CN114891701A

CN114891701A - Staphylococcus simulans HZ01, microbial inoculum and application thereof

Info

Publication number: CN114891701A
Application number: CN202210712999.7A
Authority: CN
Inventors: 李欢欢; 陈黎洪; 唐宏刚; 赵珂; 张晋
Original assignee: Zhejiang Academy of Agricultural Sciences
Current assignee: Zhejiang Academy of Agricultural Sciences
Priority date: 2022-06-22
Filing date: 2022-06-22
Publication date: 2022-08-12
Anticipated expiration: 2042-06-22
Also published as: CN116831274A; CN116875498A; CN116831273A; CN116904356A; CN114891701B

Abstract

The invention relates to the technical field of microbial food fermentation, and particularly discloses a staphylococcus simulans HZ01, a microbial inoculum and application thereof, wherein the staphylococcus simulans HZ01 has a preservation number of GDMCC NO. 62410; the simulated staphylococcus HZ01 is preserved in the Guangdong province microbial strain preservation center; the deposit name of the mimic Staphylococcus HZ01 is Staphylococcus simulans HZ 01; the simulated staphylococcus HZ01 has a preservation time of 2022, 4 months and 24 days. The invention has the characteristics of better processing adaptability, higher protease and lipase activities, better nitrate reductase activity and better flavor of the fermented meat product.

Description

Staphylococcus simulans HZ01, microbial inoculum and application thereof

Technical Field

The invention relates to the technical field of microbial food fermentation, in particular to a staphylococcus simulans HZ01, a microbial inoculum and application thereof.

Background

The microbial leaven widely used in fermented meat products at present is mainly lactic acid bacteria and Coagulase-negative staphylococcus (CNS), wherein the lactic acid bacteria are responsible for acidification of meat at the initial stage of fermentation, and reduce the pH of the raw meat and coagulate protein by producing lactic acid, thereby increasing the hardness, and in addition, the lactic acid bacteria can inhibit the growth of harmful bacteria, thereby improving the microbial safety of the fermented meat products. The CNS plays an important role in the flavor formation and the color stability of the fermented meat products. Studies have shown that staphylococci, rather than lactic acid bacteria, play an important role in the flavour development of fermented meat products. The CNS functions to reduce nitrate to nitrite and then to nitrous oxide, to prevent rancidity by peroxide decomposition, and to produce flavor and aroma compounds by proteolysis and fat hydrolysis, which play an important role in the development of the quality properties of fermented meat products and are considered to be important "flavor bacteria" of fermented meat products. The CNS in fermented meat products has abundant population diversity, such as staphylococcus xylosus (staphylococcus xylosus), staphylococcus saprophyticus (s. saprophyticus), staphylococcus equinus (s.equiorum) and staphylococcus carnosus (s.carnosu), which are the most advantageous species in traditional fermented sausages in europe, and staphylococcus xylosus (s.xylosus), staphylococcus saprophyticus (s.saprophyticus), staphylococcus epidermidis (s.epidermidis), staphylococcus squirrel (s.sciuri) and staphylococcus simulans (s.simulans), which are more advantageous in fermented meat products in chinese style. Italian law allowed staphylococcus xylosus (staphylococcus xylosus), staphylococcus carnosus (s.carnosus) and staphylococcus simulans (s.simulans) as leavening agents for the production of fermented sausages (republica italana, 1995), and china listed staphylococcus calves (s.vitulinus), staphylococcus xylosus (s.xylosus) and staphylococcus carnosus (s.carnosus) in the list of strains available for food in 2016 (department of food safety standards and supervision evaluation, china, No. 4 in 2016).

Xylosus and s. carnosus are the two CNS commercial ferments currently used most commonly in the industry. However, commercial leavening agents do not always maintain advantages in competing with local microbial populations, which results in a loss of desirable organoleptic properties of the fermented meat product, and thus screening suitable leavening agents from local microbial populations is better suited to local meat product processing environments and is more competitive due to their specific metabolic capabilities. Simulans is one of the dominant CNS populations with superior fermentation performance in chinese fermented meat products.

Disclosure of Invention

The invention provides a staphylococcus-imitated HZ01 strain, a microbial inoculum and application thereof, aiming at solving the technical problems of the existing local microbial population in the food fermentation process, and has the characteristics of good processing adaptability, high protease and lipase activities, good nitrate reductase activity and good flavor of the fermented meat product.

The first technical scheme of the invention is as follows: a staphylococcus simulans HZ01, wherein the staphylococcus simulans HZ01 has a deposit number of GDMCC NO. 62410; the simulated staphylococcus HZ01 is preserved in the Guangdong province microbial strain preservation center; the simulated staphylococcus HZ01 has a preservation unit address of No. 59 building 5 of No. 100 college of the Pieli Zhou city, Guangzhou; the deposit name of the mimic staphylococcus HZ01 is Staphyloccocussimusans HZ 01; the simulated staphylococcus HZ01 has a preservation time of 2022, 4 months and 24 days. The simulated staphylococcus HZ01 has strong metabolic capability, can keep advantages with local microbial populations in a competitive process when used as a leavening agent, has strong competitiveness, enables the fermented meat product to have ideal sensory characteristics, and can better adapt to the processing environment of local meat products; the staphylococcus HZ01 mimic has good protease activity and the ability of decomposing sarcoplasmic proteins, can degrade 58.1 percent of sarcoplasmic protein bands within 72 hours, and can obviously reduce 41.9 percent of sarcoplasmic protein bands; the simulated staphylococcus HZ01 has good nitrate reductase activity, can efficiently reduce nitrate into nitrite in the application process, and utilizes the nitrate in the fermented meat product, thereby reducing the using amount of nitrite in the meat product processing process; the simulated staphylococcus HZ01 can metabolize leucine to produce 3-methyl butyraldehyde, so that the types and the contents of volatile flavor substances are increased, and the aroma-producing property is excellent; the simulated staphylococcus HZ01 can obviously improve esters, aldehydes and acids in the fermented meat product, wherein vinyl acetate, methyl butyrate, methyl caproate, n-hexanal, acetic acid and isovaleric acid are improved most obviously, and the quality of the fermented meat product can be obviously improved.

Preferably, the gene accession number of the mimic staphylococcus HZ01 is OM758216, and the specific nucleotide sequence is shown in SEQ ID NO. 1. According to the nucleotide sequence of the staphylococcus simulans HZ01, the staphylococcus simulans HZ01 is an independent clade formed in the evolution process and is a new strain of the staphylococcus simulans.

Preferably, the determination of the sequence of the mimic staphylococcal HZ01 gene comprises the steps of,

(a) extracting total DNA imitating staphylococcus HZ01 by using a bacterial genome DNA extraction kit;

(b) performing 16s full-length amplification on the total DNA extracted in the step (a) by using a PCR reaction system;

(c) and after the 16s full-length amplification is finished, gel cutting purification is carried out, and electrophoretic sequencing is carried out, so that the detected sequence is the 16sDNA full-length sequence imitating staphylococcus HZ 01. The invention adopts the bacterial genome DNA extraction kit to extract the total DNA of the staphylococcus HZ01, the extraction is rapid and convenient, and the stability and the extraction purity are good; the PCR reaction system can quickly increase the total DNA extracted from the simulated staphylococcus HZ 01; the gel cutting purification is completed on the 16s full-length amplification sequence, so that the subsequent convenient and accurate electrophoretic sequencing is realized, and the accuracy of finally measuring the 16sDNA full-length sequence imitating staphylococcus HZ01 is ensured.

Preferably, the PCR reaction system comprises 10 XExTaqbuffer 2. mu.L, 2.5mM dNTPmix 1.6. mu.L, 5pPrimer 10.6. mu.L, 5pPrimer 20.6. mu.L, Template 2. mu.L, 5uExTaq 1. mu.L, ddH2O12.2. mu.L. The components act synergistically to rapidly increase the total DNA of the extracted staphylococcus HZ01 mimic.

Preferably, the amount of the PCR reaction system is 15. mu.L to 25. mu.L. More preferably, the amount of the PCR reaction system is 18. mu.L to 22. mu.L. According to the selection of the total DNA of the staphylococcus simulans HZ01 to be amplified, the sufficiency of the amplification and the suitability of the dosage of the total DNA of the staphylococcus simulans HZ01 are considered.

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

(b01) reacting the total DNA extracted in the step (a) in a PCR reaction system for 5min at the temperature of 95 ℃;

(b02) after the step (b01) is completed, continuing the reaction for 30s at the temperature of 95 ℃;

(b03) after the step (b02) is finished, continuing the reaction for 30s at the temperature of 55 ℃;

(b04) after the step (b03) is finished, continuing to react for 1min at the temperature of 72 ℃;

(b05) repeating the steps (b02) to (b04)24 times;

(b06) after the step (b05) is finished, extending for 10min at the temperature of 72 ℃;

(b07) after completion of step (b06), incubation at 10 ℃ temperature conditions completed the 16s full length amplification of total DNA mimicking staphylococcal HZ 01. The specific temperature and time are strictly limited, so that the completeness of the full-length amplification of the total DNA16s of the staphylococcus HZ01 can be ensured, and the completeness and the timeliness of the amplification can be considered.

The second technical scheme of the invention is as follows: the staphylococcus HZ01 mimic microbial inoculum is prepared from staphylococcus HZ 01. The mimic staphylococcus HZ01 is prepared into a microbial inoculum shape, so that the mimic staphylococcus HZ01 is more convenient to use in daily production and life.

