Application of Staphylococcus simulans HZ01 in dry-cured fermented meat products

Abstract

The present invention relates to the technical field of microbial food fermentation, and specifically discloses the application of a Staphylococcus simulans HZ01 in dry-cured fermented meat products. The preparation of the dry-cured fermented meat products includes the following steps: (1) taking the concentrated solution or powder of Staphylococcus simulans HZ01 and diluting it with edible water; (2) dissolving appropriate amounts of table salt, white sugar, and monosodium glutamate in the solution in step (1) to prepare a pickling solution; (3) taking minced meat, adding the pickling solution in step (2) to the minced meat, and 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, performing low-temperature pickling on the minced meat; (4) after the pickling is completed, subjecting the minced meat to enema, exhaust, baking, and fermentation in sequence to obtain the finished dry-cured fermented meat products. 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 in particular to application of a strain of Staphylococcus aureus HZ01 in dry-cured fermented meat products. Background Art

[0002] Currently, the microbial starter cultures widely used in fermented meat products are primarily lactic acid bacteria and coagulase-negative staphylococci (CNS). Lactic acid bacteria are responsible for the acidification of meat during the initial fermentation phase. By producing lactic acid, they lower the pH of the raw meat, coagulate proteins, and increase firmness. Furthermore, they inhibit the growth of harmful bacteria, thereby enhancing the microbial safety of fermented meat products. CNS play a crucial role in the flavor development and color stability of fermented meat products. Research has shown that staphylococci, rather than lactic acid bacteria, play a significant role in the flavor formation of fermented meat products. CNS can reduce nitrates to nitrites and then to nitrous oxide, prevent rancidity through peroxide decomposition, and produce flavor and aroma compounds through protein and fat hydrolysis. These functions play a crucial role in the development of the quality characteristics of fermented meat products, and they are considered the key "flavor bacteria" of fermented meat products. CNS in fermented meat products has rich population diversity. For example, Staphylococcus xylosus, S. saphylococcus, S. equorum and S. carnosus are the most dominant species in traditional European 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, Staphylococcus carnosus and Staphylococcus simulans to be used as starter cultures for the production of fermented sausages (Republica Italiana, 1995). In 2016, my country included Staphylococcus vitulinus, Staphylococcus xylosus and Staphylococcus carnosus in the "List of Bacteria that Can Be Used in Food" (Department of Food Safety Standards and Monitoring and Assessment, China, No. 4, 2016).

[0003] S. xylosus and S. carnosus are the two most commonly used commercial CNS starter cultures in industry. However, these commercial starter cultures cannot always maintain their advantage in competition with local microbial populations, resulting in a loss of desirable sensory properties in fermented meat products. Therefore, selecting suitable starter cultures from local microbial populations can better adapt to local meat processing environments and be more competitive due to their unique metabolic capabilities. Research has shown that S. simulans is one of the dominant CNS species with excellent fermentation performance in Chinese fermented meat products. Summary of the Invention

[0004] In order to solve the above-mentioned technical problems existing in the existing local microbial population in the food fermentation process, the present invention provides a strain imitating Staphylococcus aureus HZ01 for use in dry-cured fermented meat products. It has the characteristics of good processing adaptability, high protease and lipase activities, good nitrate reductase activity and better flavor of fermented meat products.

[0005] The first technical solution of the present invention: a strain of simulated Staphylococcus aureus HZ01, the preservation number of the simulated Staphylococcus aureus HZ01 is GDMCCNO.62410; the preservation unit of the simulated Staphylococcus aureus HZ01 is Guangdong Provincial Microbiological Culture Collection Center; the preservation name of the simulated Staphylococcus aureus HZ01 is Staphylococcus simulans HZ01; the preservation time of the simulated Staphylococcus aureus HZ01 is April 24, 2022. The mimetic Staphylococcus aureus HZ01 of the present invention has a strong metabolic capacity and can maintain an advantage in the process of competition with local microbial populations when used as a fermentation agent. It has strong competitiveness, so that the sensory characteristics of the fermented meat products are relatively ideal and can better adapt to the processing environment of local meat products. The mimetic Staphylococcus aureus HZ01 of the present invention has good protease activity and the ability to decompose sarcoplasmic protein. It can degrade 58.1% of the sarcoplasmic protein band within 72 hours and significantly reduce 41.9% of the sarcoplasmic protein band. The mimetic Staphylococcus aureus HZ01 of the present invention has good nitric acid reduction performance. The original enzyme activity can efficiently reduce nitrate to nitrite during the application process, and utilize the nitrate in the fermented meat product, thereby reducing the amount of nitrite used in the meat product processing process; the imitation Staphylococcus aureus HZ01 of the present invention can metabolize leucine to produce 3-methylbutanal, increase the types and content of volatile flavor substances, and have excellent aroma production properties; the imitation Staphylococcus aureus HZ01 of the present invention can significantly increase the esters, aldehydes and acids in the fermented meat product, among which vinyl acetate, methyl butyrate, methyl hexanoate, n-hexanal, acetic acid and isovaleric acid are increased most significantly, which can significantly improve the quality of the fermented meat product.

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

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

[0008] (a) Total DNA of Staphylococcus aureus HZ01 was extracted using a bacterial genomic DNA extraction kit;

[0009] (b) performing 16s full-length amplification of the total DNA extracted in step (a) using a PCR reaction system;

[0010] (c) After the 16s full-length amplification is completed, gel excision and purification are performed, and electrophoresis sequencing is performed. The measured sequence is the full-length 16s DNA sequence of the mimicking Staphylococcus aureus HZ01. The present invention uses a bacterial genomic DNA extraction kit to extract the total DNA of the mimicking Staphylococcus aureus HZ01. The extraction is rapid and convenient, and the extraction stability and purity are both good. The PCR reaction system can quickly increase the total DNA extracted from the mimicking Staphylococcus aureus HZ01. The gel excision and purification of the completed 16s full-length amplified sequence facilitates more convenient and accurate electrophoresis sequencing, ensuring the accuracy of the final measured full-length 16s DNA sequence of the mimicking Staphylococcus aureus HZ01.

[0011] Preferably, the PCR reaction system includes 2 μL of 10×Ex Taq buffer, 1.6 μL of 2.5 mM dNTP Mix, 10.6 μL of 5p Primer, 20.6 μL of 5p Primer, 2 μL of Template, 1 μL of 5u Ex Taq, and 12.2 μL of dd H2O. The components work synergistically to rapidly increase the total DNA extracted from the Staphylococcus aureus HZ01 strain.

[0012] Preferably, the PCR reaction system is used in an amount of 15 μL to 25 μL. More preferably, the PCR reaction system is used in an amount of 18 μL to 22 μL. The amount is selected based on the total DNA of the Staphylococcus aureus HZ01 to be amplified, taking into account both the adequacy of the total DNA amplification of the Staphylococcus aureus HZ01 and the appropriateness of the amount used.

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

[0014] (b01) reacting the total DNA extracted in step (a) in a PCR reaction system at 95° C. for 5 minutes;

[0015] (b02) After step (b01) is completed, the reaction is continued at 95°C for 30 seconds;

[0016] (b03) After step (b02) is completed, the reaction is continued at 55° C. for 30 seconds;

[0017] (b04) After step (b03) is completed, the reaction is continued at 72° C. for 1 min;

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

[0019] (b06) After step (b05) is completed, the mixture is heated at 72° C. for 10 minutes;

[0020] (b07) After step (b06), the reaction is incubated at 10°C to complete the 16s full-length amplification of the total DNA of Staphylococcus aureus HZ01. The strict temperature and time limits are designed to ensure the completeness of the amplification of the full-length 16s total DNA of Staphylococcus aureus HZ01, ensuring both completeness and timeliness.

[0021] The second technical solution of the present invention is a Staphylococcus aureus HZ01-like bacterial agent, which is prepared using Staphylococcus aureus HZ01. The Staphylococcus aureus HZ01-like bacterial agent is prepared in the form of a bacterial agent, which is more convenient for use in daily production and life.

