CN114891701B - Staphylococcus HZ 01-mimicking bacterium agent and application thereof - Google Patents
Staphylococcus HZ 01-mimicking bacterium agent and application thereof Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/44—Staphylococcus
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
The invention relates to the technical field of microbial food fermentation, and particularly discloses a staphylococcus mimicking HZ01, a microbial inoculum and application thereof, wherein the staphylococcus mimicking HZ01 has a preservation number of GDMCC NO.62410; the preservation unit of the staphylococcus mimicus HZ01 is the collection center of the microorganism strain in Guangdong province; the preservation name of the staphylococcus mimicus HZ01 is Staphylococcus simulans HZ01; the shelf life of the staphylococcus mimicus HZ01 is 2022, 4 and 24 days. The invention has the characteristics of good processing adaptability, high protease and lipase activities, good nitrate reductase activity and good flavor of the fermented meat product.
Description
Technical Field
The invention relates to the technical field of microbial food fermentation, in particular to a staphylococcus HZ 01-mimicking bacterium agent and application thereof.
Background
Microbial leavening agents which are widely used in fermented meat products at present are mainly lactobacillus and Coagulase-negative staphylococci (CNS), wherein the lactobacillus is responsible for acidification of the meat in the initial stage of fermentation, the pH value of the raw meat is reduced and protein is coagulated by generating lactic acid, the hardness is improved, and in addition, the lactobacillus can inhibit the growth of harmful bacteria, so that the microbial safety of the fermented meat products is improved. CNS plays an important role in flavor formation and color stabilization of 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. CNS can reduce nitrate to nitrite and then to nitrous oxide, can prevent rancidity by peroxide decomposition, and can produce flavors and aroma compounds by proteolysis and fat hydrolysis, which functions play an important role in the development of quality attributes of fermented meat products, considered as important "flavours" for fermented meat products. CNS in fermented meat products is rich in population diversity, such as staphylococcus xylosus (s. Saphenous), staphylococcus saprophyticus (s. Saphenous), staphylococcus equine (s. Equosum) and staphylococcus sarcococcus (s. Carnosus) are the most advantageous species in european traditional fermented sausage, whereas staphylococcus xylosus (s. Xylosus), staphylococcus saphenous (s. Saphenous), staphylococcus epidermidis (s. Sapidermidis), staphylococcus squirrel (s. Scintirus) and staphylococcus mimicus (s. Simulicus) are more advantageous in chinese fermented meat products. Italian law allows staphylococcus xylosus (staphylococcus xylosus), staphylococcus carnosus (s. Carnosus) and staphylococcus mimicus (s. Simunosus) to be used as starter for producing fermented sausage (republica italiana, 1995), staphylococcus calves (s. Vitulinus), staphylococcus xylosus (s. Xylosus) and staphylococcus carnosus (s. Carnosus) were listed in the list of bacterial species available for food (food safety standards and monitoring and evaluating department, china, 2016 No. 4) in 2016.
Xylosus and s. Carnosus are currently the two CNS commercial fermenters most commonly used in industry. However, commercial fermenters do not always remain advantageous during competition with the local microbial population, which results in a loss of desirable organoleptic properties of the fermented meat product, so screening the local microbial population for a suitable starter is better suited to the local meat product processing environment and is more competitive due to its characteristic metabolic capacity. Studies have shown that S.simulians is one of the dominant CNS populations with excellent fermentation performance in Chinese fermented meat products.
Disclosure of Invention
The invention provides a staphylococcus HZ 01-mimicking microbial agent and application thereof, which are used for solving the technical problems existing in the food fermentation process of the existing local microbial population, and the staphylococcus HZ 01-mimicking microbial agent has the characteristics of better processing adaptability, higher protease and lipase activities, better nitrate reductase activity and better flavor of fermented meat products.
The first technical scheme of the invention is as follows: a strain of staphylococcus mimicus HZ01, said staphylococcus mimicus HZ01 having a deposit number of GDMCC No.62410; the preservation unit of the staphylococcus mimicus HZ01 is the collection center of the microorganism strain in Guangdong province; the address of the staphylococcus mimicking HZ01 preservation unit is building 5 of No. 59 of No. 100 institute of Migo 100 in Guangzhou city; the preservation name of the staphylococcus mimicus HZ01 is Staphylococcus aureus HZ01; the shelf life of the staphylococcus mimicus HZ01 is 2022, 4 and 24 days. The staphylococcus HZ 01-imitating metabolic capability is strong, and when the staphylococcus HZ-imitating metabolic capability is used as a starter, the staphylococcus HZ 01-imitating metabolic capability can keep advantages with a local microorganism population in the competition process, has strong competition, ensures that the sensory characteristics of the fermented meat product are ideal, and can better adapt to the processing environment of the local meat product; the staphylococcus HZ 01-mimicking agent has good protease activity, has the capacity of decomposing sarcoplasmic proteins, can degrade 58.1% of sarcoplasmic protein strips within 72 hours, and remarkably reduces 41.9% of sarcoplasmic protein strips; the staphylococcus HZ 01-imitation 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 use level of the nitrite in the meat product processing process; the staphylococcus HZ01 can be metabolized to produce 3-methyl butyraldehyde, the variety and the content of volatile flavor substances are increased, and the aroma production property is excellent; the staphylococcus HZ01 can obviously improve esters, aldehydes and acid substances in the fermented meat product, wherein the improvement of vinyl acetate, methyl butyrate, methyl caproate, n-hexanal, acetic acid and isovaleric acid is most obvious, and the quality of the fermented meat product can be obviously improved.
Preferably, the gene accession number of the staphylococcus mimicking HZ01 is OM758216, and the specific nucleotide sequence is shown in SEQ ID NO. 1. From the nucleotide sequence of the mimicking staphylococcal HZ01, it is known that the mimicking staphylococcal HZ01 in the present invention is an independent evolutionary branch formed during evolution, and is a new strain mimicking staphylococci.
Preferably, the determination of the sequence of the mimicking the 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 (3) after the full-length amplification of 16s is finished, performing gel cutting purification, and performing electrophoresis sequencing to obtain a sequence which is the full-length sequence of the 16sDNA imitating staphylococcus HZ 01. The invention adopts the bacterial genome DNA extraction kit to extract the total DNA imitating staphylococcus HZ01, and has the advantages of quick and convenient extraction, good stability and extraction purity; the PCR reaction system can rapidly increase the total DNA extracted from the staphylococcus mimicking HZ 01; the gel cutting purification is performed on the 16s full-length amplified sequence, so that the accuracy of the finally measured 16s DNA full-length sequence imitating staphylococcus HZ01 is ensured for more convenient and accurate electrophoresis sequencing.
Preferably, the PCR reaction system comprises 10 XExTaqbuffer 2. Mu.L, 2.5mM dNTP Mix1.6. Mu.L, 5 pPrimer10.6. Mu.L, 5 pPrimer20.6. Mu.L, template 2. Mu.L, 5uExTaq 1. Mu.L, ddH2O 12.2. Mu.L. The components act synergistically, so that the total DNA of the extracted staphylococcus mimicking HZ01 can be rapidly increased.
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. The total DNA imitating the staphylococcus HZ01 is selected according to the total DNA to be amplified, and the sufficiency and the dosage suitability of the total DNA imitating the staphylococcus 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 at a temperature of 95 ℃ for 5min;
(b02) After the step (b 01) is completed, continuing to react for 30 seconds at the temperature of 95 ℃;
(b03) After step (b 02) is completed, continuing to react for 30 seconds at the temperature of 55 ℃;
(b04) After the step (b 03) is completed, continuing to react for 1min at the temperature of 72 ℃;
(b05) Repeating the steps (b 02) to (b 04) 24 times;
(b06) After step (b 05) is completed, the temperature condition of 72 ℃ is prolonged for 10min;
(b07) After the step (b 06) is completed, the temperature is kept at 10 ℃ to complete 16s full-length amplification of the total DNA imitating staphylococcus 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 simulated staphylococcus HZ01 is ensured, and the completeness and timeliness of the amplification are both considered.
The second technical scheme of the invention: a staphylococcus mimicking HZ01 bacterial agent, wherein the staphylococcus mimicking HZ01 bacterial agent is prepared by adopting staphylococcus mimicking HZ 01. The staphylococcus HZ01 is simulated to be prepared into a form of a microbial inoculum, so that the staphylococcus HZ01 is more convenient to use in daily production and life.
