Staphylococcus simulans hz01 inoculant

Abstract

The present application relates to the technical field of microbial food fermentation, and specifically discloses a Staphylococcus simulans HZ01 inoculant, preparation of the Staphylococcus simulans HZ01 inoculant comprising the following steps: (A) inoculating Staphylococcus simulans HZ01 into NB culture medium for culture to obtain a bacterial solution; (B) when the concentration of Staphylococcus simulans HZ01 in the bacterial solution obtained in step (A) is 1.0×10 8 log 10 CFU / mL to 9.9×10 9 log 10 CFU / mL, centrifugation is performed; (C) after completion of the centrifugation, sterilized skimmed milk is added for resuspension; (D) after completion of the resuspension, freeze-drying is performed to obtain a powdered Staphylococcus simulans HZ01 inoculant with a concentration of 1.0×10 10 log 10 CFU / g to 9.9×10 11 log 10 CFU / g. The present application has the characteristics of good processing adaptability, high protease and lipase activity, good nitrate reductase activity, and good flavor of fermented meat products.

Description

Technical Field

[0001] This invention relates to the field of microbial food fermentation technology, and in particular to a strain of Staphylococcus aureus HZ01 that mimics the bacteria. Background Technology

[0002] Currently, the microbial starter cultures widely used in fermented meat products are mainly lactic acid bacteria and coagulase-negative staphylococci (CNS). Lactic acid bacteria are responsible for acidification in the early stages of fermentation, lowering the pH of the raw meat and coagulating proteins by producing lactic acid, thus increasing firmness. Furthermore, lactic acid bacteria can inhibit the growth of harmful bacteria, thereby improving the microbial safety of fermented meat products. CNS plays a crucial role in the flavor formation and color stability of fermented meat products. Studies have shown that staphylococci, rather than lactic acid bacteria, play a more significant role in the flavor formation process of fermented meat products. CNS can reduce nitrates to nitrites, and then to nitrous oxide; it can prevent rancidity through peroxide decomposition; and it can produce flavor and aromatic compounds through protein and fat hydrolysis. These functions play a vital role in the formation of the quality characteristics of fermented meat products, and CNS is considered an important "flavor bacterium" in fermented meat products. CNS species exhibit rich diversity in fermented meat products. For example, Staphylococcus xylosus, Staphylococcus saphyllus, Staphylococcus equorum, and Staphylococcus carnosus are the most dominant species in traditional European fermented sausages, while Staphylococcus xylosus, Staphylococcus saphyllus, Staphylococcus epidermidis, Staphylococcus sciuri, and Staphylococcus simulans are more dominant in Chinese fermented meat products. Italian law permits Staphylococcus xylosus, Staphylococcus carnosus, and Staphylococcus simulans as starter cultures for fermented sausage production (Republica Italiana, 1995). In 2016, my country included Staphylococcus vitulinus, Staphylococcus xylosus, and Staphylococcus carnosus in the "List of Microbial Strains that Can Be Used in Food" (Department of Food Safety Standards and Monitoring and Evaluation, China, No. 4, 2016).

[0003] *S. xylosus* and *S. carnosus* are currently the two most commonly used commercial CNS fermentation agents in industry. However, commercial fermentation agents cannot always maintain an advantage in competition with native microbial populations, leading to the loss of ideal sensory characteristics in fermented meat products. Therefore, screening suitable fermentation agents from native microbial populations can better adapt to the local meat processing environment and are more competitive due to their unique metabolic capabilities. Studies have shown that *S. simulans* is one of the dominant CNS populations with excellent fermentation performance in Chinese-style fermented meat products. Summary of the Invention

[0004] In order to solve the above-mentioned technical problems existing in the food fermentation process of existing local microbial populations, this invention provides a strain of Staphylococcus aureus HZ01 that mimics the bacteria. It has the characteristics of good processing adaptability, high activity of protease and lipase, good activity of nitrate reductase, and better flavor of fermented meat products.

[0005] The first technical solution of the present invention is: a Staphylococcus simulans HZ01 strain, wherein the preservation number of the Staphylococcus simulans HZ01 is GDMCCNO.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; and the preservation date of the Staphylococcus simulans HZ01 is April 24, 2022. The *Staphylococcus aureus* HZ01 of this invention exhibits strong metabolic capacity, maintaining an advantage over local microbial populations when used as a fermentation agent. This strong competitiveness results in fermented meat products with ideal sensory characteristics, better adapting to local meat processing environments. The *Staphylococcus aureus* HZ01 of this invention also possesses good protease activity, capable of degrading sarcoplasmic proteins. Within 72 hours, it can degrade 58.1% of sarcoplasmic protein bands and significantly reduce sarcoplasmic protein bands by 41.9%. Furthermore, the *Staphylococcus aureus* HZ01 of this invention exhibits good nitrate reduction... The original enzyme activity can efficiently reduce nitrate to nitrite during application, utilizing the nitrate in fermented meat products, thereby reducing the amount of nitrite used in meat processing. The mimicking Staphylococcus aureus HZ01 of this invention can metabolize leucine to produce 3-methylbutyraldehyde, increasing the types and content of volatile flavor substances and producing excellent aroma properties. The mimicking Staphylococcus aureus HZ01 of this invention can significantly increase the esters, aldehydes and acids in fermented meat products, with the most significant increases in vinyl acetate, methyl butyrate, methyl hexanoate, n-hexanal, acetic acid and isovaleric acid, which can significantly improve the quality of fermented meat products.

[0006] Preferably, the gene accession number of the mimicked Staphylococcus HZ01 is OM758216, and its specific nucleotide sequence is shown in SEQ ID NO.1. Based on the nucleotide sequence of the mimicked Staphylococcus HZ01, it is known that the mimicked Staphylococcus HZ01 of this invention is an independent evolutionary branch formed during the evolutionary process, and is a new strain mimicking Staphylococcus.

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

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

[0009] (b) The total DNA extracted in step (a) was amplified for 16 seconds using a PCR reaction system;

[0010] (c) After the 16S full-length amplification is completed, gel purification is performed, followed by electrophoresis sequencing. The obtained sequence is the full-length 16S DNA sequence of Staphylococcus aureus HZ01. This invention uses a bacterial genomic DNA extraction kit to extract total DNA from Staphylococcus aureus HZ01, which is rapid, convenient, and exhibits good stability and purity. The PCR reaction system can rapidly increase the extracted total DNA from Staphylococcus aureus HZ01. Gel purification of the 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 Staphylococcus aureus HZ01.

[0011] Preferably, the PCR reaction system comprises 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 extracted total DNA of *Staphylococcus aureus* HZ01.

[0012] Preferably, the volume of the PCR reaction system is 15 μL to 25 μL. More preferably, the volume of the PCR reaction system is 18 μL to 22 μL. The total DNA to be amplified, mimicking Staphylococcus HZ01, is selected to balance the sufficiency of amplification of the total DNA mimicking Staphylococcus HZ01 with the appropriate amount.

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

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

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

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

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

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

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

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

[0021] The second technical solution of the present invention is a Staphylococcus aureus HZ01 microbial agent, wherein the Staphylococcus aureus HZ01 microbial agent is prepared by imitating Staphylococcus aureus HZ01. Preparing the Staphylococcus aureus HZ01 into a microbial agent form makes it more convenient for use in daily production and life.