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

[0023] (A) Staphylococcus aureus HZ01 was inoculated into NB medium to prepare a bacterial solution;

[0024] (B) After the concentration of Staphylococcus aureus HZ01 in the bacterial solution prepared in step (A) is 1.0×10 8 log 10 CFU / mL~9.9×10 9 log 10 CFU / mL was centrifuged;

[0025] (C) After centrifugation, add sterile skim milk and resuspend;

[0026] (D) After resuspension, freeze-drying was performed to obtain a concentration of 1.0×10 10 log 10 CFU / g~9.9×10 11 log 10CFU / g powdered Staphylococcus aureus HZ01 inoculum. NB medium has a better effect on the cultivation of Staphylococcus aureus HZ01; when the concentration of Staphylococcus aureus HZ01 in the bacterial solution is 1.0×10 8 log 10 CFU / mL~9.9×10 9 log 10 CFU / mL is centrifuged because the concentration of Staphylococcus aureus HZ01 at the limited concentration is already high, and the number of Staphylococcus aureus HZ01 within the limited concentration range is sufficient. If the concentration is low, the number of Staphylococcus aureus HZ01 will be insufficient, and if the concentration is high, more time will be required for cultivation; centrifugation is to better prepare the concentrated solution; adding sterilized skim milk for resuspending is to freeze-protect the cultured Staphylococcus aureus HZ01; freeze-drying is to better prepare the solid bacterial agent; the powdered Staphylococcus aureus HZ01 bacterial agent prepared by the method of the present invention has a high concentration and better performance. More preferably, when the concentration of Staphylococcus aureus HZ01 in the bacterial solution prepared in step (A) is 3.0×10 8 log 10 CFU / mL~7.9×10 9 log 10 More preferably, when the concentration of Staphylococcus aureus HZ01 in the bacterial solution prepared in step (A) is 5.0×10 8 log 10 CFU / mL~5.9×10 9 log 10 More preferably, when the concentration of Staphylococcus aureus HZ01 in the bacterial solution prepared in step (A) is 7.0×10 8 log 10 CFU / mL~3.9×10 9 log 10 More preferably, when the concentration of Staphylococcus aureus HZ01 in the bacterial solution prepared in step (A) is 9.0×10 8 log 10 CFU / mL~1.9×10 9 log 10 CFU / mL and centrifuge.

[0027] Preferably, the NB culture medium in step (A) comprises 10 g / L peptone, 3.0 g / L beef extract, and 5.0 g / L sodium chloride, so that the NB culture medium has a better culture effect on Staphylococcus aureus HZ01.

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

[0029] Preferably, the amount of NB culture medium used in step (A) is 0.5 L to 2 L. More preferably, the amount of NB culture medium used in step (A) is 1 L to 1.5 L. The amount of NB culture medium used, based on the amount of Staphylococcus aureus HZ01, has reached a defined concentration, ensuring good culture of Staphylococcus aureus HZ01.

[0030] Preferably, the culture in step (A) is static culture, which can help simulate the good proliferation of Staphylococcus aureus HZ01.

[0031] Preferably, the incubation time in step (A) is 24 to 72 hours. More preferably, the incubation time in step (A) is 36 to 60 hours. More preferably, the incubation time in step (A) is 40 to 48 hours. The incubation time limit herein is intended to ensure rapid proliferation of Staphylococcus aureus HZ01, allowing it to reach a plateau phase.

[0032] Preferably, the culture temperature in step (A) is 15°C to 65°C. More preferably, the culture temperature in step (A) is 25°C to 55°C. More preferably, the culture temperature in step (A) is 30°C to 45°C. More preferably, the culture temperature in step (A) is 35°C to 40°C. These temperature limits are more suitable for culturing Staphylococcus aureus HZ01.

[0033] Preferably, the centrifugal speed in step (B) is 5000 rpm / min to 20000 rpm / min. More preferably, the centrifugal speed in step (B) is 8000 rpm / min to 18000 rpm / min. More preferably, the centrifugal speed in step (B) is 10000 rpm / min to 15000 rpm / min. An appropriate centrifugal speed can more efficiently achieve solid-liquid separation of the bacterial cells.

[0034] Preferably, the centrifugation time in step (B) is 10 to 20 minutes. More preferably, the centrifugation time in step (B) is 12 to 18 minutes. The limited centrifugation time can fully achieve 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 and the temperature limit are used to fully ensure the activity of the Staphylococcus aureus HZ01 during the centrifugation process.

[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. More preferably, the mass concentration of the sterilized skim milk in step (C) is 0.2% m / v to 0.3% m / v. This defined concentration of sterilized skim milk provides sufficient freeze-drying protection for the cultured Staphylococcus aureus HZ01.

[0037] Preferably, the amount of sterilized skim milk used in step (C) is 1 ml to 5 ml. More preferably, the amount of sterilized skim milk used in step (C) is 2 ml to 4 ml. The amount of sterilized skim milk is limited to ensure sufficient freeze-drying protection of the cultured Staphylococcus aureus HZ01.

[0038] Preferably, the freeze-drying temperature in step (D) is between -90°C and -70°C. More preferably, the freeze-drying temperature in step (D) is between -85°C and -75°C. More preferably, the freeze-drying temperature in step (D) is between -83°C and -78°C. Limiting the freeze-drying temperature ensures sufficient and rapid drying while better ensuring the activity of the Staphylococcus aureus HZ01 in the Staphylococcus aureus HZ01 inoculum.

[0039] Preferably, the freeze-drying time in step (D) is 24 to 72 hours. More preferably, the freeze-drying time in step (D) is 36 to 60 hours. More preferably, the freeze-drying time in step (D) is 40 to 50 hours. Limiting the freeze-drying time can fully ensure complete drying.

[0040] The third technical solution of the present invention involves the use of a Staphylococcus aureus HZ01 mimic for the degradation of sarcoplasmic proteins. The Staphylococcus aureus HZ01 mimic has excellent protease activity and is capable of degrading both sarcoplasmic and myofibrillar proteins. Within 72 hours, it can degrade 58.1% of the sarcoplasmic protein band and significantly reduce the amount of sarcoplasmic protein by 41.9%.

[0041] Preferably, the detection of the degradation results of sarcoplasmic protein by simulating Staphylococcus aureus HZ01 comprises the following steps:

[0042] (S01) extracting sarcoplasmic protein and measuring the concentration of sarcoplasmic protein using a Lowry protein concentration kit;

[0043] (S02) co-incubating the sarcoplasmic protein, glucose and the simulated Staphylococcus aureus HZ01 bacterial solution in step (S01);

[0044] (S03) After the incubation is completed, the bacterial solution is centrifuged;

[0045] (S04) taking 2×SDS loading buffer and mixing it with the supernatant after centrifugation in step (S03), and then placing it in a water bath;

[0046] (S05) taking 10%-12% Bio-Rad precast gel of sarcoplasmic protein and standard protein, and respectively loading the sample in step (S04) on the 10%-12% Bio-Rad precast gel of sarcoplasmic protein and standard protein;

[0047] (S06) electrophoresis after sample loading;

[0048] (S07) After electrophoresis is complete, staining with Coomassie Brilliant Blue R-250 is performed, and the results of the staining are used to determine the degradation of sarcoplasmic proteins by the mimicking Staphylococcus aureus HZ01. The Lowry protein concentration kit can quickly and accurately determine the concentration of sarcoplasmic proteins; the addition of glucose provides a carbon source for the growth of the mimicking Staphylococcus aureus HZ01; centrifugation is used to collect the protein precipitate and eliminate interference from irrelevant substances, thereby better ensuring the accuracy of the test results; the addition of 2×SDS loading buffer is used to adjust the supernatant to a more suitable sample for testing; the water bath is used to fully unfold the protein secondary structure; 10%-12% Bio-Rad precast gel can fully separate the sarcoplasmic protein bands; standard proteins can be used as a reference; electrophoresis can fully ensure the separation of protein bands; Coomassie Brilliant Blue R-250 has a good staining effect, which can fully display the color of the sarcoplasmic protein bands; the entire detection method reflects the good degradation effect of the mimicking Staphylococcus aureus HZ01 on sarcoplasmic proteins.

[0049] Preferably, the extraction of sarcoplasmic protein comprises the following steps:

[0050] (S011) Pork is mixed with PB buffer and homogenized;

[0051] (S012) The supernatant after homogenization in step (S011) is filtered through a filter membrane and sterilized to obtain sarcoplasmic protein. The PB buffer effectively disperses the pork slurry, facilitating subsequent processing and testing of the pork slurry. The filter membrane effectively removes substandard substances in the pork slurry, ensuring that the purity of the obtained sarcoplasmic protein meets the standard. The sterilization operation further ensures the quality of the final obtained sarcoplasmic protein.

[0052] Preferably, the pork is fresh lean pork. Selecting fresh lean pork can ensure the quality of the sarcoplasmic protein finally obtained.

[0053] Preferably, the amount of pork used is 1g to 10g. More preferably, the amount of pork used is 3g to 7g. More preferably, the amount of pork used is 4g to 6g. The amount of pork used is limited by the amount of sarcoplasmic protein required and the testing requirements.

