Application of Staphylococcus simulans HZ01 in replacing part of nitrite in meat products

Abstract

The present invention relates to the technical field of microbial food fermentation, and specifically discloses the application of Staphylococcus simulans HZ01 to replace part of nitrite in meat products. The detection of the concentration of nitrite includes the following steps: (i) preparing an NB medium containing an appropriate concentration of KNO3 / NaNO3; (ii) inoculating the Staphylococcus simulans HZ01 bacterial solution into the NB medium in step (i) for cultivation; (iii) centrifuging the fermentation broth at intervals during the cultivation process and collecting the supernatant; (iv) detecting the nitrite content in the supernatant of step (iii). The present invention has the characteristics of good processing adaptability, relatively high protease and lipase activities, good nitrate reductase activity, and better flavor of the fermented meat products.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial food fermentation, and particularly relates to the application of mimicking Staphylococcus HZ01 to replace part of nitrite in meat products. Background Art

[0002] At present, the microbial starters widely used in fermented meat products are mainly lactic acid bacteria and coagulase-negative staphylococci (CNS). Among them, lactic acid bacteria are responsible for the acidification of meat in the initial stage of fermentation. By producing lactic acid, the pH value of raw meat is reduced, proteins are coagulated, and the hardness is increased. In addition, lactic acid bacteria can also inhibit the growth of harmful bacteria, thereby improving the microbial safety of fermented meat products. CNS plays an important role in the flavor formation and color stability of fermented meat products. Research shows that it is Staphylococcus, rather than lactic acid bacteria, that plays an important role in the flavor formation of fermented meat products. CNS can reduce nitrate to nitrite and then to nitrous oxide, prevent rancidity through peroxide decomposition, and produce flavor and aromatic compounds through proteolysis and lipolysis. These functions play an important role in the formation of the quality characteristics of fermented meat products and are considered important "flavor bacteria" in fermented meat products. There is a rich population diversity of CNS in fermented meat products. For example, Staphylococcus xylosus, S. saphylococcus, S. equorum, and S. carnosus are the most dominant species in European traditional fermented sausages, while S. xylosus, S. saphylococcus, S. epidermidis, S. sciuri, and S. simulans are more dominant in Chinese fermented meat products. Italian law allows Staphylococcus xylosus, S. carnosus, and S. simulans to be used as starters for fermented sausage production (Republica Italiana, 1995). In 2016, China included S. vitulinus, S. xylosus, and S. carnosus in the List of Bacterial Strains Allowed for Use in Foods (Food Safety Standards and Monitoring and Assessment Department, China, No. 4, 2016).

[0003] S. xylosus and S. carnosus are currently the two most commonly used commercial CNS starters in the industry. However, commercial starters do not always maintain an advantage in competing with local microbial populations, which leads to the loss of the ideal sensory characteristics of fermented meat products. Therefore, screening suitable starters from local microbial populations can better adapt to the local meat product processing environment and be more competitive due to their unique metabolic capabilities. Research has shown that S. simulans is one of the dominant CNS populations with excellent fermentation performance in Chinese fermented meat products. Summary of the Invention

[0004] To solve the above technical problems existing in the current local microbial populations during food fermentation, the present invention provides an application of a Staphylococcus simulans HZ01 to replace part of nitrite in meat products. It has the characteristics of good processing adaptability, relatively high protease and lipase activities, good nitrate reductase activity, and better flavor of the fermented meat products.

[0005] The first technical solution of the present invention: a Staphylococcus simulans HZ01, the preservation number of the Staphylococcus simulans HZ01 is GDMCC NO. 62410; the preservation unit of the Staphylococcus simulans HZ01 is the Guangdong Provincial Culture Collection Center of Microorganisms; the preservation name of the Staphylococcus simulans HZ01 is Staphylococcus simulans HZ01; the preservation time of the Staphylococcus simulans HZ01 is April 24, 2022. The Staphylococcus simulans HZ01 in the present invention has strong metabolic capabilities and can maintain an advantage in competing with local microbial populations when used as a starter, with strong competitiveness, making the sensory characteristics of the fermented meat products relatively ideal and being able to better adapt to the local meat product processing environment; the Staphylococcus simulans HZ01 in the present invention has good protease activity and the ability to decompose sarcoplasmic proteins, and can degrade 58.1% of the sarcoplasmic protein bands within 72 hours and significantly reduce 41.9% of the sarcoplasmic protein bands; the Staphylococcus simulans HZ01 of the present invention has good nitrate reductase activity and can efficiently reduce nitrate to nitrite during application, utilizing the nitrate in fermented meat products, thereby reducing the dosage of nitrite during meat product processing; the Staphylococcus simulans HZ01 of the present invention can metabolize leucine to produce 3-methylbutanal, increasing the types and contents of volatile flavor substances, and having excellent aroma-producing properties; the Staphylococcus simulans HZ01 of the present invention can significantly increase the contents of esters, aldehydes, and acids in fermented meat products, among which vinyl acetate, methyl butyrate, methyl hexanoate, n-hexanal, acetic acid, and isovaleric acid are increased most significantly, and can significantly improve the quality of fermented meat products.

[0006] Preferably, the gene accession number of the Staphylococcus simulans HZ01 is OM758216, and the specific nucleotide sequence is shown in SEQ ID NO.1. According to the nucleotide sequence of the Staphylococcus simulans HZ01, the Staphylococcus simulans HZ01 in the present invention is an independent evolutionary branch formed during the evolutionary process and is a new strain of the Staphylococcus simulans.

[0007] Preferably, the determination of the gene sequence of the Staphylococcus simulans HZ01 includes the following steps:

[0008] (a) Extract the total DNA of the Staphylococcus simulans HZ01 using a bacterial genomic DNA extraction kit;

[0009] (b) Use a PCR reaction system to perform 16s full-length amplification on the total DNA extracted in step (a);

[0010] (c) After the 16s full-length amplification is completed, perform gel cutting and purification, and then perform electrophoresis sequencing. The measured sequence is the full-length 16s DNA sequence of the Staphylococcus simulans HZ01. In the present invention, a bacterial genomic DNA extraction kit is used to extract the total DNA of the Staphylococcus simulans HZ01, which is fast, convenient, and has good stability and extraction purity; the PCR reaction system can quickly increase the total DNA of the Staphylococcus simulans HZ01 extracted; performing gel cutting and purification on the 16s full-length amplification sequence is for more convenient and accurate electrophoresis sequencing later, ensuring the accuracy of the final measured full-length 16s DNA sequence of the Staphylococcus simulans HZ01.

[0011] Preferably, the PCR reaction system includes 2 µL of 10×Ex Taq buffer, 1.6 µL of 2.5 mM dNTP Mix, 0.6 µL of 5p Primer 1, 0.6 µL of 5p Primer 2, 2 µL of Template, 1 µL of 5u Ex Taq, and 12.2 µL of dd H2O. Each component acts synergistically to quickly increase the total DNA of the Staphylococcus simulans HZ01 extracted.

[0012] Preferably, the dosage of the PCR reaction system is 15 µL to 25 µL. More preferably, the dosage of the PCR reaction system is 18 µL to 22 µL. It is selected according to the total DNA of the Staphylococcus simulans HZ01 to be amplified, taking into account the sufficiency of the total DNA amplification of the Staphylococcus simulans HZ01 and the appropriateness of the dosage.

[0013] Preferably, the 16s full-length amplification in the PCR reaction system includes the following steps:

[0014] (b01) React the total DNA extracted in step (a) in a PCR reaction system at a temperature of 95 °C for 5 min;

[0015] (b02) After step (b01) is completed, continue to react at a temperature of 95 °C for 30 s;

[0016] (b03) After step (b02) is completed, continue to react at a temperature of 55 °C for 30 s;

[0017] (b04) After step (b03) is completed, continue to react at a temperature of 72 °C for 1 min;

[0018] (b05) Repeat steps (b02) to (b04) 24 times;

[0019] (b06) After step (b05) is completed, extend at a temperature of 72 °C for 10 min;

[0020] (b07) After step (b06) is completed, keep it at a temperature of 10 °C to complete the amplification of the full-length 16s of the total DNA of Staphylococcus simulans HZ01. The strict limitation of the specific temperature and time is to ensure the completeness of the amplification of the full-length 16s of the total DNA of Staphylococcus simulans HZ01, taking into account both the completeness and timeliness of the amplification.

[0021] The second technical solution of the present invention: a Staphylococcus simulans HZ01 bacterial agent, which is prepared from Staphylococcus simulans HZ01. Preparing Staphylococcus simulans HZ01 into a bacterial agent form is more convenient for use in daily production and life.

[0022] Preferably, the preparation of the Staphylococcus simulans HZ01 bacterial agent includes the following steps:

[0023] (A) Inoculate Staphylococcus simulans HZ01 into NB medium for culture to obtain a bacterial liquid;

[0024] (B) When the concentration of Staphylococcus simulans HZ01 in the bacterial liquid obtained in step (A) reaches 1.0×10 8 log 10 CFU / mL to 9.9×10 9 log 10 CFU / mL, perform centrifugation;

[0025] (C) After centrifugation, add sterilized skim milk to resuspend;

[0026] (D) After resuspension, perform freeze-drying to obtain a bacterial agent with a concentration of 1.0×10 10 log 10 CFU / g to 9.9×1011 log 10 Powdery Staphylococcus simulans HZ01 bacterial agent with 10 CFU / g. NB medium has a good cultivation effect on Staphylococcus simulans HZ01; when the concentration of Staphylococcus simulans HZ01 in the bacterial liquid is 1.0×10 8 log 10 CFU / mL - 9.9×10 9 log 10 CFU / mL, centrifugation is carried out because Staphylococcus simulans HZ01 at the limited concentration already has a relatively high concentration, and the number of Staphylococcus simulans HZ01 within the limited concentration range is also sufficient. If the concentration is low, the number of Staphylococcus simulans HZ01 is insufficient, and if the concentration is high, more time is required for cultivation; centrifugation is for better preparation of the concentrated solution; adding sterilized skim milk for resuspension is for cryoprotection of the cultivated Staphylococcus simulans HZ01; freeze-drying is for better preparation of the solid bacterial agent; the powdery Staphylococcus simulans HZ01 bacterial agent prepared by the method of the present invention has a relatively high concentration and good performance. More preferably, when the concentration of Staphylococcus simulans HZ01 in the bacterial liquid obtained in step (A) is 3.0×10 8 log 10 CFU / mL - 7.9×10 9 log 10 CFU / mL, centrifugation is carried out. More preferably, when the concentration of Staphylococcus simulans HZ01 in the bacterial liquid obtained in step (A) is 5.0×10 8 log 10 CFU / mL - 5.9×10 9 log 10 CFU / mL, centrifugation is carried out. More preferably, when the concentration of Staphylococcus simulans HZ01 in the bacterial liquid obtained in step (A) is 7.0×10 8 log 10 CFU / mL - 3.9×10 9 log 10 CFU / mL, centrifugation is carried out. More preferably, when the concentration of Staphylococcus simulans HZ01 in the bacterial liquid obtained in step (A) is 9.0×10 8 log 10 CFU / mL - 1.9×10 9 log 10 CFU / mL, centrifugation is carried out.

