Staphylococcus WY58 and application thereof

Abstract

The invention relates to the technical field of microbial fermentation, in particular to staphylococcus WY58 and application thereof. The staphylococcus equorum WY58 is preserved in the Guangdong Microbial Culture Collection Center on April 3, 2025, the preservation number is GDMCC NO: 66092, and the address of the preservation unit is the 5th building, No.59 building, No.100 Courtyard, Xianlie Middle Road, Guangzhou City. The staphylococcus equorum WY58 has been preserved in the Guangdong Microbial Culture Collection Center on April 3, 2025, the preservation number is GDMCC NO: 66092. The staphylococcus does not produce ammonia, viscosity, gas and H2S, does not have amino acid decarboxylase, has catalase and has nitrate reducibility. The invention provides a novel meat product fermentation strain which is used for supplementing a strain library for meat product fermentation, and the meat product fermented by the strain has unique flavor.

Description

Technical Field

[0001] The present invention belongs to the field of microbial technology, specifically to a strain of Staphylococcus equorum WY58 and an application thereof, and more specifically to a strain of Staphylococcus equorum WY58 that can be used for fermenting meat products and an application thereof. Background Art

[0002] Fermented meat products utilize microbial or enzyme fermentation, either under natural or controlled conditions, to induce a series of biochemical and physical changes in the raw meat, ultimately resulting in products with unique flavor, color, texture, and a long shelf life. During this process, microorganisms break down and convert large organic molecules in meat into simpler smaller molecules. For example, Staphylococcus aureus metabolizes large molecules such as carbohydrates, proteins, and fats, forming small acids and aldehydes, amino acids, peptides, and fatty acids, which contribute to the flavor of meat products. Currently, the microorganisms commonly used in meat fermentation are Staphylococcus aureus and lactic acid bacteria.

[0003] Staphylococci are Gram-positive, catalase-positive bacteria. Their microscopic cells are spherical or oval, often arranged in clusters that resemble grapevines. Staphylococci are divided into two main groups based on whether they produce coagulase: coagulase-positive staphylococci (CPS) and coagulase-negative staphylococci (CNS). The former are generally pathogenic and produce enterotoxins, while the latter are generally nonpathogenic and do not produce enterotoxins.

[0004] Sausages, cold rabbit, and fermented meat products have recently become popular with consumers for their unique flavor. Existing methods also include fermented pork. For example, CN202011195550.5 describes a method for processing meat products using high-temperature fermentation technology. This method not only requires high temperatures, but also requires the addition of a fermentation agent and complex operations. Therefore, further research and development is needed to achieve better flavor effects. Summary of the Invention

[0005] The present invention aims to overcome the deficiencies in the prior art and provide a Staphylococcus aureus WY58 strain having protease activity and lipase activity, while not producing ammonia, stickiness, gas, or H2S, and having no amino acid decarboxylase, but having catalase and nitrate reducing properties. The strain can be used in sausages, cold rabbits, and other fermented meat products to enhance the fermentation effect and obtain a unique fermented flavor.

[0006] In order to achieve the above object of the invention, the specific technical solution of the present invention is:

[0007] A strain of Staphylococcus equorum WY58 was deposited in the Guangdong Provincial Microbiological Culture Collection on April 3, 2025, with the deposit number GDMCC NO: 66092. The depository address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0008] Furthermore, the Staphylococcus equorum WY58 has at least one of the following characteristics:

[0009] It has protease activity and lipase activity; it does not produce ammonia, viscosity, gas, or H2S, does not have amino acid decarboxylase, has catalase, and has nitrate reducing properties.

[0010] Furthermore, the protease activity of the Staphylococcus equorum WY58 strain is 10.51±0.64 U / mL; the lipase activity is 7.14±0.11 U / mL; the strain has good tolerance when the NaCl content is 8%; and the strain has the best acid resistance when the pH is 4.5-6.5.

[0011] The present invention also protects a product containing the Staphylococcus equorum WY58, so the product includes but is not limited to a Staphylococcus equorum WY58 bacterial suspension and a Staphylococcus equorum WY58 bacterial agent.