Preferably, the preparation of the staphylococcus mimicking HZ01 microbial agent comprises the following steps,
(A) Inoculating staphylococcus mimicus HZ01 into NB culture medium for culturing to obtain bacterial liquid;
(B) After the bacterial liquid prepared in the step (A) has the concentration of the simulated staphylococcus HZ01 of 1.0x10 8 log 10 CFU/mL～9.9×10 9 log 10 Centrifuging at CFU/mL;
(C) Adding sterilized skimmed milk for resuspension after centrifugation is completed;
(D) Freeze drying after the resuspension is completed to obtain the product with concentration of 1.0X10 10 log 10 CFU/g～9.9×10 11 log 10 The CFU/g powder mimics a staphylococcal HZ01 bacterial agent. The NB culture medium has good culture effect on the simulated staphylococcus HZ 01; when the concentration of the simulated staphylococcus HZ01 in the bacterial liquid is 1.0x10 8 log 10 CFU/mL～9.9×10 9 log 10 The centrifugation at CFU/mL is performed because the concentration of the mimicking staphylococcus HZ01 is higher, the quantity of mimicking staphylococcus HZ01 in the concentration range of the limited zone is enough, the quantity of mimicking staphylococcus HZ01 is insufficient at low concentration, and more time is needed for culture at high concentration; centrifuging to prepare a concentrate better; the sterilized skim milk is added for the purpose of freezing and protecting the cultured mimicking staphylococcus HZ 01; freeze drying is used for better preparation of solid microbial inoculum; the powdery staphylococcus HZ 01-like microbial inoculum prepared by the method has higher concentration and better performance. More preferably, the concentration of the simulated staphylococcus HZ01 in the bacterial liquid obtained in the step (A) is 3.0X10 8 log 10 CFU/mL～7.9×10 9 log 10 Centrifugation was performed at CFU/mL. More preferably, the concentration of the simulated staphylococcus HZ01 in the bacterial liquid obtained in the step (A) is 5.0X10 8 log 10 CFU/mL～5.9×10 9 log 10 Centrifugation was performed at CFU/mL. More preferably, the concentration of the simulated staphylococcus HZ01 in the bacterial liquid obtained in the step (A) is 7.0X10 8 log 10 CFU/mL～3.9×10 9 log 10 Centrifugation was performed at CFU/mL. More preferably, the concentration of the simulated staphylococcus HZ01 in the bacterial liquid obtained in the step (A) is 9.0X10 8 log 10 CFU/mL～1.9×10 9 log 10 Centrifugation was performed at CFU/mL.
Preferably, the NB medium in the step (A) comprises peptone 10g/L, beef extract 3.0g/L and sodium chloride 5.0g/L. The NB medium has better culture effect on the staphylococcus aureus HZ 01.
Preferably, the NB medium in step (A) has a pH of 7 to 7.4. More preferably, the NB medium in step (A) has a pH of 7.1 to 7.3. Culture of the model staphylococcus HZ01 provides a suitable pH environment.
Preferably, the NB medium is used in an amount of 0.5L to 2L in step (A). More preferably, the NB medium is used in an amount of 1L to 1.5L in step (A). The amount of NB medium used has reached a defined concentration based on the amount of Staphylococcus mimicus HZ01, ensuring good culture of Staphylococcus mimicus HZ 01.
Preferably, the culture in the step (a) is stationary culture. Stationary culture can be advantageous 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 defined here to ensure rapid proliferation of the mimicking staphylococcal HZ01 to achieve 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 limitation of the temperature is more suitable for the culture of the simulated staphylococcus HZ 01.
Preferably, the centrifugal speed in the step (B) is 5000rpm/min to 20000rpm/min. More preferably, the centrifugal speed in the step (B) is 8000rpm/min to 18000rpm/min. More preferably, the centrifugal speed in the step (B) is 10000rpm/min to 15000rpm/min. The proper centrifugal rotation speed can more efficiently lead the thalli to realize solid-liquid separation.
Preferably, the centrifugation time in the step (B) is 10 to 20 minutes. More preferably, the centrifugation time in the step (B) is 12 to 18 minutes. The limit of the centrifugal time can fully realize the solid-liquid separation.
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 centrifugation at low temperature is adopted, and the limitation of the centrifugation temperature is to fully ensure the activity of mimicking staphylococcus HZ01 in the centrifugation process.
Preferably, the mass concentration of the sterilized skim milk in the step (C) is 0.05 to 0.5% m/v. More preferably, the mass concentration of the sterilized skim milk in the step (C) is 0.1 to 0.4% m/v. More preferably, the mass concentration of the sterilized skim milk in the step (C) is 0.2 to 0.3% m/v. The sterilized skim milk with a limited concentration can be sufficiently freeze-dried and protected against the cultured staphylococcus mimicus HZ 01.
Preferably, the amount of sterilized skim milk used in the step (C) is 1ml to 5ml. More preferably, the amount of sterilized skim milk used in the step (C) is 2ml to 4ml. The amount of sterilized skim milk used is limited to a value sufficient to sufficiently freeze-dry and protect the cultured staphylococcus mimicus HZ 01.
Preferably, the freeze-drying temperature in the step (D) is from-90 ℃ to-70 ℃. More preferably, the freeze-drying temperature in the step (D) is from-85 ℃ to-75 ℃. More preferably, the freeze-drying temperature in the step (D) is from-83 ℃ to-78 ℃. The limiting of the freeze-drying temperature is that the staphylococcus HZ01 is fully and quickly dried, and the activity of simulating staphylococcus HZ01 in the staphylococcus HZ 01-mimicking bacterial agent is better ensured.
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: mimicking the use of staphylococcal HZ01 in degrading sarcoplasmic proteins. The staphylococcus mimicking HZ01 has good protease activity, has the capability of decomposing sarcoplasmic protein and myofibrillar protein, can degrade 58.1% of sarcoplasmic protein strips within 72 hours, and remarkably reduces 41.9% of sarcoplasmic protein strips.
Preferably, the detection of the degradation result of the sarcoplasmic protein by the staphylococcus mimicking HZ01 comprises the following steps;
(S01) extracting sarcoplasmic proteins, and measuring the concentration of the sarcoplasmic proteins by using a Lowry protein concentration kit;
(S02) taking sarcoplasmic protein, glucose and a staphylococcus mimicking HZ01 bacterial solution in the step (S01) for co-incubation;
(S03) centrifuging the bacterial liquid after incubation is completed;
(S04) mixing a 2 XSDS loading buffer with the supernatant after centrifugation in the step (S03), and carrying out water bath;
(S05) taking 10% -12% of Bio-Rad preformed mucin and standard protein, and respectively performing the sample in the step (S04) on 10% -12% of Bio-Rad preformed mucin and standard protein;
(S06) carrying out electrophoresis after loading;
(S07) staining with coomassie brilliant blue R-250 after electrophoresis is completed, and obtaining the degradation result of the staphylococcus HZ 01-mimicking sarcoplasmic protein through the staining result. The Lowry protein concentration kit can be used for rapidly and accurately measuring the concentration of sarcoplasmic proteins; the addition of glucose provides a carbon source for mimicking the growth of staphylococcal HZ 01; the centrifugation is used for collecting protein sediment and eliminating interference of irrelevant substances, so that the accuracy of a detection result is better ensured; the addition of the 2 x SDS loading buffer is to adjust the supernatant to a better detected loading sample to be detected; the water bath is used for fully expanding the secondary structure of the protein; 10% -12% of Bio-Rad preformed adhesive can fully separate sarcoplasmic protein strips; standard proteins can be used as references; electrophoresis can fully ensure the separation of protein bands; the coomassie brilliant blue R-250 has good dyeing effect, and can fully develop the sarcoplasmic protein strips; the whole detection method shows the good degradation effect of the simulated staphylococcus HZ01 on the sarcoplasmic proteins.
Preferably, the extraction of the sarcoplasmic proteins comprises the steps of,
(S011) mixing pork with PB buffer solution and homogenizing;
(S012) filtering and sterilizing the supernatant fluid after the homogenization in the step (S011) through a filter membrane to obtain the sarcoplasmic protein. The PB buffer solution can well disperse the pork serum, so that the subsequent treatment and detection of the pork serum are facilitated; the filter membrane can well remove substances which do not meet the requirements in pork serum, so that the purity of the prepared muscle pulp protein reaches the standard; the quality of the finally prepared sarcoplasmic protein is better ensured by the sterilization operation.
Preferably, the pork is fresh pork lean meat. Fresh lean pork is selected, so that the quality of the finally prepared sarcoplasmic protein can be ensured.
Preferably, the pork is used in an amount of 1g to 10g. More preferably, the pork is used in an amount of 3g to 7g. More preferably, the pork is used in an amount of 4g to 6g. The amount of pork is limited according to the required amount of sarcoplasmic protein and the detection requirement.
Preferably, the PB buffer is used in an amount of 15-50 mL. More preferably, the PB buffer is used in an amount of 20 mL-45 mL. More preferably, the PB buffer is used in an amount of 25 mL-40 mL. More preferably, the PB buffer is used in an amount of 30-35 mL. The amount of PB buffer is limited according to the amount of pork to be dispersed, and can sufficiently homogenize and disperse pork.
Preferably, the PB buffer has a concentration of 0.02mol/L. The PB buffer solution with limited concentration can better disperse pork, so that the finally prepared sarcoplasmic protein meets the subsequent processing requirements.
Preferably, the PB buffer has a pH of 5.8 to 6.6. More preferably, the PB buffer has a pH of 6.0 to 6.4. More preferably, the PB buffer has a pH of 6.2 to 6.3. The PB buffer with defined pH value can better process and disperse pork slurry.
Preferably, the homogenization rotation speed is 10000rpm/min to 15000rpm/min. Preferably, the homogenizing speed is 12000rpm/min to 14000rpm/min. The defined homogenization speed enables a more uniform mixing of pork and PB buffer.
Preferably, the homogenization time is 15min to 30min. More preferably, the homogenization time is 20min to 25min. The defined homogenization time is sufficient for homogenization of pork.
Preferably, the pore size of the filter membrane is 0.2 μm to 0.25. Mu.m. More preferably, the pore size of the filter membrane is 0.22 μm to 0.24. Mu.m. The defined pore diameter of the filter membrane can strictly filter the pork serum, thereby ensuring the quality of the finally prepared muscle pulp protein.
Preferably, the glucose is 1% glucose. 1% glucose provides the carbon source required to mimic the proliferation of staphylococcal HZ 01.
