction and color preservation with a single function; and most existing strains are antibiotic-sensitive, easily becoming ineffective in processing environments or intestinal environments with antibiotic residues.

[0006] Furthermore, in the production of high-salt fermented sauces, such as chili sauce, the raw material microbial communities are complex, the fermentation cycle is long, and the peak nitrite level persists for a long time. There is a lack of commercially available *Staphylococcus aureus* strains that can simultaneously adapt to high-salt fermentation environments, possess high nitrite degradation capabilities, strong gastrointestinal tolerance, multidrug resistance, and antioxidant activity. Therefore, developing a *Staphylococcus aureus* strain with high salt tolerance, gastric acid and bile salt tolerance, strong intestinal colonization, efficient nitrite degradation, and antioxidant effects is of significant practical importance for solving the problems of excessive nitrite and oxidative spoilage in fermented and pickled foods, and for expanding the industrial application of natural microbial fermentation agents. Summary of the Invention

[0007] In view of this, the present invention provides a drug-resistant, nitrite-reducing Staphylococcus aureus LYN3 and its application to solve the above problems.

[0008] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0009] This invention provides a Staphylococcus carnosus LYN3 strain, deposited at the Guangdong Provincial Center for Microbial Culture Collection on May 26, 2026, at the address of Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC No:68350.

[0010] The present invention also provides a method for preparing the bacterial culture of Staphylococcus carinatum LYN3, wherein the Staphylococcus carinatum LYN3 is inoculated into a fermentation medium for fermentation culture to obtain the bacterial culture.

[0011] Preferably, the fermentation culture temperature is 36~38℃ and the rotation speed is 120~220 rpm.

[0012] The present invention also provides a bacterial suspension of Staphylococcus aureus LYN3 prepared according to the preparation method described above.

[0013] Preferably, the effective viable bacteria count in the bacterial solution is 1×10⁻⁶.7 ~9×10 11 CFU / mL.

[0014] The present invention also provides the application of the aforementioned Staphylococcus aureus LYN3 or the aforementioned bacterial solution in the degradation of nitrite in food.

[0015] The present invention also provides the application of the aforementioned Staphylococcus aureus LYN3 or the aforementioned bacterial solution in the preparation of fermented and / or pickled foods.

[0016] Preferably, the fermented food includes chili sauce.

[0017] By adopting the above technical solution, this invention has the following beneficial effects: The strain is deposited at the Guangdong Provincial Microbial Culture Collection Center, with accession number GDMCC No:68349. This strain is tolerant to 10% high salt concentrations, gastric acid, and intestinal bile salts, exhibiting excellent intestinal colonization ability. It possesses good self-polymerization and cell surface hydrophobicity, and also exhibits DPPH, ABTS, and hydroxyl radical scavenging antioxidant activity. This strain can efficiently degrade nitrite in food, with a nitrite degradation rate as high as 94.44% within 24 hours. This strain can be widely used in the preparation of fermented and pickled foods, both reducing the nitrite content of the finished product and enhancing its antioxidant capacity, showing promising development and application prospects. Attached Figure Description

[0018] Figure 1 The colony morphology of strain LYN3.

[0019] Figure 2 Phylogenetic tree of strain LYN3 based on 16S rDNA sequence.

[0020] Biological Preservation Instructions

[0021] The taxonomic name of the Staphylococcus carnosus LYN3 of this invention is Staphylococcus carnosus. It is deposited at the Guangdong Provincial Center for Microbial Culture Collection on May 26, 2026, at the address of Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC No:68349. Detailed Implementation

[0022] 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.