[0012] The present invention also protects the use of the Staphylococcus equorum WY58 in fermented meat products.

[0013] Furthermore, in the application, the meat products include but are not limited to sausages and cold-eaten rabbit.

[0014] The present invention also protects a sausage, wherein the Staphylococcus equorum WY58 bacterial suspension is used during fermentation during the preparation of the sausage.

[0015] Furthermore, in the sausage, the preparation method of the Staphylococcus equorum WY58 bacterial suspension is:

[0016] Staphylococcus equorum WY58 was activated, cultured, and fermented to obtain Staphylococcus equorum WY58 fermentation liquid; the fermentation liquid was then centrifuged at 6000 rpm and 4° C. for 10 minutes, the supernatant was removed, and the liquid was washed three times with physiological saline and finally resuspended with an equal volume of physiological saline.

[0017] Furthermore, the initial strain concentration of the Staphylococcus equorum WY58 fermentation broth was 2×10 8 CFU / mL to 5×10 8 CFU / mL.

[0018] The present invention also protects a method for preparing sausages, comprising the following steps:

[0019] 1) Pretreatment: Cut the lean meat into strips and mince the fat meat; centrifuge the Staphylococcus aureus WY58 fermentation broth, remove the supernatant, wash three times with saline, and finally resuspend in an equal volume of saline and pour into a beaker;

[0020] 2) Fermentation: Weigh the required seasonings for the sausages in proportion, mix the lean meat and fat meat pretreated in step 1) with the seasonings, then add the Staphylococcus equorum WY58 suspension obtained in step 1), stir evenly, and fill and form;

[0021] 3) Air drying: The filled sausages are air dried at 15° C., 80% RH for 5 days to obtain the sausages. Other parts of the sausage preparation method not described in the specification can be made using existing technologies.

[0022] Compared with the existing technology, the beneficial effects of the present invention are:

[0023] (1) A new Staphylococcus aureus strain is provided, which has many fermentation-related characteristics and can be well used in the field of meat processing (including pork products, rabbit meat products, beef products, etc.).

[0024] (2) This strain has a good fermentation effect when used for meat product fermentation. Meat products fermented by WY58 (Staphylococcusequorum) have a unique fermentation flavor.

[0025] (3) The fermented meat product of the present invention is obtained by liquid fermentation and has high efficiency.

[0026] (4) The method for preparing the fermented meat product provided is simple and easy, the process is convenient and quick, and the product has a unique flavor. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only represent some embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0028] Figure 1 This is a diagram of a rabbit plasma coagulase experiment during the strain screening process of the present invention;

[0029] Figure 2 The left picture shows the colony morphology of Staphylococcus equorum WY58, and the right picture shows the Gram staining microscopic morphology.

[0030] Figure 3 is the growth curve of Staphylococcus equorum strain WY58;

[0031] Figure 4 Schematic diagram of the protease activity of different Staphylococci in Example 1;

[0032] Figure 5 This is a schematic diagram of the lipase activity of different Staphylococci in Example 1;

[0033] Figure 6 Schematic diagram of the salt tolerance of different Staphylococci in Example 1;

[0034] Figure 7 Schematic diagram of the acid resistance of different Staphylococci in Example 1;

[0035] Figure 8 This is a hemolysis test of Staphylococcus equorum strain WY58; (a) is the positive control Staphylococcus aureus CICC 10384, (b) is the negative control Staphylococcus xylosus CICC21445, and (c) is the WY58 strain;

[0036] Figure 9 This is a thermostable nuclease assay for Staphylococcus equorum strain WY58;

[0037] Figure 10 This is the phylogenetic tree of Staphylococcus equorum strain WY58. DETAILED DESCRIPTION

[0038] In order to make the purpose, technical solutions and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with examples. It should be understood that the specific examples described herein are merely used to explain the present invention and are not intended to limit the present invention. In addition, it should be understood that after reading the content of the present invention, those skilled in the art can make various changes and modifications to the present invention, but these equivalent forms also fall within the scope defined by the appended claims of the application.