Preferably, the amount of the staphylococcus mimicus HZ01 bacteria solution is 0.05 mL-0.5 mL. More preferably, the amount of the staphylococcus mimicus HZ01 bacteria solution is 0.1 mL-0.4 mL. More preferably, the amount of the staphylococcus mimicus HZ01 bacteria solution is 0.2 mL-0.3 mL. The dosage of the staphylococcus HZ01 bacteria liquid is determined according to the detection requirement and the dosage of the sarcoplasmic protein, so that the adequate degradation of the sarcoplasmic protein is ensured.
Preferably, the incubation time is 24 to 72 hours. More preferably, the incubation time is 36 to 60 hours. More preferably, the incubation time is 40 to 50 hours. The incubation time is defined to ensure that the incubation is sufficiently degraded.
Preferably, the amount of the 2 XSDS loading buffer is 150 to 250uL. More preferably, the amount of the 2 XSDS loading buffer is 180uL to 220uL. The amount of 2 x SDS loading buffer is limited to a sample to be tested that will allow good adjustment of the supernatant to a better test.
Preferably, the amount of the supernatant obtained after completion of the freezing is 150 to 250uL. More preferably, the amount of the supernatant obtained after completion of the freezing is 180uL to 220uL. The taking amount of the supernatant after freezing is selected according to the subsequent detection requirement.
Preferably, the water bath temperature is 90 to 98 ℃. More preferably, the water bath temperature is 93 ℃ to 96 ℃. The limitation of the water bath temperature can better fully develop the secondary structure of the protein.
Preferably, the water bath time is 3min to 10min. More preferably, the water bath time is 5min to 7min. The water bath is ensured to fully develop the secondary structure of the protein, and the excessive degradation of the protein is prevented and prevented, so that the result is influenced.
Preferably, the loading amount of 10% -12% of Bio-Rad preformed mucin is 15-25 mu L; the loading amount of the standard protein is 5-10 mu L. More preferably, the loading amount of 10% -12% of Bio-Rad preformed mucin is 17-22 mu L; the loading amount of the standard protein is 7-9 mu L. The specific loading is defined according to the concentration of the corresponding protein.
Preferably, the electrophoresis voltage is 110V. The electrophoresis voltage of 110V is the standard voltage.
Preferably, the electrophoresis time is 60min to 120min. More preferably, the electrophoresis time is 70min to 110min. More preferably, the electrophoresis time is 80min to 100min. The electrophoresis time is limited to ensure the adequate separation of protein bands.
Preferably, the dyeing time is 1 to 2 hours. More preferably, the dyeing time is 1.2 to 1.8 hours. More preferably, the dyeing time is 1.4 to 1.6 hours. The staining time was limited to ensure adequate development of the protein bands.
The fourth technical scheme of the invention: mimicking the use of staphylococcal HZ01 in the reduction of nitrate to nitrite. The staphylococcus HZ 01-imitation has good nitrate reductase activity, can reduce nitrate into nitrite well in the application process, and can utilize nitrate in the fermented meat product, so that the use amount of nitrite in the meat product processing process is reduced.
Preferably, the detection of the concentration of nitrite comprises the steps of,
preparing KNO with proper concentration 3 /NaNO 3 NB medium of (b);
(ii) inoculating a staphylococcus mimicking HZ01 broth into the NB medium of step (i) for cultivation;
(iii) intermittently centrifuging the fermentation broth during the culturing process and collecting the supernatant;
(iv) detecting the nitrite content in the supernatant of step (iii). The whole detection process is simple, and the good reduction effect of reducing nitrate into nitrite by simulating staphylococcus HZ01 is obtained conveniently and rapidly.
Preferably, said step (i) is the formulation of 0.1% KNO 3 /NaNO 3 NB medium of (a). 0.1% KNO 3 /NaNO 3 The NB medium of (3) has a better effect of expressing the nitrate reductase activity of the simulated staphylococcus HZ 01.
Preferably, the step (i) contains a KNO with a proper concentration 3 /NaNO 3 The NB medium formulation of (C) includes the following steps,
taking a proper amount of KNO (i 01) 3 /NaNO 3 And NB nutrient broth is dissolved in sterile water;
(i 02) step (i 01)Sterilizing and culturing the mixed solution to obtain KNO with proper concentration 3 /NaNO 3 NB medium of (a). From KNO 3 /NaNO 3 The NB nutrient broth contains KNO with proper concentration and is prepared from sterile water 3 /NaNO 3 The NB medium can better receive the reduction effect of the simulated staphylococcus HZ01 and better embody the effect of the simulated staphylococcus HZ01 on reducing nitrate into nitrite.
Preferably, KNO in step (i 01) 3 /NaNO 3 The dosage of (2) is 0.02 g-0.1 g. Preferably, KNO in step (i 01) 3 /NaNO 3 The dosage of (2) is 0.04 g-0.08 g. KNO (KNO) 3 /NaNO 3 The amount of (C) is used to prepare KNO with the required concentration 3 /NaNO 3 Is defined by the requirements of the NB medium.
Preferably, the NB nutrient broth in step (i 01) is used in an amount of 0.7g to 1.2g. More preferably, the NB nutrient broth in step (i 01) is used in an amount of 0.8g to 1.1g. The dosage of NB nutrient broth is used for preparing KNO with required concentration 3 /NaNO 3 Is defined by the requirements of the NB medium.
Preferably, 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 from 40mL to 60mL. More preferably, the amount of sterile water used in step (i 01) is 45 mL-55 mL. The dosage of the sterile water is used for preparing KNO with the required concentration 3 /NaNO 3 Is defined by the requirements of the NB medium.
Preferably, the sterilization temperature in 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 bacteria and spores thereof can be fully inactivated, thereby ensuring that the bacteria and spores contain a proper concentration KNO 3 /NaNO 3 Sterile quality of NB medium.
Preferably, the incubation time in step (i 02) is 15 to 30 minutes. More preferably, the incubation time in step (i 02) is 20 to 25 minutes. The culture time is limited, and KNO can be well ensured 3 /NaNO 3 And the spore and NB nutrient broth are well dissolved and mixed in sterile water。
Preferably, the inoculation amount of the step (ii) mimicking staphylococcus HZ01 bacteria solution is 0.05mL to 0.2mL. More preferably, the inoculation amount of the step (ii) mimicking staphylococcus HZ01 bacteria solution is 0.1mL to 0.15mL. The inoculum size of the staphylococcus HZ 01-like bacteria liquid is based on KNO 3 Is defined by the concentration of KNO 3 /NaNO 3 Is fully reduced.
Preferably, the culture temperature in step (ii) is 25℃to 35 ℃. More preferably, the cultivation temperature in step (ii) is 28℃to 32 ℃. The culture temperature is limited, so that the culture medium can better promote the imitation of KNO in the staphylococcus HZ01 to NB culture medium 3 /NaNO 3 Reduction is performed.
Preferably, the incubation time in step (ii) is from 12h to 36h. More preferably, the incubation time in step (ii) is 15 to 30 hours. More preferably, the incubation time in step (ii) is from 20h to 25h. The culture time is limited, so that the culture can simulate the KNO of staphylococcus HZ01 to NB culture medium 3 /NaNO 3 And fully reducing.
Preferably, the amount of the fermentation liquid used in the step (iii) is 1 mL-5 mL. More preferably, the amount of the fermentation liquid used in the step (iii) is 2 mL-4 mL. The dosage of the fermentation liquor is selected according to the detection requirement, and the requirement of checking the nitrite content can be met.
Preferably, the time interval for taking the fermentation liquid in the step (iii) is 2-6 h. More preferably, the time interval for taking the fermentation liquid in the step (iii) is 3-5 h. Taking fermentation liquor at intervals, and imitating staphylococcus HZ01 to KNO at different times 3 /NaNO 3 Multiple fermentation liquor taking can more objectively and more accurately obtain the KNO of the staphylococcus HZ01 pair 3 /NaNO 3 Is a reduction effect of (a).
Preferably, the centrifugation temperature in the step (iii) is 2 to 6 ℃. More preferably, the centrifugation temperature in step (iii) is 3 to 5 ℃. The low temperature limit on the centrifugation temperature is to ensure the safety of the activity of mimicking staphylococcal HZ01 during centrifugation.
Preferably, the centrifugal speed in the step (iii) is 5000rpm/min to 20000rpm/min. More preferably, the centrifugal speed in the step (iii) is 10000rpm/min to 15000rpm/min. The limitation of the centrifugal rotation speed is to efficiently complete the solid-liquid separation.
Preferably, said step (iv) of detecting the nitrite content of the supernatant comprises the steps of,
(iv) a proper amount of supernatant is taken and placed in a colorimetric tube;
(iv 02) adding a proper amount of sulfanilic acid solution into the colorimetric tube in the step (iv 01), uniformly mixing and standing;
(iv 03) adding a proper amount of naphthalene ethylenediamine hydrochloride solution into the solution obtained after standing in the step (iv 02);
(iv) 04) adding water to the solution obtained in the step (iv 03) until the color comparison tube is scaled, uniformly mixing and standing;
(iv 05) measuring the absorbance of the solution in step (iv 04). The sulfanilic acid solution can be subjected to diazotization reaction with nitrite in supernatant fluid, so that preparation is made for subsequent color development reaction; the addition of the naphthalene ethylenediamine hydrochloride solution can be well combined with substances after diazotization reaction to generate rose bengal solution, so that a good nitrite sample to be detected is formed; by measuring the absorbance, the nitrite content in the supernatant can be accurately and rapidly converted.
Preferably, the supernatant in the step (iv) 01) is used in an amount of 0.5 mL-1.5 mL. More preferably, the supernatant in the step (iv) 01) is used in an amount of 0.8 mL-1.2 mL. The amount of supernatant required is determined based on the detection requirements.