[0027] Preferably, the NB medium in step (A) includes 10 g / L of peptone, 3.0 g / L of beef extract, and 5.0 g / L of sodium chloride, which makes the NB medium have a better cultivation effect on Staphylococcus simulans HZ01.

[0028] Preferably, the pH value of the NB medium in step (A) is 7 to 7.4. More preferably, the pH value of the NB medium in step (A) is 7.1 to 7.3. This provides a suitable pH environment for the cultivation of Staphylococcus simulans HZ01.

[0029] Preferably, the dosage of the NB medium in step (A) is 0.5 L to 2 L. More preferably, the dosage of the NB medium in step (A) is 1 L to 1.5 L. The dosage of the NB medium has reached the limiting concentration according to the amount of Staphylococcus simulans HZ01, which can ensure the good cultivation of Staphylococcus simulans HZ01.

[0030] Preferably, the cultivation in step (A) is static cultivation. Static cultivation is beneficial to the good proliferation of Staphylococcus simulans HZ01.

[0031] Preferably, the cultivation time in step (A) is 24 h to 72 h. More preferably, the cultivation time in step (A) is 36 h to 60 h. More preferably, the cultivation time in step (A) is 40 h to 48 h. The limitation of the cultivation time here is to ensure the rapid proliferation of Staphylococcus simulans HZ01 and make it reach the plateau phase.

[0032] Preferably, the cultivation temperature in step (A) is 15°C to 65°C. More preferably, the cultivation temperature in step (A) is 25°C to 55°C. More preferably, the cultivation temperature in step (A) is 30°C to 45°C. More preferably, the cultivation temperature in step (A) is 35°C to 40°C. The limitation of the temperature here is more suitable for the cultivation of Staphylococcus simulans HZ01.

[0033] Preferably, the centrifugation speed in step (B) is 5000 rpm / min to 20000 rpm / min. More preferably, the centrifugation speed in step (B) is 8000 rpm / min to 18000 rpm / min. More preferably, the centrifugation speed in step (B) is 10000 rpm / min to 15000 rpm / min. A suitable centrifugation speed can more efficiently separate the cells from the liquid.

[0034] Preferably, the centrifugation time in step (B) is 10 min to 20 min. More preferably, the centrifugation time in step (B) is 12 min to 18 min. The limitation of the centrifugation time can fully achieve the solid-liquid separation.

[0035] Preferably, the centrifugation temperature in step (B) is 2°C to 6°C. More preferably, the centrifugation temperature in step (B) is 3°C to 5°C. Low-temperature centrifugation is used, and the limitation of the centrifugation temperature is to fully ensure the activity of Staphylococcus simulans HZ01 during centrifugation.

[0036] Preferably, the mass concentration of the sterilized skim milk in step (C) is 0.05% m / v to 0.5% m / v. More preferably, the mass concentration of the sterilized skim milk in step (C) is 0.1% m / v to 0.4% m / v. Even more preferably, the mass concentration of the sterilized skim milk in step (C) is 0.2% m / v to 0.3% m / v. The sterilized skim milk with a defined concentration can sufficiently protect Staphylococcus simulans HZ01 after cultivation during lyophilization.

[0037] Preferably, the dosage of the sterilized skim milk in step (C) is 1 ml to 5 ml. More preferably, the dosage of the sterilized skim milk in step (C) is 2 ml to 4 ml. The limitation of the dosage of the sterilized skim milk is to sufficiently protect Staphylococcus simulans HZ01 after cultivation during lyophilization.

[0038] Preferably, the lyophilization temperature in step (D) is -90°C to -70°C. More preferably, the lyophilization temperature in step (D) is -85°C to -75°C. Even more preferably, the lyophilization temperature in step (D) is -83°C to -78°C. The limitation of the lyophilization temperature can ensure sufficient and rapid drying while better guaranteeing the activity of Staphylococcus simulans HZ01 in the Staphylococcus simulans HZ01 bacterial agent.

[0039] Preferably, the lyophilization time in step (D) is 24 h to 72 h. More preferably, the lyophilization time in step (D) is 36 h to 60 h. Even more preferably, the lyophilization time in step (D) is 40 h to 50 h. The limitation of the lyophilization time can fully ensure complete drying.

[0040] The third technical solution of the present invention: The application of Staphylococcus simulans HZ01 in degrading sarcoplasmic proteins. Staphylococcus simulans HZ01 in the present invention has good protease activity and the ability to decompose sarcoplasmic proteins and myofibrillar proteins. It can degrade 58.1% of the sarcoplasmic protein bands within 72 hours and significantly reduce 41.9% of the sarcoplasmic protein bands.

[0041] Preferably, the detection of the degradation result of Staphylococcus simulans HZ01 on sarcoplasmic proteins includes the following steps;

[0042] (S01) Extract sarcoplasmic proteins and measure the concentration of sarcoplasmic proteins using a Lowry protein concentration kit;

[0043] (S02) Incubate the sarcoplasmic proteins, glucose, and the Staphylococcus simulans HZ01 bacterial solution obtained in step (S01) together;

[0044] After incubation is completed, centrifuge the bacterial solution;

[0045] Take 2×SDS loading buffer and mix it with the supernatant after centrifugation in step (S03), and then perform a water bath;

[0046] Take 10%-12% Bio-Rad precast gel myosin and standard protein, and load the samples in step (S04) onto the 10%-12% Bio-Rad precast gel myosin and standard protein respectively;

[0047] After loading, perform electrophoresis;

[0048] After electrophoresis is completed, stain with Coomassie Brilliant Blue R-250, and obtain the degradation result of myosin by Staphylococcus simulans HZ01 from the staining result. The Lowry protein concentration kit can quickly and accurately measure the myosin concentration; the addition of glucose provides a carbon source for the growth of Staphylococcus simulans HZ01; centrifugation is to collect protein precipitates and exclude the interference of irrelevant substances, so as to better ensure the accuracy of the detection result; the addition of 2×SDS loading buffer is to adjust the supernatant into a sample to be detected that is better for detection; the water bath is to fully unfold the secondary structure of the protein; the 10%-12% Bio-Rad precast gel can fully separate the myosin bands; the standard protein can be used as a reference; electrophoresis can fully ensure the separation of protein bands; the staining effect of Coomassie Brilliant Blue R-250 is good, and it can make the myosin bands fully develop color; the whole detection method reflects the good degradation effect of Staphylococcus simulans HZ01 on myosin.

[0049] Preferably, the extraction of the myosin includes the following steps:

[0050] Take pork and mix it with PB buffer and homogenize;

[0051] Filter the supernatant after homogenization in step (S011) through a filter membrane and sterilize it to obtain myosin. PB buffer can well disperse the pork slurry, facilitating subsequent processing and detection of the pork slurry; the filter membrane can well remove the substances that do not meet the requirements in the pork slurry, making the purity of the prepared myosin meet the standard; the sterilization operation better ensures the quality of the finally prepared myosin.

[0052] Preferably, the pork is fresh lean pork. Selecting fresh lean pork can ensure the quality of the finally prepared myosin.

[0053] Preferably, the amount of the pork used is 1 g to 10 g. More preferably, the amount of the pork used is 3 g to 7 g. Even more preferably, the amount of the pork used is 4 g to 6 g. The limitation of the amount of the pork is determined according to the required amount of sarcoplasmic protein and the detection requirements.

[0054] Preferably, the amount of the PB buffer used is 15 mL to 50 mL. More preferably, the amount of the PB buffer used is 20 mL to 45 mL. Even more preferably, the amount of the PB buffer used is 25 mL to 40 mL. Even more preferably, the amount of the PB buffer used is 30 mL to 35 mL. The amount of the PB buffer is determined according to the amount of the pork to be dispersed, and can fully homogenize and disperse the pork.

[0055] Preferably, the concentration of the PB buffer is 0.02 mol / L. The PB buffer with a defined concentration can better disperse the pork, so that the finally prepared sarcoplasmic protein meets the requirements of subsequent processing.

[0056] Preferably, the pH of the PB buffer is 5.8 to 6.6. More preferably, the pH of the PB buffer is 6.0 to 6.4. Even more preferably, the pH of the PB buffer is 6.2 to 6.3. The PB buffer with a defined pH value can better process and disperse the pork slurry.

[0057] Preferably, the homogenization speed is 10,000 rpm / min to 15,000 rpm / min. Preferably, the homogenization speed is 12,000 rpm / min to 14,000 rpm / min. The defined homogenization speed can make the mixture of the pork and the PB buffer more uniform.

[0058] Preferably, the homogenization time is 15 min to 30 min. More preferably, the homogenization time is 20 min to 25 min. The defined homogenization time can fully homogenize the pork.

[0059] Preferably, the pore size of the filter membrane is 0.2 µm to 0.25 µm. More preferably, the pore size of the filter membrane is 0.22 µm to 0.24 µm. The defined pore size of the filter membrane can strictly filter the pork slurry, so as to ensure the quality of the finally prepared sarcoplasmic protein.