[0039] Example 1:

[0040] Screening of Staphylococcus equorum strain WY58:

[0041] Enrichment of Staphylococci:

[0042] Take 25 g of air-dried sausage (collected in Yibin City, Sichuan Province) from a sterile sample bag, chop (mash), and homogenize in 225 mL of sterile saline. Pipette 25 mL of the homogenate into 225 mL of MSA liquid culture medium and place it in an incubator at 37°C for enrichment culture for 24 h.

[0043] Screening for Staphylococci:

[0044] The enriched culture fluid was diluted with sterile physiological saline and 10 -4 , 10 -5 , 10 -6 Spread 200 μL of three appropriate dilutions onto a solid plate containing phenol red (MSA). Incubate the plate in a 37°C incubator for 48 hours. Identify single, red, Gram-positive, and coagulase-negative colonies from the MSA. Suspected Staphylococci identified in the initial screening were tested for ammonia production, stickiness, gas production, amino acid decarboxylation, catalase, H2S production, and nitrate reduction.

[0045] Sixty-four phenol red-reactive strains were isolated from MSA solid plates, of which 33 were Gram-positive, non-spore-forming, and 15 were coagulase-negative. Staphylococci screened for meat fermentation require non-ammonia production, non-stick production, non-gas production, non-H2S production, lack of amino acid decarboxylase, presence of catalase, and nitrate reduction. Six strains were identified that met these fermentation characteristics and exhibited both protease and lipase activity. These six strains were selected for subsequent experiments (numbered RA1, ST1, ST2, WY22, WY41, and WY58).

[0046] Growth curve determination:

[0047] The preserved Staphylococcus equorum WY58 strain (referred to as strain WY58) was activated to obtain seed solution, which was inoculated into 100 mL beef extract peptone medium at a ratio of 1% (v / v). After static culture at 37°C for 24 h, the OD of each strain solution was measured using a blank beef extract peptone medium as a control. 600 , the results are as follows Figure 3 As shown in the figure, strain WY58 has been in the stable phase since 14 hours.

[0048] Protease activity assay:

[0049] The protease activity of the rescreened strains was determined by referring to the Folin method in GB / T23257.1-2023 "Quality Requirements for Enzyme Preparations Part 1: Protease Preparations". Figure 4 As shown, protease activity and HE values are positively correlated, suggesting that staphylococcal protein degradation may rely entirely on proteases. Combining protease activity and HE values in the figure reveals that strain WY58 has the highest protein degradation capacity, with an enzyme activity of 10.51 ± 0.64 U / mL. Therefore, based on protease activity, WY58 is a suitable strain for meat fermentation.

[0050] Lipase activity assay:

[0051] The lipase activity of the rescreened strains was determined according to the sodium hydroxide titration method in GB / T 2353.5-2009 "Lipase Preparations". The lipase activity and HE value of the 6 strains of Staphylococcus aureus screened were determined. The results are as follows: Figure 5 As shown. ST2 had the highest lipase activity (8.13±0.17U / mL), followed by WY58 (7.14±0.11U / mL). Figure 4 and Figure 5 It can be found that protease affects the activity of lipase, and studies have shown that protease inhibits lipase and reduces the formation of malondialdehyde. In summary, WY58 is a strain with relatively high protease activity and retains good lipase activity.

[0052] Salt tolerance determination:

[0053] The seed solution of the screened Staphylococcus aureus was prepared and inoculated into beef extract peptone containing 0%, 2%, 4%, 6%, and 8% NaCl at a ratio of 1%. The solution was cultured at 37°C for 24 hours and then the OD value at a wavelength of 600nm was measured using an ultraviolet spectrophotometer. The results are as follows. Figure 6As shown in the figure, when the NaCl content is in the range of 0-8%, the growth ability of all bacteria shows a downward trend, but the decline of strains ST1 and WY58 is smaller in this range, indicating that they may have better tolerance to salt and are suitable as starter cultures for meat products.