Preferably, the preparation of the staphylococcus HZ01 mimic microbial inoculum comprises the following steps,

(A) inoculating staphylococcus HZ01 to NB culture medium for culture to obtain bacterial liquid;

(B) the concentration of the staphylococcus HZ01 in the bacterial liquid prepared in the step (A) is 1.0 multiplied by 10 ⁸ log ₁₀ CFU/mL～9.9×10 ⁹ log ₁₀ Centrifuging at CFU/mL;

(C) after the centrifugation is finished, adding sterilized skimmed milk for resuspension;

(D) freeze drying after re-suspending to obtain the concentration of 1.0 × 10 ¹⁰ log ₁₀ CFU/g～9.9×10 ¹¹ log ₁₀ CFU/g powder mimics Staphylococcus HZ01 inoculum. The NB medium has a good culture effect on the simulated staphylococcus HZ 01; the concentration of staphylococcus HZ01 in the bacterial liquid is 1.0 × 10 ⁸ log ₁₀ CFU/mL～9.9×10 ⁹ log ₁₀ The reason for performing centrifugation at CFU/mL is that the concentration of the staphylococcus simulans HZ01 is limited to have a higher concentration, the quantity of the staphylococcus simulans HZ01 in a limited concentration range is enough, the quantity of the staphylococcus simulans HZ01 is insufficient when the concentration is low, and more time is needed for culturing when the concentration is high; centrifuging to better prepare a concentrated solution; the sterilized skim milk is added for resuspension so as to carry out freeze protection on the cultured staphylococcus simulans HZ 01; freeze drying is used for better preparing the solid microbial inoculum; the powdery staphylococcus aureus HZ01 imitating microbial inoculum prepared by the method has higher concentration and better performance. More preferably, the concentration of Staphylococcus simulans HZ01 in the bacterial liquid prepared in step (A) is 3.0X 10 ⁸ log ₁₀ CFU/mL～7.9×10 ⁹ log ₁₀ Centrifuge at CFU/mL. More preferably, the concentration of the staphylococcus HZ01 mimic in the bacterial liquid prepared in the step (A) is 5.0X 10 ⁸ log ₁₀ CFU/mL～5.9×10 ⁹ log ₁₀ Centrifuge at CFU/mL. More preferably, the concentration of Staphylococcus simulans HZ01 in the bacterial liquid prepared in step (A) is 7.0X 10 ⁸ log ₁₀ CFU/mL～3.9×10 ⁹ log ₁₀ Centrifuge at CFU/mL. More preferably, the concentration of Staphylococcus simulans HZ01 in the bacterial liquid prepared in step (A) is 9.0X 10 ⁸ log ₁₀ CFU/mL～1.9×10 ⁹ log ₁₀ Centrifuge at CFU/mL.

Preferably, the NB medium in the step (A) comprises 10g/L of peptone, 3.0g/L of beef extract and 5.0g/L of sodium chloride. So that the NB medium has better culture effect on the simulated staphylococcus HZ 01.

Preferably, the pH value of the NB medium in the step (A) is 7-7.4. More preferably, the pH value of the NB medium in the step (A) is 7.1-7.3. The culture of the staphylococcus aureus HZ01 is provided with a proper pH environment.

Preferably, the amount of NB medium used in step (A) is 0.5L to 2L. More preferably, the amount of NB medium used in step (A) is 1L to 1.5L. The amount of NB medium used had reached a defined concentration based on the amount of Staphylococcus simulans HZ01, ensuring good culture of Staphylococcus simulans HZ 01.

Preferably, the culturing in step (a) is a stationary culturing. Static culture can be beneficial to mimic good proliferation of staphylococcal HZ 01.

Preferably, the culturing time in the step (A) is 24 to 72 hours. More preferably, the culturing time in the step (A) is 36 to 60 hours. More preferably, the culturing time in the step (A) is 40 to 48 hours. The culture time is limited here to ensure that the staphylococcus HZ01 is simulated to proliferate rapidly and reach the plateau phase.

Preferably, the culture temperature in the step (A) is 15 to 65 ℃. More preferably, the culture temperature in the step (A) is 25 to 55 ℃. More preferably, the culture temperature in the step (A) is 30 to 45 ℃. More preferably, the culture temperature in the step (A) is 35 to 40 ℃. The temperature is limited here, and the culture of the Staphylococcus simulans HZ01 can be more suitably performed.

Preferably, the centrifugation speed in the step (B) is 5000 to 20000 rpm/min. More preferably, the centrifugal rotation speed in the step (B) is 8000rpm/min to 18000 rpm/min. More preferably, the centrifugal rotation speed in the step (B) is 10000 rpm/min-15000 rpm/min. The solid-liquid separation of the thalli can be realized more efficiently by proper centrifugal rotating speed.

Preferably, the centrifugation time in the step (B) is 10 to 20 min. More preferably, the centrifugation time in the step (B) is 12 to 18 min. The solid-liquid separation can be fully realized by limiting the centrifugal time.

Preferably, the centrifugation temperature in the step (B) is 2 to 6 ℃. More preferably, the centrifugation temperature in the step (B) is 3 to 5 ℃. The low-temperature centrifugation is adopted, and the centrifugation temperature is limited in order to sufficiently ensure the activity of the staphylococcus HZ01 in the centrifugation process.

Preferably, the mass concentration of the sterilized skim milk in the step (C) is 0.05% m/v to 0.5% m/v. More preferably, the mass concentration of the sterilized skim milk in the step (C) is 0.1% m/v to 0.4% m/v. More preferably, the mass concentration of the sterilized skim milk in the step (C) is 0.2% m/v to 0.3% m/v. The sterilized skim milk with the limited concentration can fully freeze-dry and protect the cultured staphylococcus aureus HZ 01.

Preferably, the sterilized skim milk used in the step (C) is 1ml to 5 ml. More preferably, the amount of the sterilized skim milk used in the step (C) is 2ml to 4 ml. The amount of sterilized skim milk is limited to that sufficient to protect cultured Staphylococcus simulans HZ01 from freeze-drying.

Preferably, the freeze-drying temperature in the step (D) is-90 ℃ to-70 ℃. More preferably, the freeze-drying temperature in the step (D) is-85 ℃ to-75 ℃. More preferably, the freeze-drying temperature in the step (D) is-83 ℃ to-78 ℃. The restriction on the freeze drying temperature is sufficient and rapid drying, and simultaneously better ensures the activity of staphylococcus HZ01 in the staphylococcus HZ 01-imitating microbial inoculum.

Preferably, the freeze-drying time in the step (D) is 24 to 72 hours. More preferably, the freeze-drying time in the step (D) is 36 to 60 hours. More preferably, the freeze-drying time in the step (D) is 40 to 50 hours. The freeze drying time is limited, and the complete drying can be fully ensured.

The third technical scheme of the invention is as follows: mimicking the use of staphylococcal HZ01 in the degradation of sarcoplasmic proteins. The staphylococcus HZ01 mimic has good protease activity, has the capability of decomposing sarcoplasmic proteins and myofibrillar proteins, can degrade 58.1 percent of sarcoplasmic protein bands within 72 hours, and obviously reduces 41.9 percent of sarcoplasmic protein bands.

Preferably, the detection of the result of simulating the degradation of sarcoplasmic proteins by staphylococcus HZ01 comprises the following steps;

(S01) extracting the sarcoplasmic proteins, and determining the concentration of the sarcoplasmic proteins by adopting a Lowry protein concentration kit;

(S02) taking and incubating the sarcoplasmic proteins, the glucose and the staphylococcus simulans HZ01 bacterial liquid in the step (S01);

(S03) centrifuging the bacterial liquid after the incubation is finished;

(S04) mixing the 2 x SDS loading buffer with the centrifuged supernatant of step (S03) and water-bathing;

(S05) taking 10% -12% of the Bio-Rad precast collagens and the standard protein, and applying the sample obtained in the step (S04) on the 10% -12% of the Bio-Rad precast collagens and the standard protein respectively;

(S06) performing electrophoresis after the sample is loaded;

(S07) after the electrophoresis is finished, staining is carried out by Coomassie brilliant blue R-250, and the result of staining simulates the degradation result of staphylococcus HZ01 on sarcoplasmic proteins. The Lowry protein concentration kit can rapidly and accurately measure the sarcoplasmic protein concentration; the addition of glucose provides a carbon source for simulating the growth of staphylococcus HZ 01; the centrifugation is used for collecting protein precipitates and eliminating the interference of irrelevant substances, so that the accuracy of a detection result is better ensured; the 2 xSDS loading buffer is added to adjust the supernatant into a sample to be detected for better detection; the water bath is used for fully developing the secondary structure of the protein; 10% -12% of Bio-Rad precast gel can fully separate sarcoplasmic protein bands; the standard protein can be used as a reference; electrophoresis can fully ensure the separation of protein bands; the dyeing effect of Coomassie brilliant blue R-250 is better, and the sarcoplasmic protein band can be fully developed; the whole detection method shows that the good degradation effect of staphylococcus HZ01 on sarcoplasmic proteins is simulated.

Preferably, the extraction of sarcoplasmic proteins comprises the steps of,

(S011) mixing pork with a PB buffer solution and homogenizing;

(S012) filtering the supernatant homogenized in the step (S011) through a filter membrane and sterilizing to obtain the sarcoplasmic protein. The PB buffer solution can well disperse the pork serous fluid, so that the subsequent treatment and detection of the pork serous fluid are facilitated; the filter membrane can well remove substances which do not meet the requirements in the pork serous fluid, so that the purity of the prepared sarcoplasmic protein reaches the standard; the sterilization operation can better ensure the quality of the sarcoplasmic protein finally prepared.

Preferably, the pork is fresh lean pork. The fresh lean pork is selected, so that the quality of the muscle pulp protein finally prepared can be ensured.

Preferably, the amount of the pork is 1g to 10 g. More preferably, the amount of the pork is 3g to 7 g. More preferably, the amount of the pork is 4g to 6 g. The limit of the pork dosage is defined according to the required muscle plasma protein dosage and the detection requirement.

Preferably, the amount of the PB buffer is 15 mL-50 mL. More preferably, the amount of the PB buffer is 20 mL-45 mL. More preferably, the amount of the PB buffer is 25 mL-40 mL. More preferably, the PB buffer is used in an amount of 30mL to 35 mL. The dosage of the PB buffer solution is limited according to the amount of pork to be dispersed, and the PB buffer solution can be used for fully homogenizing and dispersing the pork.

Preferably, the concentration of the PB buffer is 0.02 mol/L. The PB buffer solution with the limited concentration can better disperse pork, so that the finally prepared sarcoplasmic proteins meet the requirements of subsequent treatment.

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. More preferably, the pH of the PB buffer solution is 6.2-6.3. The PB buffer with limited pH value can better process and disperse the pork serous fluid.