[0054] Preferably, the amount of PB buffer used is 15 mL to 50 mL. More preferably, the amount of PB buffer used is 20 mL to 45 mL. More preferably, the amount of PB buffer used is 25 mL to 40 mL. More preferably, the amount of PB buffer used is 30 mL to 35 mL. The amount of PB buffer used is determined based on the amount of pork to be dispersed, ensuring sufficient homogenization and dispersion of the pork.

[0055] Preferably, the concentration of the PB buffer is 0.02 mol / L. The PB buffer of the defined concentration can better disperse the pork, so that the sarcoplasmic protein finally obtained 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. More preferably, the pH of the PB buffer is 6.2 to 6.3. PB buffers with defined pH values ​​can better process and disperse pork slurry.

[0057] Preferably, the homogenization speed is 10000 rpm / min to 15000 rpm / min. Preferably, the homogenization speed is 12000 rpm / min to 14000 rpm / min. The limited homogenization speed can make the pork and PB buffer mix more evenly.

[0058] Preferably, the homogenization time is 15 to 30 minutes. More preferably, the homogenization time is 20 to 25 minutes. The limited 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 limited pore size of the filter membrane can strictly filter the pork slurry, thereby ensuring the quality of the final sarcoplasmic protein.

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

[0061] Preferably, the amount of the Staphylococcus aureus HZ01 bacterial solution used is 0.05 mL to 0.5 mL. More preferably, the amount of the Staphylococcus aureus HZ01 bacterial solution used is 0.1 mL to 0.4 mL. More preferably, the amount of the Staphylococcus aureus HZ01 bacterial solution used is 0.2 mL to 0.3 mL. The amount of the Staphylococcus aureus HZ01 bacterial solution used is determined based on the test requirements and the amount of sarcoplasmic protein used to ensure sufficient degradation of sarcoplasmic protein.

[0062] Preferably, the incubation time is 24 hours to 72 hours. More preferably, the incubation time is 36 hours to 60 hours. More preferably, the incubation time is 40 hours to 50 hours. The incubation time is limited to ensure sufficient degradation during incubation.

[0063] Preferably, the amount of 2×SDS loading buffer used is 150uL to 250uL. More preferably, the amount of 2×SDS loading buffer used is 180uL to 220uL. The amount of 2×SDS loading buffer used is limited to ensure that the supernatant is well adjusted to the sample to be tested for better detection.

[0064] Preferably, the amount of the supernatant after freezing is 150uL to 250uL. More preferably, the amount of the supernatant after freezing is 180uL to 220uL. The amount of the supernatant after freezing is selected based on subsequent testing 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 limited water bath temperature can better unfold the secondary structure of the protein.

[0066] Preferably, the water bath time is 3 to 10 minutes. More preferably, the water bath time is 5 to 7 minutes. This ensures that the water bath fully unfolds the secondary structure of the protein while preventing excessive protein degradation that would affect the results.

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

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

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

[0070] Preferably, the staining time is 1 to 2 hours. More preferably, the staining time is 1.2 to 1.8 hours. More preferably, the staining time is 1.4 to 1.6 hours. The staining time is limited to ensure that the protein bands are fully developed.

[0071] The fourth technical solution of the present invention involves the use of the mimetic Staphylococcus aureus HZ01 for reducing nitrate to nitrite. The mimetic Staphylococcus aureus HZ01 of the present invention possesses excellent nitrate reductase activity and can effectively reduce nitrate to nitrite during use. This allows the utilization of nitrate in fermented meat products, thereby reducing the amount of nitrite used during meat processing.

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

[0073] (i) preparing NB medium containing appropriate concentrations of KNO3 / NaNO3;

[0074] (ii) inoculating the Staphylococcus aureus HZ01 bacterial solution into the NB medium prepared in step (i) for cultivation;

[0075] (iii) centrifuging the fermentation broth at intervals during the culturing process and collecting the supernatant;

[0076] (iv) Detection of nitrite content in the supernatant of step (iii) The entire detection process is simple and conveniently and quickly obtains a good reduction effect of nitrate to nitrite by mimicking Staphylococcus aureus HZ01.

[0077] Preferably, the step (i) is to prepare a 0.1% KNO3 / NaNO3 NB culture medium, which has a better effect on simulating the nitrate reductase activity of Staphylococcus aureus HZ01.

[0078] Preferably, the preparation of the NB culture medium containing appropriate concentrations of KNO3 / NaNO3 in step (i) comprises the following steps:

[0079] (i01) Dissolve appropriate amounts of KNO3 / NaNO3 and NB nutrient broth in sterile water;

[0080] (i02) Sterilizing and culturing the mixed solution from step (i01) yields NB culture medium containing an appropriate concentration of KNO₃ / NaNO₃. This NB culture medium, prepared from KNO₃ / NaNO₃, NB nutrient broth, and sterile water, is more receptive to the mimicking reduction effect of Staphylococcus aureus HZ01 and better demonstrates the mimicking effect of Staphylococcus aureus HZ01 in reducing nitrate to nitrite.

[0081] Preferably, the amount of KNO3 / NaNO3 used in step (i01) is 0.02g to 0.1g. Preferably, the amount of KNO3 / NaNO3 used in step (i01) is 0.04g to 0.08g. The amount of KNO3 / NaNO3 used is determined to prepare the NB culture medium with the required KNO3 / NaNO3 concentration.

[0082] Preferably, the amount of NB nutrient broth used in step (i01) is 0.7 g to 1.2 g. More preferably, the amount of NB nutrient broth used in step (i01) is 0.8 g to 1.1 g. The amount of NB nutrient broth used is determined by the preparation of the desired KNO3 / NaNO3 concentration of NB culture medium.

[0083] Preferably, the amount of sterile water used in step (i01) is 30 mL to 70 mL. More preferably, the amount of sterile water used in step (i01) is 40 mL to 60 mL. More preferably, the amount of sterile water used in step (i01) is 45 mL to 55 mL. The amount of sterile water used is limited by the requirements for preparing the NB culture medium with the required KNO3 / NaNO3 concentration.

[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 sterilization temperature is limited to fully inactivate bacteria and their spores, thereby ensuring the sterility quality of the NB medium containing an appropriate concentration of KNO3 / NaNO3.

[0085] Preferably, the incubation time in step (i02) is 15 to 30 minutes. More preferably, the incubation time in step (i02) is 20 to 25 minutes. This incubation time limit ensures that the KNO3 / NaNO3, its spores, and the NB nutrient broth are well dissolved and mixed in the sterile water.

[0086] Preferably, the inoculum volume of the Staphylococcus aureus HZ01 bacterial solution in step (ii) is 0.05 mL to 0.2 mL. More preferably, the inoculum volume of the Staphylococcus aureus HZ01 bacterial solution in step (ii) is 0.1 mL to 0.15 mL. The inoculum volume of the Staphylococcus aureus HZ01 bacterial solution is determined based on the KNO3 concentration to ensure that the KNO3 / NaNO3 is fully reduced.

[0087] Preferably, the culture temperature in step (ii) is 25° C. to 35° C. More preferably, the culture temperature in step (ii) is 28° C. to 32° C. Limiting the culture temperature can better promote the reduction of KNO3 / NaNO3 in the NB culture medium by Staphylococcus aureus HZ01.

[0088] Preferably, the incubation time in step (ii) is 12 to 36 hours. More preferably, the incubation time in step (ii) is 15 to 30 hours. More preferably, the incubation time in step (ii) is 20 to 25 hours. This limitation in incubation time ensures that the mimetic Staphylococcus aureus HZ01 fully reduces the KNO3 / NaNO3 in the NB medium.

[0089] Preferably, the amount of fermentation broth used in step (iii) is 1 mL to 5 mL. More preferably, the amount of fermentation broth used in step (iii) is 2 mL to 4 mL. The amount of fermentation broth used is selected based on the test requirements, as long as it meets the requirements for nitrite content testing.

[0090] Preferably, the interval between sampling the fermentation broth in step (iii) is 2 to 6 hours. More preferably, the interval between sampling the fermentation broth in step (iii) is 3 to 5 hours. By sampling the fermentation broth at intervals, the extent of KNO3 / NaNO3 reduction by the simulated Staphylococcus aureus HZ01 varies at different times. Multiple samplings of the fermentation broth can provide a more objective and accurate assessment of the KNO3 / NaNO3 reduction effect of the simulated Staphylococcus aureus HZ01.

[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 limit for centrifugation is to ensure the activity and safety of the Staphylococcus aureus HZ01 during the centrifugation process.

[0092] Preferably, the centrifugal speed in step (iii) is 5000 rpm / min to 20000 rpm / min. More preferably, the centrifugal speed in step (iii) is 10000 rpm / min to 15000 rpm / min. The centrifugal speed is limited to achieve efficient solid-liquid separation.