[0054] Acid resistance determination:

[0055] The seed solution of the screened Staphylococcus aureus was prepared and inoculated into beef extract peptone medium with pH values of 4.5, 5.0, 5.5, 6.0 and 6.5 at a ratio of 1%, cultured at 37°C for 24 hours, and then the OD values at a wavelength of 600nm were measured using an ultraviolet spectrophotometer. The results are as follows Figure 7 As shown in the figure, the growth ability of all bacteria in the pH range of 4.5 to 6.5 showed a trend of first increasing and then decreasing. The growth ability was the best at pH 6, among which strains ST1 and WY58 were more prominent. Meat product fermentation is an acidic environment. The previous experiment of fermenting pork showed that the fermentation pH would not be lower than 4.5 during the whole fermentation process. Figure 6 , WY58 is the most suitable starter culture.

[0056] Safety Assessment:

[0057] Hemolysis test

[0058] The activated strain was streaked onto blood agar medium, with Staphylococcus aureus CICC10384 as a positive control and Staphylococcus xylosus CICC 21445 as a negative control. After 48 hours of culture, the experimental phenomena were observed and recorded. Figure 8 As shown, a clear zone was produced around the positive control Staphylococcus aureus CICC 10384, while no clear zone was produced around the negative control Staphylococcus xylosus CICC 21445. The changes around the WY58 strain were consistent with those of the negative control, indicating that WY58 was not hemolytic.

[0059] Thermostable nuclease assay

[0060] The activated strain was inoculated onto toluidine blue-DNase agar medium, with Staphylococcus aureus CICC 10384 as a positive control and Staphylococcus xylosus CICC 21445 as a negative control. After culturing for 48 hours, the experimental phenomena were observed and recorded. Figure 9As shown, the positive control Staphylococcus aureus CICC 10384 has a light rose-colored halo around it, the negative control Staphylococcus xylosus CICC 21445 has no obvious color change around it, and the change around the WY58 strain is consistent with the negative control, so WY58 is negative for thermostable nuclease.

[0061] Virulence gene detection

[0062] After culturing WY58, cells were collected and whole-genome sequencing was performed. The VFDB database (https: / / www.mgc.ac.cn / VFs / ) was then searched for genes from the CNS strain. The results were compared with the following virulence genes: enterotoxin genes (sea, seb, sec, sed, see, seg, seh, sei, sej, sek, sel, sem, sen, seo, sep, seq, ser, seu); drug resistance genes (blaZ, mecA); leukocytolytic genes (pvl); abscisin genes (eta, etb, etd); biofilm genes (icaA, icaD); hemolysin genes (hla, hlb, hld); and decarboxylase genes (tdc, hdc, odc, ldc).

[0063] The whole genome of the WY58 strain was sequenced by Shanghai Jie Li Biotechnology Co., Ltd., and the relevant virulence genes were compared in the VFDB database. The comparison found that the gene fragments of the WY58 strain did not have enterotoxin genes, drug-resistant genes, leukocyte-killing genes, shedding genes, biofilm genes, hemolysin genes, and decarboxylase genes.

[0064] Bacterial morphological characteristics identification

[0065] The selected strains were streaked onto beef jerky peptone solid medium and cultured at 37°C for 48 hours, and then their colony morphology and Gram staining characteristics were observed. Figure 2 As shown, the colonies of strain WY58 are small and round, white, flat, moist, with smooth surface and neat edges; and the microscopic morphology under 100x oil microscope is Gram-positive, spherical, arranged singly, in pairs or in multiple grape-like shapes, and without spores.

[0066] Physiological and biochemical identification

[0067] With reference to Bergey's Manual of Bacterial Identification and Manual of Identification of Common Bacterial Systems, physiological and biochemical tests such as muscarinic fermentation, mannitol fermentation, mannose fermentation, fructose fermentation, xylose fermentation, lactose fermentation, maltose fermentation, sucrose fermentation, urea test, and nitrate reduction test were performed on the screened bacteria. The specific results are shown in Table 1.

[0068] Table 1 Physiological and biochemical identification results of Staphylococcus aureus WY58

[0069] Experimental Project Experimental results Mushroom Candy d Mannitol - Mannose + fructose + lactose + Xylose - maltose + sucrose + urea + VP reaction +

[0070] Note: "+" indicates more than 90% positive, "-" indicates more than 90% negative, and "d" indicates 11-89% positive.