[0060] Preferably, the glucose is 1% glucose. 1% glucose can provide the carbon source required for the proliferation of Staphylococcus HZ01.

[0061] Preferably, the dosage of the Staphylococcus simulans HZ01 bacterial solution is 0.05 mL to 0.5 mL. More preferably, the dosage of the Staphylococcus simulans HZ01 bacterial solution is 0.1 mL to 0.4 mL. Even more preferably, the dosage of the Staphylococcus simulans HZ01 bacterial solution is 0.2 mL to 0.3 mL. The dosage of the Staphylococcus simulans HZ01 bacterial solution is determined according to the detection requirements and the dosage of the sarcoplasmic protein to ensure sufficient degradation of the sarcoplasmic protein.

[0062] Preferably, the incubation time is 24 h to 72 h. More preferably, the incubation time is 36 h to 60 h. Even more preferably, the incubation time is 40 h to 50 h. The limitation of the incubation time is to ensure sufficient degradation during incubation.

[0063] Preferably, the dosage of the 2×SDS loading buffer is 150 μL to 250 μL. More preferably, the dosage of the 2×SDS loading buffer is 180 μL to 220 μL. The dosage limitation of the 2×SDS loading buffer is to be able to adjust the supernatant into a sample to be detected for better detection.

[0064] Preferably, the dosage of the supernatant after freezing is 150 μL to 250 μL. More preferably, the dosage of the supernatant after freezing is 180 μL to 220 μL. The dosage of the supernatant after freezing is selected according to subsequent detection requirements.

[0065] Preferably, the water bath temperature is 90 °C to 98 °C. More preferably, the water bath temperature is 93 °C to 96 °C. The limitation of the water bath temperature can better fully unfold the secondary structure of the protein.

[0066] Preferably, the water bath time is 3 min to 10 min. More preferably, the water bath time is 5 min to 7 min. While ensuring that the water bath fully unfolds the secondary structure of the protein, it prevents excessive degradation of the protein and affects the results.

[0067] Preferably, the loading amount of the sarcoplasmic protein on the 10%-12% Bio-Rad precast gel is 15 μL to 25 μL; the loading amount of the standard protein is 5 μL to 10 μL. More preferably, the loading amount of the sarcoplasmic protein on the 10%-12% Bio-Rad precast gel is 17 μL to 22 μL; the loading amount of the standard protein is 7 μL to 9 μL. The limitation of the specific loading amount is determined according to the concentration of the corresponding protein.

[0068] Preferably, the electrophoresis voltage is 110 V. The electrophoresis voltage of 110 V is the standard voltage.

[0069] Preferably, the electrophoresis time is 60 min to 120 min. More preferably, the electrophoresis time is 70 min to 110 min. Even more preferably, the electrophoresis time is 80 min to 100 min. The limitation of the electrophoresis time ensures the sufficient separation of protein bands.

[0070] Preferably, the staining time is 1 h to 2 h. More preferably, the staining time is 1.2 h to 1.8 h. Even more preferably, the staining time is 1.4 h to 1.6 h. The limitation of the staining time is to ensure the sufficient color development of protein bands.

[0071] The fourth technical solution of the present invention: The application of Staphylococcus simulans HZ01 to replace part of nitrite in meat products. Staphylococcus simulans HZ01 of the present invention has good nitrate reductase activity and can reduce nitrate to nitrite well during the application process, and can utilize the nitrate in fermented meat products, thereby reducing the dosage of nitrite in the meat product processing.

[0072] Preferably, the detection of the nitrite concentration includes the following steps

[0073] (i) Prepare NB medium containing an appropriate concentration of KNO 3 / NaNO 3 ;

[0074] (ii) Inoculate the Staphylococcus simulans HZ01 bacterial solution into the NB medium in step (i) and culture;

[0075] (iii) Centrifuge the fermentation broth at intervals during the culture process and collect the supernatant;

[0076] (iv) Detect the nitrite content in the supernatant of step (iii). The whole detection process is simple and can quickly obtain the good reduction effect of Staphylococcus simulans HZ01 reducing nitrate to nitrite.

[0077] Preferably, step (i) is to prepare 0.1% KNO 3 / NaNO 3 NB medium. 0.1% KNO 3 / NaNO 3 NB medium has a better effect on reflecting the nitrate reductase activity of Staphylococcus simulans HZ01.

[0078] Preferably, the preparation of the NB medium containing an appropriate concentration of KNO 3 / NaNO 3 in step (i) includes the following steps

[0079] (i01) Take an appropriate amount of KNO 3 / NaNO3 Dissolve it and NB nutrient broth in sterile water;

[0080] (ⅰ02) After sterilizing and culturing the mixture in step (ⅰ01), an NB medium containing an appropriate concentration of KNO 3 / NaNO 3 is obtained. The NB medium prepared from KNO 3 / NaNO 3 , NB nutrient broth and sterile water and containing an appropriate concentration of KNO 3 / NaNO 3 can better accept the reduction effect of Staphylococcus simulans HZ01 and better reflect the effect of Staphylococcus simulans HZ01 reducing nitrate to nitrite.

[0081] Preferably, the dosage of KNO 3 / NaNO 3 in step (ⅰ01) is 0.02 g to 0.1 g. Preferably, the dosage of KNO 3 / NaNO 3 in step (ⅰ01) is 0.04 g to 0.08 g. The dosage of KNO 3 / NaNO 3 is limited according to the requirement of preparing an NB medium with a required concentration of KNO 3 / NaNO 3 .

[0082] Preferably, the dosage of NB nutrient broth in step (ⅰ01) is 0.7 g to 1.2 g. More preferably, the dosage of NB nutrient broth in step (ⅰ01) is 0.8 g to 1.1 g. The dosage of NB nutrient broth is limited according to the requirement of preparing an NB medium with a required concentration of KNO 3 / NaNO 3 .

[0083] Preferably, the dosage of sterile water in step (ⅰ01) is 30 mL to 70 mL. More preferably, the dosage of sterile water in step (ⅰ01) is 40 mL to 60 mL. More preferably, the dosage of sterile water in step (ⅰ01) is 45 mL to 55 mL. The dosage of sterile water is limited according to the requirement of preparing an NB medium with a required concentration of KNO 3 / NaNO 3 .

[0084] Preferably, the sterilization temperature in step (ⅰ02) is 115 °C to 130 °C. More preferably, the sterilization temperature in step (ⅰ02) is 120 °C to 125 °C. The limitation of the sterilization temperature can fully inactivate bacteria and their spores, thus ensuring an appropriate concentration of KNO 3 / NaNO3 The aseptic quality of NB medium.

[0085] Preferably, the incubation time in step (ⅰ02) is 15 min to 30 min. More preferably, the incubation time in step (ⅰ02) is 20 min to 25 min. The limitation of the incubation time can well ensure that KNO 3 / NaNO 3 and its spores and NB nutrient broth are well dissolved and mixed in sterile water.

[0086] Preferably, the inoculation amount of the Staphylococcus aureus HZ01-like bacterial solution in step (ⅱ) is 0.05 mL to 0.2 mL. More preferably, the inoculation amount of the Staphylococcus aureus HZ01-like bacterial solution in step (ⅱ) is 0.1 mL to 0.15 mL. The inoculation amount of the Staphylococcus aureus HZ01-like bacterial solution is limited according to the concentration of KNO 3 to ensure that KNO 3 / NaNO 3 is fully reduced.

[0087] Preferably, the incubation temperature in step (ⅱ) is 25 °C to 35 °C. More preferably, the incubation temperature in step (ⅱ) is 28 °C to 32 °C. The limitation of the incubation temperature can better promote the reduction of KNO 3 / NaNO 3 in the NB medium by Staphylococcus aureus HZ01-like.

[0088] Preferably, the incubation time in step (ⅱ) is 12 h to 36 h. More preferably, the incubation time in step (ⅱ) is 15 h to 30 h. More preferably, the incubation time in step (ⅱ) is 20 h to 25 h. The limitation of the incubation time can ensure the full reduction of KNO 3 / NaNO 3 in the NB medium by Staphylococcus aureus HZ01-like.

[0089] Preferably, the amount of the fermentation broth taken in step (ⅲ) is 1 mL to 5 mL. More preferably, the amount of the fermentation broth taken in step (ⅲ) is 2 mL to 4 mL. The amount of the fermentation broth taken is selected according to the detection requirements, as long as it can meet the inspection requirements for the nitrite content.

[0090] Preferably, the time interval for taking the fermentation broth in step (ⅲ) is 2 h to 6 h. More preferably, the time interval for taking the fermentation broth in step (ⅲ) is 3 h to 5 h. By taking the fermentation broth at intervals, Staphylococcus aureus HZ01-like reduces KNO 3 / NaNO 3The degree of reduction is different. Taking the fermentation broth multiple times can more objectively and accurately obtain the reduction effect of Staphylococcus simulans HZ01 on KNO 3 / NaNO 3 .

[0091] Preferably, the centrifugation temperature in step (iii) is 2°C to 6°C. More preferably, the centrifugation temperature in step (iii) is 3°C to 5°C. The low-temperature limitation of the centrifugation temperature is to ensure the safety of the activity of Staphylococcus simulans HZ01 during centrifugation.

[0092] Preferably, the centrifugation speed in step (iii) is 5000 rpm / min to 20000 rpm / min. More preferably, the centrifugation speed in step (iii) is 10000 rpm / min to 15000 rpm / min. The limitation of the centrifugation speed is to efficiently complete the solid-liquid separation.