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

[0023] (A) The staphylococcus mimicry strain HZ01 was inoculated into NB medium and cultured to obtain a bacterial culture;

[0024] (B) The concentration of the staphylococcus HZ01-mimicking bacterial culture obtained in step (A) is 1.0 × 10⁻⁶. 8 log 10 CFU / mL ~9.9×10 9 log 10 Centrifuge at CFU / mL;

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

[0026] (D) After resuspension, freeze-drying is performed to obtain a concentration of 1.0 × 10⁻⁶. 10 log 10 CFU / g ~ 9.9 × 10 11 log 10A powdered Staphylococcus aureus HZ01 microbial agent at CFU / g. NB medium showed good culturing effect on Staphylococcus aureus HZ01; the concentration of Staphylococcus aureus HZ01 in the bacterial solution was 1.0 × 10⁻⁶. 8 log 10 CFU / mL ~9.9×10 9 log 10 Centrifugation at CFU / mL is performed because the concentration of *Staphylococcus mimicryl* HZ01 at the specified concentration is already high, and the number of *Staphylococcus mimicryl* HZ01 within the specified concentration range is sufficient. A lower concentration would result in insufficient *Staphylococcus mimicryl* HZ01, while a higher concentration would require more cultivation time. Centrifugation is for better preparation of the concentrate. Resuspension with sterilized skim milk is for cryoprotection of the cultured *Staphylococcus mimicryl* HZ01. Freeze-drying is for better preparation of the solid bacterial agent. The powdered *Staphylococcus mimicryl* HZ01 bacterial agent obtained by the method of this invention has a high concentration and good performance. More preferably, the concentration of *Staphylococcus mimicryl* HZ01 in the bacterial solution obtained in step (A) is 3.0 × 10⁻⁶. 8 log 10 CFU / mL ~ 7.9 × 10 9 log 10 Centrifugation is performed at a concentration of CFU / mL. More preferably, the concentration of the staphylococcal HZ01-mimicking bacteria in the bacterial culture obtained in step (A) is 5.0 × 10⁻⁶. 8 log 10 CFU / mL ~5.9×10 9 log 10 Centrifugation is performed at a concentration of CFU / mL. More preferably, the concentration of the staphylococcal HZ01-mimicking bacteria in the bacterial culture obtained in step (A) is 7.0 × 10⁻⁶. 8 log 10 CFU / mL ~3.9×10 9 log 10 Centrifugation is performed at a concentration of CFU / mL. More preferably, the concentration of the staphylococcal HZ01-mimicking bacteria in the bacterial culture obtained in step (A) is 9.0 × 10⁻⁶. 8 log 10 CFU / mL ~1.9×10 9 log 10 Centrifuge at CFU / mL.

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

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

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

[0030] Preferably, the culture in step (A) is a static culture. Static culture is beneficial for mimicking the good proliferation of Staphylococcus aureus HZ01.

[0031] Preferably, the culture time in step (A) is 24h to 72h. More preferably, the culture time in step (A) is 36h to 60h. Even more preferably, the culture time in step (A) is 40h to 48h. The limitation on the culture time here is to ensure that the rapid proliferation of Staphylococcus aureus HZ01 is mimicked, allowing it to reach the 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. Even more preferably, the culture temperature in step (A) is 30°C to 45°C. Even more preferably, the culture temperature in step (A) is 35°C to 40°C. These temperature limitations are more suitable for culturing *Staphylococcus aureus* 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. Even more preferably, the centrifugation speed in step (B) is 10000 rpm / min to 15000 rpm / min. A suitable centrifugation speed can achieve solid-liquid separation of the bacterial cells more efficiently.

[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. Limiting the centrifugation time ensures sufficient 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. Using low-temperature centrifugation and limiting the centrifugation temperature is to ensure sufficient mimicry of the activity of 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. 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 at this specific concentration can adequately protect the cultured Staphylococcus mimicus HZ01 through freeze-drying.

[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 limitation on the amount of sterilized skim milk is to ensure sufficient freeze-drying protection of the cultured Staphylococcus mimicry strain HZ01.

[0038] Preferably, the freeze-drying temperature in step (D) is -90℃ to -70℃. More preferably, the freeze-drying temperature in step (D) is -85℃ to -75℃. Even more preferably, the freeze-drying temperature in step (D) is -83℃ to -78℃. Limiting the freeze-drying temperature ensures sufficient and rapid drying while better preserving the activity of the Staphylococcus aureus HZ01-mimicking agent.

[0039] Preferably, the freeze-drying time in step (D) is 24h to 72h. More preferably, the freeze-drying time in step (D) is 36h to 60h. Even more preferably, the freeze-drying time in step (D) is 40h to 50h. Limiting the freeze-drying time ensures complete drying.

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

[0041] Preferably, the detection of the degradation results of sarcoplasmic proteins by Staphylococcus aureus HZ01 includes the following steps;

[0042] (S01) Sarcoplasmic proteins were extracted and their concentrations were determined using a Lowry protein concentration kit;

[0043] (S02) Take the sarcoplasmic protein, glucose and the Staphylococcus mimicus HZ01 bacterial solution from step (S01) and incubate them together;

[0044] (S03) Centrifuge the bacterial solution after incubation is complete;

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

[0046] (S05) Take 10%-12% of Bio-Rad pre-made collagen sarcoplasmic protein and standard protein, and apply the sample from step (S04) to the 10%-12% Bio-Rad pre-made collagen sarcoplasmic protein and standard protein respectively.

[0047] (S06) Electrophoresis is performed after sample loading;

[0048] (S07) After electrophoresis, staining with Coomassie Brilliant Blue R-250 was performed to obtain the degradation results of sarcoplasmic proteins by *Staphylococcus aureus* HZ01. The Lowry protein concentration kit can rapidly and accurately determine the concentration of sarcoplasmic proteins; the addition of glucose provides a carbon source for the growth of *Staphylococcus aureus* HZ01; centrifugation is to collect protein precipitates and eliminate interference from irrelevant substances, thereby better ensuring the accuracy of the detection results; the addition of 2×SDS loading buffer is to adjust the supernatant to a more suitable sample for detection; water bath is to allow the secondary structure of proteins to fully develop; 10%-12% Bio-Rad precast gel can fully separate 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 and can fully develop sarcoplasmic protein bands; the entire detection method demonstrates the good degradation effect of *Staphylococcus aureus* HZ01 on sarcoplasmic proteins.

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

[0050] (S011) Mix pork with PB buffer and homogenize;

[0051] (S012) After homogenizing in step (S011), the supernatant is filtered through a filter membrane and sterilized to obtain sarcoplasmic protein. PB buffer can effectively disperse pork slurry, facilitating subsequent processing and testing; the filter membrane can effectively remove unsuitable substances from the pork slurry, ensuring the purity of the obtained sarcoplasmic protein meets the standards; and the sterilization process further guarantees the quality of the final sarcoplasmic protein.

[0052] Preferably, the pork is fresh lean pork. Using fresh lean pork ensures the quality of the final sarcoplasmic protein.

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

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

[0055] Preferably, the concentration of the PB buffer is 0.02 mol / L. A specific concentration of PB buffer allows for better dispersion of pork, ensuring that the resulting sarcoplasmic proteins meet the requirements for subsequent processing.

[0056] Preferably, the pH of the PB buffer solution is 5.8–6.6. More preferably, the pH of the PB buffer solution is 6.0–6.4. Even more preferably, the pH of the PB buffer solution is 6.2–6.3. PB buffer solutions with defined pH values ​​can better process and disperse pork slurry.

[0057] Preferably, the homogenization speed is 10,000 rpm / min to 15,000 rpm / min. More preferably, the homogenization speed is 12,000 rpm / min to 14,000 rpm / min. This specific homogenization speed allows for a more uniform mixing of the pork and PB buffer.