[0071] Molecular biology identification:

[0072] After the screened strains were cultured in beef extract peptone liquid medium for 24 hours, 50 mL of culture medium was taken and centrifuged at 4°C and 6000 r / min for 10 minutes to collect the bacteria. The total DNA extraction, 16S rDNA amplification and sequencing of the collected bacteria were completed by Shanghai Jie Li Biotechnology Co., Ltd. After proofreading the 16S rRNA splicing sequence obtained by sequencing, BLAST analysis was performed with the GenBank database of NCBI. The species relationship of the strains was determined based on sequence homology, and the neighbor-joining (NJ) method in MEGA 11 software was used to draw a phylogenetic tree. The results showed that the WY58 strain had a high similarity with most species of Staphylococcus. Some of the bacteria were selected and Macrococcus lamaes R 16089 of the genus Macrococcus was used as an exogenous bacterium. The phylogenetic tree constructed with MEGA11 was as follows: Figure 10 The WY58 strain is in the same clade as Staphylococcus equorum PA231 (accession number: NR_027520.1) with a confidence level of 99%. Therefore, the WY58 strain is identified as Staphylococcus equorum. The 16S rRNA splicing sequence of the strain is shown in SEQ ID NO: 1:

[0073] TGCGGCGTGCTATACATGCAAGTCGAGCGAACGGATAAGGAGCTTGCTCCTTTGAAGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTACCTATAAGACTGGAATAACTTCGGGAAACCGGAGCTAATGCCGGATAACATTTGGAACCGCATGGTTCTAAAGTAAAAGATGGTTTTGCTATCACTTATAGATGGACCCGCGCCGTATTAGCTAGTTGGTAAGGTAACGGCTTACCAAGGCAACGATACGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAACTCTGTTATTAGGGAAGAACAAATGCGTAAGTAACTGTGCGCATCTTGACGGTACCTAATCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGAATTATTGGGCGTAAAGCGCGCGTAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCACGGCTCAACCGTGGAGGGTCATTGGAAACTGGGAAACTTGAGTACAGAAGAGGAAAGTGGAATTCCATGTGTAGCGGTGAAATGCGCAGAGATATGGAGGAACACCAGTGGCGAAGGCGACTTTCTGGTCTGTAACTGACGCTGATGTGCGAAAGCGTGGGGATCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGACCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAAATCTTGACATCCTTTGAAAACTCTAGAGATAGAGCCTTCCCCTTCGGGGGACAAAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCTTAAACTTAGTTGCCAGCATTTAGTTGGGCACTCTAGGTTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGATTTGGGCT ACACACGTGCTACAATGGACAATACAAAGGGCAGCTAAACCGCGAGGTCATGCAAATCCCATAAAGTTGTTCTCAGTTCGGATTGTAGTCTGCAACTCGACTACATGAAGCTGGAATCGCTAGTAA TCGTAGATCAGCATGCTACGGTGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGCCGGTGGAGTAACCATTTATGGAGCTAGCCTCGAAGTGAA.

[0074] Strain preservation

[0075] The screened Staphylococcus equorum WY58 was deposited in the Guangdong Provincial Microbial Culture Collection on April 3, 2025, with the deposit number GDMCC NO: 66092. The depository address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0076] Example 2

[0077] This embodiment discloses a sausage processing process, comprising the following steps:

[0078] Preparation of fermentation liquid → Preparation of bacterial powder → Pretreatment of raw meat → Adding seasoning → Inoculation → Mixing → Sausage filling → Fermentation and air drying → Storage

[0079] S1. Preparation of fermentation broth

[0080] (1) Staphylococcus equi WY58 expansion medium: 1000 mL of water, 5.0 g of beef extract, 10.0 g of peptone, 5.0 g of yeast extract, 10.0 g of glucose, 5.0 g of lactose, and 5.0 g of sodium chloride. After dissolving all ingredients in distilled water, adjust the pH to 6.5 and dispense into ten 250 mL conical flasks, 100 mL per flask. Add glass beads and sterilize at 121°C for 20 min. Remove from the autoclave and cool until ready for use.