Preferably, the homogenizing speed is 10000rpm/min to 15000 rpm/min. Preferably, the homogenizing rotation speed is 12000 rpm/min-14000 rpm/min. The limited homogenizing rotating speed can ensure that the pork and the PB buffer solution are mixed more uniformly.

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

Preferably, the pore size of the filter membrane is 0.2 to 0.25. mu.m. More preferably, the pore size of the filter membrane is 0.22 to 0.24. mu.m. The limited aperture of the filter membrane can strictly filter the pork serous fluid, thereby ensuring the quality of the finally prepared sarcoplasmic protein.

Preferably, the glucose is 1% glucose. 1% glucose can provide the carbon source needed to mimic proliferation of staphylococcal HZ 01.

Preferably, the amount of the bacterial liquid of the staphylococcus aureus HZ01 is 0.05 mL-0.5 mL. More preferably, the amount of the bacterial liquid of the staphylococcus aureus HZ01 is 0.1-0.4 mL. More preferably, the amount of the bacterial liquid of the staphylococcus aureus HZ01 is 0.2 mL-0.3 mL. The dosage of the staphylococcus HZ 01-imitating bacterial liquid is determined according to the detection requirement and the dosage of the sarcoplasmic proteins, and the sufficient degradation of the sarcoplasmic proteins is ensured.

Preferably, the incubation time is 24h to 72 h. More preferably, the incubation time is 36 to 60 hours. More preferably, the incubation time is 40h to 50 h. The incubation time is defined to ensure that the incubation is sufficiently degraded.

Preferably, the amount of the 2 XSDS loading buffer is 150uL to 250 uL. More preferably, the amount of the 2 XSDS loading buffer taken is 180uL to 220 uL. The amount of 2 xSDS loading buffer is limited to the sample to be tested which is good enough to adjust the supernatant for better detection.

Preferably, the amount of the supernatant after freezing is 150uL to 250 uL. More preferably, the amount of the supernatant after freezing is 180uL to 220 uL. And selecting the supernatant after freezing according to the subsequent detection requirements.

Preferably, the temperature of the water bath is 90-98 ℃. More preferably, the water bath temperature is 93 ℃ to 96 ℃. The temperature of the water bath is limited, so that the secondary structure of the protein can be fully developed.

Preferably, the water bath time is 3min to 10 min. More preferably, the water bath time is 5min to 7 min. The water bath ensures that the secondary structure of the protein is fully expanded, and prevents the protein from being excessively degraded to influence the result.

Preferably, the loading amount of 10-12% of Bio-Rad prefabricated collagen is 15-25 muL; the loading amount of the standard protein is 5-10 mu L. More preferably, the loading amount of 10% -12% of Bio-Rad prefabricated collagen is 17-22 muL; the loading amount of the standard protein is 7-9 mu L. The specific amount is defined according to the concentration of the corresponding protein.

Preferably, the electrophoresis voltage is 110V. The electrophoretic voltage of 110V is a standard voltage.

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

Preferably, the dyeing time is 1 to 2 hours. More preferably, the dyeing time is 1.2h to 1.8 h. More preferably, the dyeing time is 1.4h to 1.6 h. The staining time is defined to ensure that the protein bands are sufficiently colored.

The fourth technical scheme of the invention is as follows: mimicking the use of staphylococcal HZ01 in the reduction of nitrate to nitrite. The simulated staphylococcus HZ01 has good nitrate reductase activity, can well reduce nitrate into nitrite in the application process, and can utilize the nitrate in fermented meat products, thereby reducing the using amount of nitrite in the meat product processing process.

Preferably, the detection of the nitrite concentration comprises the steps of,

preparing a mixture containing KNO at a proper concentration ₃ /NaNO ₃ The NB medium of (1);

(ii) inoculating the staphylococcus simulans HZ01 bacterial liquid into the NB medium in the step (i) for culture;

(iii) centrifuging the fermentation broth at intervals during the cultivation and collecting the supernatant;

(iv) measuring the nitrite content of the supernatant from step (iii). The whole detection process is simple, and the good reduction effect of simulating staphylococcus HZ01 to reduce nitrate into nitrite can be obtained conveniently and quickly.

Preferably, step (i) is carried out by formulating 0.1% KNO ₃ /NaNO ₃ NB medium of (1). 0.1% KNO ₃ /NaNO ₃ The NB medium of (1) showed a better effect in mimicking the nitrate reductase activity of Staphylococcus HZ 01.

Preferably, said step (i) comprises a suitable concentration of KNO ₃ /NaNO ₃ The preparation of NB medium of (1) comprises the following steps,

taking appropriate amount of KNO ₃ /NaNO ₃ And NB nutrient broth dissolved in sterile water;

sterilizing and culturing the mixed solution obtained in the step (i 01) to obtain the mixed solution containing KNO with proper concentration ₃ /NaNO ₃ NB medium of (1). By KNO ₃ /NaNO ₃ NB nutrient broth and sterile water containing KNO in appropriate concentration ₃ /NaNO ₃ The NB culture medium can better receive the reduction effect of the simulated staphylococcus HZ01, and better shows the effect of the simulated staphylococcus HZ01 on reducing nitrate into nitrite.

Preferably, KNO in said step (i 01) ₃ /NaNO ₃ The dosage of the compound is 0.02 g-0.1 g. Preferably, KNO in said step (i 01) ₃ /NaNO ₃ The dosage of the composition is 0.04g to 0.08 g. KNO ₃ /NaNO ₃ In order to formulate the desired concentration of KNO ₃ /NaNO ₃ The NB medium of (1).

Preferably, the amount of NB nutrient broth used in step (i 01) is from 0.7g to 1.2 g. More preferably, the amount of NB nutrient broth used in step (i 01) is from 0.8g to 1.1 g. The NB nutrient broth is used in an amount to formulate the desired concentration of KNO ₃ /NaNO ₃ The NB medium of (1).

Has good effectOptionally, the amount of sterile water used in step (i 01) is 30 mL-70 mL. More preferably, the amount of sterile water used in step (i 01) is 40mL to 60 mL. More preferably, the amount of sterile water used in step (i 01) is 45mL to 55 mL. The sterile water is used in an amount to formulate the desired concentration of KNO ₃ /NaNO ₃ The NB medium of (1).

Preferably, the sterilization temperature in the step (i 02) is 115 ℃ to 130 ℃. More preferably, the sterilization temperature in step (i 02) is 120 ℃ to 125 ℃. The sterilization temperature is limited, and the bacteria and spores thereof can be fully inactivated, thereby ensuring that the composition contains KNO with proper concentration ₃ /NaNO ₃ The sterile quality of NB medium (1).

Preferably, the culturing time in the step (i 02) is 15 to 30 min. More preferably, the culturing time in the step (i 02) is 20min to 25 min. The culture time is limited, so that KNO can be well guaranteed ₃ /NaNO ₃ And its spores and NB nutrient broth were well mixed in sterile water.

Preferably, the amount of the bacterial suspension of the Staphylococcus simulans HZ01 inoculated in the step (ii) is 0.05 mL-0.2 mL. More preferably, the amount of the bacterial suspension of the Staphylococcus simulans HZ01 inoculated in step (ii) is 0.1 mL-0.15 mL. The inoculum size of the staphylococcus HZ 01-imitating bacterial liquid is determined according to KNO ₃ The concentration of (B) is limited, and KNO can be ensured ₃ /NaNO ₃ Is fully reduced.

Preferably, the incubation temperature in step (ii) is from 25 ℃ to 35 ℃. More preferably, the incubation temperature in step (ii) is from 28 ℃ to 32 ℃. The limitation of the culture temperature can better promote the simulation of staphylococcus HZ01 on KNO in NB medium ₃ /NaNO ₃ And (4) carrying out reduction.

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. The culture time is limited to ensure that staphylococcus HZ01 is imitated to KNO in NB medium ₃ /NaNO ₃ And fully reducing.

Preferably, the amount of the fermentation broth used in step (iii) is 1mL to 5 mL. More preferably, the amount of the fermentation broth used in the step (iii) is 2mL to 4 mL. The taking amount of the fermentation liquor is selected according to the detection requirement, so long as the requirement for checking the nitrite content can be met.

Preferably, the time interval for taking the fermentation broth in step (iii) is 2 to 6 hours. More preferably, the time interval for taking the fermentation broth in said step (iii) is 3 to 5 hours. The fermentation broth was taken at intervals and simulated at different times for staphylococcal HZ01 vs KNO ₃ /NaNO ₃ The reduction degrees of the simulated staphylococcus HZ01 to KNO can be obtained more objectively and accurately by taking the fermentation liquor for multiple times ₃ /NaNO ₃ The reducing effect of (3).

Preferably, the centrifugation temperature in the step (iii) is 2 to 6 ℃. More preferably, the centrifugation temperature in the step (iii) is 3 to 5 ℃. The low temperature limit of the centrifugation temperature is to ensure the safety of the activity of the staphylococcus HZ01 during the centrifugation.

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

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

(iv 01) taking a proper amount of supernatant and placing the supernatant in a colorimetric tube;

(iv 02) adding a proper amount of sulfanilic acid solution into the colorimetric tube obtained in the step (iv 01), uniformly mixing, and standing;

(iv 03) adding a proper amount of naphthyl ethylenediamine hydrochloride solution into the solution after standing in the step (iv 02);

(iv 04) adding water to the solution obtained in the step (iv 03) to the scale of the colorimetric tube, uniformly mixing and standing;

(iv 05) measuring the absorbance of the solution in the step (iv 04). The aminobenzene sulfonic acid solution and nitrite in the supernatant can carry out diazotization reaction, so that preparation is made for subsequent color development reaction; the naphthyl ethylenediamine hydrochloride solution can be well combined with substances subjected to diazotization reaction to generate rose bengal solution, so that a good nitrite sample to be tested is formed; through the determination of the absorbance, the content of the nitrite in the supernatant can be accurately and quickly converted.

Preferably, the amount of the supernatant in step (iv 01) is 0.5 mL-1.5 mL. More preferably, the amount of the supernatant in step (iv 01) is 0.8 mL-1.2 mL. The dosage requirement of the supernatant is determined according to the detection requirement.