[0093] Preferably, the step (iv) of detecting the content of nitrite in the supernatant comprises the following steps:

[0094] (iv01) taking an appropriate amount of the supernatant and placing it in a colorimetric tube;

[0095] (iv02) adding an appropriate amount of p-aminobenzenesulfonic acid solution to the colorimetric tube of step (iv01), mixing and then allowing to stand;

[0096] (iv03) adding an appropriate amount of naphthylethylenediamine hydrochloride solution to the solution after standing in step (iv02);

[0097] (iv04) adding water to the solution in step (iv03) up to the mark on the colorimetric tube, mixing and allowing to stand;

[0098] (iv05) Determine the absorbance of the solution in step (iv04). The p-aminobenzenesulfonic acid solution can undergo a diazotization reaction with the nitrite in the supernatant, preparing for the subsequent color development reaction; the addition of the naphthylethylenediamine hydrochloride solution can effectively combine with the substance after the diazotization reaction to generate a rose red solution, thereby forming a good nitrite test sample; by measuring the absorbance, the nitrite content in the supernatant can be accurately and quickly converted.

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

[0100] Preferably, the size of the colorimetric tube in step (iv01) is 20 mL to 30 mL. More preferably, the size of the colorimetric tube in step (iv01) is 25 mL. The size of the colorimetric tube is limited so that the nitrite content in the sample can be accurately and quickly calculated after the volume is adjusted to the scale.

[0101] Preferably, the amount of sulfanilic acid solution added 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 amount of sulfanilic acid solution added 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 amount of sulfanilic acid solution added and the concentration of the sulfanilic acid solution are both set according to the detection requirements and the amount of supernatant required, both for the purpose of more accurately and quickly calculating the nitrite content in the sample.

[0102] Preferably, the solution in step (iv02) is allowed to stand for 3 to 5 minutes. More preferably, the solution in step (iv02) is allowed to stand for 3.5 to 4.5 minutes. The limitation on the standing time is to allow the solution to return to calmness after the diazotization reaction, thereby maintaining a better state for subsequent reactions.

[0103] Preferably, the amount of the naphthylethylenediamine hydrochloride solution added in step (iv03) is 0.2 mL to 1 mL, and the concentration of the naphthylethylenediamine hydrochloride solution is 1 g / L to 5 g / L. More preferably, the amount of the naphthylethylenediamine hydrochloride solution added in step (iv03) is 0.5 mL to 0.8 mL, and the concentration of the naphthylethylenediamine hydrochloride solution is 2 g / L to 4 g / L. The amount of the naphthylethylenediamine hydrochloride solution added and the concentration of the naphthylethylenediamine hydrochloride solution are both set according to the detection requirements and the requirements of the color development reaction, both for the purpose of more accurately and quickly calculating the nitrite content in the sample.

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

[0105] Preferably, step (iv05) comprises using a 1 cm cuvette, adjusting the cuvette's zero point with a zero tube, taking an appropriate amount of the solution from step (iv04), and measuring the absorbance at a wavelength of 538 nm. The cuvette specifications, zero point adjustment, and wavelength setting are all based on the specific 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 involves the use of Staphylococcus aureus HZ01 in dry-cured fermented meat products. This Staphylococcus aureus HZ01 can significantly increase the levels of esters, aldehydes, and acids in fermented meat products, with vinyl acetate, methyl butyrate, methyl hexanoate, n-hexanal, acetic acid, and isovaleric acid being the most significantly increased, significantly improving 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 liquid or powder of Staphylococcus aureus HZ01 and dilute it with drinking water;

[0109] (2) dissolving appropriate amounts of salt, sugar, and sodium glutamate in the solution prepared in step (1) to prepare a pickling solution;

[0110] (3) Take minced meat and add the pickling solution in step (2) to the minced meat until the concentration of Staphylococcus aureus HZ01 in the minced meat reaches 1.0×107 log 10 CFU / g~9.9×10 8 log 10 CFU / g, the minced meat was marinated at low temperature;

[0111] (4) After the pickling is completed, the minced meat is sequentially subjected to sausage filling, degassing, baking and fermentation to obtain the finished dry-cured fermented meat product. Appropriate amounts of salt, sugar, sodium glutamate and concentrated liquid / powder of Staphylococcus aureus HZ01 are prepared together to form a curing liquid, which achieves a better curing effect on minced meat. The minced meat is directly flavored after curing, and there is no need to season the subsequent meat products; low-temperature curing can effectively maintain the activity and safety of Staphylococcus aureus HZ01; the cured minced meat is sequentially subjected to sausage filling, degassing, baking and fermentation, and the dry-cured and fermented meat product prepared after the process sequence is fragrant, nutritious and of excellent quality; after drying and then fermentation, the activity of Staphylococcus aureus HZ01 is still present after drying and further exerts its effect during the fermentation process, indicating that the Staphylococcus aureus HZ01 of the present invention has high heat resistance; in the dry-cured and fermented meat product prepared after treatment with Staphylococcus aureus HZ01, the content of esters, aldehydes and acids is significantly increased, among which the increase in vinyl acetate, methyl butyrate, methyl hexanoate, n-hexanal, acetic acid and isovaleric acid is the most significant.

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

[0113] Preferably, the pickling liquid in step (2) comprises the following components in parts by weight:

[0114] The curing liquid contains 1-5 parts salt, 3-8 parts sugar, 0.2-0.8 parts sodium glutamate, 2-5 parts water, and an appropriate amount of Staphylococcus aureus HZ01 concentrate / powder. The defined ratios of salt, sugar, sodium glutamate, water, and Staphylococcus aureus HZ01 concentrate / powder ensure a nutritionally balanced curing liquid. The components interact synergistically, effectively curing the minced meat. The minced meat is immediately flavorful after curing, eliminating the need for subsequent seasoning of the meat product. Appropriate amounts of spices can also be added as needed.

[0115] Preferably, the pickling liquid in step (2) comprises the following components in parts by weight:

[0116] 2-4 parts salt, 4-7 parts sugar, 0.3-0.7 parts sodium glutamate, 3-4 parts water, and an appropriate amount of Staphylococcus aureus HZ01 concentrate / powder. The defined ratio of salt, sugar, sodium glutamate, water, and Staphylococcus aureus HZ01 concentrate / powder results in a nutritionally balanced marinade. The components work synergistically, effectively curing the minced meat, ensuring the meat is immediately flavorful and eliminating the need for subsequent seasoning.

[0117] Preferably, the pickling liquid in step (2) comprises the following components in parts by weight:

[0118] 2-4 parts salt, 5-6 parts sugar, 0.4-0.6 parts sodium glutamate, 3-4 parts water, and an appropriate amount of Staphylococcus aureus HZ01 concentrate / powder. The defined ratio of salt, sugar, sodium glutamate, water, and Staphylococcus aureus HZ01 concentrate / powder results in a nutritionally balanced marinade. The components work synergistically, effectively curing the minced meat, ensuring the meat is immediately flavorful and eliminating the need for subsequent seasoning.

[0119] Preferably, the mass ratio of fat to lean meat in the minced meat is 1-3:6-9. More preferably, the mass ratio of fat to lean meat in the minced meat is 2:7-8. This defined mass ratio of fat to lean meat in the minced meat results in a higher quality dry-cured and fermented meat product.

[0120] Preferably, the curing temperature in step (3) is 2° C. to 6° C. More preferably, the curing temperature in step (3) is 3° C. to 5° C. Curing at low temperature ensures that the Staphylococcus aureus HZ01 always maintains good activity and safety during the curing process.

[0121] Preferably, the marinating time in step (3) is 48 hours to 72 hours. More preferably, the marinating time in step (3) is 55 hours to 65 hours. The limited marinating time can well ensure that the minced meat is fully marinated and flavored.

[0122] Preferably, the baking temperature in step (4) is 50° C. to 65° C. More preferably, the baking temperature in step (4) is 55° C. to 60° C. The baking temperature is limited to effectively dry the sausage while not significantly affecting the activity of the Staphylococcus aureus HZ01, so that the Staphylococcus aureus HZ01 can still function during the subsequent fermentation process.

[0123] Preferably, the baking time in step (4) is 48 hours to 72 hours. More preferably, the baking time in step (4) is 55 hours to 65 hours. The limitation of the baking time can fully dry the sausage.

[0124] Preferably, the fermentation temperature in step (iv) is 20° C. to 30° C. More preferably, the fermentation temperature in step (iv) is 23° C. to 27° C. The limitation on the fermentation temperature is also for the purpose of maintaining good activity of the mimetic Staphylococcus aureus HZ01.