[0093] Preferably, the detection of the nitrite content in the supernatant in step (iv) includes the following steps

[0094] (iv01) Take an appropriate amount of supernatant and place it in a colorimetric tube;

[0095] (iv02) Add an appropriate amount of sulfanilic acid solution to the colorimetric tube in step (iv01), mix well and let it stand;

[0096] (iv03) Add an appropriate amount of hydrochloric acid naphthylenediamine solution to the solution after standing in step (iv02);

[0097] (iv04) After adding water to the solution in step (iv03) to the scale of the colorimetric tube, mix well and let it stand;

[0098] (iv05) Measure the absorbance of the solution in step (iv04). The sulfanilic acid solution can undergo a diazotization reaction with the nitrite in the supernatant, preparing for the subsequent color reaction; the addition of the hydrochloric acid naphthylenediamine solution can combine well with the substance after the diazotization reaction to form a rose-red solution, thus forming a good nitrite test sample; by measuring the absorbance, the content of nitrite in the supernatant can be accurately and quickly converted.

[0099] Preferably, the amount of the supernatant used in step (iv01) is 0.5 mL to 1.5 mL. More preferably, the amount of the supernatant used in step (iv01) is 0.8 mL to 1.2 mL. The requirement for the amount of the supernatant is determined according to the detection requirements.

[0100] Preferably, the specification of the colorimetric tube in step (ⅳ01) is 20 mL to 30 mL. More preferably, the specification of the colorimetric tube in step (ⅳ01) is 25 mL. The limitation of the specification of the colorimetric tube is to accurately and quickly calculate the nitrite content in the sample after volume fixation to the scale.

[0101] Preferably, the addition amount of the sulfanilic acid solution in step (ⅳ02) is 0.5 mL to 2 mL, and the concentration of the sulfanilic acid solution is 3 g / L to 5 g / L. More preferably, the addition amount of the sulfanilic acid solution in step (ⅳ02) is 0.1 mL to 1.5 mL, and the concentration of the sulfanilic acid solution is 3.5 g / L to 4.5 g / L. The addition amount of the sulfanilic acid solution and the concentration of the sulfanilic acid solution are both set according to the detection requirements and the dosage requirements of the supernatant, in order to more accurately and quickly calculate the nitrite content in the sample.

[0102] Preferably, the standing time of the solution in step (ⅳ02) is 3 min to 5 min. More preferably, the standing time of the solution in step (ⅳ02) is 3.5 min to 4.5 min. The limitation of the standing time is to let the solution after the diazotization reaction return to calm and maintain a better state for the subsequent reaction.

[0103] Preferably, the addition amount of the N-(1-Naphthyl)ethylenediamine dihydrochloride solution in step (ⅳ03) is 0.2 mL to 1 mL, and the concentration of the N-(1-Naphthyl)ethylenediamine dihydrochloride solution is 1 g / L to 5 g / L. More preferably, the addition amount of the N-(1-Naphthyl)ethylenediamine dihydrochloride solution in step (ⅳ03) is 0.5 mL to 0.8 mL, and the concentration of the N-(1-Naphthyl)ethylenediamine dihydrochloride solution is 2 g / L to 4 g / L. The addition amount of the N-(1-Naphthyl)ethylenediamine dihydrochloride solution and the concentration of the N-(1-Naphthyl)ethylenediamine dihydrochloride solution are both set according to the detection requirements and the requirements of the color reaction, in order to more accurately and quickly calculate the nitrite content in the sample.

[0104] Preferably, the standing time in step (ⅳ04) is 10 min to 20 min. More preferably, the standing time in step (ⅳ04) is 13 min to 17 min. The limitation of the standing time here is to maintain a good state for the subsequent measurement of absorbance.

[0105] Preferably, step (ⅳ05) is to use a 1 cm colorimetric cup, adjust the zero point of the colorimetric cup with the zero tube, take an appropriate amount of the solution in step (ⅳ04), and measure the absorbance at a wavelength of 538 nm. The limitation of the colorimetric cup specification, zero point adjustment, and wavelength setting are all based on the specific situation requirements of nitrite in the supernatant, in order to more accurately and quickly calculate the nitrite content in the sample.

[0106] The fifth technical solution of the present invention: Application of the Staphylococcus simulans HZ01 in dry-cured fermented meat products. The Staphylococcus simulans HZ01 of the present invention can significantly increase the contents of esters, aldehydes and acids in fermented meat products. Among them, vinyl acetate, methyl butyrate, methyl hexanoate, n-hexanal, acetic acid and isovaleric acid are increased most significantly, which can significantly improve the quality of fermented meat products.

[0107] Preferably, the preparation of the dry-cured fermented meat product comprises the following steps.

[0108] (1) Take the concentrated solution or powder of Staphylococcus simulans HZ01 and dilute it with edible water.

[0109] (2) Take appropriate amounts of salt, white sugar and monosodium glutamate and dissolve them in the solution in step (1) to prepare a pickling solution.

[0110] (3) Take minced meat, add the pickling solution in step (2) to the minced meat. When the concentration of Staphylococcus simulans HZ01 in the minced meat reaches 1.0×10 7 log 10 CFU / g to 9.9×10 8 log 10 CFU / g, carry out low-temperature pickling on the minced meat.

[0111] (4) After pickling is completed, subject the minced meat to enema, exhaust, baking and fermentation in sequence to obtain the finished dry-cured fermented meat product. Appropriate amounts of salt, white sugar, monosodium glutamate and the concentrated solution / powder of Staphylococcus simulans HZ01 are used together to prepare a pickling solution, which makes the pickling effect on the minced meat better, and the minced meat is directly flavored after pickling, without the need to season the subsequent meat products; low-temperature pickling can effectively maintain the activity and safety of Staphylococcus simulans HZ01; the finished dry-cured fermented meat product prepared by sequentially subjecting the pickled minced meat to enema, exhaust, baking and fermentation has a fragrance, is rich in nutrition and has excellent quality; among them, after drying and then fermentation, the activity of Staphylococcus simulans HZ01 still exists after drying and further plays a role during the fermentation process, indicating that Staphylococcus simulans HZ01 in the present invention has high heat resistance; in the finished dry-cured fermented meat product prepared after being treated with Staphylococcus simulans HZ01, the contents of esters, aldehydes and acids are significantly increased, and among them, vinyl acetate, methyl butyrate, methyl hexanoate, n-hexanal, acetic acid and isovaleric acid are increased most significantly.

[0112] Preferably, the concentration of the Staphylococcus simulans HZ01 bacterial agent in step (3) is 3.0×10 7 log 10 CFU / g to 7.9×10 8 log 10CFU / g. More preferably, the concentration of the Staphylococcus aureus HZ01-like bacterium agent in step (iii) is 5.0×10 7 log 10 CFU / g to 5.9×10 8 log 10 CFU / g. More preferably, the concentration of the Staphylococcus aureus HZ01-like bacterium agent in step (iii) is 7.0×10 7 log 10 CFU / g to 3.9×10 8 log 10 CFU / g. More preferably, the concentration of the Staphylococcus aureus HZ01-like bacterium agent in step (iii) is 9.0×10 7 log 10 CFU / g to 1.9×10 8 log 10 CFU / g. The limitation of the concentration of the Staphylococcus aureus HZ01-like bacterium agent is to form a good ratio with salt, white sugar and monosodium glutamate, so as to better marinate the subsequent minced meat to be processed and play a better role in the subsequent marinating and fermenting processes of the minced meat.

[0113] Preferably, the marinating liquid in step (ii) comprises the following components by weight,

[0114] 1 - 5 parts of salt, 3 - 8 parts of white sugar, 0.2 - 0.8 part of monosodium glutamate, 2 - 5 parts of edible water, and an appropriate amount of Staphylococcus aureus HZ01 concentrated liquid / powder. Limiting the ratio of the parts of salt, white sugar, monosodium glutamate, edible water and Staphylococcus aureus HZ01 concentrated liquid / powder makes the prepared marinating liquid nutritionally balanced, and the components can synergistically act on each other to play a good role in marinating the minced meat. After marinating the minced meat, it is directly flavored, and there is no need to season the subsequent meat products. Spices can also be added in appropriate amounts according to needs.

[0115] Preferably, the marinating liquid in step (ii) comprises the following components by weight,

[0116] 2 - 4 parts of salt, 4 - 7 parts of white sugar, 0.3 - 0.7 part of monosodium glutamate, 3 - 4 parts of edible water, and an appropriate amount of Staphylococcus aureus HZ01 concentrated liquid / powder. Limiting the ratio of the parts of salt, white sugar, monosodium glutamate, edible water and Staphylococcus aureus HZ01 concentrated liquid / powder makes the prepared marinating liquid more nutritionally balanced, and the components can synergistically act on each other to play a good role in marinating the minced meat. After marinating the minced meat, it is directly flavored, and there is no need to season the subsequent meat products.

[0117] Preferably, the marinating liquid in step (ii) comprises the following components by weight,

[0118] 2 - 4 parts of table salt, 5 - 6 parts of white sugar, 0.4 - 0.6 parts of sodium glutamate, 3 - 4 parts of edible water, and an appropriate amount of Staphylococcus simulans HZ01 concentrate / powder. By limiting the ratio of table salt, white sugar, sodium glutamate, edible water, and Staphylococcus simulans HZ01 concentrate / powder, the prepared pickling solution has a more balanced nutrition, and each component can synergistically act on each other, playing a good role in pickling the meat paste. After pickling the meat paste, it is directly flavored, and there is no need to season the subsequent meat products again.

[0119] Preferably, the mass ratio of fat to lean meat in the meat paste is 1 - 3:6 - 9. More preferably, the mass ratio of fat to lean meat in the meat paste is 2:7 - 8. By limiting the mass ratio of fat to lean meat in the meat paste, the quality of the final dry - cured and fermented meat product is better.

[0120] Preferably, the pickling temperature in step (three) is 2°C - 6°C. More preferably, the pickling temperature in step (three) is 3°C - 5°C. Conducting low - temperature pickling ensures that Staphylococcus simulans HZ01 always maintains good activity and safety during the pickling process.

[0121] Preferably, the pickling time in step (three) is 48h - 72h. More preferably, the pickling time in step (three) is 55h - 65h. Limiting the pickling time can ensure that the meat paste is fully pickled and flavored.

[0122] Preferably, the baking temperature in step (four) is 50°C - 65°C. More preferably, the baking temperature in step (four) is 55°C - 60°C. By limiting the baking temperature, while effectively drying the enema, it does not overly affect the activity of Staphylococcus simulans HZ01, enabling Staphylococcus simulans HZ01 to still play a role in the subsequent fermentation process.