[0058] Preferably, the homogenization time is 15 min to 30 min. More preferably, the homogenization time is 20 min to 25 min. The limited homogenization time ensures sufficient homogenization of the pork.

[0059] Preferably, the filter membrane has a pore size of 0.2 μm to 0.25 μm. More preferably, the filter membrane has a pore size of 0.22 μm to 0.24 μm. The defined pore size allows for strict filtration of the pork slurry, thereby ensuring the quality of the final sarcoplasmic protein.

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

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

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

[0063] Preferably, the volume of the 2×SDS loading buffer is 150 μL to 250 μL. More preferably, the volume of the 2×SDS loading buffer is 180 μL to 220 μL. The volume of the 2×SDS loading buffer is limited to ensure that the supernatant is well adjusted to a more suitable sample for detection.

[0064] Preferably, the amount of supernatant used after freezing is 150 μL to 250 μL. More preferably, the amount of supernatant used after freezing is 180 μL to 220 μL. The amount of supernatant used after freezing is selected according to the 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. Limiting the water bath temperature allows for better development of the protein's secondary structure.

[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 secondary structure of the protein is fully developed during the water bath, while preventing excessive protein degradation that could affect the results.

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

[0068] Preferably, the electrophoresis voltage is 110V. 110V is the standard electrophoresis 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. Limiting the electrophoresis time ensures sufficient separation of protein bands.

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

[0071] The fourth technical solution of the present invention: mimicking the application of Staphylococcus aureus HZ01 in the reduction of nitrate to nitrite. The mimicking Staphylococcus aureus HZ01 of the present invention has good nitrate reductase activity, and can effectively reduce nitrate to nitrite during application. This allows the utilization of nitrate in fermented meat products, thereby reducing the amount of nitrite used in meat processing.

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

[0073] (i) Prepare NB medium containing an appropriate concentration of KNO3 / NaNO3;

[0074] (ii) Inoculate the Staphylococcus mimicry HZ01 bacterial culture into the NB medium in step (i) and culture it.

[0075] (iii) During the culture process, the fermentation broth is centrifuged at intervals and the supernatant is collected;

[0076] (iv) Detection steps (iii) Nitrite content in the supernatant. The entire detection process is simple, convenient, and quick, demonstrating a good reduction effect that mimics Staphylococcus aureus HZ01 in reducing nitrate to nitrite.

[0077] Preferably, step (i) involves preparing 0.1% KNO3 / NaNO3 NB medium. The 0.1% KNO3 / NaNO3 NB medium provides a better mimicry of the nitrate reductase activity of Staphylococcus aureus HZ01.

[0078] Preferably, the preparation of NB culture medium containing an appropriate concentration of KNO3 / NaNO3 in step (i) includes the following steps.

[0079] (i) Dissolve an appropriate amount of KNO3 / NaNO3 and NB nutrient broth in sterile water;

[0080] (i02) After sterilizing and culturing the mixture from step (i01), NB medium containing an appropriate concentration of KNO3 / NaNO3 is obtained. NB medium containing an appropriate concentration of KNO3 / NaNO3, prepared from KNO3 / NaNO3, NB nutrient broth, and sterile water, can better mimic the reduction action of Staphylococcus aureus HZ01, and better demonstrates the effect of mimicking 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 is limited to meet the requirements for preparing NB culture medium with the required concentration of KNO3 / NaNO3.

[0082] Preferably, the amount of NB nutrient broth used in step (i01) is 0.7g to 1.2g. More preferably, the amount of NB nutrient broth used in step (i01) is 0.8g to 1.1g. The amount of NB nutrient broth is limited to meet the requirements for preparing NB culture medium with a required concentration of KNO3 / NaNO3.

[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. Even more preferably, the amount of sterile water used in step (i01) is 45 mL to 55 mL. The amount of sterile water is limited to meet the requirements for preparing NB culture medium with a required concentration of KNO3 / NaNO3.

[0084] Preferably, the sterilization temperature in step (i02) is 115℃ to 130℃. More preferably, the sterilization temperature in step (i02) is 120℃ to 125℃. This limitation on the sterilization temperature ensures sufficient inactivation of bacteria and their spores, thereby guaranteeing the sterility of the NB culture medium containing an appropriate concentration of KNO3 / NaNO3.

[0085] Preferably, the culture time in step (i02) is 15 min to 30 min. More preferably, the culture time in step (i02) is 20 min to 25 min. This limitation on the culture time ensures good dissolution and mixing of KNO3 / NaNO3 and its spores with the NB nutrient broth in sterile water.

[0086] Preferably, in step (ii), the inoculation volume of the simulated Staphylococcus HZ01 bacterial suspension is 0.05 mL to 0.2 mL. More preferably, in step (ii), the inoculation volume of the simulated Staphylococcus HZ01 bacterial suspension is 0.1 mL to 0.15 mL. The inoculation volume of the simulated Staphylococcus HZ01 bacterial suspension is limited according to the concentration of KNO3 to ensure that 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 better promotes the reduction of KNO3 / NaNO3 in NB medium by Staphylococcus aureus HZ01.

[0088] Preferably, the culture time in step (ii) is 12h to 36h. More preferably, the culture time in step (ii) is 15h to 30h. Even more preferably, the culture time in step (ii) is 20h to 25h. Limiting the culture time ensures that the reduction of KNO3 / NaNO3 in NB medium is fully achieved by mimicking Staphylococcus aureus HZ01.

[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 according to the testing requirements, as long as it meets the requirements for testing nitrite content.

[0090] Preferably, the time interval for taking fermentation broth samples in step (iii) is 2 to 6 hours. More preferably, the time interval for taking fermentation broth samples in step (iii) is 3 to 5 hours. Taking fermentation broth at intervals allows for a more objective and accurate assessment of the reduction effect of Staphylococcus aureus HZ01 on KNO3 / NaNO3.

[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. This low centrifugation temperature is to ensure the safety of mimicking the activity of Staphylococcus aureus 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. This limitation on the centrifugation speed is to efficiently achieve solid-liquid separation.

[0093] Preferably, step (iv) for detecting the nitrite content in the supernatant 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 p-aminobenzenesulfonic acid solution to the colorimetric tube in step (ⅳ01), mix well and let stand;

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

[0097] (ⅳ04) Add water to the colorimetric tube mark after adding the solution from step (ⅳ03), mix well and let 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 colorimetric reaction; the addition of naphthylethylenediamine hydrochloride solution can effectively combine with the diazotized substance to generate a rose-red solution, thus forming a good nitrite sample; by measuring the absorbance, the nitrite content in the supernatant can be accurately and quickly determined.

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

[0100] Preferably, the colorimetric tube in step (iv01) is 20 mL to 30 mL. More preferably, the colorimetric tube in step (iv01) is 25 mL. This limitation on the colorimetric tube size is to ensure that the nitrite content in the sample can be accurately and quickly calculated after subsequent volume adjustment to the mark.

[0101] Preferably, in step (iv02), the amount of p-aminobenzenesulfonic acid solution added is 0.5 mL to 2 mL, and the concentration of the p-aminobenzenesulfonic acid solution is 3 g / L to 5 g / L. More preferably, in step (iv02), the amount of p-aminobenzenesulfonic acid solution added is 0.1 mL to 1.5 mL, and the concentration of the p-aminobenzenesulfonic acid solution is 3.5 g / L to 4.5 g / L. Both the amount of p-aminobenzenesulfonic acid solution added and its concentration are set according to the detection requirements and the required amount of supernatant, all for the purpose of more accurate and rapid calculation of the nitrite content in the sample.