Preferably, the standard of the colorimetric tube in the step (iv 01) is 20mL to 30 mL. More preferably, the size of the colorimetric tube in the step (iv 01) is 25 mL. The specification of the colorimetric tube is limited, so that the nitrite content in the sample can be accurately and rapidly calculated after the volume is fixed to the scale.

Preferably, the addition amount of the sulfanilic acid solution in the step (iv 02) is 0.5-2 mL, and the concentration of the sulfanilic acid solution is 3-5 g/L. More preferably, the sulfanilic acid solution in step (iv 02) is added in an amount of 0.1 mL-1.5 mL, and the concentration of the sulfanilic acid solution is 3.5 g/L-4.5 g/L. The addition amount of the sulfanilic acid solution and the concentration of the sulfanilic acid solution are set according to the detection requirement and the dosage requirement of the supernatant, and the nitrite content in the sample is calculated more accurately and rapidly.

Preferably, the standing time of the solution in the step (iv 02) is 3-5 min. More preferably, the standing time of the solution in the step (iv 02) is 3.5-4.5 min. The standing time is limited to allow the solution to return to a calm state after the diazotization reaction occurs, and to maintain a better state for the subsequent reaction.

Preferably, the amount of the naphthyl ethylenediamine hydrochloride solution added in the step (iv 03) is 0.2 mL-1 mL, and the concentration of the naphthyl ethylenediamine hydrochloride solution is 1 g/L-5 g/L. More preferably, the amount of the naphthyl ethylenediamine hydrochloride solution added in the step (iv 03) is 0.5-0.8 mL, and the concentration of the naphthyl ethylenediamine hydrochloride solution is 2-4 g/L. The addition amount of the naphthyl ethylenediamine hydrochloride solution and the concentration of the naphthyl ethylenediamine hydrochloride solution are set according to the detection requirement and the requirement of the color reaction, and the purpose is to calculate the nitrite content in the sample more accurately and rapidly.

Preferably, the standing time in the step (iv 04) is 10 to 20 min. More preferably, the standing time in the step (iv 04) is 13min to 17 min. Here, the term "standing time" is defined as a state in which the absorbance is kept good for the subsequent measurement.

Preferably, in the step (iv 05), a 1cm cuvette is used, the zero point of the cuvette is adjusted using a zero tube, and an appropriate amount of the solution in the step (iv 04) is taken, and the absorbance is measured at a wavelength of 538 nm. The specification limitation, zero point adjustment and wavelength setting of the comparison color cup are all required according to the specific situation of nitrite in the supernatant, so that the nitrite content in the sample can be calculated more accurately and rapidly.

The fifth technical scheme of the invention is as follows: the use of staphylococcus HZ01 in dry-curing fermented meat products was simulated. The simulated staphylococcus HZ01 can obviously improve esters, aldehydes and acids in the fermented meat product, wherein vinyl acetate, methyl butyrate, methyl caproate, n-hexanal, acetic acid and isovaleric acid are improved most obviously, and the quality of the fermented meat product can be obviously improved.

Preferably, the preparation of the dry-cured, fermented meat product comprises the steps of,

firstly, taking concentrated solution or powder imitating staphylococcus HZ01, and diluting with edible water;

secondly, dissolving a proper amount of salt, white sugar and sodium glutamate in the solution obtained in the first step to prepare a pickling solution;

thirdly, taking the meat paste, adding the pickling liquid in the second step into the meat paste until the concentration of the staphylococcus HZ01 in the meat paste reaches 1.0 multiplied by 10 ⁷ log ₁₀ CFU/g～9.9×10 ⁸ log ₁₀ When the concentration is CFU/g, pickling the meat paste at low temperature;

and (IV) after the pickling is finished, sequentially performing sausage filling, air exhausting, baking and fermentation on the meat paste to obtain the dry-pickled fermented meat product finished product. The proper amount of salt, white sugar, sodium glutamate and the concentrated solution/powder imitating staphylococcus HZ01 are prepared into the pickling liquid together, so that the pickling effect on the meat emulsion is better, the meat emulsion is directly tasty after being pickled, and the subsequent meat products do not need to be seasoned; the low-temperature pickling can effectively keep the activity and safety of the staphylococcus HZ 01; the meat paste after pickling is sequentially subjected to sausage filling, air exhausting, baking and fermentation, and the finished product of the dry-pickled fermented meat product which is processed by the process sequence has the advantages of fragrance, rich nutrition and excellent quality; the staphylococcus aureus HZ01 still has activity after being dried and plays a further role in the fermentation process, and the simulated staphylococcus HZ01 has higher heat resistance; the content of esters, aldehydes and acids in the finished product of the dry-cured fermented meat product prepared by the simulated staphylococcus HZ01 treatment is obviously improved, wherein the content of vinyl acetate, methyl butyrate, methyl caproate, n-hexanal, acetic acid and isovaleric acid is obviously improved most.

Preferably, the concentration of the staphylococcus HZ01 simulated microbial inoculum in the step (III) is 3.0 multiplied by 10 ⁷ log ₁₀ CFU/g～7.9×10 ⁸ log ₁₀ CFU/g. More preferably, the concentration of the staphylococcus HZ 01-imitating microbial inoculum in the step (three) is 5.0 multiplied by 10 ⁷ log ₁₀ CFU/g～5.9×10 ⁸ log ₁₀ CFU/g. More preferably, the concentration of the staphylococcus HZ 01-imitating microbial inoculum in the step (three) is 7.0 multiplied by 10 ⁷ log ₁₀ CFU/g～3.9×10 ⁸ log ₁₀ CFU/g. More preferably, the concentration of the staphylococcus HZ 01-imitating microbial inoculum in the step (three) is 9.0 multiplied by 10 ⁷ log ₁₀ CFU/g～1.9×10 ⁸ log ₁₀ CFU/g. The concentration of the staphylococcus aureus HZ 01-imitating microbial inoculum is limited to form a good proportion with salt, white sugar and sodium glutamate, so that the subsequent meat paste to be treated can be better pickled, and the meat paste can play a better role in the subsequent pickling and fermentation processes.

Preferably, the pickling solution in the step (II) comprises the following components in parts by weight,

1-5 parts of table salt, 3-8 parts of white sugar, 0.2-0.8 part of sodium glutamate, 2-5 parts of edible water and a proper amount of staphylococcus HZ01 simulation concentrated solution/powder. The proportion of the salt, the white sugar, the sodium glutamate, the edible water and the simulated staphylococcus HZ01 concentrated solution/powder is limited, so that the prepared pickling liquid is balanced in nutrition, the components can mutually act synergistically, the pickling liquid plays a good role in pickling the meat paste, the meat paste is directly tasty after being pickled, and the subsequent meat product does not need to be seasoned. Wherein, a proper amount of spice can be added according to the requirement.

2-4 parts of table salt, 4-7 parts of white sugar, 0.3-0.7 part of sodium glutamate, 3-4 parts of edible water and a proper amount of staphylococcus HZ01 simulating concentrated solution/powder. The proportion of the salt, the white sugar, the sodium glutamate, the edible water and the staphylococcus HZ01 imitating concentrated solution/powder is limited, so that the prepared pickling solution has more balanced nutrition, the components can mutually act synergistically, the pickling of the meat paste has good effect, the meat paste is directly tasty after being pickled, and the subsequent meat product does not need to be seasoned.

2-4 parts of table salt, 5-6 parts of white sugar, 0.4-0.6 part of sodium glutamate, 3-4 parts of edible water and a proper amount of staphylococcus HZ01 simulating concentrated solution/powder. The proportion of the salt, the white sugar, the sodium glutamate, the edible water and the staphylococcus HZ01 imitating concentrated solution/powder is limited, so that the prepared pickling solution has more balanced nutrition, the components can mutually act synergistically, the pickling of the meat paste has good effect, the meat paste is directly tasty after being pickled, and the subsequent meat product does not need to be seasoned.

Preferably, the mass ratio of the fat to the lean meat in the meat paste is 1-3: 6-9. More preferably, the mass ratio of fat to lean meat in the meat emulsion is 2: 7-8. The mass ratio of fat to lean meat in the meat paste is limited, so that the quality of the final dry-cured fermented meat product is better.

Preferably, the salting temperature in the step (three) is 2 to 6 ℃. More preferably, the salting temperature in the step (three) is 3 to 5 ℃. The low-temperature pickling is carried out, so that the staphylococcus HZ01 can be simulated in the pickling process, and the good activity and safety can be kept.

Preferably, the salting time in the step (three) is 48 to 72 hours. More preferably, the salting time in the step (three) is 55 to 65 hours. The limitation on the curing time can well ensure that the meat paste is fully cured and tasty.

Preferably, the baking temperature in the step (IV) is 50 to 65 ℃. More preferably, the baking temperature in the step (IV) is 55-60 ℃. The limitation of the baking temperature can not influence the activity of the staphylococcus HZ01 too much while effectively baking the sausage, so that the staphylococcus HZ01 can still play a role in the subsequent fermentation process.

Preferably, the baking time in the step (four) is 48 to 72 hours. More preferably, the baking time in the step (IV) is 55 to 65 hours. The limitation on the baking time can fully dry the sausage.

Preferably, the fermentation temperature in the step (IV) is 20 to 30 ℃. More preferably, the fermentation temperature in the step (IV) is 23 to 27 ℃. The temperature of the fermentation is also limited in order to maintain good activity in the simulant staphylococcus HZ 01.

Preferably, the fermentation time in the step (iv) is 1 week to 2 weeks. More preferably, the fermentation time in the step (iv) is 8 to 10 days. The limit on the fermentation time can ensure that the staphylococcus HZ01 is simulated to more completely ferment the dried sausage.

Preferably, the preparation method of the staphylococcus simulans HZ01 concentrate comprises the steps of inoculating a single colony of purified and refrigerated staphylococcus simulans HZ01 into a sterilized NB liquid medium for culture, and enabling the concentration of staphylococcus simulans HZ01 in the NB liquid medium to reach 1.0 x 10 ⁷ log ₁₀ CFU/mL～5.0×10 ⁷ log ₁₀ And when the concentration is CFU/mL, preparing a concentrated solution. The concentrated solution is prepared for better preparing the pickling solution in the follow-up process, so that the meat paste is well subjected to the follow-up pickling and fermentation processes; wherein the concentration of the simulated staphylococcus HZ01 in the concentrated solution is limited in order to meet the processing requirements of meat emulsion in the subsequent process and the finally prepared dried meatThe finished product of the salted and fermented meat product has better quality.