[0125] Preferably, the fermentation time in step (4) is 1 to 2 weeks. More preferably, the fermentation time in step (4) is 8 to 10 days. Limiting the fermentation time can ensure that the dried sausage is fermented more completely by the simulated Staphylococcus aureus HZ01.

[0126] Preferably, the concentrated solution of the mimetic Staphylococcus aureus HZ01 is prepared by inoculating a purified and refrigerated single colony of the mimetic Staphylococcus aureus HZ01 into a sterilized NB liquid culture medium for cultivation. 7 log 10 CFU / mL~5.0×10 7 log 10 CFU / mL, a concentrate is prepared. The purpose of preparing the concentrate is to better prepare the curing liquid, thereby optimizing the subsequent curing and fermentation of the minced meat. The concentration of the Staphylococcus aureus HZ01 mimic in the concentrate is limited to meet the processing requirements of the minced meat in subsequent steps and to ensure that the final dry-cured and fermented meat product has better quality.

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

[0128] Preferably, the amount of NB liquid culture medium used during the preparation of the concentrate is 40 mL to 60 mL. More preferably, the amount of NB liquid culture medium used during the preparation of the concentrate is 45 mL to 55 mL. The amount of NB liquid culture medium used is determined based on the concentration of the Staphylococcus aureus HZ01 to be cultured and the final volume of the concentrate.

[0129] Preferably, the incubation time during the preparation of the concentrate is 12 to 36 hours. More preferably, the incubation time during the preparation of the concentrate is 15 to 35 hours. More preferably, the incubation time during the preparation of the concentrate is 20 to 30 hours. The incubation time limit herein is to ensure that the Staphylococcus aureus HZ01 is well cultured and that the concentration of the Staphylococcus aureus HZ01 reaches a plateau.

[0130] Preferably, the culture temperature during the preparation of the concentrate is 25°C to 35°C. More preferably, the culture temperature during the preparation of the concentrate is 28°C to 32°C. The culture temperature is limited herein to create optimal temperature conditions for the successful cultivation of Staphylococcus aureus HZ01 in the sterilized NB liquid culture medium, allowing the Staphylococcus aureus HZ01 to rapidly reach a desired concentration in the concentrate.

[0131] Preferably, the culture in the concentrate preparation process is static culture, which is suitable for simulating the growth characteristics of Staphylococcus aureus HZ01.

[0132] Preferably, the Staphylococcus aureus HZ01 strain tolerates a pH of 4 to 8 during fermentation. More preferably, the Staphylococcus aureus HZ01 strain tolerates a pH of 5 to 7 during fermentation. Staphylococcus aureus HZ01 strain can grow in acidic, neutral, and alkaline conditions, and exhibits excellent acid and alkaline resistance over a wide range.

[0133] Preferably, the Staphylococcus aureus HZ01 strain tolerates nitrites in the range of 0 to 150 mg / kg during fermentation. More preferably, the Staphylococcus aureus HZ01 strain tolerates nitrites in the range of 50 to 100 mg / kg during fermentation. The Staphylococcus aureus HZ01 strain tolerates nitrites in a wide range of amounts during fermentation, allowing for a wide range of nitrite additions to be used according to actual needs during fermentation.

[0134] Preferably, the Staphylococcus aureus HZ01 mimic has a salt tolerance of 3% to 12% during fermentation. More preferably, the Staphylococcus aureus HZ01 mimic has a salt tolerance of 5% to 10%. More preferably, the Staphylococcus aureus HZ01 mimic has a salt tolerance of 6% to 8%. The wide range of salt tolerance of Staphylococcus aureus HZ01 during fermentation allows for a wide range of salt additions during fermentation, depending on actual needs.

[0135] Preferably, the Staphylococcus aureus HZ01 can tolerate a temperature of 20°C to 40°C during fermentation. Preferably, the Staphylococcus aureus HZ01 can tolerate a temperature of 25°C to 35°C during fermentation. The Staphylococcus aureus HZ01 can tolerate a wide temperature range during fermentation, allowing the fermentation temperature to be adjusted within a wide range according to actual needs.

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

[0137] (1) The mimetic Staphylococcus aureus HZ01 has a strong metabolic capacity and can maintain an advantage in the competition with local microbial populations when used as a fermentation agent. It has strong competitiveness, making the sensory characteristics of the fermented meat products more ideal and better adapted to the local meat processing environment;

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

[0139] (3) The mimicking Staphylococcus aureus HZ01 has good nitrate reductase activity, which can efficiently reduce nitrate to nitrite during the application process, utilizing the nitrate in fermented meat products, thereby reducing the amount of nitrite used in the meat processing process;

[0140] (4) The imitation Staphylococcus aureus HZ01 can metabolize leucine to produce 3-methylbutyraldehyde, increasing the types and contents of volatile flavor substances and producing excellent aroma properties;

[0141] (5) Imitation of Staphylococcus aureus HZ01 can significantly increase the esters, aldehydes and acids in fermented meat products, among which vinyl acetate, methyl butyrate, methyl hexanoate, n-hexanal, acetic acid and isovaleric acid increase the most significantly, which can significantly improve the quality of fermented meat products. BRIEF DESCRIPTION OF THE DRAWINGS

[0142] Figure 1 This is a colony morphology diagram of the present invention imitating Staphylococcus HZ01;

[0143] Figure 2This is a Gram staining result diagram of the present invention imitating Staphylococcus aureus HZ01;

[0144] Figure 3 This is a comparison diagram of the simulated Staphylococcus aureus HZ01 and other Staphylococcus aureus strains in the present invention;

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

[0146] Figure 5 This is a pH tolerance fermentation characteristic curve diagram of the simulated Staphylococcus aureus HZ01 in the present invention;

[0147] Figure 6 This is a fermentation characteristic curve diagram of nitrite tolerance of Staphylococcus aureus HZ01 in the present invention;

[0148] Figure 7 This is a curve diagram of the salt tolerance fermentation characteristics of the imitation Staphylococcus aureus HZ01 in the present invention;

[0149] Figure 8 This is a temperature tolerance fermentation characteristic curve of the simulated Staphylococcus aureus HZ01 in the present invention;

[0150] Figure 9 This is an SDS-PAGE image of the present invention that simulates the decomposition of sarcoplasmic protein by Staphylococcus HZ01;

[0151] Figure 10 The present invention simulates the Staphylococcus HZ01 strain using NO3 under anaerobic conditions - Converted to NO2 – The histogram of the situation. DETAILED DESCRIPTION

[0152] The present invention will be further described below with reference to the accompanying drawings and examples, but they are not intended to limit 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 Microbiological 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 imitation Staphylococcus HZ01 bacterial agent is prepared by imitating Staphylococcus HZ01;

[0155] The preparation of the Staphylococcus aureus HZ01 bacterial agent includes the following steps:

[0156] (A) inoculating Staphylococcus aureus HZ01 into NB culture medium to prepare a bacterial solution; the NB culture medium in step (A) comprises 10 g / L peptone, 3.0 g / L beef extract, and 5.0 g / L sodium chloride; the pH value of the NB culture medium in step (A) is 7 to 7.4; the amount of NB culture medium in step (A) is 0.5 L to 2 L; the culture in step (A) is a static culture; the culture time in step (A) is 24 hours to 72 hours; the culture temperature in step (A) is 25° C. to 35° C.;

[0157] (B) After the concentration of Staphylococcus aureus HZ01 in the bacterial solution prepared in step (A) is 1.0×10 8 log 10 CFU / mL~9.9×10 9 log 10 CFU / mL; the centrifugal speed in step (B) is 5000 rap / min to 20000 rap / min; the centrifugal time in step (B) is 10 min to 20 min; the centrifugal temperature in step (B) is 2°C to 6°C;

[0158] (C) After centrifugation, sterilized skim milk is added to resuspend the mixture; the mass concentration of the sterilized skim milk in step (C) is 0.05% m / v to 0.5% m / v; the amount of sterilized skim milk used in step (C) is 1 ml to 5 ml;

[0159] (D) After resuspension, freeze-drying was performed to obtain a concentration of 1.0×10 10 log 10 CFU / g~9.9×10 11 log 10 CFU / g of powdered imitation Staphylococcus aureus HZ01 bacterial agent; the freeze-drying temperature in step (D) is -90°C to -70°C; and the freeze-drying time in step (D) is 24h to 72h.