[0102] Preferably, the settling time of the solution in step (iv02) is 3 to 5 minutes. More preferably, the settling time of the solution in step (iv02) is 3.5 to 4.5 minutes. The limited settling time is to allow the solution to return to a calm state after the diazotization reaction, thus maintaining a better condition for subsequent reactions.

[0103] Preferably, in step (iv03), the amount of naphthylethylenediamine hydrochloride solution added 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, in step (iv03), the amount of naphthylethylenediamine hydrochloride solution added is 0.5 mL to 0.8 mL, and the concentration of the naphthylethylenediamine hydrochloride solution is 2 g / L to 4 g / L. Both the amount of naphthylethylenediamine hydrochloride solution added and its concentration are set according to the detection requirements and the requirements of the colorimetric reaction, in order to more accurately and quickly calculate the nitrite content in the sample.

[0104] Preferably, the settling time in step (iv04) is 10 min to 20 min. More preferably, the settling time in step (iv04) is 13 min to 17 min. This limitation on the settling time is to ensure a good condition for subsequent absorbance measurement.

[0105] Preferably, step (iv05) involves using a 1cm cuvette, adjusting the zero point of the cuvette with a zero tube, taking an appropriate amount of the solution from step (iv04), and measuring the absorbance at a wavelength of 538nm. The limitations on the cuvette specifications, zero-point adjustment, and wavelength setting are all based on the specific requirements of nitrite levels in the supernatant, in order to more accurately and quickly calculate the nitrite content in the sample.

[0106] The fifth technical solution of this invention: The application of mimicking Staphylococcus HZ01 in dry-cured fermented meat products. The mimicking Staphylococcus HZ01 of this invention can significantly increase the levels of esters, aldehydes, and acids in fermented meat products, with the most significant increases in vinyl acetate, methyl butyrate, methyl hexanoate, n-hexanal, acetic acid, and isovaleric acid, thus significantly improving the quality of fermented meat products.

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

[0108] (a) Take the concentrated solution or powder that imitates Staphylococcus aureus HZ01 and dilute it with drinking water;

[0109] (ii) Dissolve appropriate amounts of salt, sugar and sodium glutamate in the solution in step (i) to make a pickling solution;

[0110] (III) Take the minced meat and add the marinade from step (II) to the minced meat until the concentration of Staphylococcus aureus HZ01 in the minced meat reaches 1.0 × 10⁻⁶.7 log 10 CFU / g ~ 9.9 × 10 8 log 10 When the concentration of CFU / g is reached, the minced meat is marinated at a low temperature.

[0111] (iv) After marinating, the minced meat is stuffed into sausages, degassed, dried and fermented in sequence to obtain the dry-cured fermented meat product. A marinating solution is prepared by combining appropriate amounts of salt, sugar, monosodium glutamate, and a concentrated / powdered solution of Staphylococcus aureus HZ01, resulting in better marinating of minced meat. The minced meat is directly seasoned after marinating, eliminating the need for further seasoning of the meat products. Low-temperature marinating effectively maintains the activity and safety of Staphylococcus aureus HZ01. The marinated minced meat is then sequentially stuffed, degassed, dried, and fermented. The resulting dry-cured fermented meat product is fragrant, nutritious, and of excellent quality. The fact that the Staphylococcus aureus HZ01 remains active after drying and continues to play a role during fermentation indicates that the Staphylococcus aureus HZ01 in this invention has high heat resistance. The dry-cured fermented meat product treated with Staphylococcus aureus HZ01 shows a significant increase in the content of esters, aldehydes, and acids, with the most significant increases in vinyl acetate, methyl butyrate, methyl hexanoate, n-hexanal, acetic acid, and isovaleric acid.

[0112] Preferably, the concentration of the Staphylococcus aureus HZ01 inoculum in step (iii) is 3.0 × 10⁻⁶. 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 × 10⁻⁶. 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 × 10⁻⁶. 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 × 10⁻⁶ CFU / g. 7 log 10 CFU / g ~ 1.9 × 10 8 log 10The concentration limit for the Staphylococcus aureus HZ01 inoculant is set at CFU / g to ensure a good ratio with salt, sugar, and monosodium glutamate, thereby improving the subsequent curing of the minced meat and allowing it to play a better role in the curing and fermentation processes.

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

[0114] The mixture consists of 1-5 parts salt, 3-8 parts sugar, 0.2-0.8 parts monosodium glutamate (MSG), 2-5 parts water, and an appropriate amount of Staphylococcus aureus HZ01 concentrate / powder. The precise proportions of salt, sugar, MSG, water, and Staphylococcus aureus HZ01 concentrate / powder ensure a nutritionally balanced marinade. The components work synergistically to effectively marinate minced meat, allowing it to absorb the flavor directly without requiring further seasoning of the meat products. Spices can also be added as needed.

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

[0116] The mixture consists of 2-4 parts salt, 4-7 parts sugar, 0.3-0.7 parts monosodium glutamate (MSG), 3-4 parts water, and an appropriate amount of Staphylococcus aureus HZ01 concentrate / powder. The precise proportions of salt, sugar, MSG, water, and Staphylococcus aureus HZ01 concentrate / powder ensure a more balanced nutritional profile in the marinating solution. The components work synergistically to effectively marinate the minced meat, allowing it to absorb the flavor directly without requiring further seasoning of the meat products.

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

[0118] The mixture consists of 2-4 parts salt, 5-6 parts sugar, 0.4-0.6 parts monosodium glutamate (MSG), 3-4 parts water, and an appropriate amount of Staphylococcus aureus HZ01 concentrate / powder. The precise proportions of salt, sugar, MSG, water, and Staphylococcus aureus HZ01 concentrate / powder ensure a more balanced nutritional profile in the marinating solution. The components work synergistically to effectively marinate the minced meat, allowing it to absorb the flavor directly without requiring further seasoning of the meat products.

[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. Limiting the mass ratio of fat to lean meat in the minced meat results in a better quality final product of dry-cured fermented meat.

[0120] Preferably, the pickling temperature in step (iii) is 2℃ to 6℃. More preferably, the pickling temperature in step (iii) is 3℃ to 5℃. Low-temperature pickling ensures that the bacteria maintain good activity and safety during the pickling process, mimicking Staphylococcus aureus HZ01.

[0121] Preferably, the marinating time in step (iii) is 48h to 72h. More preferably, the marinating time in step (iii) is 55h to 65h. Limiting the marinating time ensures that the minced meat is fully marinated and flavorful.

[0122] Preferably, the drying temperature in step (iv) is 50℃~65℃. More preferably, the drying temperature in step (iv) is 55℃~60℃. This limitation on the drying temperature ensures effective drying of the sausages without excessively affecting the activity of the mimic Staphylococcus aureus HZ01, allowing the mimic Staphylococcus aureus HZ01 to continue functioning in the subsequent fermentation process.

[0123] Preferably, the drying time in step (iv) is 48h to 72h. More preferably, the drying time in step (iv) is 55h to 65h. Limiting the drying time ensures that the sausages are thoroughly dried.

[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. This limitation on the fermentation temperature is also intended to maintain good activity mimicking Staphylococcus aureus HZ01.

[0125] Preferably, the fermentation time in step (iv) is 1 to 2 weeks. More preferably, the fermentation time in step (iv) is 8 to 10 days. Limiting the fermentation time ensures more complete fermentation of the dried enemas by mimicking Staphylococcus aureus HZ01.