Preferably, the refrigeration temperature in the preparation process of the concentrated solution is 2-6 ℃. More preferably, the refrigeration temperature in the preparation process of the concentrated solution is 3-5 ℃. The refrigeration temperature is limited to ensure the activity of the staphylococcus HZ01 to be safe.

Preferably, the amount of NB liquid medium used in the preparation of the concentrate is 40 mL-60 mL. More preferably, the amount of NB liquid medium used in the preparation of the concentrate is 45 mL-55 mL. The amount of NB broth used is defined herein based on the concentration of the Staphylococcus simulans HZ01 to be cultured and the amount of concentrate to be finally prepared.

Preferably, the culture time in the preparation process of the concentrated solution is 12-36 h. More preferably, the culture time in the preparation process of the concentrated solution is 15 to 35 hours. More preferably, the culture time in the preparation process of the concentrated solution is 20 to 30 hours. The culture time is limited to ensure that the staphylococcus simulans HZ01 is cultured well and the staphylococcus simulans HZ01 reaches the plateau.

Preferably, the culture temperature in the preparation process of the concentrated solution is 25-35 ℃. More preferably, the culture temperature during the preparation of the concentrated solution is 28 ℃ to 32 ℃. The culture temperature is defined here to create a good temperature condition that allows good culture of Staphylococcus simulans HZ01 in the sterilized NB broth, allowing Staphylococcus simulans HZ01 to reach the desired concentration of concentrate quickly.

Preferably, the culture in the preparation of the concentrated solution is a static culture. The resting culture suitably mimics the growth characteristics of staphylococcal HZ 01.

Preferably, the tolerance pH of the staphylococcus HZ01 in the fermentation process is 4-8. More preferably, the tolerance pH of the staphylococcus HZ01 in the fermentation process is 5-7. The simulated staphylococcus HZ01 can grow under acidic, neutral and alkaline conditions, and the simulated staphylococcus HZ01 has good acid and alkaline resistance effect and wide range.

Preferably, the nitrite tolerance amount of the staphylococcus aureus HZ01 in the fermentation process is 0-150 mg/kg. More preferably, the nitrite tolerance of the staphylococcus HZ01 in the fermentation process is 50 mg/kg-100 mg/kg. The simulated staphylococcus HZ01 has a wide range of nitrite tolerance in the fermentation process, so that the nitrite dosage can be added in a wide range according to actual needs in the fermentation process.

Preferably, the salt tolerance of the staphylococcus HZ01 in the fermentation process is 3-12%. More preferably, the salt tolerance of the staphylococcus HZ01 simulant in the fermentation process is 5-10%. More preferably, the salt tolerance of the staphylococcus HZ01 in the fermentation process is 6-8%. The salt tolerance range of the simulated staphylococcus HZ01 in the fermentation process is wide, so that the salt dosage can be added in a wide range according to actual requirements in the fermentation process.

Preferably, the temperature of the simulated staphylococcus HZ01 is 20-40 ℃ during the fermentation process. Preferably, the simulated staphylococcal HZ01 has a tolerance temperature of 25-35 ℃ during fermentation. The tolerance temperature range of the staphylococcus HZ01 in the fermentation process is wide, so that the temperature can be adjusted in a wide temperature range according to actual needs in the fermentation process.

The invention has the following beneficial effects:

(1) the simulated staphylococcus HZ01 has strong metabolic capability, can keep advantages with local microbial populations in a competitive process when used as a leavening agent, has strong competitiveness, enables the fermented meat product to have ideal sensory characteristics, and can better adapt to the processing environment of local meat products;

(2) the simulated staphylococcus HZ01 has good protease activity and the ability of decomposing sarcoplasmic proteins, can degrade 58.1 percent of sarcoplasmic protein bands within 72 hours and obviously reduce 41.9 percent of sarcoplasmic protein bands;

(3) the simulated staphylococcus HZ01 has good nitrate reductase activity, can efficiently reduce nitrate into nitrite in the application process, and utilizes the nitrate in the fermented meat product, thereby reducing the using amount of nitrite in the meat product processing process;

(4) the simulated staphylococcus HZ01 can metabolize leucine to generate 3-methyl butyraldehyde, so that the types and the content of volatile flavor substances are increased, and the aroma-producing property is excellent;

(5) the simulated staphylococcus HZ01 can obviously improve esters, aldehydes and acids in the fermented meat product, wherein vinyl acetate, methyl butyrate, methyl caproate, n-hexanal, acetic acid and isovaleric acid are improved most obviously, and the quality of the fermented meat product can be improved obviously.

Drawings

FIG. 1 is a colony morphology of a mock staphylococcal bacterium HZ01 according to the invention;

FIG. 2 is a graph of the results of gram staining of a mock staphylococcal bacterium HZ01 according to the present invention;

FIG. 3 is a graph of the alignment of a mock staphylococcal strain of HZ01 with other staphylococcal strains in accordance with the invention;

FIG. 4 is a graph of the evolution of a Staphylococcus simulans HZ01 in accordance with the present invention;

FIG. 5 is a graph of pH tolerant fermentation performance of the present invention mimicking Staphylococcus HZ 01;

FIG. 6 is a graph of nitrite tolerance fermentation performance of a simulated staphylococcal HZ01 according to the invention;

FIG. 7 is a graph of salt tolerance fermentation performance of a simulated staphylococcal HZ01 according to the present invention;

FIG. 8 is a graph of temperature tolerant fermentation performance of the present invention mimicking Staphylococcus HZ 01;

FIG. 9 is an SDS-PAGE pattern mimicking the breakdown of sarcoplasmic proteins by Staphylococcus HZ01 in accordance with the present invention;

FIG. 10 shows the NO utilization of Staphylococcus aureus HZ01 strain under anaerobic conditions in accordance with the present invention ₃ ^- Conversion to NO ₂ ^– Histogram of the situation.

Detailed Description

The invention is further illustrated by the following figures and examples, which are not to be construed as limiting the invention.

A strain of staphylococcus simulans HZ01, wherein the staphylococcus simulans HZ01 has the deposit number of GDMCCNO.62410; the simulated staphylococcus HZ01 has a preservation unit of Guangdong province microbial strain preservation center; the simulated staphylococcus HZ01 has a preservation unit address of No. 59 building 5 of No. 100 college of the Pieli Zhou city, Guangzhou; the deposit name of the mimic staphylococcus HZ01 is StaphylococcussilansHZ 01; the simulated staphylococcus HZ01 has a preservation time of 2022, 4 months and 24 days.

The staphylococcus HZ01 imitating microbial inoculum is prepared from staphylococcus HZ01 imitating microbial inoculum;

the preparation of the staphylococcus HZ01 mimic microbial inoculum comprises the following steps,

(A) inoculating staphylococcus HZ01 to NB culture medium for culture to obtain bacterial liquid; the NB culture medium in the step (A) comprises 10g/L of peptone, 3.0g/L of beef extract and 5.0g/L of sodium chloride; the pH value of the NB culture medium in the step (A) is 7-7.4; the dosage of the NB culture medium in the step (A) is 0.5L-2L; the culture in the step (A) is static culture; the culture time in the step (A) is 24-72 h; the culture temperature in the step (A) is 25-35 ℃;

(B) the concentration of the staphylococcus HZ01 in the bacterial liquid prepared in the step (A) is 1.0 multiplied by 10 ⁸ log ₁₀ CFU/mL～9.9×10 ⁹ log ₁₀ Centrifuging at CFU/mL; the centrifugal rotating speed in the step (B) is 5000rap/min to 20000 rap/min; the centrifugation time in the step (B) is 10-20 min; the centrifugal temperature in the step (B) is 2-6 ℃;

(C) after the centrifugation is finished, adding sterilized skimmed milk for resuspension; the mass concentration of the sterilized skim milk in the step (C) is 0.05-0.5% m/v; the dosage of the sterilized skim milk in the step (C) is 1ml to 5 ml;

(D) freeze drying after re-suspending to obtain the concentration of 1.0 × 10 ¹⁰ log ₁₀ CFU/g～9.9×10 ¹¹ log ₁₀ CFU/g of powdered mimic staphylococcal HZ01 inoculum; the freeze drying temperature in the step (D) is-90 ℃ to-70 ℃; the freeze drying time in the step (D) is 24-72 h.

The gene accession number of the mimic staphylococcus HZ01 is OM758216, and the specific nucleotide sequence is shown in SEQ ID NO. 1;

the determination of the sequence of the mimic staphylococcal HZ01 gene comprises the following steps,

(b) performing 16s full-length amplification on the total DNA extracted in the step (a) by using a PCR reaction system; the PCR reaction system comprises 10 XEx Taqbuffer2 μ L, 2.5mM dNTPmix1.6 μ L, 5pPrimer10.6 μ L, 5pPrimer20.6 μ L, Template2 μ L, 5uEx Taq1 μ L, ddH2O12.2 μ L; the dosage of the PCR reaction system is 15-25 mu L;

the 16s full-length amplification in the PCR reaction system comprises the following steps,

(b02) after the step (b01) is finished, continuing the reaction for 30s at the temperature of 95 ℃;

(b05) repeating the steps (b02) to (b04)24 times;

(b07) after the step (b06) is finished, carrying out heat preservation at the temperature of 10 ℃, and finishing 16s full-length amplification of the total DNA of the simulated staphylococcus HZ 01;

(c) and after the 16s full-length amplification is finished, gel cutting purification is carried out, and electrophoretic sequencing is carried out, so that the detected sequence is the 16sDNA full-length sequence imitating staphylococcus HZ 01.