[0023] Example 1

[0024] 1. Isolation and purification of LYN3 strain

[0025] Handmade chili sauce from Sanming, Fujian Province, was selected. 1g of the chili sauce sample was weighed under sterile conditions, added to 9mL of water, and mixed thoroughly using a pipette to obtain the microbial sample. This microbial sample was then gently mixed using a pipette to obtain the stock solution. The stock solution was serially diluted to prepare 10... -1 10 -2 10 -3 10 -4 10 -5 10 -6 10 -7 10 -8 The bacterial suspension was prepared and spread onto LB agar plates using the plate spread method. The plates were then incubated for 24 hours. After incubation, colony morphology was observed. Based on the morphology, colonies were selected and purified by continuous streak subculturing onto fresh LB agar plates. This process was repeated until a pure culture with consistent morphology was obtained.

[0026] 2. Identification of LYN3 strain

[0027] (1) Microbiological characteristics

[0028] The obtained strain was inoculated onto LB agar plates and incubated at 37°C for 48 hours. Colony characteristics were then observed, such as... Figure 1 As shown. The colonies of this strain are round, about 2 mm in diameter. They are round, raised, with neat edges, smooth and moist surfaces, and appear opaque milky white or pale yellow.

[0029] (2) Molecular biological characteristics

[0030] Single colonies were picked and cultured in LB liquid medium at 37°C and 200 rpm for 24 h with shaking. Using the bacterial culture as a template, the 16S rDNA sequence was amplified using universal primers 27F / 1492R.

[0031] The PCR amplification reaction system was 25 μL, including 12.5 μL of 2×Taq PCR Master Mix, 1.0 μL of upstream primer 27F (10 μmol / L), 1.0 μL of downstream primer 1492R (10 μmol / L), 1.0 μL of bacterial culture, and 9.5 μL of ddH2O.

[0032] PCR amplification conditions: 94℃ pre-denaturation for 5 min, 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 90 s, 35 cycles; final extension at 72℃ for 10 min; amplified products were stored at 4℃. The amplified products were separated and identified by 1% agarose gel electrophoresis. The PCR products were sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. The 16S rDNA of strain LYN3 is shown in SEQ ID NO.1.

[0033]

[0034] BLAST homology alignment of the 16S rDNA sequence showed that this strain shared 99.79% similarity with *Staphylococcus carnosus*. A phylogenetic tree was constructed using the Neighbor-Joining (NJ) method with MEGA 6.0 software. The position of strain LYN3 in the constructed library is shown below. Figure 2 As shown, this strain was named Staphylococcus aureus LYN3.

[0035] Example 2

[0036] (1) NaCl tolerance test of LYN3 strain

[0037] Centrifuge the culture medium of well-grown strains to collect bacterial cells. Inoculate the cells with 2% (v / v) NaCl in LB liquid culture medium containing 4%, 7%, and 10% (w / v) NaCl. Incubate at 37°C. Take samples at 0h and 3h. Dilute the samples serially with physiological saline and spread them on LB plates for colony counting. Calculate the survival rate of the strains. The results are shown in Table 1.

[0038] Strain survival rate (%) = N t / N0×100 (Formula 1), where N t N is the colony count after 3 hours, and N0 is the colony count after 0 hours.

[0039] Table 1. NaCl tolerance of strain LYN3

[0040] strain survival rate 169.08 133.03 129.90

[0041] As shown in Table 1, when the NaCl content reaches 10%, the survival rate of strain LYN3 is as high as 133.03%, indicating that the strain has a strong salt tolerance.

[0042] (2) Bile salt tolerance assay of LYN3 strain

[0043] Take 1 mL of LYN3 strain culture medium with good growth, centrifuge, collect the bacterial cells, and resuspend them in LB liquid medium with bile salt content of 0.0%, 0.3%, and 0.5% (w / v), respectively. Incubate the LYN3 strain suspension at 37°C, and take samples at 0 h and 3 h. Dilute the sample solution serially with physiological saline, spread it on LB plates, and count the colonies. The survival rate of the strain was calculated in the same way as above. The results are shown in Table 2.

[0044] Table 2. Bile salt tolerance of strain LYN3

[0045] strain survival rate 161.50 47.50 10.74

[0046] As shown in Table 2, when the bile salt concentration is 0.5%, the survival rate of strain LYN3 is still maintained at 10.74%, indicating that the strain has a certain bile salt tolerance and can adapt to the intestinal bile salt stress environment.