[0126] Preferably, the method for preparing the concentrated solution of *Staphylococcus aureus* HZ01 is as follows: a single colony of purified and refrigerated *Staphylococcus aureus* HZ01 is inoculated into sterilized NB liquid culture medium and cultured until the concentration of *Staphylococcus aureus* HZ01 in the NB liquid culture medium reaches 1.0 × 10⁻⁶. 7 log 10 CFU / mL ~5.0×10 7 log 10 The concentrate is obtained when the concentration reaches CFU / mL. The purpose of preparing the concentrate is to better prepare the marinade for subsequent processes, thereby facilitating the subsequent marinating and fermentation of the minced meat. The limitation on the concentration of Staphylococcus aureus HZ01 in the concentrate is to meet the processing requirements of the minced meat in subsequent steps, and to ensure that the final dry-cured 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. This limitation on the refrigeration temperature is to ensure the safety and activity of the mimicking Staphylococcus aureus HZ01.

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

[0129] Preferably, the culture time during the preparation of the concentrate is 12h to 36h. More preferably, the culture time during the preparation of the concentrate is 15h to 35h. Even more preferably, the culture time during the preparation of the concentrate is 20h to 30h. The limitation on the culture time here is to ensure proper culture of *Staphylococcus mimicus* HZ01, until the concentration of *Staphylococcus mimicus* HZ01 reaches a plateau.

[0130] Preferably, the culture temperature during the preparation of the concentrate is 25℃ to 35℃. More preferably, the culture temperature during the preparation of the concentrate is 28℃ to 32℃. This limitation on the culture temperature is to create favorable temperature conditions so that *Staphylococcus mimicus* HZ01 can be well cultured in sterilized NB liquid medium, allowing *Staphylococcus mimicus* HZ01 to quickly reach the required concentration of concentrate.

[0131] Preferably, the culture process during the preparation of the concentrate is a static culture. Static culture is suitable for mimicking the growth characteristics of Staphylococcus aureus HZ01.

[0132] Preferably, the simulated Staphylococcus aureus HZ01 tolerates a pH of 4–8 during fermentation. More preferably, the simulated Staphylococcus aureus HZ01 tolerates a pH of 5–7 during fermentation. The simulated Staphylococcus aureus HZ01 can grow under acidic, neutral, and alkaline conditions, exhibiting good and wide-ranging acid and alkali tolerance.

[0133] Preferably, the nitrite tolerance level of the mimic *Staphylococcus aureus* HZ01 during fermentation is 0–150 mg / kg. More preferably, the nitrite tolerance level of the mimic *Staphylococcus aureus* HZ01 during fermentation is 50 mg / kg–100 mg / kg. The mimic *Staphylococcus aureus* HZ01 exhibits a wide tolerance range for nitrite content during fermentation, allowing for the adjustment of nitrite dosage within a broad range based on actual needs.

[0134] Preferably, the salt tolerance of the mimicked Staphylococcus HZ01 during fermentation is 3% to 12%. More preferably, the salt tolerance of the mimicked Staphylococcus HZ01 during fermentation is 5% to 10%. Even more preferably, the salt tolerance of the mimicked Staphylococcus HZ01 during fermentation is 6% to 8%. The mimicked Staphylococcus HZ01 exhibits a wide range of salt tolerance during fermentation, allowing for a greater range of salt additions as needed during the fermentation process.

[0135] Preferably, the temperature tolerance of the mimicked Staphylococcus HZ01 during fermentation is 20℃ to 40℃. More preferably, the temperature tolerance of the mimicked Staphylococcus HZ01 during fermentation is 25℃ to 35℃. The mimicked Staphylococcus HZ01 exhibits a wide temperature tolerance range during fermentation, allowing for adjustment within a relatively large temperature range according to actual needs during the fermentation process.

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

[0137] (1) It mimics the strong metabolic capacity of Staphylococcus aureus HZ01. When used as a fermentation agent, it can maintain its advantage in the competition with local microbial populations and has strong competitiveness. This makes 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, which has the ability to degrade sarcoplasmic proteins. It can degrade 58.1% of sarcoplasmic protein bands within 72 hours and significantly reduce sarcoplasmic protein bands by 41.9%.

[0139] (3) It mimics the good nitrate reductase activity of Staphylococcus aureus HZ01, which can efficiently reduce nitrate to nitrite during application and utilize the nitrate in fermented meat products, thereby reducing the amount of nitrite used in meat processing.

[0140] (4) It can mimic Staphylococcus HZ01 to metabolize leucine to produce 3-methylbutyraldehyde, which increases the types and contents of volatile flavor substances and produces excellent aroma properties.

[0141] (5) Imitation of Staphylococcus aureus HZ01 can significantly increase the esters, aldehydes and acids in fermented meat products. Among them, the increase of vinyl acetate, methyl butyrate, methyl hexanoate, n-hexanal, acetic acid and isovaleric acid is the most significant, which can significantly improve the quality of fermented meat products. Attached Figure Description

[0142] Figure 1 This is a diagram illustrating the colony morphology of Staphylococcus aureus HZ01 as described in this invention.

[0143] Figure 2This is a Gram staining result diagram of the imitation of Staphylococcus HZ01 in this invention;

[0144] Figure 3 This is a comparison diagram of the results of mimicking Staphylococcus HZ01 with other Staphylococcus strains in this invention;

[0145] Figure 4 This is an evolutionary relationship diagram of the imitation of Staphylococcus aureus HZ01 in this invention;

[0146] Figure 5 This is a curve illustrating the pH tolerance fermentation characteristics of Staphylococcus aureus HZ01 in this invention;

[0147] Figure 6 This is a graph showing the fermentation characteristics of Staphylococcus aureus HZ01 in terms of nitrite tolerance, as described in this invention.

[0148] Figure 7 This is a graph showing the salt tolerance fermentation characteristics of Staphylococcus aureus HZ01 in this invention.

[0149] Figure 8 This is a curve illustrating the temperature tolerance fermentation characteristics of Staphylococcus aureus HZ01 in this invention;

[0150] Figure 9 This is an SDS-PAGE image of the biochemical degradation of sarcoplasmic proteins by Staphylococcus aureus HZ01 in this invention.

[0151] Figure 10 This invention mimics the anaerobic conditions of Staphylococcus aureus strain HZ01, utilizing NO3. - Converted to NO2 – The situation is shown in the bar chart. Detailed Implementation

[0152] The present invention will be further described below with reference to the accompanying drawings and embodiments, but this should not be construed as limiting the present invention.

[0153] A strain of Staphylococcus simulans HZ01 has been deposited. The accession number of Staphylococcus simulans HZ01 is GDMCC NO.62410. The depositary institution for Staphylococcus simulans HZ01 is Guangdong Provincial Center for Microbial Culture Collection. The accession name for Staphylococcus simulans HZ01 is Staphylococcus simulans HZ01. The deposit date for Staphylococcus simulans HZ01 is April 24, 2022.

[0154] The Staphylococcus HZ01 inoculum agent is prepared by imitating Staphylococcus HZ01.