[0123] Preferably, the baking time in step (four) is 48h - 72h. More preferably, the baking time in step (four) is 55h - 65h. Limiting the baking time can fully dry the enema.

[0124] Preferably, the fermentation temperature in step (four) is 20°C - 30°C. More preferably, the fermentation temperature in step (four) is 23°C - 27°C. Limiting the fermentation temperature is also considered to maintain the good activity of Staphylococcus simulans HZ01.

[0125] Preferably, the fermentation time in step (four) is 1 week - 2 weeks. More preferably, the fermentation time in step (four) is 8 days - 10 days. Limiting the fermentation time can ensure that Staphylococcus simulans HZ01 ferments the dried enema more completely.

[0126] Preferably, the method for preparing the concentrated solution of Staphylococcus sp. HZ01 is to inoculate the purified and refrigerated single colony of Staphylococcus sp. HZ01 into the sterilized NB liquid medium for cultivation. When the concentration of Staphylococcus sp. HZ01 in the NB liquid medium reaches 1.0×10 7 log 10 CFU / mL to 5.0×10 7 log 10 CFU / mL, the concentrated solution is obtained. The preparation of the concentrated solution is for better preparation of the pickling solution in the subsequent process, so as to better pickle and ferment the meat paste in the subsequent process; the limitation of the concentration of Staphylococcus sp. HZ01 in the concentrated solution is to meet the processing requirements of the meat paste in the subsequent process, in order to obtain a dry-cured and fermented meat product with better quality in the end.

[0127] Preferably, the refrigeration temperature during the preparation of the concentrated solution is 2°C to 6°C. More preferably, the refrigeration temperature during the preparation of the concentrated solution is 3°C to 5°C. The limitation of the refrigeration temperature is to ensure the activity and safety of Staphylococcus sp. HZ01.

[0128] Preferably, the amount of NB liquid medium used during the preparation of the concentrated solution is 40 mL to 60 mL. More preferably, the amount of NB liquid medium used during the preparation of the concentrated solution is 45 mL to 55 mL. The limitation of the amount of NB liquid medium here is required according to the concentration of Staphylococcus sp. HZ01 to be cultured and the amount of the finally prepared concentrated solution.

[0129] Preferably, the cultivation time during the preparation of the concentrated solution is 12 h to 36 h. More preferably, the cultivation time during the preparation of the concentrated solution is 15 h to 35 h. More preferably, the cultivation time during the preparation of the concentrated solution is 20 h to 30 h. The limitation of the cultivation time here is to ensure good cultivation of Staphylococcus sp. HZ01 and the concentration of Staphylococcus sp. HZ01 reaches the plateau phase.

[0130] Preferably, the cultivation temperature during the preparation of the concentrated solution is 25°C to 35°C. More preferably, the cultivation temperature during the preparation of the concentrated solution is 28°C to 32°C. The limitation of the cultivation temperature here is to create a good temperature condition so that Staphylococcus sp. HZ01 can be well cultured in the sterilized NB liquid medium and quickly reach the concentrated solution with the required concentration.

[0131] Preferably, the cultivation during the preparation of the concentrated solution is static cultivation. Static cultivation is suitable for the growth characteristics of Staphylococcus sp. HZ01.

[0132] Preferably, the Staphylococcus simulans HZ01 has a tolerance pH of 4 to 8 during fermentation. More preferably, the Staphylococcus simulans HZ01 has a tolerance pH of 5 to 7 during fermentation. The Staphylococcus simulans HZ01 can grow under acidic, neutral, and alkaline conditions, and has good acid and alkali tolerance effects and a wide range.

[0133] Preferably, the tolerance amount of nitrite of the Staphylococcus simulans HZ01 during fermentation is 0 to 150 mg / kg. More preferably, the tolerance amount of nitrite of the Staphylococcus simulans HZ01 during fermentation is 50 mg / kg to 100 mg / kg. The Staphylococcus simulans HZ01 has a wide range of tolerance to nitrite content during fermentation, so that the dosage of nitrite can be added in a large range according to actual needs during fermentation.

[0134] Preferably, the salt tolerance of the Staphylococcus simulans HZ01 during fermentation is 3% to 12%. More preferably, the salt tolerance of the Staphylococcus simulans HZ01 during fermentation is 5% to 10%. More preferably, the salt tolerance of the Staphylococcus simulans HZ01 during fermentation is 6% to 8%. The Staphylococcus simulans HZ01 has a wide range of salt tolerance during fermentation, so that the salt dosage can be added in a large range according to actual needs during fermentation.

[0135] Preferably, the tolerance temperature of the Staphylococcus simulans HZ01 during fermentation is 20°C to 40°C. Preferably, the tolerance temperature of the Staphylococcus simulans HZ01 during fermentation is 25°C to 35°C. The Staphylococcus simulans HZ01 has a wide range of tolerance temperatures during fermentation, so that it can be adjusted within a large temperature range according to actual needs during fermentation.

[0136] The present invention has the following beneficial effects:

[0137] (1) The Staphylococcus simulans HZ01 has strong metabolic ability. When used as a starter, it can maintain an advantage in the competition with local microbial populations, has strong competitiveness, makes the sensory characteristics of the fermented meat products more ideal, and can better adapt to the local meat product processing environment;

[0138] (2) The Staphylococcus simulans HZ01 has good protease activity and the ability to decompose sarcoplasmic proteins. It can degrade 58.1% of the sarcoplasmic protein bands within 72 hours and significantly reduce 41.9% of the sarcoplasmic protein bands;

[0139] (3) The Staphylococcus simulans HZ01 has good nitrate reductase activity and can efficiently reduce nitrate to nitrite during application, utilizing the nitrate in fermented meat products, thereby reducing the dosage of nitrite during meat product processing;

[0140] (4) Staphylococcus simulans HZ01 can metabolize leucine to produce 3-methylbutyraldehyde, increasing the variety and content of volatile flavor substances and having excellent flavor-producing characteristics.

[0141] (5) Staphylococcus simulans HZ01 can significantly increase the contents of esters, aldehydes and acids in fermented meat products. Among them, vinyl acetate, methyl butyrate, methyl hexanoate, n-hexanal, acetic acid and isovaleric acid are increased most significantly, which can significantly improve the quality of fermented meat products. Brief Description of the Drawings

[0142] Figure 1 is the colony morphology diagram of Staphylococcus simulans HZ01 in the present invention;

[0143] Figure 2 is the Gram staining result diagram of Staphylococcus simulans HZ01 in the present invention;

[0144] Figure 3 is the comparison result diagram of Staphylococcus simulans HZ01 and other Staphylococcus strains in the present invention;

[0145] Figure 4 is the evolutionary relationship diagram of Staphylococcus simulans HZ01 in the present invention;

[0146] Figure 5 is the fermentation characteristic curve diagram of Staphylococcus simulans HZ01 with respect to pH tolerance in the present invention;

[0147] Figure 6 is the fermentation characteristic curve diagram of Staphylococcus simulans HZ01 with respect to nitrite tolerance in the present invention;

[0148] Figure 7 is the fermentation characteristic curve diagram of Staphylococcus simulans HZ01 with respect to salt tolerance in the present invention;

[0149] Figure 8 is the fermentation characteristic curve diagram of Staphylococcus simulans HZ01 with respect to temperature tolerance in the present invention;

[0150] Figure 9 is the SDS-PAGE diagram of Staphylococcus simulans HZ01 decomposing sarcoplasmic proteins in the present invention;

[0151] Figure 10 is the bar chart of the situation of Staphylococcus simulans HZ01 strain utilizing NO 3 - being converted to NO 2 – in the present invention under anaerobic conditions. Detailed Embodiments

[0152] The present invention will be further described below in conjunction with the accompanying drawings and embodiments, but it shall not be used as a basis for limiting the present invention.

[0153] A strain of Staphylococcus simulans HZ01, the preservation number of Staphylococcus simulans HZ01 is GDMCC NO.62410; the preservation unit of Staphylococcus simulans HZ01 is Guangdong Provincial Microbial Culture Collection Center; the preservation name of Staphylococcus simulans HZ01 is Staphylococcus simulans HZ01; the preservation time of Staphylococcus simulans HZ01 is April 24, 2022.

[0154] The Staphylococcus simulans HZ01 bacterial agent is prepared from Staphylococcus simulans HZ01.

[0155] The preparation of the Staphylococcus simulans HZ01 bacterial agent includes the following steps.

[0156] (A) Inoculate Staphylococcus simulans HZ01 into NB medium for culture to obtain a bacterial solution; the NB medium in step (A) includes 10 g / L of peptone, 3.0 g / L of beef extract, and 5.0 g / L of sodium chloride; the pH value of the NB medium in step (A) is 7 - 7.4; the dosage of the NB medium in step (A) is 0.5 L - 2 L; the culture in step (A) is static culture; the culture time in step (A) is 24 h - 72 h; the culture temperature in step (A) is 25°C - 35°C.

[0157] (B) Centrifuge when the concentration of Staphylococcus simulans HZ01 in the bacterial solution obtained in step (A) is 1.0×10 8 log 10 CFU / mL - 9.9×10 9 log 10 CFU / mL; the centrifugation speed in step (B) is 5000 - 20000 rap / min; the centrifugation time in step (B) is 10 min - 20 min; the centrifugation temperature in step (B) is 2°C - 6°C.

[0158] (C) After centrifugation, add sterilized skim milk for resuspension; the mass concentration of the sterilized skim milk in step (C) is 0.05% m / v - 0.5% m / v; the dosage of the sterilized skim milk in step (C) is 1 ml - 5 ml.

[0159] (D) After resuspension, perform freeze-drying to obtain a bacterial agent with a concentration of 1.0×10 10 log 10 CFU / g - 9.9×10 11 log 10Powdery Staphylococcus simulans HZ01 bactericide at CFU / g; the freeze-drying temperature in step (D) is -90°C to -70°C; the freeze-drying time in step (D) is 24h to 72h.