[0047] (3) Determination of acid resistance of LYN3 strain

[0048] Take 1 mL of the culture medium of the strain with good growth, centrifuge, collect the bacterial cells, and resuspend them in LB liquid culture medium with pH 3.0, 4.0, and 5.0, respectively. Incubate at 37℃, and take samples at 0 h and 3 h. Dilute the samples serially with physiological saline, spread them on LB plates, and count the colonies. The survival rate of the strain was calculated in the same way as above, and the results are shown in Table 3.

[0049] Table 3. Acid resistance of strains

[0050] strain survival rate 63.87 109.09 118.30

[0051] As shown in Table 3, the survival rate of strain LYN3 was 63.87% under strongly acidic conditions of pH 3.0, indicating that the strain has good acid resistance and can tolerate the acidic stress environment of gastric acid.

[0052] (4) Tests on the tolerance of LYN3 strain to artificial gastric and intestinal fluids

[0053] The culture medium of LYN3 strain grown to the logarithmic phase was centrifuged, and the bacterial cells were collected. The cells were resuspended in simulated gastric and intestinal fluids and incubated at 37℃ and 150 rpm on a shaker. Samples of simulated gastric fluid were collected at 0 h and 2.5 h, and samples of simulated intestinal fluid were collected at 0 h, 2 h, and 4 h. The samples were serially diluted with physiological saline, plated, and colony counted. The survival rate of the strain was calculated using the same method as above. The results showed that the survival rate of LYN3 strain after 2.5 h in simulated gastric fluid was 65.64%; the survival rates after 2 h and 4 h in simulated intestinal fluid were 73.32% and 69.58%, respectively. This indicates that the strain can tolerate the acidic stress of gastric fluid, successfully reach the intestine, and survive stably in the intestinal environment, demonstrating good gastrointestinal tolerance.

[0054] (5) Determination of surface hydrophobicity of LYN3 strain

[0055] The culture medium of the strain with good growth was centrifuged at 4500 rpm and 4℃ for 5 min. The supernatant was discarded, and the bacterial cells were collected. The bacterial cells were then resuspended in PBS buffer, and the process was repeated 2-3 times. The concentration of the strain was adjusted with PBS buffer so that the absorbance of the bacterial suspension at 600 nm was 0.6 ± 0.02, which was recorded as A0. Then, 3 mL of the bacterial suspension was taken, 1 mL of ethyl acetate / xylene was added, and the mixture was mixed well. The mixture was allowed to stand at room temperature for 20 min until liquid phase separation occurred. The absorbance of the aqueous phase was measured at 600 nm and recorded as A. The hydrophobicity of the strain surface was calculated according to Formula 2. The experiment was repeated three times, and the average value was taken. The results are shown in Table 4.

[0056] Strains' surface hydrophobicity (%) = (1-A0 / A)×100 (Formula 2)

[0057] Table 4. Surface hydrophobicity of strains (%)

[0058] Hydrophobicity (%) 26.39 45.31

[0059] The data in Table 4 show that strain LYN3 exhibits certain surface hydrophobicity to both xylene and ethyl acetate, indicating that the strain possesses strong cell surface hydrophobicity, which is beneficial for its adhesion and colonization in the intestinal mucosa.

[0060] (6) Determination of self-aggregation of LYN3 strain

[0061] The culture medium of the strain with good growth was centrifuged to collect the bacterial cells. The cells were then washed with PBS and the strain concentration was adjusted so that the absorbance of the bacterial suspension at 600 nm was 0.6 ± 0.02, denoted as A0. The bacterial suspension was incubated at 37℃. The absorbance of the upper bacterial suspension was measured at 600 nm after 2 h, 4 h, and 24 h, and denoted as A. The self-aggregation of the strain was calculated according to the formula. The experiment was repeated three times, and the average value was taken. The results are shown in Table 5.