[0081] (2) Strain expansion culture On a sterile clean bench, use an inoculation loop to make two loops of Staphylococcus aureus WY58 on the slant culture medium into a conical flask (with glass beads) containing 100 mL of fermentation liquid. Then, the inoculated culture liquid was placed in a 37°C constant temperature incubator and cultured at 180 rpm for 24 h to obtain the fermentation stock solution. The initial strain concentration was 2×10 8 CFU / mL.

[0082] (3) Preparation of fermentation liquid: On a sterile clean bench, the fermentation stock liquid was inoculated into the expanded culture of Staphylococcus equi WY58 according to a certain inoculation ratio. The inoculated culture was then placed in a 37°C constant temperature incubator and cultured at 180 rpm for 24 h to obtain the fermentation liquid, i.e., Staphylococcus equi fermentation liquid.

[0083] S2.Production process

[0084] (1) Pre-processing of raw meat: cut lean meat into strips and mince fat (the ratio of lean meat to fat is 8:2);

[0085] (2) Adding seasonings: Add spices and seasonings (2% liquor, 2.3% salt, 1.3% chili pepper, 0.5% Sichuan peppercorns, 1.2% rock sugar powder, 0.4% black pepper powder, 0.1% ginger powder);

[0086] (3) Inoculation: 50 mL of Staphylococcus equi WY58 suspension (centrifuged at 6000 rpm, 4°C for 10 min) was removed from the supernatant, washed three times with normal saline, and finally resuspended with an equal volume of normal saline and poured into a beaker;

[0087] (4) Mixing: Mix the resuspended bacterial suspension with various seasonings and stir evenly;

[0088] (5) Sausage enema: Use a sausage enema to fill and shape the sausage;

[0089] (6) Fermentation and air drying: The filled sausages are air-dried at 15°C for 5 days and at a humidity of 80% rh. At the end of air drying, the raw meat should lose more than 30% of its weight and the moisture content should be less than 60%;

[0090] (7) Storage: Store at 4°C.

[0091] Example 3 Flavor Determination of Sausage

[0092] Headspace solid phase microextraction (HS-SPME)

[0093] Weigh 5 g of sample and place it in a 20 mL headspace vial. Add 3 μL of internal standard solution (2% n-amyl acetate) and seal the vial for subsequent volatile substance determination. HS-SPME conditions: Extraction head 50 / 30 μm DVB

[0094] / CAR / PDMS; constant temperature equilibration at 80°C for 10 min, extraction with an extraction head for 30 min (80°C), and then desorption of the extraction head at the injection port (230°C, 3 min).

[0095] Gas chromatography-mass spectrometry (GC-MS)

[0096] GC conditions: chromatographic column TG-WAXMSB (30 m × 0.25 mm, 0.25 μm); temperature program: inlet temperature 230°C, splitless mode, initial column temperature 40°C, hold for 3 min, increase to 210°C at 5°C / min, hold for 5 min; carrier gas (He) (purity >99.999%), flow rate 1.0 mL / min.

[0097] Mass spectrometry conditions: electron bombardment ion source; electron energy 70 eV; transfer line temperature 230 °C; ion source temperature 250 °C;

[0098] Interface temperature 210°C; activation voltage 1.2 kV; full scan mode, mass scan range m / z 40-500; scan time 2 s;

[0099] EI ion source. Qualitative identification was performed by comparing the mass spectrum database NIST 2011, with a match greater than 700 as the basis for identification. Semi-quantitative analysis was performed using the peak area and internal standard concentration of the obtained compound as the basis for semi-quantitative analysis.

[0100] The types, quantities and contents of volatile flavor compounds in sausage products without WY58 strain inoculation (CK group), sausage products fermented with WY58 strain (T1 group) and sausage products fermented with commercial strains (T2) are shown in Table 2.

[0101] Table 2 Relative content of volatile flavor substances in sausages Unit: μg / g

[0102]

[0103]

[0104] Note: “-” means not detected.