[0160] The gene accession number of Staphylococcus simulans HZ01 is OM758216, and the specific nucleotide sequence is as shown in SEQ ID NO.1;

[0161] The determination of the gene sequence of Staphylococcus simulans HZ01 includes the following steps,

[0162] (a) Extract the total DNA of Staphylococcus simulans HZ01 using a bacterial genomic DNA extraction kit;

[0163] (b) Use a PCR reaction system to perform 16s full-length amplification on the total DNA extracted in step (a); the PCR reaction system includes 2µL of 10×Ex Taq buffer, 1.6µL of 2.5mM dNTP Mix, 0.6 µL of 5p Primer 1, 0.6µL of 5p Primer2, 2µL of Template, 1µL of 5u Ex Taq, and 12.2µL of dd H2O; the dosage of the PCR reaction system is 15µL to 25µL;

[0164] The 16s full-length amplification in the PCR reaction system includes the following steps,

[0165] (b01) React the total DNA extracted in step (a) in the PCR reaction system at 95°C for 5 min;

[0166] (b02) After step (b01) is completed, continue to react at 95°C for 30 s;

[0167] (b03) After step (b02) is completed, continue to react at 55°C for 30 s;

[0168] (b04) After step (b03) is completed, continue to react at 72°C for 1 min;

[0169] (b05) Repeat steps (b02) to (b04) 24 times;

[0170] (b06) After step (b05) is completed, extend at 72°C for 10 min;

[0171] (b07) After step (b06) is completed, keep it at 10°C for incubation, and the 16s full-length amplification of the total DNA of Staphylococcus simulans HZ01 is completed;

[0172] (c) After 16s full-length amplification is completed, perform gel cutting and purification, and then perform electrophoresis sequencing. The measured sequence is the full-length 16s DNA sequence of Staphylococcus simulans HZ01.

[0173] Application of Staphylococcus simulans HZ01 in degrading sarcoplasmic proteins;

[0174] Detection of the degradation result of sarcoplasmic proteins by Staphylococcus simulans HZ01 includes the following steps;

[0175] (S01) Extract sarcoplasmic proteins and measure the concentration of sarcoplasmic proteins using a Lowry protein concentration kit;

[0176] The extraction of sarcoplasmic proteins includes the following steps,

[0177] (S011) Mix pork with PB buffer and homogenize; the pork is fresh lean pork; the amount of pork used is 1g - 10g; the amount of PB buffer used is 15mL - 50mL; the concentration of PB buffer is 0.02mol / L; the pH of PB buffer is 5.8 - 6.6; the homogenization speed is 10000rpm / min - 15000rpm / min; the homogenization time is 15min - 30min;

[0178] (S012) After filtering and sterilizing the supernatant obtained in step (S011) through a filter membrane, sarcoplasmic proteins are obtained; the pore size of the filter membrane is 0.2µm - 0.25µm;

[0179] (S02) Incubate the sarcoplasmic proteins, glucose, and Staphylococcus simulans HZ01 bacterial solution obtained in step (S01) together; the glucose is 1% glucose; the amount of Staphylococcus simulans HZ01 bacterial solution used is 0.05mL - 0.5mL; the incubation time is 24h - 72h;

[0180] (S03) Centrifuge the bacterial solution after incubation is completed;

[0181] (S04) Mix 2×SDS loading buffer with the supernatant after centrifugation in step (S03) and perform water bath; the amount of 2×SDS loading buffer taken is 150uL - 250uL; the amount of the supernatant after centrifugation taken is 150uL - 250uL; the water bath temperature is 90℃ - 98℃; the water bath time is 3min - 10min;

[0182] (S05) Take 10%-12% Bio-Rad precast gel sarcoplasmic proteins and standard proteins, and load the samples in step (S04) onto the 10%-12% Bio-Rad precast gel sarcoplasmic proteins and standard proteins respectively; the loading amount for the 10%-12% Bio-Rad precast gel sarcoplasmic proteins is 15μL - 25μL; the loading amount for the standard protein is 5μL - 10μL.

[0183] After loading the sample, electrophoresis is carried out; the electrophoresis voltage is 110 V; the electrophoresis time is 60 min to 120 min;

[0184] After the electrophoresis is completed, it is stained with Coomassie Brilliant Blue R-250, and the degradation result of sarcoplasmic protein by Staphylococcus simulans HZ01 is obtained through the staining result; the staining time is 1 h to 2 h.

[0185] Application of Staphylococcus simulans HZ01 to replace part of nitrite in meat products;

[0186] The detection of the concentration of nitrite includes the following steps,

[0187] (i) Prepare NB medium containing an appropriate concentration of KNO 3 / NaNO 3 ; step (i) is to prepare 0.1% KNO 3 / NaNO 3 NB medium;

[0188] In step (i), the preparation of NB medium containing an appropriate concentration of KNO 3 / NaNO 3 includes the following steps,

[0189] (i01) Take an appropriate amount of KNO 3 / NaNO 3 and NB nutrient broth and dissolve them in sterile water; in step (i01), the dosage of KNO 3 / NaNO 3 is 0.02 g to 0.1 g; the dosage of NB nutrient broth in step (i01) is 0.7 g to 1.2 g; the dosage of sterile water in step (i01) is 30 mL to 70 mL;

[0190] (i02) After sterilizing and culturing the mixture in step (i01), the NB medium containing an appropriate concentration of KNO 3 / NaNO 3 is obtained; the sterilization temperature in step (i02) is 115 °C to 130 °C; the culturing time in step (i02) is 15 min to 30 min;

[0191] (ii) Inoculate the Staphylococcus simulans HZ01 bacterial solution into the NB medium in step (i) for culturing; in step (ii), the inoculation amount of the Staphylococcus simulans HZ01 bacterial solution is 0.05 mL to 0.2 mL; the culturing temperature in step (ii) is 25 °C to 35 °C; the culturing time in step (ii) is 12 h to 36 h;

[0192] (ⅲ) During the cultivation process, take the fermentation broth at intervals, centrifuge it, and collect the supernatant; in step (ⅲ), the amount of the fermentation broth taken is 1 mL to 5 mL; the time interval for taking the fermentation broth in step (ⅲ) is 2 h to 6 h; the centrifugation temperature in step (ⅲ) is 2 °C to 6 °C; the centrifugation speed in step (ⅲ) is 5000 rap / min to 20000 rap / min;

[0193] (ⅳ) Detect the nitrite content in the supernatant of step (ⅲ);

[0194] The detection of the nitrite content in the supernatant in step (ⅳ) includes the following steps,

[0195] (ⅳ01) Take an appropriate amount of the supernatant and place it in a colorimetric tube; in step (ⅳ01), the amount of the supernatant taken is 0.5 mL to 1.5 mL; the specification of the colorimetric tube in step (ⅳ01) is 20 mL to 30 mL;

[0196] (ⅳ02) Add an appropriate amount of sulfanilic acid solution to the colorimetric tube in step (ⅳ01), mix well, and let it stand; in step (ⅳ02), the added amount of the sulfanilic acid solution is 0.5 mL to 2 mL, and the concentration of the sulfanilic acid solution is 3 g / L to 5 g / L; the standing time of the solution in step (ⅳ02) is 3 min to 5 min;

[0197] (ⅳ03) Add an appropriate amount of hydrochloric acid naphthyl ethylenediamine solution to the solution after standing in step (ⅳ02); in step (ⅳ03), the added amount of the hydrochloric acid naphthyl ethylenediamine solution is 0.2 mL to 1 mL, and the concentration of the hydrochloric acid naphthyl ethylenediamine solution is 1 g / L to 5 g / L;

[0198] (ⅳ04) After adding water to the solution in step (ⅳ03) to the scale of the colorimetric tube, mix well and let it stand; the standing time in step (ⅳ04) is 10 min to 20 min;

[0199] (ⅳ05) Measure the absorbance of the solution in step (ⅳ04); step (ⅳ05) is to use a 1 cm colorimetric cup, adjust the zero point of the colorimetric cup with the zero tube, take an appropriate amount of the solution in step (ⅳ04), and measure the absorbance at a wavelength of 538 nm.

[0200] The application of Staphylococcus simulans HZ01 in dry-cured fermented meat products;

[0201] The preparation of dry-cured fermented meat products includes the following steps,

[0202] (i) Take the concentrated solution / powder of Staphylococcus simulans HZ01 and dilute it with drinking water;

[0203] The preparation method of the concentrated solution of Staphylococcus simulans HZ01 is as follows: inoculate the purified and refrigerated single colony of Staphylococcus simulans HZ01 into the sterilized NB liquid medium for cultivation. When the concentration of Staphylococcus simulans HZ01 in the NB liquid medium reaches 1.0×10 7 log 10 CFU / mL to 5.0×10 7 log 10 CFU / mL, the concentrated solution is obtained. The refrigeration temperature during the preparation of the concentrated solution is 2℃ - 6℃. The dosage of the NB liquid medium during the preparation of the concentrated solution is 40mL - 60mL. The cultivation time during the preparation of the concentrated solution is 12h - 36h. The cultivation temperature during the preparation of the concentrated solution is 25℃ - 35℃. The cultivation during the preparation of the concentrated solution is static cultivation;

[0204] (2) Dissolve appropriate amounts of table salt, white sugar, and monosodium glutamate in the solution in step (1) to make a pickling solution;

[0205] The pickling solution in step (2) includes the following components by weight:

[0206] 1 - 5 parts of table salt, 3 - 8 parts of white sugar, 0.2 - 0.8 part of monosodium glutamate, 2 - 5 parts of edible water, 1 - 5 parts of the concentrated solution / powder of Staphylococcus simulans HZ01;

[0207] (3) Take minced meat, add the pickling solution in step (2) to the minced meat. When the concentration of Staphylococcus simulans HZ01 in the minced meat reaches 1.0×10 7 log 10 CFU / g to 9.9×10 8 log 10 CFU / g, carry out low - temperature pickling on the minced meat. Based on the mass of the minced meat, the concentration of the Staphylococcus simulans HZ01 bacterial agent in step (3) is 3.0×10 7 log 10 CFU / g to 7.9×10 8 log 10 CFU / g. The mass ratio of fat to lean meat in the minced meat is 1 - 3:6 - 9. The pickling temperature in step (3) is 2℃ - 6℃. The pickling time in step (3) is 48h - 72h;

[0208] (4) After the marinating is completed, the minced meat is successively subjected to enema, exhaust, baking and fermentation to obtain the finished product of dry-cured and fermented meat products; the baking temperature in step (4) is 50°C to 65°C; the baking time in step (4) is 48h to 72h; the fermentation temperature in step (4) is 20°C to 30°C; the fermentation time in step (4) is 1 week to 2 weeks; the tolerated pH of Staphylococcus simulans HZ01 during fermentation is 4 to 8; the tolerated nitrite amount of Staphylococcus simulans HZ01 during fermentation is 0 to 150 mg / kg; the salt tolerance of Staphylococcus simulans HZ01 during fermentation is 3% to 12%; the tolerated temperature of Staphylococcus simulans HZ01 during fermentation is 20°C to 40°C.