Preferably, in the step (iv 01), the cuvette is of a size of 20 mL-30 mL. More preferably, the cuvette in step (iv 01) has a format of 25mL. The specification of the contrasting color 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 amount of the sulfanilic acid solution added in the step (iv 02) is 0.5 to 2mL, and the concentration of the sulfanilic acid solution is 3 to 5g/L. More preferably, the amount of the sulfanilic acid solution added in the step (iv 02) is 0.1 to 1.5mL, and the concentration of the sulfanilic acid solution is 3.5 to 4.5g/L. The adding amount of the sulfanilic acid solution and the concentration of the sulfanilic acid solution are set according to the detection requirement and the requirement of the dosage of the supernatant, and the nitrite content in the sample is calculated more accurately and rapidly.
Preferably, the solution in the step (iv 02) is allowed to stand for 3 to 5 minutes. More preferably, the solution in step (iv 02) is allowed to stand for 3.5 to 4.5 minutes. The standing time is limited in order to allow the solution after diazotization to be calm and to keep a better state for the subsequent reaction.
Preferably, the amount of the naphthalene diamine hydrochloride solution added in the step (iv 03) is 0.2 mL-1 mL, and the concentration of the naphthalene diamine hydrochloride solution is 1 g/L-5 g/L. More preferably, the amount of the naphthalene diamine hydrochloride solution added in the step (iv 03) is 0.5 mL-0.8 mL, and the concentration of the naphthalene diamine hydrochloride solution is 2 g/L-4 g/L. The adding amount of the naphthalene ethylenediamine hydrochloride solution and the concentration of the naphthalene ethylenediamine hydrochloride solution are set according to the detection requirement and the requirement of the color reaction, so that the nitrite content in the sample can be calculated more accurately and rapidly.
Preferably, the standing time in the step (iv 04) is 10 to 20 minutes. More preferably, the standing time in the step (iv 04) is 13 to 17 minutes. The term "standing time" is defined herein as a state in which the absorbance is kept good for the subsequent measurement.
Preferably, the step (iv 05) is carried out by adjusting the zero point of the cuvette with a zero tube using a cuvette of 1cm, taking an appropriate amount of the solution in the step (iv 04), and measuring the absorbance at a wavelength of 538 nm. The specification of the contrasting cuvette, zero point adjustment and wavelength setting are all required according to the specific condition of nitrite in supernatant liquid, so that the nitrite content in a sample can be calculated more accurately and rapidly.
The fifth technical scheme of the invention: mimicking the use of staphylococcal HZ01 in dry salted fermented meat products. The staphylococcus HZ01 can obviously improve esters, aldehydes and acid substances in the fermented meat product, wherein the improvement of vinyl acetate, methyl butyrate, methyl caproate, n-hexanal, acetic acid and isovaleric acid is most obvious, and the quality of the fermented meat product can be obviously improved.
Preferably, the preparation of the dry salted fermented meat product comprises the steps of,
firstly, taking concentrated solution or powder of the simulated staphylococcus HZ01, and diluting with edible water;
dissolving proper amount of salt, white sugar and sodium glutamate in the solution obtained in the step (one) to prepare a pickling solution;
thirdly, taking meat emulsion, adding the pickling solution in the second step into the meat emulsion, and keeping the concentration of staphylococcus HZ01 in the meat emulsion to be 1.0x10 7 log 10 CFU/g～9.9×10 8 log 10 When CFU/g is carried out, the meat emulsion is salted at a low temperature;
and fourthly, after the pickling is finished, sequentially performing sausage filling, air exhausting, baking and fermentation on the minced meat to obtain a finished product of the dry-pickled fermented meat product. The salt, the white sugar, the sodium glutamate and the concentrated solution/powder imitating staphylococcus HZ01 are prepared into the pickling solution together in proper parts, so that the pickling effect on the meat paste is better, the meat paste is directly tasty after being pickled, and the subsequent meat products do not need to be seasoned again; the activity safety of the staphylococcus HZ01 can be effectively kept by low-temperature pickling; the meat paste after pickling is subjected to sausage filling, air exhaust, baking and fermentation in sequence, and the finished product of the dry pickled fermented meat product prepared by the process sequence has the advantages of fragrance, rich nutrition and excellent quality; wherein the staphylococcus HZ 01-mimicking activity still exists after drying and further plays a role in the fermentation process, which indicates that the staphylococcus HZ 01-mimicking activity 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 treatment of the staphylococcus-simulated HZ01 is obviously improved, wherein the improvement of vinyl acetate, methyl butyrate, methyl caproate, n-hexanal, acetic acid and isovaleric acid is most obvious.
Preferably, the concentration of the simulated staphylococcus HZ01 bacteria agent in the step (three) is 3.0x10 7 log 10 CFU/g～7.9×10 8 log 10 CFU/g. More preferably, the concentration of the simulated staphylococcus HZ01 bacteria agent in the step (three) is 5.0x10 7 log 10 CFU/g～5.9×10 8 log 10 CFU/g. More preferably, the concentration of the simulated staphylococcus HZ01 bacteria agent in the step (three) is 7.0x10 7 log 10 CFU/g～3.9×10 8 log 10 CFU/g. More preferably, the concentration of the simulated staphylococcus HZ01 bacteria agent in the step (three) is 9.0x10 7 log 10 CFU/g～1.9×10 8 log 10 CFU/g. The concentration of the staphylococcus HZ 01-mimicking microbial inoculum is limited, so that a good proportion is formed with salt, white sugar and sodium glutamate, so that the subsequent meat paste to be treated can be better salted, and the subsequent salting and fermentation processes of the meat paste can be better played.
Preferably, the pickling solution in the step (II) comprises the following components in parts by weight,
1 to 5 parts of salt, 3 to 8 parts of white sugar, 0.2 to 0.8 part of sodium glutamate, 2 to 5 parts of edible water and a proper amount of staphylococcus HZ01 concentrated solution/powder. The proportion of the salt, the white sugar, the sodium glutamate, the edible water and the staphylococcus HZ01 simulated concentrated solution/powder is limited, so that the prepared pickling solution has balanced nutrition, the components can be mutually cooperated to play a good role in pickling the meat emulsion, the meat emulsion is directly tasty after being pickled, and the subsequent meat product is not required to be seasoned. Wherein, a proper amount of perfume can be added according to the need.
Preferably, the pickling solution in the step (II) comprises the following components in parts by weight,
2-4 parts of 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 concentrated solution/powder. The proportion of the table salt, the white sugar, the sodium glutamate, the edible water and the staphylococcus HZ01 simulated concentrated solution/powder is limited, so that the nutrition of the prepared pickling solution is more balanced, the components can be mutually cooperated, a good effect is exerted on pickling of the meat emulsion, the meat emulsion is directly tasty after being pickled, and the subsequent meat product is not required to be seasoned.
Preferably, the pickling solution in the step (II) comprises the following components in parts by weight,
2-4 parts of 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 concentrated solution/powder. The proportion of the table salt, the white sugar, the sodium glutamate, the edible water and the staphylococcus HZ01 simulated concentrated solution/powder is limited, so that the nutrition of the prepared pickling solution is more balanced, the components can be mutually cooperated, a good effect is exerted on pickling of the meat emulsion, the meat emulsion is directly tasty after being pickled, and the subsequent meat product is not required to be seasoned.
Preferably, the mass ratio of fat to lean meat in the meat emulsion 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 emulsion is limited, so that the quality of the finished dry-cured fermented meat product is better.
Preferably, the curing temperature in the step (three) is 2 to 6 ℃. More preferably, the curing temperature in the step (three) is 3 to 5 ℃. The low-temperature pickling is carried out, so that the staphylococcus HZ01 is imitated in the pickling process and has good activity and safety all the time.
Preferably, the pickling time in the step (three) is 48 to 72 hours. More preferably, the pickling time in the step (three) is 55 to 65 hours. The limitation of the curing time can well ensure that the minced meat is fully cured and tasty.
Preferably, the baking temperature in the step (four) is 50 to 65 ℃. More preferably, the baking temperature in the step (four) is 55 to 60 ℃. The limitation of the baking temperature can not excessively influence the activity of the staphylococcus HZ01 during the effective baking of 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 (four) is 55 to 65 hours. The sausage can be fully dried by limiting the drying time.
Preferably, the fermentation temperature in the step (four) is 20 to 30 ℃. More preferably, the fermentation temperature in the step (four) is 23 to 27 ℃. The fermentation temperature is also limited in order to maintain good activity in mimicking staphylococcal HZ 01.
Preferably, the fermentation time in the step (four) is 1 to 2 weeks. More preferably, the fermentation time in the step (four) is 8 to 10 days. The limitation of fermentation time can ensure that the dried sausage is completely fermented by mimicking staphylococcus HZ 01.
Preferably, the preparation method of the concentrated solution of the staphylococcus mimicking HZ01 comprises the steps of inoculating purified and refrigerated single staphylococcus mimicking HZ01 colonies into a sterilized NB liquid culture medium for culture until the concentration of the staphylococcus mimicking HZ01 in the NB liquid culture medium reaches 1.0x10 7 log 10 CFU/mL～5.0×10 7 log 10 CFU/mL, a concentrated solution is obtained. The preparation of the concentrated solution is to prepare the pickling solution better later, so that the meat paste is well subjected to the subsequent pickling and fermentation processes; the concentration of the staphylococcus HZ01 in the concentrated solution is limited, so that the method is suitable for the processing requirements of minced meat in the subsequent working procedures, and the finished product of the dry-cured fermented meat product is better in quality.