The application of staphylococcus HZ01 in degrading sarcoplasmic proteins is simulated;

the detection of the result of simulating the degradation of the sarcoplasmic proteins by the staphylococcus HZ01 comprises the following steps;

the extraction of sarcoplasmic proteins comprises the following steps,

(S011) mixing pork with a PB buffer solution and homogenizing; pork is fresh lean pork; the using amount of the pork is 1g to 10 g; the dosage of the PB buffer solution is 15 mL-50 mL; the concentration of the PB buffer solution is 0.02 mol/L; the pH value of the PB buffer solution is 5.8-6.6; the homogenate rotating speed is 10000 rpm/min-15000 rpm/min; homogenizing for 15-30 min;

(S012) filtering the supernatant homogenized in the step (S011) through a filter membrane and sterilizing to obtain sarcoplasmic protein; the aperture of the filter membrane is 0.2-0.25 μm;

(S02) taking and incubating the sarcoplasmic proteins, the glucose and the staphylococcus simulans HZ01 bacterial liquid in the step (S01); the glucose is 1% glucose; the using amount of the staphylococcus HZ01 imitating bacterial liquid is 0.05 mL-0.5 mL; the incubation time is 24-72 h;

(S03) centrifuging the bacterial liquid after the incubation is finished;

(S04) mixing the 2 x SDS loading buffer with the centrifuged supernatant of step (S03) and water-bathing; the sampling amount of the 2 xSDS loading buffer solution is 150 uL-250 uL; the amount of the supernatant after centrifugation is 150 uL-250 uL; the water bath temperature is 90-98 ℃; the water bath time is 3min to 10 min;

(S05) taking 10% -12% of the Bio-Rad precast collagens and the standard protein, and applying the sample obtained in the step (S04) on the 10% -12% of the Bio-Rad precast collagens and the standard protein respectively; the sample loading amount of 10-12% of Bio-Rad prefabricated collagen is 15-25 mu L; the loading amount of the standard protein is 5-10 mu L.

(S06) performing electrophoresis after the sample is loaded; the electrophoresis voltage is 110V; the electrophoresis time is 60 min-120 min;

(S07) after the electrophoresis is finished, staining the protein with Coomassie brilliant blue R-250, and obtaining the degradation result of the simulated staphylococcus HZ01 on the sarcoplasmic protein according to the staining result; the dyeing time is 1-2 h.

Mimicking the use of staphylococcus HZ01 in the reduction of nitrate to nitrite;

the detection of the nitrite concentration comprises the following steps,

preparing a mixture containing KNO at a proper concentration ₃ /NaNO ₃ The NB medium of (1); step (i) is to prepare 0.1% KNO ₃ /NaNO ₃ The NB medium of (1);

the appropriate concentration of KNO is contained in the step (i) ₃ /NaNO ₃ The preparation of NB medium of (1) comprises the following steps,

taking appropriate amount of KNO ₃ /NaNO ₃ And NB nutrient broth dissolved in sterile water; KNO in step (i 01) ₃ /NaNO ₃ The dosage of the composition is 0.02g to 0.1 g; the dosage of the NB nutrient broth in the step (i 01) is 0.7 g-1.2 g; the using amount of the sterile water in the step (i 01) is 30 mL-70 mL;

sterilizing and culturing the mixed solution obtained in the step (i 01) to obtain the mixed solution containing KNO with proper concentration ₃ /NaNO ₃ The NB medium of (1); the sterilization temperature in the step (i 02) is 115-130 ℃; the culture time in the step (i 02) is 15-30 min;

(ii) inoculating the staphylococcus simulans HZ01 bacterial liquid into the NB medium in the step (i) for culture; in the step (ii), the inoculation amount of the staphylococcus HZ01 simulant bacterial liquid is 0.05 mL-0.2 mL; the culture temperature in the step (ii) is 25-35 ℃; the culture time in the step (ii) is 12-36 h;

(iii) centrifuging the fermentation broth at intervals during the cultivation and collecting the supernatant; taking the fermentation liquor in the step (iii) in an amount of 1-5 mL; the time interval of taking the fermentation liquor in the step (iii) is 2-6 h; the centrifugation temperature in the step (iii) is 2-6 ℃; the centrifugal rotating speed in the step (iii) is 5000 rap/min-20000 rap/min;

(iv) detecting the nitrite content of the supernatant of step (iii);

the step (iv) of detecting the content of the nitrite in the supernatant comprises the following steps,

(iv 01) taking a proper amount of supernatant and placing the supernatant in a colorimetric tube; the dosage of the supernatant in the step (iv 01) is 0.5 mL-1.5 mL; the specification of the colorimetric tube in the step (iv 01) is 20 mL-30 mL;

(iv 02) adding a proper amount of sulfanilic acid solution into the colorimetric tube obtained in the step (iv 01), uniformly mixing, and standing; in the step (iv 02), the addition amount of the sulfanilic acid solution is 0.5-2 mL, and the concentration of the sulfanilic acid solution is 3-5 g/L; the standing time of the solution in the step (iv 02) is 3-5 min;

(iv 03) adding a proper amount of naphthyl ethylenediamine hydrochloride solution into the solution after standing in the step (iv 02); in the step (iv 03), the addition amount of the naphthyl ethylenediamine hydrochloride solution is 0.2 mL-1 mL, and the concentration of the naphthyl ethylenediamine hydrochloride solution is 1 g/L-5 g/L;

(iv 04) adding water to the solution obtained in the step (iv 03) to the scale of the colorimetric tube, uniformly mixing and standing; the standing time in the step (iv 04) is 10-20 min;

(iv 05) measuring the absorbance of the solution in the step (iv 04); step (iv 05) is to use a 1cm cuvette, adjust the zero point of the cuvette with a zero tube, take an appropriate amount of the solution in step (iv 04), and measure the absorbance at the wavelength of 538 nm.

Mimicking the use of staphylococcus HZ01 in dry-cured fermented meat products;

the preparation of the dry-cured fermented meat product comprises the following steps,

firstly, taking concentrated solution/powder imitating staphylococcus HZ01, and diluting with edible water;

the preparation method of the concentrate of the staphylococcus HZ01 comprises the steps of inoculating a single colony of purified and refrigerated staphylococcus HZ01 in a sterilized NB liquid culture medium for culture until the concentration of staphylococcus HZ01 in the NB liquid culture medium reaches 1.0 multiplied by 10 ⁷ log ₁₀ CFU/mL～5.0× 10 ⁷ log ₁₀ Preparing a concentrated solution when the concentration is CFU/mL; the refrigeration temperature in the preparation process of the concentrated solution is 2-6 ℃; the dosage of the NB liquid culture medium in the preparation process of the concentrated solution is 40 mL-60 mL; the culture time in the preparation process of the concentrated solution is 12-36 h; the culture temperature in the preparation process of the concentrated solution is 25-35 ℃; the culture in the preparation process of the concentrated solution is static culture;

the pickling liquid in the step (II) comprises the following components in parts by weight,

1-5 parts of table salt, 3-8 parts of white sugar, 0.2-0.8 part of sodium glutamate, 2-5 parts of edible water and 1-5 parts of staphylococcus HZ01 simulation concentrated solution/powder;

thirdly, taking the meat paste, adding the pickling liquid in the second step into the meat paste, and waiting for the simulated grape in the meat pasteThe concentration of coccus HZ01 reaches 1.0X 10 ⁷ log ₁₀ CFU/g～9.9×10 ⁸ log ₁₀ When the concentration is CFU/g, pickling the meat paste at low temperature; the concentration of the staphylococcus HZ 01-imitating microbial inoculum in the step (three) is 3.0 multiplied by 10 according to the mass of the meat paste ⁷ log ₁₀ CFU/g～7.9×10 ⁸ log ₁₀ CFU/g. The mass ratio of fat to lean meat in the meat paste is 1-3: 6-9; the pickling temperature in the step (III) is 2-6 ℃; the pickling time in the step (III) is 48-72 h;

after the pickling is finished, sequentially performing sausage filling, air exhausting, baking and fermentation on the meat paste to obtain a dry-pickled fermented meat product finished product; the drying temperature in the step (four) is 50-65 ℃; the baking time in the step (four) is 48-72 h; the fermentation temperature in the step (four) is 20-30 ℃; the fermentation time in the step (IV) is 1-2 weeks; simulating the tolerance pH of staphylococcus HZ01 in the fermentation process to be 4-8; the nitrite tolerance amount of the simulated staphylococcus HZ01 in the fermentation process is 0-150 mg/kg; the salt tolerance of the simulated staphylococcus HZ01 in the fermentation process is 3-12%; the tolerance temperature of the staphylococcus HZ01 in the fermentation process is 20-40 ℃.

Example 1:

a staphylococcus HZ01 bacterial agent is simulated, the strain is named as Staphyloccocussimus HZ01, and the preparation method comprises the following steps,

(A) inoculating staphylococcus HZ01 to NB culture medium for culture to obtain bacterial liquid; the NB culture medium in the step (A) comprises 10g/L of peptone, 3.0g/L of beef extract and 5.0g/L of sodium chloride; the pH value of the NB medium in the step (A) is 7.2 +/-0.2; the dosage of the NB culture medium in the step (A) is 1L; the culture in the step (A) is static culture; the culture time in the step (A) is 48 h; the culture temperature in the step (A) is 30 ℃;

(B) the concentration of the staphylococcus HZ01 in the bacterial liquid prepared in the step (A) is 2.5 multiplied by 10 ⁹ log ₁₀ Centrifuging at CFU/mL; the centrifugal rotating speed in the step (B) is 10000 rap/min; the centrifugation time in the step (B) is 15 min; the centrifugation temperature in the step (B) is 4 ℃;

(C) after the centrifugation is finished, adding sterilized skimmed milk for resuspension; the mass concentration of the sterilized skim milk in the step (C) is 0.1% m/v; the dosage of the sterilized skim milk in the step (C) is 2 ml;

(D) freeze drying after re-suspending to obtain the concentration of 2.0 × 10 ¹¹ log ₁₀ CFU/g of powdered mimic staphylococcal HZ01 inoculum; the freeze drying temperature in the step (D) is-80 ℃; the freeze-drying time in step (D) was 48 h.