[0209] Example 1: Staphylococcus simulans HZ01 bacterial agent. This strain is named Staphylococcus simulans HZ01, and its preparation includes the following steps.

[0210] (A) Inoculate Staphylococcus simulans HZ01 into NB medium for cultivation to obtain a bacterial solution; the NB medium in step (A) includes 10 g / L of peptone, 3.0 g / L of beef extract and 5.0 g / L of sodium chloride; the pH value of the NB medium in step (A) is 7.2 ± 0.2; the dosage of the NB medium in step (A) is 1 L; the cultivation in step (A) is static cultivation; the cultivation time in step (A) is 48 h; the cultivation temperature in step (A) is 30°C.

[0211] (B) Centrifuge when the concentration of Staphylococcus simulans HZ01 in the bacterial solution obtained in step (A) reaches 2.5×10 9 log 10 CFU / mL; the centrifugation speed in step (B) is 10000 rap / min; the centrifugation time in step (B) is 15 min; the centrifugation temperature in step (B) is 4°C.

[0212] (C) After centrifugation is completed, add sterilized skim milk for resuspension; the mass concentration of the sterilized skim milk in step (C) is 0.1% m / v; the dosage of the sterilized skim milk in step (C) is 2 ml.

[0213] (D) After resuspension is completed, perform freeze-drying to obtain a powdery Staphylococcus simulans HZ01 bacterial agent with a concentration of 2.0×10 11 log 10 CFU / g; the freeze-drying temperature in step (D) is -80°C; the freeze-drying time in step (D) is 48 h.

[0214] Example 2: The CNKI gene accession number of the Staphylococcus aureus HZ01 mimic is OM758216, and the preservation number at the Guangdong Provincial Microbial Culture Collection Center (GDMCC) is 62410. The specific nucleotide sequence is shown in SEQ ID NO.1:

[0215]

[0216] The determination of the gene sequence of Staphylococcus simulans HZ01 includes the following steps:

[0217] (a)Extract the total DNA of Staphylococcus simulans HZ01 using a bacterial genomic DNA extraction kit;

[0218] (b)Perform 16S full-length amplification on the total DNA extracted in step (a) using a PCR reaction system; the PCR reaction system includes 2 μL of 10×Ex Taq buffer, 1.6 μL of 2.5 mM dNTP Mix, 0.6 μL of 5p Primer 1, 0.6 μL of 5p Primer2, 2 μL of Template, 1 μL of 5u Ex Taq, and 12.2 μL of dd H2O; the dosage of the PCR reaction system is 20 μL;

[0219] The 16S full-length amplification in the PCR reaction system includes the following steps:

[0220] (b01)React the total DNA extracted in step (a) in the PCR reaction system at 95 °C for 5 min for pre-denaturation;

[0221] (b02)After step (b01) is completed, continue to react at 95 °C for 30 s;

[0222] (b03)After step (b02) is completed, continue to react at 55 °C for 30 s;

[0223] (b04)After step (b03) is completed, continue to react at 72 °C for 1 min;

[0224] (b05)Repeat steps (b02) to (b04) 24 times;

[0225] (b06)After step (b05) is completed, extend at 72 °C for 10 min;

[0226] (b07)After step (b06) is completed, incubate at 10 °C to complete the 16S full-length amplification of the total DNA of Staphylococcus simulans HZ01;

[0227] (c)After the 16S full-length amplification is completed, perform gel cutting and purification, and perform electrophoresis sequencing using a horizontal electrophoresis instrument. The measured sequence is the 16S DNA full-length sequence of Staphylococcus simulans HZ01, 1437 bp, as Figure 1 and Figure 2 shown. The similarity of this strain to the strain S. simulans MR1 (CP015642.1) with the closest genetic relationship is asFigure 3 As shown, 99.65%, during the process of evolution, forms an independent evolutionary branch as shown in Figure 4 and is a new strain that mimics Staphylococcus.

[0228] Example 3: As shown in Figure 5 , Figure 6 , Figure 7 and Figure 8 , the Staphylococcus simulans HZ01 can tolerate fermentation conditions with a pH value of 5 - 8, nitrite of 150 mg / kg, salt content of 3% - 9%, and temperature of 20°C - 35°C.

[0229] The preparation method of the concentrated solution of Staphylococcus simulans HZ01 is as follows: Pick a purified single colony of Staphylococcus simulans HZ01 stored in a 4°C refrigerator and inoculate it into 50 mL of sterilized NB liquid medium. Incubate it statically at 30°C for 24 hours until its concentration reaches 3.0×10 7 log 10 CFU / mL, and this culture solution is the concentrated solution. Prepare 50 mL of NB liquid medium with pH values of 4, 5, 6, 7, 8, salt contents of 0%, 3%, 6%, 9%, 12%, and nitrite concentrations of 0, 50 mg / kg, 100 mg / kg, 150 mg / kg respectively. Inoculate 1 mL of the concentrated solution into the NB medium under different culture conditions and incubate at 30°C for 24 hours. Measure the OD value every 3 hours to verify the optimal fermentation conditions of Staphylococcus simulans HZ01. Prepare 50 mL of NB liquid medium, inoculate 1 mL of the concentrated solution, verify the OD value curve of strain HZ01 at different temperatures, and determine the temperature growth range of HZ01. The results show that the growth range of HZ01 is relatively wide and it can grow under the conditions of pH value 5 - 8, nitrite 0 - 150 mg / kg, salt content 3% - 9%, and temperature 20°C - 35°C.

[0230] Example 4: Application of Staphylococcus simulans HZ01 in degrading sarcoplasmic proteins; As shown in Figure 9 and Figure 10 , Staphylococcus simulans HZ0 can secrete highly active protease and can degrade 58.1% of the sarcoplasmic protein bands within 72 hours and significantly reduce 41.9% of the sarcoplasmic protein bands;

[0231] The detection of the degradation result of Staphylococcus simulans HZ01 on sarcoplasmic proteins includes the following steps;

[0232] (S01) Extract sarcoplasmic proteins and measure the concentration of sarcoplasmic proteins using a Lowry protein concentration kit;

[0233] The extraction of sarcoplasmic proteins includes the following steps,

[0234] (S011) Mix pork with PB buffer and homogenize; the pork is fresh lean pork; the amount of pork used is 2 g; the amount of PB buffer used is 20 mL; the concentration of PB buffer is 0.02 mol / L; the pH of PB buffer is 6.5; the homogenization speed is 13,000 rpm / min; the homogenization time is 20 min;

[0235] (S012) After filtering and sterilizing the supernatant after homogenization in step (S011) through a filter membrane, myosin protein is obtained; the pore size of the filter membrane is 0.22 µm;

[0236] (S02) Incubate the myosin protein, glucose, and Staphylococcus simulans HZ01 bacterial solution from step (S01) together; the glucose is 1% glucose; the amount of Staphylococcus simulans HZ01 bacterial solution used is 0.1 mL; the incubation time is 12 h to 36 h;

[0237] (S03) Take 2 mL of the bacterial solution and centrifuge it every 24 hours;

[0238] (S04) Mix 2×SDS loading buffer with the supernatant after centrifugation in step (S03), and perform a water bath; the amount of 2×SDS loading buffer taken is 200 µL; the amount of the supernatant after centrifugation taken is 200 µL; the water bath temperature is 95 °C; the water bath time is 5 min;

[0239] (S05) Take 10%-12% Bio-Rad precast gel myosin protein and standard protein, and load the samples in step (S04) onto the 10%-12% Bio-Rad precast gel myosin protein and standard protein respectively; the loading volume for the 10%-12% Bio-Rad precast gel myosin protein is 20 µL; the loading volume for the standard protein is 8 µL.

[0240] (S06) Perform electrophoresis after loading; the electrophoresis voltage is 110 V; the electrophoresis time is 90 min;

[0241] (S07) After electrophoresis is completed, stain with Coomassie Brilliant Blue R-250, and obtain the degradation result of Staphylococcus simulans HZ01 on the myosin protein bands through the staining result; the staining time is 1.5 h.