Preferably, the refrigerating temperature in the preparation process of the concentrated solution is 2-6 ℃. More preferably, the refrigerating temperature in the preparation process of the concentrated solution is 3-5 ℃. The refrigerating temperature is limited to ensure the safety of the activity of the staphylococcus HZ 01.
Preferably, the dosage of NB liquid culture medium in the preparation process of the concentrated solution is 40 mL-60 mL. More preferably, the NB liquid medium is used in an amount of 45 mL-55 mL in the preparation process of the concentrated solution. Here the limit of the amount of NB broth is required depending on the concentration of the mimicking Staphylococcus HZ01 to be cultivated and the amount of concentrate to be formulated finally.
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 15-35 hours. More preferably, the culture time in the preparation process of the concentrated solution is 20-30 hours. The culture time is defined herein to ensure that the concentration of the mimetic staphylococcal HZ01 reaches the plateau when the mimetic staphylococcal HZ01 is cultured well.
Preferably, the culture temperature in the preparation process of the concentrated solution is 25-35 ℃. More preferably, the culture temperature in the preparation process of the concentrated solution is 28-32 ℃. The culture temperature is defined here in order to create a good temperature condition that allows good culture of the mimicking staphylococcal HZ01 in the post-sterilization NB liquid medium, allowing the mimicking staphylococcal HZ01 to reach the desired concentration of concentrate rapidly.
Preferably, the culture in the preparation process of the concentrated solution is stationary culture. The resting culture suitably mimics the growth characteristics of staphylococcal HZ 01.
Preferably, the tolerance pH of the staphylococcus mimicus HZ01 during fermentation is 4 to 8. More preferably, the tolerance pH of the mimicking staphylococcal HZ01 during fermentation is in the range of 5 to 7. The staphylococcus HZ01 can be simulated to grow under acidic, neutral and alkaline conditions, and the staphylococcus HZ01 is simulated to have good acid and alkaline resistance effect and wide range.
Preferably, the amount of nitrite resistant in the fermentation process of the staphylococcus mimicus HZ01 is 0 to 150mg/kg. More preferably, the amount of nitrite resistant in the fermentation process of the staphylococcus mimicus HZ01 is 50mg/kg to 100mg/kg. The tolerance nitrite content range of the staphylococcus HZ01 in the fermentation process is simulated to be wider, so that the nitrite can be added in a larger range according to actual needs in the fermentation process.
Preferably, the salt tolerance of the staphylococcus mimicus HZ01 in the fermentation process is 3-12%. More preferably, the salt tolerance of the staphylococcus mimicus HZ01 in the fermentation process is 5 to 10 percent. More preferably, the salt tolerance of the staphylococcus mimicus HZ01 in the fermentation process is 6 to 8 percent. The salt tolerance range of the staphylococcus HZ01 in the fermentation process is simulated to be wider, so that the salt can be added in a larger range according to actual needs in the fermentation process.
Preferably, the tolerance temperature of the staphylococcus mimicus HZ01 in the fermentation process is 20-40 ℃. Preferably, the tolerance temperature of the staphylococcus mimicus HZ01 in the fermentation process is 25-35 ℃. The tolerance temperature range of the staphylococcus HZ01 is wide in the fermentation process, so that the staphylococcus HZ01 can be regulated in a large temperature range according to actual needs in the fermentation process.
The invention has the following beneficial effects:
(1) The staphylococcus HZ 01-imitating metabolism is strong, and when the staphylococcus HZ-imitating metabolism is used as a starter, the staphylococcus HZ-imitating metabolism can keep advantages with a local microorganism population in the competition process, has strong competitiveness, ensures that the sensory characteristics of the fermented meat product are ideal, and can better adapt to the processing environment of the local meat product;
(2) The staphylococcus HZ01 has good protease activity, has the capacity of decomposing sarcoplasmic proteins, can degrade 58.1% of sarcoplasmic protein strips within 72 hours, and remarkably reduces 41.9% of sarcoplasmic protein strips;
(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 use amount of the nitrite in the meat product processing process;
(4) The staphylococcus HZ01 can be imitated to metabolize leucine to generate 3-methyl butyraldehyde, so that the variety and the content of volatile flavor substances are increased, and the aroma production property is excellent;
(5) The staphylococcus HZ01 can be imitated to remarkably improve esters, aldehydes and acid substances in the fermented meat product, wherein the improvement of vinyl acetate, methyl butyrate, methyl caproate, n-hexanal, acetic acid and isovaleric acid is most remarkable, and the quality of the fermented meat product can be remarkably improved.
Drawings
FIG. 1 is a colony morphology of a simulated staphylococcus HZ01 according to the present invention;
FIG. 2 is a graph of the results of gram staining of a simulated staphylococcus HZ01 in the present invention;
FIG. 3 is a graph of the results of a comparison of a simulated staphylococcal HZ01 strain with other staphylococcal strains in the invention;
FIG. 4 is a graph of the evolutionary relationship of a simulated staphylococcal HZ01 in the invention;
FIG. 5 is a graph of pH fermentation resistance characteristics of a simulated staphylococcus HZ01 in the present invention;
FIG. 6 is a graph of the fermentation characteristics of the simulated Staphylococcus HZ01 tolerant to nitrite amounts in the present invention;
FIG. 7 is a graph showing the salt tolerance fermentation characteristics of a staphylococcus aureus HZ01 in the present invention;
FIG. 8 is a graph of temperature fermentation resistance characteristics of a simulated staphylococcus HZ01 in the present invention;
FIG. 9 is a SDS-PAGE of simulated staphylococcal HZ01 resolved sarcoplasmic proteins according to the invention;
FIG. 10 is a graph of the use of NO by the Staphylococcus HZ 01-mimicking strain under anaerobic conditions of the present invention 3 - Conversion to NO 2 – Is a bar graph of the case of (2).
Detailed Description
The invention is further illustrated by the following figures and examples, which are not intended to be limiting.
A strain of staphylococcus HZ01, the preservation number of which is GDMCCNO.62410; the collection unit mimicking staphylococcal HZ01 is the collection of microorganism strains in guangdong province; the address of the staphylococcus mimicking HZ01 preservation unit is building 5 of No. 59 of No. 100 institute of Migo 100 in Guangzhou city; the preservation name of the staphylococcus mimicus HZ01 is Staphylococcus aureus HZ01; the shelf life of the mimicking staphylococcal HZ01 was 2022, 4 months and 24 days.
A staphylococcus HZ 01-mimicking agent, wherein the staphylococcus HZ 01-mimicking agent is prepared by mimicking staphylococcus HZ01;
the preparation of the staphylococcus mimicking HZ01 microbial inoculum comprises the following steps,
(A) Inoculating staphylococcus mimicus HZ01 into NB culture medium for culturing to obtain bacterial liquid; the NB medium in the step (A) comprises 10g/L peptone, 3.0g/L beef extract and 5.0g/L 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 stationary culture; the culture time in the step (A) is 24-72 hours; the culture temperature in the step (A) is 25-35 ℃;
(B) After the bacterial liquid prepared in the step (A) has the concentration of the simulated staphylococcus HZ01 of 1.0x10 8 log 10 CFU/mL～9.9×10 9 log 10 Centrifuging at CFU/mL; the centrifugal rotating speed in the step (B) is 5000-20000 rap/min; the centrifugation time in the step (B) is 10-20 min; the centrifugation temperature in the step (B) is 2-6 ℃;
(C) Adding sterilized skimmed milk for resuspension after centrifugation is completed; 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 1 ml-5 ml;
(D) Freeze drying after the resuspension is completed to obtain the product with concentration of 1.0X10 10 log 10 CFU/g～9.9×10 11 log 10 CFU/g powdered mimicking staphylococcal HZ01 bacteria; 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 imitating staphylococcus HZ01 is OM758216, and the specific nucleotide sequence is shown as SEQ ID NO. 1;
the determination of the sequence mimicking the 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; the PCR reaction system included 10 XEx Taqbuffer 2. Mu.L, 2.5mM dNTP Mix1.6. Mu.L, 5 pPrimer10.6. Mu.L, 5 pPrimer20.6. Mu.L, template 2. Mu.L, 5uExTaq 1. Mu.L, ddH2O 12.2. Mu.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,
(b01) Reacting the total DNA extracted in the step (a) in a PCR reaction system at a temperature of 95 ℃ for 5min;
(b02) After the step (b 01) is completed, continuing to react for 30 seconds at the temperature of 95 ℃;
(b03) After step (b 02) is completed, continuing to react for 30 seconds at the temperature of 55 ℃;
(b04) After the step (b 03) is completed, continuing to react for 1min at the temperature of 72 ℃;
(b05) Repeating the steps (b 02) to (b 04) 24 times;
(b06) After step (b 05) is completed, the temperature condition of 72 ℃ is prolonged for 10min;
(b07) After the step (b 06) is finished, preserving heat at the temperature of 10 ℃ to finish 16s full-length amplification of the total DNA imitating staphylococcus HZ 01;
(c) And (3) after the full-length amplification of 16s is finished, performing gel cutting purification, and performing electrophoresis sequencing to obtain a sequence which is the full-length sequence of the 16sDNA imitating staphylococcus HZ 01.