[0062] Strain self-aggregation (%) = (1-A0 / A)×100 (Formula 3)

[0063] Table 5. Strains' self-aggregation rate (%)

[0064] Strain self-aggregation (%) 38.85 43.14 69.67

[0065] The data in Table 5 show that the self-aggregation ability of strain LYN3 continuously increases over time, reaching 69.67% after 24 hours, indicating that it has good self-aggregation performance and strong intestinal adhesion potential.

[0066] Example 3. Determination of antioxidant activity of LYN3 strain

[0067] Take the culture medium of the strain with good growth condition, centrifuge, collect the bacterial cells, resuspend in PBS buffer, and adjust the bacterial cell concentration to 10. 7 10 8 10 9 The CFU / mL concentration was used to determine the antioxidant activity of the strain.

[0068] (1) Take 1 mL of bacterial suspension and add 1 mL of freshly prepared DPPH ethanol solution (0.2 mM). After vortexing and mixing, react at 37°C in the dark for 30 min. Measure the absorbance at 517 nm using an ELISA reader and record it as A. 517s Use PBS buffer instead of bacterial suspension and add 1 mL of DPPH ethanol solution as a blank; record the absorbance as A. 517b Using ethanol instead of DPPH ethanol solution, 1 mL of bacterial suspension was added as a control, and its absorbance was recorded as A. 517c The DPPH free radical scavenging ability of the strain was calculated according to Formula 4. The experiment was repeated three times, and the results showed that the DPPH free radical scavenging rate of strain LYN3 was 43.04%.

[0069] DPPH free radical scavenging rate of strain (%) = (A 517b +A 517c -A 517s ) / A 517c ×100 (Formula 4)

[0070] (2) Mix equal volumes of ABTS (7.4 mM) and K2S2O8 (2.6 mM), adjust the absorbance to 734 nm with anhydrous ethanol, and measure the absorbance to be 0.7 ± 0.02 to obtain the ABTS working solution. Add 0.2 mL of bacterial suspension to 0.8 mL of ABTS working solution, vortex until homogeneous, and react at 37°C in the dark for 10 min. Measure the absorbance at 734 nm using a microplate reader and record it as A. 734s ; 0.8 mL of ABTS working solution was added to replace the bacterial suspension as a blank, and its absorbance was recorded as A. 734b Using ethanol instead of ABTS working solution, 0.2 mL of bacterial suspension was added as a control, and its absorbance was recorded as A. 734c The ABTS free radical scavenging ability of the strain was calculated according to Formula 5. The experiment was repeated three times, and the results showed that the ABTS scavenging rate of strain LYN3 was 33.44%.

[0071] ABTS clearance rate of strain (%) = (A 734b +A 734c -A 734s ) / A 734c ×100 (Formula 5)

[0072] (3) Take 1 mL of bacterial suspension (effective viable count 10) 8Add 1 mL of o-phenanthroline (2.5 mM) solution and 1 mL of PBS buffer to a solution containing cfu / mL. Vortex until homogeneous, then add 1 mL of FeSO4 (25 mM) solution. Vortex again, then add 1 mL of H2O2 (3 mM). Incubate at 37°C in the dark for 30 min. Measure the absorbance at 736 nm and record it as A. 736s ; Use PBS buffer instead of bacterial suspension as a blank, and record its absorbance as A. 736b Using deionized water instead of FeSO4 (2.5 mM) solution as a control, its absorbance is recorded as A. 736c The hydroxyl radical scavenging capacity of the strain was calculated according to Formula 6. The experiment was repeated three times, and the results showed that the hydroxyl radical scavenging rate of strain LYN3 was 58.34%.

[0073] Strain hydroxyl radical scavenging rate (%) = (A 736b +A 736c -A 736s ) / A 736c ×100 (Formula 6)

[0074] Table 6. Results of antioxidant activity assay for strain LYN3

[0075] 43.04% 33.44% 58.34%

[0076] As shown in Table 6, the DPPH scavenging rate of strain LYN3 was 43.04%, the ABTS scavenging rate was 33.44%, and the ·OH scavenging rate was 58.34%, indicating that strain LYN3 has excellent antioxidant properties.