[0242] Example 5: Application of Staphylococcus simulans HZ01 replacing part of nitrite in meat products;

[0243] The activity detection of nitrite includes the following steps,

[0244] (i) Prepare NB medium containing an appropriate concentration of KNO 3 ; step (i) is to prepare 0.1% KNO 3 NB medium;

[0245] In step (i), the preparation of NB medium containing an appropriate concentration of KNO 3 includes the following steps:

[0246] (i01) Take an appropriate amount of KNO 3 and NB nutrient broth and dissolve them in sterile water; the amount of KNO 3 used in step (i01) is 0.05 g; the amount of NB nutrient broth used in step (i01) is 0.9 g; the amount of sterile water used in step (i01) is 50 mL;

[0247] After sterilizing and culturing the mixture in step (i01), the NB medium containing an appropriate concentration of KNO 3 is obtained; the sterilization temperature in step (i02) is 121 °C; the culturing time in step (i02) is 20 min;

[0248] In step (ii), inoculate the Staphylococcus simulans HZ01 bacterial liquid into the NB medium in step (i) and culture it; the inoculation amount of the Staphylococcus simulans HZ01 bacterial liquid in step (ii) is 0.1 mL; the culturing temperature in step (ii) is 30 °C; the culturing time in step (ii) is 24 h;

[0249] In step (iii), take the fermentation broth at intervals during the culturing process, centrifuge it, and collect the supernatant; the amount of the fermentation broth taken in step (iii) is 2 mL; the time interval for taking the fermentation broth in step (iii) is 4 h; the centrifugation temperature in step (iii) is 4 °C; the centrifugation speed in step (iii) is 10,000 rpm;

[0250] In step (iv), detect the nitrite content in the supernatant in step (iii);

[0251] The detection of the nitrite content in the supernatant in step (iv) includes the following steps:

[0252] (iv01) Take an appropriate amount of the supernatant and place it in a stoppered colorimetric tube; the amount of the supernatant used in step (iv01) is 1 mL; the specification of the colorimetric tube in step (iv01) is 25 mL;

[0253] (iv02) Add an appropriate amount of sulfanilic acid solution to the colorimetric tube in step (iv01), mix well, and let it stand; the amount of the sulfanilic acid solution added in step (iv02) is 1 mL, and the concentration of the sulfanilic acid solution is 4 g / L; the standing time of the solution in step (iv02) is 3 min - 5 min;

[0254] (iv03) Add an appropriate amount of hydrochloric acid naphthyl ethylenediamine solution to the solution after standing in step (iv02); the amount of the hydrochloric acid naphthyl ethylenediamine solution added in step (iv03) is 0.5 mL, and the concentration of the hydrochloric acid naphthyl ethylenediamine solution is 2 g / L;

[0255] (iv04) Add water to the solution in step (iv03) to the scale of the colorimetric tube, mix well and let stand; the standing time in step (iv04) is 15 min;

[0256] (iv05) Measure the absorbance of the solution in step (iv04); step (iv05) is to use a 1 cm cuvette, adjust the zero point of the cuvette with the zero tube, take an appropriate amount of the solution in step (iv04), measure the absorbance at a wavelength of 538 nm, and at the same time, perform a reagent blank and a standard curve.

[0257] Example 6: Application of Staphylococcus simulans HZ01 in dry-cured fermented meat products;

[0258] The preparation of dry-cured fermented meat products includes the following steps.

[0259] (I) Take the concentrated solution of Staphylococcus simulans HZ01 and dilute it with drinking water;

[0260] The preparation method of the concentrated solution of Staphylococcus simulans HZ01 is to pick a purified single colony of Staphylococcus simulans HZ01 stored in a 4 °C refrigerator and inoculate it into a 50 mL sterilized NB liquid medium, and culture it statically at 30 °C for 24 hours until its concentration reaches 3.0×10 7 log 10 CFU / mL, and this culture solution is the concentrated solution.

[0261] (II) Take appropriate amounts of table salt, white sugar and monosodium glutamate and dissolve them in the solution in step (I) to make a pickling solution;

[0262] The pickling solution in step (II) includes the following components by weight:

[0263] 2.5 parts of table salt, 4 parts of white sugar, 0.4 part of monosodium glutamate, 2.5 parts of drinking water, and 2.5 parts of the concentrated solution of Staphylococcus simulans HZ01;

[0264] (III) Take minced meat, add the pickling solution in step (II) to the minced meat, and when the concentration of Staphylococcus simulans HZ01 in the minced meat reaches 8.0×10 7 log 10 CFU / g to 9.9×10 8 log 10 CFU / g, perform low-temperature pickling on the minced meat; the mass ratio of fat to lean meat in the minced meat is 2:8; the pickling temperature in step (III) is 4 °C; the pickling time in step (III) is 48 h to 72 h;

[0265] (4) After the marinating is completed, the minced meat is successively subjected to enema, and there are no requirements for the length and diameter of the enema; degassing, baking and fermentation are carried out to obtain the finished product of dry-cured fermented meat products; the baking temperature in step (4) is 52°C to 54°C; the baking time in step (4) is 48h to 72h; the fermentation temperature in step (4) is 20°C to 30°C; the fermentation time in step (4) is 1 week to 2 weeks; the tolerated pH of Staphylococcus simulans HZ01 during fermentation is 4 to 8; the tolerated nitrite amount of Staphylococcus simulans HZ01 during fermentation is 0 to 150 mg / kg; the salt tolerance of Staphylococcus simulans HZ01 during fermentation is 3% to 12%; the tolerated temperature of Staphylococcus simulans HZ01 during fermentation is 20°C to 35°C.

[0266] The physicochemical properties of fermented meat products inoculated with Staphylococcus simulans HZ01 are shown in Table 1:

[0267] Table 1: Physicochemical properties of fermented meat products inoculated with Staphylococcus simulans

[0268]

[0269] As shown in Table 1, the amount of nitrite in meat products can be reduced.

[0270] The volatile compound compositions of fermented meat products with different treatments are shown in Table 2:

[0271] Table 2: Volatile compound compositions of fermented meat products with different treatments (AU*10 5 )

[0272]

[0273]

[0274] As can be seen from Table 2, Staphylococcus simulans HZ01 can significantly increase the contents of esters, aldehydes and acids in fermented meat products. Among them, vinyl acetate, methyl butyrate, methyl hexanoate, n-hexanal, acetic acid and isovaleric acid are increased most significantly, which can significantly improve the quality of fermented meat products.

Claims

1. Application of Staphylococcus simulans HZ01 in replacing part of nitrite in meat products, Characterized in that: The preservation number of the Staphylococcus simulans HZ01 is GDMCC NO.62410; the preservation unit of the Staphylococcus simulans HZ01 is Guangdong Provincial Microbial Culture Collection Center; the preservation name of the Staphylococcus simulans HZ01 is Staphylococcus simulans HZ01; the preservation time of the Staphylococcus simulans HZ01 is April 24, 2022; The detection of the concentration of nitrite includes the following steps, (ⅰ)Prepare NB medium containing an appropriate concentration of KNO 3 / NaNO 3 . (ii) Inoculate the Staphylococcus simulans HZ01 bacterial solution into the NB medium in step (i) and culture; (iii) Centrifuge the fermentation broth at intervals during the culture process and collect the supernatant; (iv) Detect the nitrite content in the supernatant in step (iii).

2. The application of Staphylococcus simulans HZ01 in replacing part of nitrite in meat products according to claim 1, Characterized in that: The preparation of the NB medium containing an appropriate concentration of KNO 3 / NaNO 3 includes the following steps: (ⅰ01) Take an appropriate amount of KNO 3 / NaNO 3 and NB nutrient broth and dissolve them in sterile water; After sterilizing and culturing the mixture in step (i01), an NB medium containing an appropriate concentration of KNO 3 / NaNO 3 is obtained.

3. The application of Staphylococcus simulans HZ01 in replacing part of nitrite in meat products according to claim 2, Characterized in that: The dosage of KNO 3 / NaNO 3 in the step (ⅰ01) is 0.02 g to 0.1 g; the dosage of NB nutrient broth in the step (ⅰ01) is 0.7 g to 1.2 g; the dosage of sterile water in the step (ⅰ01) is 30 mL to 70 mL.

4. The application of Staphylococcus simulans HZ01 in replacing part of nitrite in meat products according to claim 2, Characterized in that: The sterilization temperature in step (i02) is 115°C to 130°C; the culture time in step (i02) is 15 min to 30 min.

5. The application of Staphylococcus simulans HZ01 in replacing part of nitrite in meat products according to claim 1, Characterized in that: The inoculation amount of the Staphylococcus simulans HZ01 bacterial solution in step (ii) is 0.05 mL to 0.2 mL; the culture temperature in step (ii) is 25°C to 35°C; the culture time in step (ii) is 12 h to 36 h.

6. The application of Staphylococcus simulans HZ01 in replacing part of nitrite in meat products according to claim 1, Characterized in that: The amount of the fermentation broth taken in step (iii) is 1 mL to 5 mL; the time interval for taking the fermentation broth in step (iii) is 2 h to 6 h; the centrifugation temperature in step (iii) is 2°C to 6°C; the centrifugation speed in step (iii) is 5000 rpm / min to 20000 rpm / min.

7. The application of Staphylococcus simulans HZ01 in replacing part of nitrite in meat products according to claim 1, Characterized in that: The detection of the nitrite content in the supernatant in step (iv) includes the following steps, (iv01) Take an appropriate amount of the supernatant and place it in a colorimetric tube; (iv02) Add an appropriate amount of sulfanilic acid solution to the colorimetric tube in step (iv01), mix well and let stand; (iv03) Add an appropriate amount of hydrochloric acid naphthyl ethylenediamine solution to the solution after standing in step (iv02); (iv04) Add water to the solution in step (iv03) to the scale of the colorimetric tube, mix well and let stand; (iv05) Measure the absorbance of the solution in step (iv04).

8. Use of Staphylococcus aureus HZ01 mimetic to replace part of nitrite in meat products according to claim 7, Characterized in that: The dosage of the supernatant in step (ⅳ01) is 0.5 mL to 1.5 mL; the specification of the colorimetric tube in step (ⅳ01) is 20 mL to 30 mL.

9. Use of Staphylococcus aureus HZ01 mimetic to replace part of nitrite in meat products according to claim 7, Characterized in that: The addition amount of sulfanilic acid solution in step (ⅳ02) is 0.5 mL to 2 mL, and the concentration of the sulfanilic acid solution is 3 g / L to 5 g / L; the standing time of the solution in step (ⅳ02) is 3 min to 5 min.

10. Use of Staphylococcus aureus HZ01 mimetic to replace part of nitrite in meat products according to claim 7, Characterized in that: The addition amount of N-(1-Naphthyl)ethylenediamine dihydrochloride solution in step (ⅳ03) is 0.2 mL to 1 mL, and the concentration of the N-(1-Naphthyl)ethylenediamine dihydrochloride solution is 1 g / L to 5 g / L; the standing time in step (ⅳ04) is 10 min to 20 min.

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