Mimicking the use of staphylococcal HZ01 in degrading sarcoplasmic proteins;
the detection of the degradation result of the staphylococcus HZ01 on the sarcoplasmic protein comprises the following steps of;
(S01) extracting sarcoplasmic proteins, and measuring the concentration of the sarcoplasmic proteins by using a Lowry protein concentration kit;
the extraction of sarcoplasmic proteins comprises the steps of,
(S011) mixing pork with PB buffer solution and homogenizing; pork is fresh lean pork; the pork is used in an amount of 1 g-10 g; the dosage of the PB buffer solution is 15-50 mL; the concentration of PB buffer solution is 0.02mol/L; the pH value of the PB buffer solution is 5.8-6.6; the homogenizing rotating speed is 10000 rpm/min-15000 rpm/min; the homogenizing time is 15 min-30 min;
(S012) filtering and sterilizing the supernatant fluid homogenized in the step (S011) through a filter membrane to obtain sarcoplasmic protein; the pore diameter of the filter membrane is 0.2-0.25 mu m;
(S02) taking sarcoplasmic protein, glucose and a staphylococcus mimicking HZ01 bacterial solution in the step (S01) for co-incubation; glucose is 1% glucose; the dosage of the staphylococcus HZ01 bacteria liquid is 0.05 mL-0.5 mL; the incubation time is 24-72 hours;
(S03) centrifuging the bacterial liquid after incubation is completed;
(S04) mixing a 2 XSDS loading buffer with the supernatant after centrifugation in the step (S03), and carrying out water bath; the taking amount of the 2 XSDS loading buffer solution is 150-250 uL; the dosage of the supernatant after centrifugation is 150 uL-250 uL; the water bath temperature is 90-98 ℃; the water bath time is 3 min-10 min;
(S05) taking 10% -12% of Bio-Rad preformed mucin and standard protein, and respectively performing the sample in the step (S04) on 10% -12% of Bio-Rad preformed mucin and standard protein; the loading amount of 10% -12% of Bio-Rad preformed mucin is 15 mu L-25 mu L; the loading amount of the standard protein is 5-10 mu L.
(S06) carrying out electrophoresis after loading; the electrophoresis voltage is 110V; the electrophoresis time is 60 min-120 min;
(S07) dyeing with coomassie brilliant blue R-250 after electrophoresis is completed, and obtaining a degradation result of the staphylococcus HZ 01-simulated sarcoplasmic protein through a dyeing result; the dyeing time is 1-2 h.
Mimicking the use of staphylococcal HZ01 in the reduction of nitrate to nitrite;
the detection of the concentration of nitrite comprises the steps of,
preparing KNO with proper concentration 3 /NaNO 3 NB medium of (b); step (i) is to prepare 0.1% KNO 3 /NaNO 3 NB medium of (b);
in step (i) a proper concentration KNO is contained 3 /NaNO 3 The NB medium formulation of (C) includes the following steps,
taking a proper amount of KNO (i 01) 3 /NaNO 3 And NB nutrient broth is dissolved in sterile water; KNO in step (i 01) 3 /NaNO 3 The dosage of (2) is 0.02 g-0.1 g; the NB nutrient broth in step (i 01) is used in an amount of 0.7g to 1.2g; the dosage 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 KNO with proper concentration 3 /NaNO 3 NB medium of (b); 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 a staphylococcus mimicking HZ01 broth into the NB medium of step (i) for cultivation; step (ii) the inoculation amount of the simulated staphylococcus HZ01 bacteria 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) intermittently centrifuging the fermentation broth during the culturing process and collecting the supernatant; the dosage of the fermentation liquor in the step (iii) is 1 mL-5 mL; taking fermentation liquor in the step (iii) at a time interval of 2-6 h; the centrifugation temperature in step (iii) is 2-6 ℃; the centrifugal rotation speed in the step (iii) is 5000-20000 rap/min;
(iv) detecting the nitrite content in the supernatant of step (iii);
step (iv) detecting the nitrite content in the supernatant comprises the following steps,
(iv) a proper amount of supernatant is taken and placed in a colorimetric tube; the dosage of the supernatant in the step (iv 01) is 0.5 mL-1.5 mL; in the step (iv 01), the specification of the colorimetric tube is 20-30 mL;
(iv 02) adding a proper amount of sulfanilic acid solution into the colorimetric tube in the step (iv 01), uniformly mixing and standing; in the step (iv 02), the adding 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 naphthalene ethylenediamine hydrochloride solution into the solution obtained after standing in the step (iv 02); in the step (iv 03), the adding amount of the naphthalene diamine hydrochloride solution is 0.2-1 mL, and the concentration of the naphthalene diamine hydrochloride solution is 1-5 g/L;
(iv) 04) adding water to the solution obtained in the step (iv 03) until the color comparison tube is scaled, uniformly mixing and standing; the standing time in the step (iv 04) is 10 min-20 min;
(iv 05) measuring the absorbance of the solution in step (iv 04); in the step (iv 05), a 1cm cuvette is used, the zero point of the cuvette is adjusted by a zero tube, and an appropriate amount of the solution in the step (iv 04) is taken and absorbance is measured at a wavelength of 538 nm.
Mimicking the use of staphylococcal HZ01 in dry salted fermented meat products;
the preparation of the dry salted fermented meat product comprises the steps of,
firstly, taking concentrated solution/powder of the simulated staphylococcus HZ01, and diluting the concentrated solution/powder by using edible water;
concentrated solution of staphylococcus HZ01The preparation method comprises inoculating purified and refrigerated single colony of simulated staphylococcus HZ01 into sterilized NB liquid culture medium, and culturing until the concentration of simulated staphylococcus HZ01 in NB liquid culture medium reaches 1.0X10 7 log 10 CFU/mL～5.0× 10 7 log 10 When CFU/mL is carried out, the concentrated solution is prepared; the refrigerating temperature in the preparation process of the concentrated solution is 2-6 ℃; the dosage of 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 ℃; culturing in the preparation process of the concentrated solution is stationary culturing;
Dissolving proper amount of salt, white sugar and sodium glutamate in the solution obtained in the step (one) to prepare a pickling solution;
the pickling solution in the step (II) comprises the following components in parts by weight,
1 to 5 parts of salt, 3 to 8 parts of white sugar, 0.2 to 0.8 part of sodium glutamate, 2 to 5 parts of edible water and 1 to 5 parts of concentrated solution/powder imitating staphylococcus HZ 01;
thirdly, taking meat emulsion, adding the pickling solution in the second step into the meat emulsion, and keeping the concentration of staphylococcus HZ01 in the meat emulsion to be 1.0x10 7 log 10 CFU/g～9.9×10 8 log 10 When CFU/g is carried out, the meat emulsion is salted at a low temperature; the concentration of the simulated staphylococcus HZ01 bacteria agent in the step (three) is 3.0x10 based on the mass of the meat emulsion 7 log 10 CFU/g～7.9×10 8 log 10 CFU/g. The mass ratio of the fat to the lean meat in the meat emulsion is 1-3:6-9; the curing temperature in the step (III) is 2-6 ℃; the pickling time in the step (III) is 48-72 hours;
fourthly, after curing, sequentially performing sausage filling, air exhausting, baking and fermentation on the minced meat to obtain a finished product of the dry cured and fermented meat product; the baking temperature in the step (four) is 50-65 ℃; the baking time in the step (IV) is 48-72 hours; the fermentation temperature in the step (four) is 20-30 ℃; the fermentation time in the step (four) is 1 to 2 weeks; the tolerance pH of the staphylococcus HZ01 during fermentation is simulated to be 4-8; the tolerance nitrite quantity of the staphylococcus HZ01 during the fermentation process is simulated to be 0-150 mg/kg; the salt tolerance of the staphylococcus HZ01 in the fermentation process is 3-12%; the tolerance temperature of the staphylococcus HZ01 during the fermentation process is simulated to be 20-40 ℃.
Example 1:
mimicking a staphylococcus HZ01 inoculum, designated Staphylococcus aureus HZ01, the preparation comprising the steps of,
(A) Inoculating staphylococcus mimicus HZ01 into NB culture medium for culturing to obtain bacterial liquid; the NB medium in the step (A) comprises 10g/L peptone, 3.0g/L beef extract and 5.0g/L sodium chloride; the NB medium in step (A) had a pH of 7.2.+ -. 0.2; the NB medium was used in 1L in step (A); the culture in the step (A) is stationary culture; the incubation time in step (a) was 48 hours; the culture temperature in step (A) was 30 ℃;
(B) After the bacterial liquid prepared in the step (A) has the concentration of the simulated staphylococcus HZ01 of 2.5X10 9 log 10 Centrifuging at CFU/mL; the centrifugal rotating speed in the step (B) is 10000rap/min; the centrifugation time in step (B) is 15min; the centrifugation temperature in step (B) is 4 ℃;
(C) Adding sterilized skimmed milk for resuspension after centrifugation is completed; the mass concentration of the sterilized skim milk in the step (C) is 0.1 percent m/v; the amount of sterilized skim milk in the step (C) is 2ml;
(D) Freeze drying after the resuspension is completed to obtain the product with concentration of 2.0X10 11 log 10 CFU/g powdered mimicking staphylococcal HZ01 bacteria; the freeze-drying temperature in step (D) is-80 ℃; the lyophilization time in step (D) was 48h.