[0155] The preparation of a Staphylococcus aureus HZ01 inoculum includes the following steps:

[0156] (A) Inoculate Staphylococcus aureus HZ01 into NB medium and culture to obtain bacterial suspension; the NB medium in step (A) includes 10 g / L peptone, 3.0 g / L beef extract and 5.0 g / L sodium chloride; the pH of the NB medium in step (A) is 7-7.4; the volume of 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℃-35℃;

[0157] (B) The concentration of the staphylococcus HZ01-mimicking bacterial culture obtained in step (A) is 1.0 × 10⁻⁶. 8 log 10 CFU / mL ~9.9×10 9 log 10 Centrifuge at CFU / mL; the centrifugation speed in step (B) is 5000 rap / min to 20000 rap / min; the centrifugation time in step (B) is 10 min to 20 min; the centrifugation temperature in step (B) is 2℃ to 6℃;

[0158] (C) After centrifugation, add sterilized skim milk to resuspend; the mass concentration of 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 is performed to obtain a concentration of 1.0 × 10⁻⁶. 10 log 10 CFU / g ~ 9.9 × 10 11 log 10 The powdered agent, containing CFU / g, mimics Staphylococcus aureus HZ01; the freeze-drying temperature in step (D) is -90℃ to -70℃; the freeze-drying time in step (D) is 24h to 72h.

[0160] The gene that mimics Staphylococcus HZ01 has accession number OM758216, and its specific nucleotide sequence is shown in SEQ ID NO.1;

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

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

[0163] (b) The total DNA extracted in step (a) was amplified for 16 seconds using a PCR reaction system. The PCR reaction system consisted of 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 volume of the PCR reaction system was 15 μL to 25 μL.

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

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

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

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

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

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

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

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

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

[0173] The application of Staphylococcus aureus HZ01 in degrading sarcoplasmic proteins was imitated.

[0174] The detection of degradation results of sarcoplasmic proteins by mimicking Staphylococcus aureus HZ01 includes the following steps;

[0175] (S01) Sarcoplasmic proteins were extracted and their concentrations were determined 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 to 10g; the amount of PB buffer used is 15mL to 50mL; the concentration of PB buffer is 0.02mol / L; the pH of PB buffer is 5.8 to 6.6; the homogenization speed is 10000rpm / min to 15000rpm / min; the homogenization time is 15min to 30min.

[0178] (S012) After homogenizing the supernatant in step (S011) through a filter membrane and sterilizing it, sarcoplasmic protein is obtained; the pore size of the filter membrane is 0.2μm~0.25μm;

[0179] (S02) Take the sarcoplasmic protein, glucose and the Staphylococcus mimicus HZ01 bacterial solution from step (S01) and incubate them together; the glucose is 1% glucose; the amount of Staphylococcus mimicus HZ01 bacterial solution is 0.05 mL to 0.5 mL; the incubation time is 24 h to 72 h;

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

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

[0182] (S05) Take 10%-12% of Bio-Rad pre-formed sarcosin and standard protein, and load the samples from step (S04) onto the 10%-12% Bio-Rad pre-formed sarcosin and standard protein respectively; the loading volume of the 10%-12% Bio-Rad pre-formed sarcosin is 15μL-25μL; the loading volume of the standard protein is 5μL-10μL.

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

[0184] (S07) After electrophoresis, stain with Coomassie Brilliant Blue R-250 to obtain the results of the degradation of sarcoplasmic proteins by Staphylococcus aureus HZ01; the staining time is 1h to 2h.

[0185] Inspired by the application of Staphylococcus aureus HZ01 in the reduction of nitrate to nitrite;

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

[0187] (i) Prepare NB medium containing an appropriate concentration of KNO3 / NaNO3; step (i) is to prepare NB medium containing 0.1% KNO3 / NaNO3;

[0188] The preparation of NB medium containing an appropriate concentration of KNO3 / NaNO3 in step (i) includes the following steps:

[0189] (i01) Dissolve an appropriate amount 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) After sterilizing and culturing the mixture in step (i01), NB medium containing an appropriate concentration of KNO3 / NaNO3 is obtained; the sterilization temperature in step (i02) is 115℃~130℃; the culturing time in step (i02) is 15min~30min;

[0191] (ii) Inoculate the Staphylococcus mimicryl HZ01 bacterial suspension into the NB medium in step (i) and culture it; the inoculation volume of the Staphylococcus mimicryl HZ01 bacterial suspension in step (ii) is 0.05 mL to 0.2 mL; the culture temperature in step (ii) is 25℃ to 35℃; the culture time in step (ii) is 12 h to 36 h;

[0192] (iii) During the culture process, take the fermentation broth at intervals, centrifuge and collect the supernatant; the amount of fermentation broth used 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 ℃ to 6 ℃; the centrifugation speed in step (iii) is 5000 rap / min to 20000 rap / min;

[0193] (iv) Detection steps (iii) Nitrite content in the supernatant;

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

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

[0196] (ⅳ02) Add an appropriate amount of p-aminobenzenesulfonic acid solution to the colorimetric tube in step (ⅳ01), mix well and let 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 standing time of the solution in step (ⅳ02) is 3 min to 5 min;

[0197] (ⅳ03) Add 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) After adding water to the colorimetric tube to the mark in step (ⅳ03), mix well and let 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 1cm cuvette, adjust the zero point of the cuvette with the zero tube, take an appropriate amount of the solution in step (ⅳ04), and measure the absorbance at a wavelength of 538nm.

[0200] The application of Staphylococcus aureus HZ01 in dry-cured fermented meat products was imitated.

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

[0202] (a) Take the concentrated solution / powder that imitates Staphylococcus aureus HZ01 and dilute it with drinking water;

[0203] The method for preparing the concentrated solution of Staphylococcus aureus HZ01 is as follows: a single colony of purified and refrigerated Staphylococcus aureus HZ01 is inoculated into sterilized NB liquid medium and cultured until the concentration of Staphylococcus aureus HZ01 in the NB liquid medium reaches 1.0 × 10⁻⁶. 7 log 10 CFU / mL~5.0×

[0204] 10 7 log 10 The concentrate is obtained when the concentration is CFU / mL; the refrigeration temperature during the concentrate preparation process is 2℃~6℃; the volume of NB liquid culture medium used during the concentrate preparation process is 40mL~60mL; the incubation time during the concentrate preparation process is 12h~36h; the incubation temperature during the concentrate preparation process is 25℃~35℃; the incubation during the concentrate preparation process is static incubation.

[0205] (ii) Dissolve appropriate amounts of salt, sugar and sodium glutamate in the solution in step (i) to make a pickling solution;

[0206] The marinating liquid in step (II) comprises the following components by weight:

[0207] 1-5 parts salt, 3-8 parts white sugar, 0.2-0.8 parts monosodium glutamate, 2-5 parts drinking water, and 1-5 parts concentrated / powder of Staphylococcus aureus HZ01.

[0208] (III) Take the minced meat and add the marinade from step (II) to the minced meat until the concentration of Staphylococcus aureus HZ01 in the minced meat reaches 1.0 × 10⁻⁶. 7 log 10 CFU / g ~ 9.9 × 10 8 log 10 At CFU / g, the minced meat is marinated at low temperature; based on the weight of the minced meat, the concentration of the Staphylococcus aureus HZ01 inoculant in step (iii) is 3.0 × 10⁻⁶. 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 marinating temperature in step (III) is 2℃-6℃; the marinating time in step (III) is 48h-72h;

[0209] (IV) After marinating, the minced meat is sequentially stuffed into sausages, degassed, dried, and fermented to obtain the finished dry-cured fermented meat product; the drying temperature in step (IV) is 50℃~65℃; the drying time in step (IV) is 48h~72h; the fermentation temperature in step (IV) is 20℃~30℃; the fermentation time in step (IV) is 1 week~2 weeks; the pH tolerance of mimicking Staphylococcus aureus HZ01 during fermentation is 4~8; the nitrite tolerance of mimicking Staphylococcus aureus HZ01 during fermentation is 0~150mg / kg; the salt tolerance of mimicking Staphylococcus aureus HZ01 during fermentation is 3%~12%; the temperature tolerance of mimicking Staphylococcus aureus HZ01 during fermentation is 20℃~40℃.