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

[0161] The determination of the gene sequence of Staphylococcus aureus HZ01 includes the following steps:

[0162] (a) Total DNA of Staphylococcus aureus HZ01 was extracted using a bacterial genomic DNA extraction kit;

[0163] (b) Performing 16s full-length amplification of 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, 10.6 μL of 5p Primer, 20.6 μL of 5p Primer, 2 μL of Template, 1 μL of 5u Ex Taq, and 12.2 μL of dd H2O; the amount of the PCR reaction system used is 15 μL to 25 μL;

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

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

[0166] (b02) After step (b01) is completed, the reaction is continued at 95°C for 30 seconds;

[0167] (b03) After step (b02) is completed, the reaction is continued at 55° C. for 30 seconds;

[0168] (b04) After step (b03) is completed, the reaction is continued at 72° C. for 1 min;

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

[0170] (b06) After step (b05) is completed, the mixture is heated at 72° C. for 10 minutes;

[0171] (b07) After step (b06) is completed, the mixture is kept at 10° C. to complete the 16s full-length amplification of the total DNA of Staphylococcus aureus HZ01;

[0172] (c) After the 16s full-length amplification is completed, gel purification is performed, and electrophoresis sequencing is performed. The measured sequence is the full-length 16s DNA sequence that mimics Staphylococcus aureus HZ01.

[0173] Mimicking the application of Staphylococcus HZ01 in degrading sarcoplasmic proteins;

[0174] The detection of the degradation results of sarcoplasmic protein by simulating Staphylococcus aureus HZ01 includes the following steps:

[0175] (S01) extracting sarcoplasmic protein and measuring the concentration of sarcoplasmic protein using a Lowry protein concentration kit;

[0176] The extraction of sarcoplasmic protein includes the following steps:

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

[0178] (S012) filtering the supernatant after homogenization in step (S011) through a filter membrane and sterilizing it to obtain sarcoplasmic protein; the filter membrane pore size is 0.2 μm to 0.25 μm;

[0179] (S02) taking the sarcoplasmic protein, glucose and the simulated Staphylococcus aureus HZ01 bacterial solution in step (S01) and incubating them together; the glucose is 1% glucose; the amount of the simulated Staphylococcus aureus HZ01 bacterial solution is 0.05 mL to 0.5 mL; the incubation time is 24 h to 72 h;

[0180] (S03) After the incubation is completed, the bacterial solution is centrifuged;

[0181] (S04) taking 2×SDS loading buffer and mixing it with the supernatant after centrifugation in step (S03), and placing it in a water bath; the amount of 2×SDS loading buffer used is 150uL to 250uL; the amount of the supernatant after centrifugation is 150uL to 250uL; the water bath temperature is 90°C to 98°C; the water bath time is 3min to 10min;

[0182] (S05) Take 10%-12% Bio-Rad precast gel myoplasmic protein and standard protein, and load the sample in step (S04) on the 10%-12% Bio-Rad precast gel myoplasmic protein and standard protein respectively; the loading amount of 10%-12% Bio-Rad precast gel myoplasmic protein is 15μL~25μL; the loading amount of standard protein is 5μL~10μL.

[0183] (S06) After loading the sample, electrophoresis is performed; the electrophoresis voltage is 110 V; the electrophoresis time is 60 min to 120 min;

[0184] (S07) After the electrophoresis is completed, staining is performed with Coomassie Brilliant Blue R-250, and the degradation results of sarcoplasmic protein by staphylococcus HZ01 are obtained through the staining results; the staining time is 1 hour to 2 hours.

[0185] Mimicking the application of Staphylococcus aureus HZ01 in reducing nitrate to nitrite;

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

[0187] (i) preparing NB culture medium containing appropriate concentrations of KNO3 / NaNO3; step (i) preparing NB culture medium containing 0.1% KNO3 / NaNO3;

[0188] The preparation of the NB culture medium containing appropriate concentrations of KNO3 / NaNO3 in step (i) comprises the following steps:

[0189] (i01) dissolving appropriate amounts of KNO3 / NaNO3 and NB nutrient broth in sterile water; the amount of KNO3 / NaNO3 used in step (i01) is 0.02g to 0.1g; the amount of NB nutrient broth used in step (i01) is 0.7g to 1.2g; the amount of sterile water used in step (i01) is 30mL to 70mL;

[0190] (i02) sterilizing and culturing the mixed solution in step (i01) to obtain NB culture medium containing appropriate concentrations of KNO3 / NaNO3; the sterilization temperature in step (i02) is 115°C to 130°C; and the culturing time in step (i02) is 15 min to 30 min;

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

[0192] (iii) during the culturing process, the fermentation broth is centrifuged at intervals and the supernatant is collected; the amount of the fermentation broth taken in step (iii) is 1 mL to 5 mL; the time interval between 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) detecting the nitrite content in the supernatant of step (iii);

[0194] Step (iv) detecting the content of nitrite in the supernatant comprises the following steps:

[0195] (iv01) taking an appropriate amount of supernatant and placing it in a colorimetric tube; the amount of supernatant used 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;

[0196] (iv02) adding an appropriate amount of p-aminobenzenesulfonic acid solution to the colorimetric tube of step (iv01), mixing and then allowing to stand; the amount of p-aminobenzenesulfonic acid solution added in step (iv02) is 0.5 mL to 2 mL, and the concentration of p-aminobenzenesulfonic acid solution is 3 g / L to 5 g / L; the solution is allowed to stand for 3 min to 5 min in step (iv02);

[0197] (iv03) adding an appropriate amount of naphthylethylenediamine hydrochloride solution to the solution after standing in step (iv02); the amount of naphthylethylenediamine hydrochloride solution added in step (iv03) is 0.2 mL to 1 mL, and the concentration of naphthylethylenediamine hydrochloride solution is 1 g / L to 5 g / L;

[0198] (iv04) adding water to the solution in step (iv03) up to the mark on the colorimetric tube, mixing and allowing to stand; the standing time in step (iv04) is 10 min to 20 min;

[0199] (iv05) Determine 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 a zero tube, take an appropriate amount of the solution in step (iv04), and measure the absorbance at a wavelength of 538 nm.

[0200] Application of mimicking Staphylococcus aureus HZ01 in dry-cured fermented meat products;

[0201] The preparation of dry-cured fermented meat products comprises the following steps:

[0202] (1) Take the concentrated liquid / powder of Staphylococcus aureus HZ01 and dilute it with drinking water;

[0203] The concentrated solution of Staphylococcus aureus HZ01 is prepared by inoculating a purified and refrigerated single colony of Staphylococcus aureus HZ01 into a sterilized NB liquid medium for cultivation. 7 log 10 CFU / mL~5.0×

[0204] 10 7 log 10 CFU / mL, the concentrate is prepared; the refrigeration temperature during the preparation of the concentrate is 2°C to 6°C; the amount of NB liquid culture medium used during the preparation of the concentrate is 40mL to 60mL; the incubation time during the preparation of the concentrate is 12h to 36h; the incubation temperature during the preparation of the concentrate is 25°C to 35°C; the incubation during the preparation of the concentrate is static culture;

[0205] (2) dissolving appropriate amounts of salt, sugar, and sodium glutamate in the solution prepared in step (1) to prepare a pickling solution;

[0206] The pickling liquid in step (2) comprises the following components in parts by weight:

[0207] 1-5 parts of table salt, 3-8 parts of sugar, 0.2-0.8 parts of sodium glutamate, 2-5 parts of drinking water, 1-5 parts of Staphylococcus aureus HZ01 concentrate / powder;

[0208] (3) Take minced meat and add the pickling solution in step (2) to the minced meat until the concentration of Staphylococcus aureus HZ01 in the minced meat reaches 1.0×10 7 log 10 CFU / g~9.9×10 8 log 10 CFU / g, the minced meat was marinated at low temperature; based on the mass of the minced meat, the concentration of the Staphylococcus aureus HZ01 agent in step (iii) was 3.0×10 7 log 10 CFU / g~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 curing temperature in step (3) is 2°C to 6°C; the curing time in step (3) is 48h to 72h;

[0209] (4) After the salting is completed, the minced meat is sequentially subjected to sausage filling, degassing, baking and fermentation to obtain a finished dry-salted fermented meat product; 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 pH tolerance of the simulated Staphylococcus aureus HZ01 during the fermentation process is 4 to 8; the nitrite tolerance of the simulated Staphylococcus aureus HZ01 during the fermentation process is 0 to 150 mg / kg; the salt tolerance of the simulated Staphylococcus aureus HZ01 during the fermentation process is 3% to 12%; the temperature tolerance of the simulated Staphylococcus aureus HZ01 during the fermentation process is 20°C to 40°C.