Example 2:
the CNKI gene imitating staphylococcus HZ01 has accession number OM758216, and the collection number of the Guangdong province microorganism strain collection (GDMCC) is 62410, and the specific nucleotide sequence is shown in SEQ ID NO. 1:
GCTATACATGCAAGTCGAGCGAACAGACGAGGAGCTTGCTCCTCTGACGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTAC CTATAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATAATACATGAAACCGCATGGTTTCATGATGAAAGACGGTTTTGCTGTCACTTATAGATGGACCCGCGGCGTATTAGCTAGTTGGTAAGGTAACGGCTTACCAAGGCAACGATACGTAGCCGACCTGAGAGGGTGATCG GCCACACTGGAACTGAGGACACGGTCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGGCGAAAGCCTGACGGAGCAACGCCGCGTGAGGTGATGAAGGTCTTCGGATCGTAAAACTCTGTTATTAGGGAAGAACAAGGGTGTAAGTAACTGTGCATCCCCTTGACGGTACCTAATCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGAATTATTGGGCGTAAAGCGCG CGTAGGCGGTTTTTTAAGTCTGATGTGAAAGCCCACGGCTCAACCGTGGAGGGTCATTGGAAACTGGAAAACTTGAGTGCAGAAGAGGAAAGTGGAATTCCATGTGTAGCGGTGAAATGCGCAGAGATATGGAGGAACACCAGTGGCGAAGGCGACTTTCTGGTCTGCAACTGACGCTGATG TGCGAAAGCGTGGGGATCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGCCGCAAGGCTGAAACTCAAAGGAATTGACGGGGACCCGCACAAGCGG TGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAAATCTTGACATCCTTTGACAACTCTAGAGATAGAGCTTTCCCCTTCGGGGGACAAAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTAAGCTT AGTTGCCAGCATTAAGTTGGGCACTCTAAGTTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGGATGACGTCAAATCATCATGCCCCTTATGATTTGGGCTACACACGTGCTACAATGGACGGTACAAAGGGCAGCGAACCCGCGAGGTCAAGCAAATCCCATAAAGCCGTTCTCAGTTCG GATTGTAGTCTGCAACTCGACTACATGAAGCTGGAATCGCTAGTAATCGTAGATCAGCATGCTACGGTGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGCCGGTGGAGTAACCTTTTAGGAACTAGCCGTCGAAGGTGACA
the determination of the sequence mimicking the 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; the PCR reaction system included 10 XEx Taqbuffer 2. Mu.L, 2.5mM dNTP Mix1.6. Mu.L, 5 pPrimer10.6. Mu.L, 5 pPrimer20.6. Mu.L, template 2. Mu.L, 5uExTaq 1. Mu.L, ddH2O 12.2. Mu.L; the dosage of the PCR reaction system is 20 mu L;
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 at a temperature of 95 ℃ for 5min to perform pre-denaturation;
(b02) After the step (b 01) is completed, continuing to react for 30 seconds at the temperature of 95 ℃;
(b03) After step (b 02) is completed, continuing to react for 30 seconds at the temperature of 55 ℃;
(b04) After the step (b 03) is completed, continuing to react for 1min at the temperature of 72 ℃;
(b05) Repeating the steps (b 02) to (b 04) 24 times;
(b06) After step (b 05) is completed, the temperature condition of 72 ℃ is prolonged for 10min;
(b07) After the step (b 06) is finished, preserving heat at the temperature of 10 ℃ to finish 16s full-length amplification of the total DNA imitating staphylococcus HZ 01;
(c) After the full-length amplification of 16s is completed, gel cutting and purification are carried out, a horizontal electrophoresis instrument is used for electrophoresis sequencing, the measured sequence is the full-length sequence of 16sDNA imitating staphylococcus HZ01, 1437bp is shown in figures 1 and 2, the similarity of the strain and the strain S.simulians MR1 (CP 015642.1) with the closest relativity is 99.65 percent shown in figure 3, an independent evolution branch shown in figure 4 is formed in the evolution process, and the strain is a new strain imitating staphylococcus.
Example 3:
as shown in figures 5,6,7 and 8, the staphylococcus HZ01 is simulated to resist the fermentation condition that the pH value is 5-8, the nitrite is 150mg/kg, the salinity is 3-9% and the temperature is 20-35 ℃.
A concentrated solution of Staphylococcus mimicus HZ01 is prepared by selecting purified single colony of Staphylococcus mimicus HZ01 stored in refrigerator at 4deg.C, inoculating to 50mL sterilized NB liquid culture medium, and standing at 30deg.C for 24 hr until its concentration reaches 3.0X10 7 log 10 CFU/mL, the culture solution is concentrated solution. 50mL of NB liquid medium with pH value of 4,5,6,7 and 8, salinity of 0%,3%,6%,9%,12% and nitrite concentration of 0, 50mg/kg, 100mg/kg and 150mg/kg was prepared respectively, 1mL of the concentrate was inoculated into NB medium under different culture conditions, cultured at 30℃for 24 hours, OD value was measured every 3 hours, and the optimum fermentation conditions mimicking Staphylococcus HZ01 were verified. 50mL of NB liquid culture medium is prepared, 1mL of concentrated solution is inoculated, OD value curves of the strain HZ01 at different temperatures are verified, and the temperature growth range of the HZ01 is determined. The results show that HZ01 The growth range is wider, and the growth can be carried out under the conditions of pH value of 5-8, nitrite of 0-150 mg/kg, salinity of 3-9% and temperature of 20-35 ℃.
Example 4:
mimicking the use of staphylococcal HZ01 in degrading sarcoplasmic proteins; as shown in fig. 9 and 10, mimicking staphylococcal HZ0 can secrete highly active proteases, degrade 58.1% of the sarcoplasmic protein bands within 72 hours, and significantly reduce 41.9% of the sarcoplasmic protein bands;
the detection of the degradation result of the staphylococcus HZ01 on the sarcoplasmic protein comprises the following steps of;
(S01) extracting sarcoplasmic proteins, and measuring the concentration of the sarcoplasmic proteins by using a Lowry protein concentration kit;
the extraction of sarcoplasmic proteins comprises the steps of,
(S011) mixing pork with PB buffer solution and homogenizing; pork is fresh lean pork; the pork is used in an amount of 2g; the PB buffer was used in an amount of 20mL; the concentration of PB buffer solution is 0.02mol/L; the pH of PB buffer was 6.5; the homogenization rotation speed is 13000rpm/min; the homogenization time is 20min;
(S012) filtering and sterilizing the supernatant fluid homogenized in the step (S011) through a filter membrane to obtain sarcoplasmic protein; the pore diameter of the filter membrane is 0.22 mu m;
(S02) taking sarcoplasmic protein, glucose and a staphylococcus mimicking HZ01 bacterial solution in the step (S01) for co-incubation; glucose is 1% glucose; the dosage of the staphylococcus HZ01 bacteria liquid is 0.1mL; the incubation time is 12-36 h;
(S03) taking 2mL of bacterial liquid and centrifuging at intervals of 24 hours;
(S04) mixing a 2 XSDS loading buffer with the supernatant after the centrifugation in the step (S03), and carrying out water bath; the amount of the sample buffer solution applied to the 2 XSDS is 200uL; the supernatant after centrifugation was taken in an amount of 200uL; the water bath temperature is 95 ℃; the water bath time is 5min;
(S05) taking 10% -12% of Bio-Rad preformed mucin and standard protein, and respectively taking the samples in the step (S04) on 10% -12% of Bio-Rad preformed mucin and standard protein; the loading amount of 10% -12% of Bio-Rad preformed mucin is 20 mu L; the loading amount of the standard protein was 8. Mu.L.
(S06) carrying out electrophoresis after loading; the electrophoresis voltage is 110V; the electrophoresis time is 90min;
(S07) dyeing with coomassie brilliant blue R-250 after electrophoresis is completed, and obtaining a degradation result of the sarcoplasmic protein band by simulating staphylococcus HZ01 through a dyeing result; the dyeing time was 1.5h.
Example 5:
mimicking the use of staphylococcal HZ01 in the reduction of nitrate to nitrite;
the detection of nitrite activity comprises the steps of,
preparing KNO with proper concentration 3 NB medium of (b); step (i) is to prepare 0.1% KNO 3 NB medium of (b);
In step (i) a proper concentration KNO is contained 3 The NB medium formulation of (C) includes the following steps,
taking a proper amount of KNO (i 01) 3 And NB nutrient broth is dissolved in sterile water; KNO in step (i 01) 3 The dosage of (2) is 0.05g; the amount of NB nutrient broth in step (i 01) was 0.9g; the amount of sterile water used in step (i 01) is 50mL;
sterilizing and culturing the mixed solution obtained in the step (i 01) to obtain the KNO with proper concentration 3 NB medium of (b); the sterilization temperature in step (i 02) is 121 ℃; the incubation time in step (i 02) was 20min;
(ii) inoculating a staphylococcus mimicking HZ01 broth into the NB medium of step (i) for cultivation; step (ii) the inoculation amount of the simulated staphylococcus HZ01 bacteria liquid is 0.1mL; the incubation temperature in step (ii) is 30 ℃; the incubation time in step (ii) is 24 hours;
(iii) intermittently centrifuging the fermentation broth during the culturing process and collecting the supernatant; the amount of the fermentation liquor taken in the step (iii) is 2mL; taking fermentation liquor in the step (iii) at a time interval of 4 hours; the centrifugation temperature in step (iii) is 4 ℃; the centrifugal speed in the step (iii) is 10000rap/min;
(iv) detecting the nitrite content in the supernatant of step (iii);
step (iv) detecting the nitrite content in the supernatant comprises the following steps,
(iv) a proper amount of supernatant is taken and placed in a colorimetric tube with a plug; the amount of the supernatant in the step (iv 01) is 1mL; the specification of the colorimetric tube in the step (iv 01) is 25mL;
(iv 02) adding a proper amount of sulfanilic acid solution into the colorimetric tube in the step (iv 01), uniformly mixing and standing; in the step (iv 02), the adding amount of the sulfanilic acid solution is 1mL, and the concentration of the sulfanilic acid solution is 4g/L; the standing time of the solution in the step (iv 02) is 3-5 min;
(iv 03) adding a proper amount of naphthalene ethylenediamine hydrochloride solution into the solution obtained after standing in the step (iv 02); in the step (iv 03), the adding amount of the naphthalene diamine hydrochloride solution is 0.5mL, and the concentration of the naphthalene diamine hydrochloride solution is 2g/L;
(iv) 04) adding water to the solution obtained in the step (iv 03) until the color comparison tube is scaled, uniformly mixing and standing; the standing time in step (iv 04) is 15min;
(iv 05) measuring the absorbance of the solution in step (iv 04); and (iv) using a 1cm cuvette, regulating the zero point of the cuvette by using a zero pipe, taking a proper amount of the solution in the step (iv) 04), measuring absorbance at the wavelength of 538nm, and simultaneously making a reagent blank and a standard curve.