[0210] Example 1:

[0211] A microbial agent mimicking Staphylococcus HZ01, named Staphylococcus simulans HZ01, is prepared by the following steps.

[0212] (A) Staphylococcus aureus HZ01 was inoculated into NB medium and cultured to obtain a bacterial suspension; the NB medium in step (A) consisted of 10 g / L peptone, 3.0 g / L beef extract and 5.0 g / L sodium chloride; the pH of the NB medium in step (A) was 7.2 ± 0.2; the volume of NB medium used in step (A) was 1 L; the culture in step (A) was a static culture; the culture time in step (A) was 48 h; the culture temperature in step (A) was 30 °C.

[0213] (B) The concentration of the staphylococcal HZ01-mimicking bacterial culture obtained in step (A) is 2.5 × 10⁻⁶. 9 log 10 Centrifuge at CFU / mL; the centrifugation speed in step (B) is 10000 RPM / min; the centrifugation time in step (B) is 15 min; the centrifugation temperature in step (B) is 4℃;

[0214] (C) After centrifugation, add sterilized skim milk to resuspend; the mass concentration of 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 is performed to obtain a concentration of 2.0 × 10⁻⁶. 11 log 10 The powdered agent, containing CFU / g, mimics Staphylococcus aureus HZ01; the freeze-drying temperature in step (D) is -80℃; the freeze-drying time in step (D) is 48h.

[0216] Example 2:

[0217] The CNKI accession number for the gene mimicking Staphylococcus HZ01 is OM758216, and the accession number at the Guangdong Provincial Microbial Culture Collection Center (GDMCC) is 62410. The specific nucleotide sequence is shown in SEQ ID NO.1.

[0218]

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

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

[0221] (b) The total DNA extracted in step (a) was amplified for 16 seconds using a PCR reaction system. The PCR reaction system consisted of 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 total volume of the PCR reaction system was 20 μL.

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

[0223] (b01) The total DNA extracted in step (a) was pre-denatured by reacting it in a PCR reaction system at 95°C for 5 min.

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

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

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

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

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

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

[0230] (c) After the full-length 16S amplification is complete, gel purification is performed, followed by electrophoresis sequencing using a horizontal electrophoresis apparatus. The obtained sequence is the full-length 16S DNA sequence of Staphylococcus aureus HZ01, 1437 bp. Figure 1 and Figure 2 As shown, the similarity between this strain and its closest phylogenetic relative, S. simulans MR1 ​​(CP015642.1), is as follows: Figure 3 The 99.65% shown, formed independent species during the evolutionary process, such as... Figure 4 The evolutionary branch shown is a new strain that mimics Staphylococcus aureus.

[0231] Example 3:

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

[0233] The method for preparing the concentrated solution of Staphylococcus aureus HZ01 is as follows: a single colony of purified Staphylococcus aureus HZ01 stored at 4℃ is inoculated into 50 mL of sterile NB liquid medium and incubated statically at 30℃ for 24 hours until the concentration reaches 3.0 × 10⁻⁶. 7 log 10 The concentration was CFU / mL, and this culture medium was the concentrated solution. 50 mL of NB liquid medium was prepared with pH values ​​of 4, 5, 6, 7, and 8; salinity of 0%, 3%, 6%, 9%, and 12%; and nitrite concentrations of 0, 50 mg / kg, 100 mg / kg, and 150 mg / kg, respectively. 1 mL of the concentrated solution was inoculated into each of the NB mediums under different culture conditions and incubated at 30°C for 24 hours. The OD value was measured every 3 hours to verify the optimal fermentation conditions for mimicking Staphylococcus aureus HZ01. 50 mL of NB liquid medium was prepared, and 1 mL of the concentrated solution was inoculated into it. The OD value curves of strain HZ01 at different temperatures were verified to determine the temperature growth range of HZ01. The results showed that HZ01 has a wide growth range, and it can grow under conditions of pH 5–8, nitrite 0–150 mg / kg, salinity 3%–9%, ​​and temperature 20°C–35°C.

[0234] Example 4:

[0235] Mimicking the application of Staphylococcus aureus HZ01 in the degradation of sarcoplasmic proteins; such as Figure 9 and Figure 10 As shown, mimicking Staphylococcus HZ0 can secrete highly active proteases, which can degrade 58.1% of sarcoplasmic protein bands within 72 hours and significantly reduce sarcoplasmic protein bands by 41.9%.

[0236] The detection of degradation results of sarcoplasmic proteins by mimicking Staphylococcus aureus HZ01 includes the following steps;

[0237] (S01) Sarcoplasmic proteins were extracted and their concentrations were determined using a Lowry protein concentration kit;

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

[0239] (S011) Mix pork with PB buffer and homogenize; the pork is fresh lean pork; the amount of pork used is 2g; the amount of PB buffer used is 20mL; the concentration of PB buffer is 0.02mol / L; the pH of PB buffer is 6.5; the homogenization speed is 13000rpm / min; the homogenization time is 20min.

[0240] (S012) After homogenizing the supernatant in step (S011) through a filter membrane and sterilizing it, sarcoplasmic protein is obtained; the filter membrane pore size is 0.22 μm;

[0241] (S02) Take the sarcoplasmic protein, glucose and the imitation Staphylococcus HZ01 bacterial solution from step (S01) and incubate them together; the glucose is 1% glucose; the volume of the imitation Staphylococcus HZ01 bacterial solution is 0.1 mL; the incubation time is 12 h to 36 h;

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

[0243] (S04) Take 2×SDS loading buffer and mix it with the supernatant after centrifugation in step (S03), and place it in a water bath; the volume of 2×SDS loading buffer is 200uL; the volume of supernatant after centrifugation is 200uL; the water bath temperature is 95℃; the water bath time is 5min;

[0244] (S05) Take 10%-12% of Bio-Rad pre-formed sarcosin and standard protein, and load the samples from step (S04) onto the 10%-12% Bio-Rad pre-formed sarcosin and standard protein respectively; the loading volume of 10%-12% Bio-Rad pre-formed sarcosin is 20 μL; the loading volume of standard protein is 8 μL.

[0245] (S06) After loading the sample, perform electrophoresis; the electrophoresis voltage is 110V; the electrophoresis time is 90min;

[0246] (S07) After electrophoresis, staining was performed with Coomassie Brilliant Blue R-250. The staining results were used to determine the degradation of sarcoplasmic protein bands by Staphylococcus aureus HZ01. The staining time was 1.5 h.