[0210] Example 1:

[0211] The preparation of the Staphylococcus simulans HZ01 bacterial agent, which is named Staphylococcus simulans HZ01, comprises the following steps:

[0212] (A) Inoculating Staphylococcus aureus HZ01 into NB medium to prepare a bacterial solution; the NB medium in step (A) comprises 10 g / L peptone, 3.0 g / L beef extract, and 5.0 g / L sodium chloride; the pH value of the NB medium in step (A) is 7.2±0.2; the amount of NB medium used in step (A) is 1 L; the culture in step (A) is a static culture; the culture time in step (A) is 48 hours; and the culture temperature in step (A) is 30°C;

[0213] (B) After the concentration of Staphylococcus aureus HZ01 in the bacterial solution prepared in step (A) is 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;

[0214] (C) After centrifugation, sterilized skim milk is added to resuspend the mixture; the mass concentration of the sterilized skim milk in step (C) is 0.1% m / v; the amount of sterilized skim milk used in step (C) is 2 ml;

[0215] (D) After resuspension, freeze-drying was performed to obtain a concentration of 2.0×10 11 log 10 CFU / g of powdered imitation Staphylococcus aureus HZ01 bacterial agent; the freeze-drying temperature in step (D) is -80°C; and the freeze-drying time in step (D) is 48h.

[0216] Example 2:

[0217] The CNKI gene accession number of the Staphylococcus aureus HZ01 is OM758216, the Guangdong Provincial Microbiological Culture Collection Center (GDMCC) deposit number is 62410, and the specific nucleotide sequence is shown in SEQ ID NO.1:

[0218]

[0219] The determination of the gene sequence of Staphylococcus aureus HZ01 includes the following steps:

[0220] (a) Total DNA of Staphylococcus aureus HZ01 was extracted using a bacterial genomic DNA extraction kit;

[0221] (b) Performing 16s full-length amplification of 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, 10.6 μL of 5p Primer, 20.6 μL of 5p Primer, 2 μL of Template, 1 μL of 5u Ex Taq, and 12.2 μL of dd H2O; the amount of the PCR reaction system is 20 μL;

[0222] The 16s full-length amplification in the PCR reaction system includes the following steps:

[0223] (b01) pre-denaturing the total DNA extracted in step (a) in a PCR reaction system at 95° C. for 5 minutes;

[0224] (b02) After step (b01) is completed, the reaction is continued at 95°C for 30 seconds;

[0225] (b03) After step (b02) is completed, the reaction is continued at 55° C. for 30 seconds;

[0226] (b04) After step (b03) is completed, the reaction is continued at 72° C. for 1 min;

[0227] (b05) Repeat steps (b02) to (b04) 24 times;

[0228] (b06) After step (b05) is completed, the mixture is heated at 72° C. for 10 minutes;

[0229] (b07) After step (b06) is completed, the mixture is kept at 10° C. to complete the 16s full-length amplification of the total DNA of Staphylococcus aureus HZ01;

[0230] (c) After the 16s full-length amplification is completed, gel cutting and purification are performed, and electrophoresis sequencing is performed using a horizontal electrophoresis instrument. The measured sequence is the full-length 16s DNA sequence imitating Staphylococcus HZ01, 1437 bp, such as Figure 1 and Figure 2 As shown, the similarity between this strain and the closest related strain S. simulans MR1 ​​(CP015642.1) is as follows Figure 3 As shown in 99.65%, in the process of evolution formed independent Figure 4 The evolutionary branch shown mimics a new strain of Staphylococcus aureus.

[0231] Example 3:

[0232] like Figure 5 、 Figure 6 、 Figure 7 and Figure 8 As shown, the simulated Staphylococcus aureus HZ01 can tolerate the fermentation conditions of pH 5-8, nitrite 150 mg / kg, salt 3%-9%, and temperature 20℃-35℃.

[0233] The concentrated solution of Staphylococcus aureus HZ01 was prepared by picking a single colony of purified Staphylococcus aureus HZ01 stored in a refrigerator at 4°C and inoculating it into 50 mL of sterilized NB liquid culture medium. The culture was then allowed to stand at 30°C for 24 hours until the concentration reached 3.0×10 7 log 10 CFU / mL, and this culture medium is the concentrate. 50 mL of NB liquid medium with pH values ​​of 4, 5, 6, 7, and 8, salinity levels of 0%, 3%, 6%, 9%, and 12%, and nitrite concentrations of 0, 50 mg / kg, 100 mg / kg, and 150 mg / kg were prepared. One mL of the concentrate was inoculated into NB medium containing different culture conditions and incubated at 30°C for 24 hours. OD values ​​were measured every three hours to verify the optimal fermentation conditions for Staphylococcus aureus HZ01. 50 mL of NB liquid medium was prepared and inoculated with 1 mL of the concentrate. OD curves of strain HZ01 at different temperatures were then verified to determine the temperature range of HZ01 growth. The results showed that HZ01 had a wide growth range, growing at pH values ​​of 5 to 8, nitrite levels of 0 to 150 mg / kg, salinity levels of 3% to 9%, and temperatures of 20°C to 35°C.

[0234] Example 4:

[0235] The application of mimicking Staphylococcus HZ01 in degrading sarcoplasmic proteins; Figure 9 and Figure 10 As shown, the mimic Staphylococcus HZ0 can secrete highly active proteases, which can degrade 58.1% of the sarcoplasmic protein bands within 72 hours and significantly reduce 41.9% of the sarcoplasmic protein bands;

[0236] The detection of the degradation results of sarcoplasmic protein by simulating Staphylococcus aureus HZ01 includes the following steps:

[0237] (S01) extracting sarcoplasmic protein and measuring the concentration of sarcoplasmic protein using a Lowry protein concentration kit;

[0238] The extraction of sarcoplasmic protein includes the following steps:

[0239] (S011) Pork was mixed with PB buffer and homogenized; the pork was fresh lean pork; the amount of pork was 2 g; the amount of PB buffer was 20 mL; the concentration of PB buffer was 0.02 mol / L; the pH of PB buffer was 6.5; the homogenization speed was 13000 rpm / min; and the homogenization time was 20 min;

[0240] (S012) filtering the supernatant after homogenization in step (S011) through a filter membrane and sterilizing it to obtain sarcoplasmic protein; the filter membrane pore size is 0.22 μm;

[0241] (S02) taking the sarcoplasmic protein, glucose and the simulated Staphylococcus aureus HZ01 bacterial solution in step (S01) and incubating them together; the glucose is 1% glucose; the amount of the simulated Staphylococcus aureus HZ01 bacterial solution is 0.1 mL; and the incubation time is 12 h to 36 h;

[0242] (S03) Centrifuge 2 mL of bacterial solution every 24 hours;

[0243] (S04) taking 2×SDS loading buffer and mixing it with the supernatant after centrifugation in step (S03), and placing it in a water bath; the amount of 2×SDS loading buffer used is 200 μL; the amount of supernatant after centrifugation is 200 μL; the water bath temperature is 95° C.; the water bath time is 5 min;

[0244] (S05) Take 10%-12% Bio-Rad precast gel myoplasmic protein and standard protein, and load the sample in step (S04) on the 10%-12% Bio-Rad precast gel myoplasmic protein and standard protein respectively; the loading amount for 10%-12% Bio-Rad precast gel myoplasmic protein is 20 μL; the loading amount for standard protein is 8 μL.

[0245] (S06) After loading, electrophoresis was performed; the electrophoresis voltage was 110 V; and the electrophoresis time was 90 min;

[0246] (S07) After the electrophoresis is completed, staining is performed with Coomassie Brilliant Blue R-250, and the degradation results of the sarcoplasmic protein bands by staphylococcus HZ01 are obtained based on the staining results; the staining time is 1.5 hours.

[0247] Example 5:

[0248] Mimicking the application of Staphylococcus aureus HZ01 in reducing nitrate to nitrite;

[0249] The activity test of nitrite includes the following steps:

[0250] (i) preparing NB culture medium containing an appropriate concentration of KNO3; step (i) preparing NB culture medium containing 0.1% KNO3;

[0251] The preparation of the NB culture medium containing an appropriate concentration of KNO3 in step (i) comprises the following steps:

[0252] (ⅰ01) Take appropriate amounts of KNO3 and NB nutrient broth and dissolve them in sterile water; the amount of KNO3 used in step (ⅰ01) is 0.05g; the amount of NB nutrient broth used in step (ⅰ01) is 0.9g; the amount of sterile water used in step (ⅰ01) is 50mL;

[0253] (i02) sterilizing and culturing the mixed solution in step (i01) to obtain NB culture medium containing an appropriate concentration of KNO3; the sterilization temperature in step (i02) is 121°C; and the culturing time in step (i02) is 20 minutes;

[0254] (ii) inoculating the Staphylococcus aureus HZ01 bacterial solution into the NB medium in step (i) for cultivation; the inoculation volume of the Staphylococcus aureus HZ01 bacterial solution in step (ii) is 0.1 mL; the cultivation temperature in step (ii) is 30° C.; and the cultivation time in step (ii) is 24 h;