Example 2:

the CNKI gene accession number of the simulated staphylococcus HZ01 is OM758216, the preservation number of Guangdong province microbial culture Collection (GDMCC) is 62410, and the specific nucleotide sequence is shown as SEQ ID NO. 1:

GCTATACATGCAAGTCGAGCGAACAGACGAGGAGCTTGCTCCTCTGACGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTAC CTATAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATAATACATGAAACCGCATGGTTTCATGATGAAAGACGGTTTTGCTGT CACTTATAGATGGACCCGCGGCGTATTAGCTAGTTGGTAAGGTAACGGCTTACCAAGGCAACGATACGTAGCCGACCTGAGAGGGTGATCG GCCACACTGGAACTGAGGACACGGTCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGGCGAAAGCCTGACGGAGCAACG CCGCGTGAGGTGATGAAGGTCTTCGGATCGTAAAACTCTGTTATTAGGGAAGAACAAGGGTGTAAGTAACTGTGCATCCCCTTGACGGTAC CTAATCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGAATTATTGGGCGTAAAGCGCG CGTAGGCGGTTTTTTAAGTCTGATGTGAAAGCCCACGGCTCAACCGTGGAGGGTCATTGGAAACTGGAAAACTTGAGTGCAGAAGAGGAAA GTGGAATTCCATGTGTAGCGGTGAAATGCGCAGAGATATGGAGGAACACCAGTGGCGAAGGCGACTTTCTGGTCTGCAACTGACGCTGATG TGCGAAAGCGTGGGGATCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTT AGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGCCGCAAGGCTGAAACTCAAAGGAATTGACGGGGACCCGCACAAGCGG TGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAAATCTTGACATCCTTTGACAACTCTAGAGATAGAGCTTTCCCCTTCG GGGGACAAAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTAAGCTT AGTTGCCAGCATTAAGTTGGGCACTCTAAGTTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGGATGACGTCAAATCATCATGCCCCTTA TGATTTGGGCTACACACGTGCTACAATGGACGGTACAAAGGGCAGCGAACCCGCGAGGTCAAGCAAATCCCATAAAGCCGTTCTCAGTTCG GATTGTAGTCTGCAACTCGACTACATGAAGCTGGAATCGCTAGTAATCGTAGATCAGCATGCTACGGTGAATACGTTCCCGGGTCTTGTAC ACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGCCGGTGGAGTAACCTTTTAGGAACTAGCCGTCGAAGGTGACA

(b) performing 16s full-length amplification on the total DNA extracted in the step (a) by using a PCR reaction system; the PCR reaction system comprises 10 XEx Taqbuffer2 μ L, 2.5mM dNTPmix1.6 μ L, 5pPrimer10.6 μ L, 5pPrimer20.6 μ L, Template2 μ L, 5uEx Taq1 μ L, ddH2O12.2 μ L; the dosage of the PCR reaction system is 20 mu L;

(b01) reacting the total DNA extracted in the step (a) in a PCR reaction system at the temperature of 95 ℃ for 5min, and performing pre-denaturation;

(b05) repeating the steps (b02) to (b04)24 times;

(c) after 16s full-length amplification is completed, gel cutting purification is carried out, and electrophoresis sequencing is carried out by using a horizontal electrophoresis apparatus, the determined sequence is the 16sDNA full-length sequence of the staphylococcus mimic HZ01, 1437bp is shown in figure 1 and figure 2, the similarity of the strain and the strain S.simulans MR1(CP015642.1) with the closest genetic relationship is 99.65% as shown in figure 3, and an independent evolutionary branch as shown in figure 4 is formed in the evolutionary process and is a new strain of the staphylococcus mimic.

Example 3:

as shown in FIG. 5, FIG. 6, FIG. 7 and FIG. 8, the simulated staphylococci HZ01 can tolerate the fermentation conditions of pH 5-8, nitrite 150mg/kg, salt content 3-9% and temperature 20-35 ℃.

A concentrate of Staphylococcus simulans HZ01 is prepared by selecting purified Staphylococcus simulans HZ01 single colony stored in refrigerator at 4 deg.C, inoculating into 50mL sterilized NB liquid medium, and standing at 30 deg.C for 24 hr to reach concentration of 3.0 × 10 ⁷ log ₁₀ CFU/mL, the culture solution is a concentrated solution. Respectively preparing 50mL NB liquid culture media with pH values of 4, 5, 6, 7 and 8, salinity of 0%, 3%, 6%, 9% and 12% and nitrite concentrations of 0, 50mg/kg, 100mg/kg and 150mg/kg, inoculating 1mL of concentrated solution into NB culture media with different culture conditions, culturing at 30 ℃ for 24 hours, measuring OD value every 3 hours, and verifying the optimum fermentation condition of simulated staphylococcus HZ 01. 50mL of NB liquid medium was prepared, 1mL of the concentrated solution was inoculated, the OD value curve of the strain HZ01 at different temperatures was verified, and the temperature growth range of HZ01 was determined. The result shows that the HZ01 has a wide growth range and can grow under the conditions of pH value of 5-8, nitrite content of 0-150 mg/kg, salt content of 3-9% and temperature of 20-35 ℃.

Example 4:

mimicking the use of staphylococcal HZ01 in the degradation of sarcoplasmic proteins; as shown in fig. 9 and 10, mimicking that staphylococcal HZ0 can secrete highly active protease, degrading 58.1% of the sarcoplasmic protein band within 72 hours and significantly reducing 41.9% of the sarcoplasmic protein band;

the extraction of sarcoplasmic proteins comprises the following steps,

(S011) mixing pork with a PB buffer solution and homogenizing; pork is fresh lean pork; the using amount of pork is 2 g; the dosage of the PB buffer solution is 20 mL; the concentration of the PB buffer solution is 0.02 mol/L; the pH of the PB buffer was 6.5; the rotating speed of the homogenate is 13000 rpm/min; homogenizing for 20 min;

(S012) filtering the supernatant homogenized in the step (S011) through a filter membrane and sterilizing to obtain sarcoplasmic protein; the aperture of the filter membrane is 0.22 mu m;

(S02) taking and incubating the sarcoplasmic proteins, the glucose and the staphylococcus simulans HZ01 bacterial liquid in the step (S01); the glucose is 1% glucose; the dosage of the staphylococcus HZ01 imitating bacterial liquid is 0.1 mL; the incubation time is 12-36 h;

(S03) taking 2mL of bacterial liquid at intervals of 24 hours and centrifuging;

(S04) mixing the 2 x SDS loading buffer with the supernatant after the centrifugation of the step (S03), and water-bathing; the taking amount of the 2 xSDS loading buffer solution is 200 uL; the amount of the supernatant after centrifugation is 200 uL; the water bath temperature was 95 ℃; the water bath time is 5 min;

(S05) taking 10% -12% of the Bio-Rad prefabricated collagen and the standard protein, and respectively adding the sample in the step (S04) on the taken 10% -12% of the Bio-Rad prefabricated collagen and the standard protein; the loading amount of 10-12% of Bio-Rad prefabricated collagen is 20 mu L; the loading of standard protein was 8. mu.L.

(S06) performing electrophoresis after the sample is loaded; the electrophoresis voltage is 110V; the electrophoresis time is 90 min;

(S07) after the electrophoresis is finished, staining the protein band with Coomassie brilliant blue R-250, and obtaining the degradation result of the simulated staphylococcus HZ01 on the sarcoplasmic protein band through the staining result; the dyeing time was 1.5 h.

Example 5:

the detection of nitrite activity comprises the following steps,

preparing a mixture containing KNO at a proper concentration ₃ The NB medium of (1); step (i) is to prepare 0.1% KNO ₃ The NB medium of (1);

the appropriate concentration of KNO is contained in the step (i) ₃ The preparation of NB medium of (1) comprises the following steps,

taking appropriate amount of KNO ₃ And NB nutrient broth dissolved in sterile water; KNO in step (i 01) ₃ The dosage of the medicine is 0.05 g; use of NB nutrient broth in step (i 01)The amount was 0.9 g; the using amount of the sterile water in the step (i 01) is 50 mL;

sterilizing and culturing the mixed solution obtained in the step (i 01) to obtain the mixed solution containing KNO with proper concentration ₃ The NB medium of (1); the sterilization temperature in the step (i 02) is 121 ℃; the culture time in the step (i 02) is 20 min;

(ii) inoculating the staphylococcus simulans HZ01 bacterial liquid into the NB medium in the step (i) for culture; in the step (ii), the inoculation amount of the staphylococcus HZ01 simulant bacterial liquid is 0.1 mL; the culture temperature in step (ii) is 30 ℃; the culture time in step (ii) is 24 h;

(iii) centrifuging the fermentation broth at intervals during the cultivation and collecting the supernatant; the taking amount of the fermentation liquor in the step (iii) is 2 mL; the time interval of taking the fermentation liquor in the step (iii) is 4 h; the centrifugation temperature in step (iii) was 4 ℃; the centrifugal rotating speed in the step (iii) is 10000 rap/min;

(iv) detecting the nitrite content of the supernatant of step (iii);

(iv 01) taking a proper amount of supernatant and placing the supernatant into a colorimetric tube with a plug; the dosage of the supernatant in the step (iv 01) is 1 mL; the specification of the colorimetric tube in the step (iv 01) is 25 mL;

(iv 02) adding a proper amount of sulfanilic acid solution into the colorimetric tube obtained in the step (iv 01), uniformly mixing, and standing; in the step (iv 02), the addition amount of the sulfanilic acid solution is 1mL, and the concentration of the sulfanilic acid solution is 4 g/L; the standing time of the solution in the step (iv 02) is 3-5 min;

(iv 03) adding a proper amount of naphthyl ethylenediamine hydrochloride solution into the solution after standing in the step (iv 02); in the step (iv 03), the addition amount of the naphthyl ethylenediamine hydrochloride solution is 0.5mL, and the concentration of the naphthyl ethylenediamine hydrochloride solution is 2 g/L;

(iv 04) adding water to the solution obtained in the step (iv 03) to the scale of the colorimetric tube, uniformly mixing and standing; the standing time in the step (iv 04) is 15 min;

(iv 05) measuring the absorbance of the solution in the step (iv 04); and (iv 05) using a 1cm cuvette, adjusting the zero point of the cuvette by using a zero tube, taking a proper amount of the solution in the step (iv 04), measuring the absorbance at the wavelength of 538nm, and simultaneously making a reagent blank and a standard curve.