[0247] Example 5:

[0248] Inspired by the application of Staphylococcus aureus HZ01 in the reduction of nitrate to nitrite;

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

[0250] (i) Prepare NB medium containing an appropriate concentration of KNO3; step (i) is to prepare NB medium containing 0.1% KNO3;

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

[0252] (i01) Dissolve an appropriate amount of KNO3 and NB nutrient broth 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) After sterilizing and culturing the mixture in step (i01), NB medium containing an appropriate concentration of KNO3 is obtained; the sterilization temperature in step (i02) is 121℃; the culturing time in step (i02) is 20min;

[0254] (ii) Inoculate the Staphylococcus mimicryl HZ01 bacterial suspension into the NB medium in step (i) and culture it; the inoculation volume of the Staphylococcus mimicryl HZ01 bacterial suspension in step (ii) is 0.1 mL; the culture temperature in step (ii) is 30℃; the culture time in step (ii) is 24 h;

[0255] (iii) During the culture process, the fermentation broth is centrifuged at intervals and the supernatant is collected; the amount of fermentation broth used in step (iii) is 2 mL; the time interval for taking fermentation broth in step (iii) is 4 h; the centrifugation temperature in step (iii) is 4 ℃; the centrifugation speed in step (iii) is 10000 rap / min;

[0256] (iv) Detection steps (iii) Nitrite content in the supernatant;

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

[0258] (ⅳ01) Take an appropriate amount of supernatant and place it in a stoppered colorimetric tube; the amount of supernatant used in step (ⅳ01) is 1 mL; the size of the colorimetric tube in step (ⅳ01) is 25 mL;

[0259] (ⅳ02) Add an appropriate amount of p-aminobenzenesulfonic acid solution to the colorimetric tube in step (ⅳ01), mix well and let 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 standing time of the solution in step (ⅳ02) is 3 min to 5 min.

[0260] (ⅳ03) Add 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) After adding water to the colorimetric tube to the mark in step (ⅳ03), mix well and let stand; the standing time in step (ⅳ04) is 15 min;

[0262] (ⅳ05) Determine the absorbance of the solution in step (ⅳ04); Step (ⅳ05) is to use a 1cm cuvette, adjust the zero point of the cuvette with the zero tube, take an appropriate amount of the solution in step (ⅳ04), and measure the absorbance at a wavelength of 538nm, while simultaneously preparing a reagent blank and a standard curve.

[0263] Example 6:

[0264] The application of Staphylococcus aureus HZ01 in dry-cured fermented meat products was imitated.

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

[0266] (a) Take the concentrated solution that mimics Staphylococcus aureus HZ01 and dilute it with drinking water;

[0267] The method for preparing the concentrated solution of Staphylococcus aureus HZ01 is as follows: a single colony of purified Staphylococcus aureus HZ01 stored at 4℃ is inoculated into 50 mL of sterile NB liquid medium and incubated statically at 30℃ for 24 hours until the concentration reaches 3.0 × 10⁻⁶. 7 log 10 The concentration is CFU / mL, and the culture medium is the concentrated solution.

[0268] (ii) Dissolve appropriate amounts of salt, sugar and sodium glutamate in the solution in step (i) to make a pickling solution;

[0269] The marinating liquid in step (II) comprises the following components by weight:

[0270] 2.5 parts salt, 4 parts sugar, 0.4 parts monosodium glutamate, 2.5 parts drinking water, and 2.5 parts concentrated solution of Staphylococcus aureus HZ01.

[0271] (III) Take the minced meat and add the marinade from step (II) to the minced meat until the concentration of Staphylococcus aureus HZ01 in the minced meat reaches 8.0 × 10⁻⁶. 7 log 10 CFU / g ~ 9.9 × 10 8 log 10 When 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 (III) is 4℃; the marinating time in step (III) is 48h~72h.

[0272] (IV) After marinating, stuff the minced meat into sausages in sequence. The length and diameter of the sausages are not required. Exhaust, dry, and ferment to obtain the finished dry-cured fermented meat product. The drying temperature in step (IV) is 52℃~54℃. The drying time in step (IV) is 48h~72h. The fermentation temperature in step (IV) is 20℃~30℃. The fermentation time in step (IV) is 1 week~2 weeks. The pH tolerance of the simulated Staphylococcus HZ01 during fermentation is 4~8. The nitrite tolerance of the simulated Staphylococcus HZ01 during fermentation is 0~150mg / kg. The salt tolerance of the simulated Staphylococcus HZ01 during fermentation is 3%~12%. The temperature tolerance of the simulated Staphylococcus HZ01 during fermentation is 20℃~35℃.

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

[0274] Table 1: Physicochemical properties of meat products fermented using Staphylococcus aureus inoculation

[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 used in meat products can be reduced.

[0277] The volatile compound composition of fermented meat products with different processing methods is 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, mimicking Staphylococcus aureus HZ01 can significantly increase the levels of esters, aldehydes, and acids in fermented meat products. Among these, the increases in vinyl acetate, methyl butyrate, methyl hexanoate, n-hexanal, acetic acid, and isovaleric acid are the most significant, thus significantly improving the quality of fermented meat products.

Claims

1. A method for preparing a Staphylococcus simulans HZ01 inoculant, characterized by: The Staphylococcus simulans HZ01 inoculant was prepared using Staphylococcus simulans HZ01 as a mimic; the accession number of the Staphylococcus simulans HZ01 is GDMCC NO.62410; the depositary institution of the Staphylococcus simulans HZ01 is Guangdong Provincial Center for Microbial Culture Collection; the accession name of the Staphylococcus simulans HZ01 is Staphylococcus simulans HZ01; the accession date of the Staphylococcus simulans HZ01 is April 24, 2022. The preparation of the Staphylococcus aureus HZ01-mimicking agent includes the following steps: (A) The staphylococcus mimicry strain HZ01 was inoculated into NB medium and cultured to obtain a bacterial culture; (B) The concentration of the staphylococcus HZ01-mimicking bacterial culture obtained in step (A) is 1.0 × 10⁻⁶. 8 log 10 CFU / mL ~ 9.9 × 10 9 log 10 Centrifuge at CFU / mL; (C) After centrifugation, add sterilized skim milk to resuspend; (D) After resuspension, freeze-drying is performed to obtain a concentration of 1.0 × 10⁻⁶. 10 log 10 CFU / g ~ 9.9 × 10 11 log 10 A powdered agent containing CFU / g that mimics Staphylococcus aureus HZ01.

2. The method for preparing the Staphylococcus aureus HZ01 inoculum according to claim 1, characterized in that: 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.

3. The method for preparing the Staphylococcus aureus HZ01 inoculum according to claim 1, characterized in that: In step (A), the pH value of the NB medium is 7 to 7.

4.

4. The method for preparing the Staphylococcus aureus HZ01 inoculum according to claim 1, characterized in that: The amount of NB culture medium used in step (A) is 0.5L to 2L; the culture in step (A) is a static culture.

5. The method for preparing the Staphylococcus aureus HZ01 inoculum according to claim 1, characterized in that: The culture time in step (A) is 24h to 72h; the culture temperature in step (A) is 15℃ to 65℃.

6. The method for preparing the Staphylococcus aureus HZ01 agent according to claim 1, characterized in that: The centrifugation speed in step (B) is 5000 rpm / min to 20000 rpm / min.

7. The method for preparing the Staphylococcus aureus HZ01 inoculum according to claim 1, characterized in that: The centrifugation time in step (B) is 10 min to 20 min; the centrifugation temperature in step (B) is 2℃ to 6℃.

8. The method for preparing the Staphylococcus aureus HZ01 inoculum according to claim 1, characterized in that: In step (C), the mass concentration of the sterilized skim milk is 0.05%m / v to 0.5%m / v.

9. The method for preparing the Staphylococcus aureus HZ01 inoculum agent according to claim 1, characterized in that: The amount of sterilized skim milk used in step (C) is 1ml to 5ml.

10. The method for preparing the Staphylococcus aureus HZ01 agent according to claim 1, characterized in that: The freeze-drying temperature in step (D) is -90℃ to -70℃; the freeze-drying time in step (D) is 24h to 72h.

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