[0255] (iii) during the culturing process, the fermentation broth was centrifuged at intervals and the supernatant was collected; the amount of fermentation broth taken in step (iii) was 2 mL; the time interval between taking the fermentation broth in step (iii) was 4 hours; the centrifugation temperature in step (iii) was 4° C.; the centrifugation speed in step (iii) was 10,000 rap / min;

[0256] (iv) detecting the nitrite content in the supernatant of step (iii);

[0257] Step (iv) detecting the content of nitrite in the supernatant comprises the following steps:

[0258] (iv01) taking an appropriate amount of the supernatant and placing it in a stoppered colorimetric tube; the amount of the supernatant in step (iv01) is 1 mL; the specification of the colorimetric tube in step (iv01) is 25 mL;

[0259] (iv02) adding an appropriate amount of p-aminobenzenesulfonic acid solution to the colorimetric tube of step (iv01), mixing and then allowing to stand; the amount of p-aminobenzenesulfonic acid solution added in step (iv02) is 1 mL, and the concentration of p-aminobenzenesulfonic acid solution is 4 g / L; the solution in step (iv02) is allowed to stand for 3 min to 5 min;

[0260] (iv03) adding an appropriate amount of naphthylethylenediamine hydrochloride solution to the solution after standing in step (iv02); the amount of naphthylethylenediamine hydrochloride solution added in step (iv03) is 0.5 mL, and the concentration of naphthylethylenediamine hydrochloride solution is 2 g / L;

[0261] (iv04) adding water to the solution in step (iv03) up to the mark on the colorimetric tube, mixing and allowing to stand; the standing time in step (iv04) is 15 minutes;

[0262] (iv05) Determine 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 a zero tube, take an appropriate amount of the solution in step (iv04), measure the absorbance at a wavelength of 538 nm, and simultaneously make a reagent blank and a standard curve.

[0263] Example 6:

[0264] Application of mimicking Staphylococcus aureus HZ01 in dry-cured fermented meat products;

[0265] The preparation of dry-cured fermented meat products comprises the following steps:

[0266] (1) Take the concentrated solution of Staphylococcus aureus HZ01 and dilute it with drinking water;

[0267] The concentrated solution of Staphylococcus aureus HZ01 was prepared by picking a single colony of purified Staphylococcus aureus HZ01 stored in a refrigerator at 4°C and inoculating it into 50 mL of sterilized NB liquid culture medium. The culture was then allowed to stand at 30°C for 24 hours until the concentration reached 3.0×10 7 log 10 CFU / mL, the culture medium is the concentrated medium.

[0268] (2) dissolving appropriate amounts of salt, sugar, and sodium glutamate in the solution prepared in step (1) to prepare a pickling solution;

[0269] The pickling liquid in step (2) comprises the following components in parts by weight:

[0270] 2.5 parts of table salt, 4 parts of sugar, 0.4 parts of sodium glutamate, 2.5 parts of drinking water, 2.5 parts of Staphylococcus aureus HZ01 concentrate;

[0271] (3) Take minced meat and add the pickling solution in step (2) to the minced meat until the concentration of Staphylococcus aureus HZ01 in the minced meat reaches 8.0×10 7 log 10 CFU / g~9.9×10 8 log 10 CFU / g, the minced meat is marinated at low temperature; the mass ratio of fat to lean meat in the minced meat is 2:8; the marinating temperature in step (3) is 4° C.; and the marinating time in step (3) is 48 h to 72 h;

[0272] (4) After the pickling is completed, the minced meat is stuffed into sausages in turn, and the length and diameter of the sausages are not required; the meat is vented, baked and fermented to obtain the finished dry-salted fermented meat product; 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 pH tolerance of the simulated Staphylococcus aureus HZ01 during the fermentation process is 4 to 8; the nitrite tolerance of the simulated Staphylococcus aureus HZ01 during the fermentation process is 0 to 150 mg / kg; the salt tolerance of the simulated Staphylococcus aureus HZ01 during the fermentation process is 3% to 12%; the temperature tolerance of the simulated Staphylococcus aureus HZ01 during the fermentation process is 20°C to 35°C.

[0273] The physicochemical properties of the fermented meat products inoculated with Staphylococcus aureus HZ01 are shown in Table 1:

[0274] Table 1: Physicochemical properties of fermented meat products inoculated with Staphylococcus aureus

[0275] index comparison HZ01 pH <![CDATA[5.53±0.02 a ]]> <![CDATA[5.63±0.08 a ]]> <![CDATA[a w ]]> <![CDATA[0.81±0.03 a ]]> <![CDATA[0.77±0.03 a ]]> <![CDATA[Staphylococcus concentration (log 10 CFU / g)]]> <![CDATA[7.58±0.89 a ]]> <![CDATA[7.34±0.86 a ]]> Nitrite concentration (mg / kg) <![CDATA[15.8±0.10 b ]]> <![CDATA[38.4±0.43 a ]]> L* <![CDATA[36.0±1.32 a ]]> <![CDATA[35.2±1.89 a ]]> a* <![CDATA[8.56±2.92 a ]]> <![CDATA[6.08±0.69 b ]]> b* <![CDATA[7.31±1.56 b ]]> <![CDATA[9.86±1.42 a ]]>

[0276] As shown in Table 1, the amount of nitrite in meat products can be reduced.

[0277] The volatile compound compositions of fermented meat products with different treatments are shown in Table 2:

[0278] Table 2: Volatile compound composition of fermented meat products with different treatments (AU*10 5 )

[0279]

[0280]

[0281] As shown in Table 2, the imitation Staphylococcus aureus HZ01 can significantly increase the 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, which can significantly improve the quality of fermented meat products.

Claims

1. Application of Staphylococcus simulans HZ01 in dry-cured fermented meat products, characterized in that: 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; The preparation of the dry-cured fermented meat product includes the following steps: (1) Take the concentrated solution or powder of Staphylococcus simulans HZ01 and dilute it with edible water; (2) Take appropriate amounts of salt, white sugar and monosodium glutamate and dissolve them in the solution in step (1) to make a pickling solution; (3) Take minced meat, add the pickling solution in step (2) to the minced meat, and when the concentration of Staphylococcus simulans HZ01 in the minced meat reaches 3.0×10 7 CFU / g to 7.9×10 8 CFU / g, conduct low-temperature pickling on the minced meat; (4) After pickling is completed, the meat paste is successively subjected to enema, exhaust, baking and fermentation to obtain the finished dry-cured fermented meat product.

2. Use of Staphylococcus sp. HZ01 according to claim 1 in dry-cured fermented meat products, characterized in that: The pickling solution in step (2) includes the following components by weight: 1-5 parts of salt, 3-8 parts of white sugar, 0.2-0.8 parts of monosodium glutamate, 2-5 parts of edible water, 1-5 parts of the concentrated solution / powder of Staphylococcus simulans HZ01.

3. Use of Staphylococcus sp. HZ01 according to claim 1 in dry-cured fermented meat products, characterized in that: The mass ratio of fat to lean meat in the meat paste is 1-3:6-9.

4. Use of Staphylococcus sp. HZ01 according to claim 1 in dry-cured fermented meat products, characterized in that: The pickling temperature in step (3) is 2°C to 6°C; the pickling time in step (3) is 48h to 72h.

5. Use of Staphylococcus sp. HZ01 as claimed in claim 1 in dry-cured fermented meat products, characterized in that: 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.

6. Use of the staphylococcus aureus HZ01 according to claim 2 in dry-cured fermented meat products, characterized in that: 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 CFU / g to 5.0×10 7 CFU / g, the concentrated solution is obtained.

7. Use of the staphylococcus aureus HZ01 according to claim 6 in dry-cured fermented meat products, characterized in that: The refrigeration temperature during the preparation of the concentrated solution is 2°C to 6°C; the dosage of NB liquid medium during the preparation of the concentrated solution is 40mL to 60mL; the culture time during the preparation of the concentrated solution is 12h to 36h; the culture temperature during the preparation of the concentrated solution is 25°C to 35°C.

8. Use of Staphylococcus sp. HZ01 according to claim 1 in dry-cured fermented meat products, characterized in that: The pH tolerance of Staphylococcus simulans HZ01 during fermentation is 4 to 8; the nitrite tolerance of Staphylococcus simulans HZ01 during fermentation is 0 to 150mg / kg.

9. Use of Staphylococcus sp. HZ01 according to claim 1 in dry-cured fermented meat products, characterized in that: The salt tolerance of Staphylococcus simulans HZ01 during fermentation is 3% to 12%; the temperature tolerance of Staphylococcus simulans HZ01 during fermentation is 20°C to 40°C.

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