[0077] Example 4. Nitrite degradation rate of strain LYN3

[0078] The nitrite content was determined using the ethylenediamine hydrochloride method. A culture medium of a well-growing strain was taken, centrifuged, and the bacterial cells were collected. The bacterial concentration was adjusted to 10⁻⁶. 9 CFU / mL was inoculated into LB broth with a final sodium nitrite concentration of 0.25 mg / mL at a 2% (v / v) inoculation rate and incubated at 37°C. A blank control without bacterial culture medium was used. Bacterial culture was collected at 6 h, 12 h, and 24 h, centrifuged, and the bacterial cells were discarded. The supernatant was used as the sample solution, and the nitrite content was determined using the ethylenediamine hydrochloride method. The results are shown in Table 7.

[0079] Table 7. Nitrite degradation rates at 6h, 12h, and 24h

[0080] Nitrite degradation rate (%) 3.22±0.77ab 39.00±0.02b 94.44±1.79a

[0081] Note: Different lowercase letters in the same line indicate significant differences between groups (P < 0.05), the same applies below.

[0082] As shown in Table 7, the degradation effect of nitrite by strain LYN3 gradually increased with the extension of culture time, and the nitrite degradation rate reached 94.44% after 24 h, demonstrating excellent nitrite degradation potential.

[0083] Example 5. Drug resistance of LYN3 strain

[0084] Take 10 μL of the culture medium of the strain with good growth condition and spread it on LB agar plate. Use tweezers to place the drug susceptibility test strips of penicillin, ampicillin, ceftriaxone, gentamicin, tetracycline, erythromycin, lincomycin and chloramphenicol in the center of the plate. Incubate statically in a 37℃ incubator. After the inhibition zone appears, measure its diameter with vernier calipers and evaluate the drug susceptibility. The results are shown in Table 8.

[0085] Table 8. Drug resistance of LYN3 strain

[0086]

[0087] The results in Table 8 show that LYN3 is only moderately sensitive to tetracycline, and is resistant to gentamicin, lincomycin, chloramphenicol, erythromycin, ampicillin, penicillin and ceftriaxone, exhibiting multidrug resistance characteristics.

[0088] As can be seen from the above embodiments, the present invention provides a drug-resistant, nitrite-reducing Staphylococcus aureus LYN3 and its application. The Lactobacillus rhamnosus LRX1 of the present invention has excellent salt resistance, antioxidant, nitrite degradation performance and gastrointestinal tolerance characteristics.

[0089] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A strain of Staphylococcus aureus ( Staphylococcus carnosus LYN3, characterized in that, It is deposited at the Guangdong Provincial Center for Microbial Culture Collection on May 26, 2026, at the address of Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC No:68349.

2. The method for preparing the bacterial culture of Staphylococcus aureus LYN3 according to claim 1, characterized in that, The Staphylococcus aureus LYN3 was inoculated into a fermentation medium for fermentation culture to obtain the bacterial solution.

3. The preparation method according to claim 2, characterized in that, The fermentation culture temperature is 36~38℃, and the rotation speed is 120~220 rpm.

4. The Staphylococcus aureus LYN3 bacterial suspension prepared by the preparation method according to claim 2 or 3.

5. The bacterial solution according to claim 4, characterized in that, The effective viable bacteria count in the bacterial solution is 1×10⁻⁶. 7 ~9×10 11 CFU / mL.

6. The application of Staphylococcus aureus LYN3 as described in claim 1 or the bacterial solution as described in claim 5 in the degradation of nitrite in food.

7. The use of Staphylococcus aureus LYN3 as described in claim 1 or the bacterial solution as described in claim 5 in the preparation of fermented and / or pickled foods.

8. The application according to claim 7, characterized in that, The fermented food includes chili sauce.

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