Example 6:
mimicking the use of staphylococcal HZ01 in dry salted fermented meat products;
the preparation of the dry salted fermented meat product comprises the steps of,
Firstly, taking concentrated solution of the simulated staphylococcus HZ01, and diluting the concentrated solution with edible water;
a concentrated solution of Staphylococcus mimicus HZ01 is prepared by selecting purified single colony of Staphylococcus mimicus HZ01 stored in refrigerator at 4deg.C, inoculating to 50mL sterilized NB liquid culture medium, and standing at 30deg.C for 24 hr until its concentration reaches 3.0X10 7 log 10 CFU/mL, the culture solution is concentrated solution.
Dissolving proper amount of salt, white sugar and sodium glutamate in the solution obtained in the step (one) to prepare a pickling solution;
the pickling solution in the step (II) comprises the following components in parts by weight,
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 concentrate;
thirdly, taking meat emulsion, adding the pickling solution in the second step into the meat emulsion, and keeping the concentration of staphylococcus HZ01 in the meat emulsion to be 8.0x10 7 log 10 CFU/g～9.9×10 8 log 10 When CFU/g is carried out, the meat emulsion is salted at a low temperature; the mass ratio of fat to lean meat in the meat emulsion is 2:8; the curing temperature in the step (III) is 4 ℃; the pickling time in the step (III) is 48-72 hours;
fourthly, after curing is finished, the minced meat is sequentially subjected to sausage, and the length and the diameter of the sausage are not required; exhausting, baking and fermenting to obtain the dry-cured fermented meat product; the baking temperature in the step (four) is 52-54 ℃; the baking time in the step (IV) is 48-72 hours; the fermentation temperature in the step (four) is 20-30 ℃; the fermentation time in the step (four) is 1 to 2 weeks; the tolerance pH of the staphylococcus HZ01 during fermentation is simulated to be 4-8; the tolerance nitrite quantity of the staphylococcus HZ01 during the fermentation process is simulated to be 0-150 mg/kg; the salt tolerance of the staphylococcus HZ01 in the fermentation process is 3-12%; the tolerance temperature of the staphylococcus HZ01 during the fermentation process is simulated to be 20-35 ℃.
Physicochemical properties of the simulated staphylococcal HZ01 inoculated fermented meat product are shown in table 1:
table 1: physicochemical properties of a fermented meat product mimicking staphylococcal inoculation
| 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 |
| Staphylococcal 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 can be reduced.
The volatile compound compositions of the different treated fermented meat products are shown in table 2:
table 2: volatile compound composition (AU. 10) of differently treated fermented meat products 5 )
From Table 2, it can be seen that mimicking staphylococcal HZ01 can significantly improve esters, aldehydes and acids in fermented meat products, with the most significant improvements in vinyl acetate, methyl butyrate, methyl caproate, n-hexanal, acetic acid and isovaleric acid, and can significantly improve the quality of the fermented meat products.
Sequence listing
<110> academy of agricultural sciences in Zhejiang province
<120> A staphylococcus mimicking 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 (8)
1. A strain mimicking staphylococcal bacterium (Staphylococcus simulans) HZ01, characterized by: the preservation number of the staphylococcus mimicus HZ01 is GDMCC NO.62410; the preservation unit of the staphylococcus mimicus HZ01 is the collection center of the microorganism strain in Guangdong province; the preservation name of the staphylococcus mimicus HZ01 is Staphylococcus simulans HZ01; the shelf life of the staphylococcus mimicus HZ01 is 2022, 4 and 24 days.
2. The staphylococcus HZ01 bacterium agent is characterized in that: the staphylococcus mimicus HZ01 bacterial agent is prepared by adopting staphylococcus mimicus HZ01 in the method of claim 1;
the preparation of the staphylococcus mimicus HZ01 microbial inoculum comprises the following steps,
(A) Inoculating staphylococcus mimicus HZ01 into NB culture medium for culturing to obtain bacterial liquid;
(B) After the bacterial liquid prepared in the step (A) has the concentration of the simulated staphylococcus HZ01 of 1.0x10 8 log 10 CFU/mL～9.9×10 9 log 10 Centrifuging at CFU/mL;
(C) Adding sterilized skimmed milk for resuspension after centrifugation is completed;
(D) Freeze drying after the resuspension is completed to obtain the product with concentration of 1.0X10 10 log 10 CFU/g～9.9×10 11 log 10 The CFU/g powder mimics a staphylococcal HZ01 bacterial agent.
3. Use of staphylococcal mimetic HZ01 according to claim 1 for degrading sarcoplasmic proteins.
4. Use of staphylococcus mimicking HZ01 in degrading sarcoplasmic proteins according to claim 3, characterized in that: the detection of the degradation result of the staphylococcus HZ01 on the sarcoplasmic protein comprises the following steps of;
(S01) extracting sarcoplasmic proteins, and measuring the concentration of the sarcoplasmic proteins by using a Lowry protein concentration kit;
(S02) taking sarcoplasmic protein, glucose and a staphylococcus mimicking HZ01 bacterial solution in the step (S01) for co-incubation;
(S03) centrifuging the bacterial liquid after incubation is completed;
(S04) mixing a 2 XSDS loading buffer with the supernatant after centrifugation in the step (S03), and carrying out water bath;
(S05) taking 10% -12% of Bio-Rad preformed mucin and standard protein, and respectively performing the sample in the step (S04) on 10% -12% of Bio-Rad preformed mucin and standard protein;
(S06) carrying out electrophoresis after loading;
(S07) dyeing with coomassie brilliant blue R-250 after electrophoresis is completed, and obtaining a degradation result of the staphylococcus HZ 01-simulated sarcoplasmic protein through a dyeing result;
the extraction of sarcoplasmic proteins comprises the steps of,
(S011) mixing pork with PB buffer solution and homogenizing;
(S012) filtering and sterilizing the supernatant fluid after the homogenization in the step (S011) through a filter membrane to obtain the sarcoplasmic protein.
5. Use of staphylococcus mimicus HZ01 according to claim 1 for reducing nitrate to nitrite.
6. Use of staphylococcus mimicking HZ01 in reducing nitrate to nitrite according to claim 5, wherein: the detection of the concentration of nitrite comprises the steps of,
preparing KNO with proper concentration 3 /NaNO 3 NB medium of (b);
(ii) inoculating a staphylococcus mimicking HZ01 broth into the NB medium of step (i) for cultivation;
(iii) intermittently centrifuging the fermentation broth during the culturing process and collecting the supernatant;
(iv) detecting the nitrite content in the supernatant of step (iii);
the step (iv) of detecting the nitrite content in the supernatant comprises the steps of,
(iv) a proper amount of supernatant is taken and placed in a colorimetric tube;
(iv 02) adding a proper amount of sulfanilic acid solution into the colorimetric tube in the step (iv 01), uniformly mixing and standing;
(iv 03) adding a proper amount of naphthalene ethylenediamine hydrochloride solution into the solution obtained after standing in the step (iv 02);
(iv) 04) adding water to the solution obtained in the step (iv 03) until the color comparison tube is scaled, uniformly mixing and standing;
(iv 05) measuring the absorbance of the solution in step (iv 04).
7. Use of staphylococcus mimicus HZ01 according to claim 1 in dry marinated fermented meat products.
8. Use of staphylococcus mimicking HZ01 in dry salted fermented meat products according to claim 7, characterized in that: the preparation of the dry salted fermented meat product comprises the following steps,
firstly, taking concentrated solution or powder of the simulated staphylococcus HZ01, and diluting with edible water;
Dissolving proper amount of salt, white sugar and sodium glutamate in the solution obtained in the step (one) to prepare a pickling solution;
thirdly, taking meat emulsion, adding the pickling solution in the second step into the meat emulsion, and keeping the concentration of staphylococcus HZ01 in the meat emulsion to be 1.0x10 7 log 10 CFU/g～9.9×10 8 log 10 When CFU/g is carried out, the meat emulsion is salted at a low temperature;
and fourthly, after the pickling is finished, sequentially performing sausage filling, air exhausting, baking and fermentation on the minced meat to obtain a finished product of the dry-pickled fermented meat product.
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| CN202310812671.7A CN116831273A (en) | 2022-06-22 | 2022-06-22 | Application of mimicking staphylococcus HZ01 to substitution of partial nitrite in meat products |
| CN202210712999.7A CN114891701B (en) | 2022-06-22 | 2022-06-22 | Staphylococcus HZ 01-mimicking bacterium agent and application thereof |
| CN202310812660.9A CN116904356A (en) | 2022-06-22 | 2022-06-22 | Staphylococcus mimicus HZ01 bacteria agent |
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