Example 6:

mimicking the use of staphylococcus HZ01 in dry-cured fermented meat products;

firstly, taking a concentrated solution imitating staphylococcus HZ01, and diluting the concentrated solution with edible water;

the preparation method of the concentrate of Staphylococcus simulans HZ01 comprises selecting purified Staphylococcus simulans HZ01 single colony stored in refrigerator at 4 deg.C, inoculating into 50mL sterilized NB liquid culture medium, and standing and culturing at 30 deg.C for 24 hr to reach concentration of 3.0 × 10 ⁷ log ₁₀ CFU/mL, the culture solution is a concentrated solution.

2.5 parts of salt, 4 parts of white sugar, 0.4 part of sodium glutamate, 2.5 parts of edible water and 2.5 parts of staphylococcus HZ01 simulated concentrated solution;

thirdly, taking the meat paste, adding the pickling liquid in the second step into the meat paste until the concentration of the staphylococcus HZ01 in the meat paste reaches 8.0 multiplied by 10 ⁷ log ₁₀ CFU/g～9.9×10 ⁸ log ₁₀ When the concentration is CFU/g, pickling the meat paste at low temperature; the mass ratio of fat to lean meat in the meat paste is 2: 8; the pickling temperature in the step (three) is 4 ℃; the pickling time in the step (III) is 48-72 h;

fourthly, after the pickling is finished, sequentially performing sausage filling on the meat paste, wherein the length and the diameter of the sausage are not required; exhausting, drying and fermenting to obtain a finished product of the dry-cured fermented meat product; the drying temperature in the step (four) is 52-54 ℃; the baking time in the step (four) is 48-72 h; the fermentation temperature in the step (IV) is 20-30 ℃; the fermentation time in the step (IV) is 1-2 weeks; simulating the tolerance pH of staphylococcus HZ01 in the fermentation process to be 4-8; the nitrite tolerance amount of the simulated staphylococcus HZ01 in the fermentation process is 0-150 mg/kg; the salt tolerance of the simulated staphylococcus HZ01 in the fermentation process is 3-12%; the tolerance temperature of the simulated staphylococcus HZ01 in the fermentation process is 20-35 ℃.

The physicochemical properties of the fermented meat product inoculated with simulated staphylococcus HZ01 are shown in table 1:

table 1: physical and chemical characteristics of simulated staphylococcus inoculated fermented meat product

Index (I)	Control	HZ01
			pH	5.53±0.02 ^a	5.63±0.08 ^a
a _w	0.81±0.03 ^a	0.77±0.03 ^a
			Staphylococcus concentration (log) ₁₀ CFU/g)	7.58±0.89 ^a	7.34±0.86 ^a
Nitrite concentration (mg/kg)	15.8±0.10 ^b	38.4±0.43 ^a
			L*	36.0±1.32 ^a	35.2±1.89 ^a
a*	8.56±2.92 ^a	6.08±0.69 ^b
			b*	7.31±1.56 ^b	9.86±1.42 ^a

As shown in Table 1, the amount of nitrite in the meat product was reduced.

The volatile compound composition of the different treated fermented meat products is shown in table 2:

table 2: composition of volatile compounds (AU 10) of differently processed fermented meat products ⁵ )

As can be seen from Table 2, the simulated staphylococcus HZ01 can significantly improve esters, aldehydes and acids in the fermented meat product, wherein the vinyl acetate, methyl butyrate, methyl caproate, n-hexanal, acetic acid and isovaleric acid are improved most significantly, and the quality of the fermented meat product can be significantly improved.

Sequence listing

<110> Zhejiang province academy of agricultural sciences

<120> staphylococcus simulans HZ01, microbial inoculum and application thereof

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1444

<212> DNA

<213> Staphylococcus simulans

<400> 1

gctatacatg caagtcgagc gaacagacga ggagcttgct cctctgacgt tagcggcgga 60

cgggtgagta acacgtgggt aacctaccta taagactggg ataactccgg gaaaccgggg 120

ctaataccgg ataatacatg aaaccgcatg gtttcatgat gaaagacggt tttgctgtca 180

cttatagatg gacccgcggc gtattagcta gttggtaagg taacggctta ccaaggcaac 240

gatacgtagc cgacctgaga gggtgatcgg ccacactgga actgaggaca cggtccagac 300

tcctacggga ggcagcagta gggaatcttc cgcaatgggc gaaagcctga cggagcaacg 360

ccgcgtgagg tgatgaaggt cttcggatcg taaaactctg ttattaggga agaacaaggg 420

tgtaagtaac tgtgcatccc cttgacggta cctaatcaga aagccacggc taactacgtg 480

ccagcagccg cggtaatacg taggtggcaa gcgttatccg gaattattgg gcgtaaagcg 540

cgcgtaggcg gttttttaag tctgatgtga aagcccacgg ctcaaccgtg gagggtcatt 600

ggaaactgga aaacttgagt gcagaagagg aaagtggaat tccatgtgta gcggtgaaat 660

gcgcagagat atggaggaac accagtggcg aaggcgactt tctggtctgc aactgacgct 720

gatgtgcgaa agcgtgggga tcaaacagga ttagataccc tggtagtcca cgccgtaaac 780

gatgagtgct aagtgttagg gggtttccgc cccttagtgc tgcagctaac gcattaagca 840

ctccgcctgg ggagtacggc cgcaaggctg aaactcaaag gaattgacgg ggacccgcac 900

aagcggtgga gcatgtggtt taattcgaag caacgcgaag aaccttacca aatcttgaca 960

tcctttgaca actctagaga tagagctttc cccttcgggg gacaaagtga caggtggtgc 1020

atggttgtcg tcagctcgtg tcgtgagatg ttgggttaag tcccgcaacg agcgcaaccc 1080

ttaagcttag ttgccagcat taagttgggc actctaagtt gactgccggt gacaaaccgg 1140

aggaaggtgg gggatgacgt caaatcatca tgccccttat gatttgggct acacacgtgc 1200

tacaatggac ggtacaaagg gcagcgaacc cgcgaggtca agcaaatccc ataaagccgt 1260

tctcagttcg gattgtagtc tgcaactcga ctacatgaag ctggaatcgc tagtaatcgt 1320

agatcagcat gctacggtga atacgttccc gggtcttgta cacaccgccc gtcacaccac 1380

gagagtttgt aacacccgaa gccggtggag taacctttta ggaactagcc gtcgaaggtg 1440

aca 1444

Claims

1. A staphylococcus HZ01 mimic is characterized in that: the simulated staphylococcus HZ01 has a preservation number of GDMCC NO. 62410; the simulated staphylococcus HZ01 is preserved in the Guangdong province microbial strain preservation center; the deposit name of the mimic Staphylococcus HZ01 is Staphylococcus simulans HZ 01; the simulated staphylococcus HZ01 has a preservation time of 2022, 4 months and 24 days.

2. The staphylococcus aureus HZ01 mimic according to claim 1, wherein: the gene accession number of the mimic staphylococcus HZ01 is OM758216, and the specific nucleotide sequence is shown in SEQ ID NO. 1.

3. The staphylococcus aureus HZ01 mimic according to claim 2, wherein: the determination of the sequence of the mimic staphylococcal HZ01 gene comprises the following steps,

(c) after the 16s full-length amplification is finished, gel cutting purification is carried out, and electrophoresis sequencing is carried out, wherein the obtained sequence is the 16s DNA full-length sequence imitating staphylococcus HZ 01;

(b05) repeating the steps (b02) to (b04)24 times;

(b07) after completion of step (b06), incubation at 10 ℃ temperature conditions completed the 16s full length amplification of total DNA mimicking staphylococcal HZ 01.

4. A staphylococcus HZ01 mimic microbial inoculum, which is characterized in that: the staphylococcus simulans HZ01 microbial inoculum is prepared by adopting staphylococcus simulans HZ01 in claim 1;

(D) freeze drying after re-suspending to obtain the concentration of 1.0 × 10 ¹⁰ log ₁₀ CFU/g～9.9×10 ¹¹ log ₁₀ CFU/g powder mimics Staphylococcus HZ01 inoculum.

5. Mimicking the use of staphylococcal HZ01 in the degradation of sarcoplasmic proteins.

6. The use of a staphylococcus aureus HZ01 mimic according to claim 5 for degrading sarcoplasmic proteins, wherein: the detection of the result of simulating the degradation of the sarcoplasmic proteins by the staphylococcus HZ01 comprises the following steps;

(S02) incubating the sarcoplasmic proteins, glucose and the staphylococcus simulans HZ01 bacterial liquid in the step (S01);

(S03) centrifuging the bacterial liquid after the incubation is finished;

(S06) performing electrophoresis after the sample is loaded;

(S07) after the electrophoresis is finished, staining the protein with Coomassie brilliant blue R-250, and obtaining the degradation result of the simulated staphylococcus HZ01 on the sarcoplasmic protein according to the staining result;

the extraction of the sarcoplasmic proteins comprises the following steps,

(S011) mixing pork with a PB buffer solution and homogenizing;

(S012) filtering the supernatant homogenized in the step (S011) through a filter membrane and sterilizing to obtain the sarcoplasmic protein.

7. Mimicking the use of staphylococcal HZ01 in the reduction of nitrate to nitrite.

8. The use of staphylococcus simulans HZ01 as claimed in claim 7 for the reduction of nitrate to nitrite, wherein: the detection of the nitrite concentration comprises the following steps,

(iv) detecting the nitrite content of the supernatant of step (iii);

the step (iv) for detecting the content of the nitrite in the supernatant comprises the following steps,

(iv 03) adding a proper amount of naphthyl ethylenediamine hydrochloride solution into the solution after the standing in the step (iv 02);

(iv 05) measuring the absorbance of the solution in the step (iv 04).

9. The use of staphylococcus HZ01 in dry-curing fermented meat products was simulated.

10. The use of the simulated staphylococcal HZ01 in dry-cured fermented meat products according to claim 9, wherein: the preparation of the dry-cured fermented meat product comprises the following steps,

and (IV) after the pickling is finished, sequentially performing sausage filling, air exhausting, baking and fermentation on the meat paste to obtain the dry-pickled fermented meat product finished product.