[0105] As shown in Table 2 , 30, 30, and 23 volatile flavor compounds were detected in the sausages of the T1, T2, and CK treatment groups, respectively, including esters, ketones, aldehydes, alcohols, acids, and other hydrocarbons.

[0106] Aldehydes: Aldehydes are a typical flavor component of fat degradation. Due to their low threshold, they contribute more significantly to the overall flavor. They primarily originate from the oxidation of unsaturated fatty acids such as oleic and linoleic acids and are the most important flavor compounds in sausage products. The total aldehyde content in the T1 group was higher than in the T2 and CK groups, with higher levels of 2,4-decadienal, 2,4-decanedial, hexanal, nonanal, and 4-anisaldehyde. Nonanal contributes more to the flavor of meat, producing aromas of carnation, citrus, and laurel. The higher nonanal content in the T1 group compared to the CK group may be due to the addition of a bacterial starter.

[0107] Esters: Ester compounds usually have a floral and fruity aroma, and the odor detection threshold is very low. They can make people feel pleasant and contribute greatly to the flavor of sausages. 13 ester substances were detected in the CK group, while 14 ester substances were detected in the T1 and T2 groups. This may be because the pork in the sausage is rich in palmitic acid and oleic acid during the processing of the sausage, which mainly exists in the form of triglycerides in the adipose tissue. During the processing of sausages (such as pickling, fermentation, and air-drying), fat is gradually hydrolyzed into free fatty acids under lipase or acidic environment, and esterification reaction occurs with the added liquor to produce the corresponding ethyl esters. Ethyl oleate has a floral, fruity and creamy aroma, ethyl palmitate has a faint waxy, fruity and creamy aroma, and octyl acetate has a faint floral, fruity and green aroma.

[0108] Alcohols: The threshold of alcohols is higher than that of aldehydes and has little effect on the flavor of meat products. However, alcohols are precursors of aldehyde and ketone compounds and mainly play an additive modification role in flavor.

[0109] Phenols: Phenolic substances (such as rosmarinic acid and eugenol) can effectively scavenge free radicals, slow fat oxidation, and prevent the sausage from developing a rancid taste. The presence of 2-methoxy-3-(2-propenyl)phenol and 3-allylguaiacol in the T1 sausages may be small amounts of phenolic substances produced by Staphylococcus aureus during fermentation.

[0110] Acids: Acids promote the formation of esters, which mainly come from the oxidative degradation of glycerol and phospholipids and the Maillard reaction. Among them, acetic acid is the product of staphylococcal fermentation of sugars, which gives fermented meat products a sour vinegar taste. The large number of staphylococci in the T1 and T2 groups increased the acid production capacity, so the T1 and T2 groups contained acetic acid, while the CK group did not.

[0111] In summary, the comparison of characteristic flavor substances in sausages of CK group, T2 group and T1 group shows that the WY58 strain fermented sausages contributes more to the volatile flavor substances.

[0112] The above-described embodiments merely represent specific implementation methods of the present application. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of protection of the present application. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the technical concept of the present application, and all such variations and improvements fall within the scope of protection of the present application.

[0113] This background section is provided to generally present the context of the invention, and the work of the presently named inventors, the work to the extent described in this background section, and aspects of the description in this section that did not constitute prior art at the time of filing are neither explicitly nor implicitly admitted to be prior art to the present invention.

[0114] The above-described embodiments merely represent specific implementation methods of the present application. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of protection of the present application. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the technical concept of the present application, and all such variations and improvements fall within the scope of protection of the present application.

[0115] This background section is provided to generally present the context of the invention, and the work of the presently named inventors, the work to the extent described in this background section, and aspects of the description in this section that did not constitute prior art at the time of filing are neither explicitly nor implicitly admitted to be prior art to the present invention.

Claims

1. A strain of Staphylococcus equorum WY58 was deposited in the Guangdong Provincial Microbiological Culture Collection on April 3, 2025, with the deposit number GDMCC NO: 66092. The depository address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

2. The Staphylococcus equorum WY58 according to claim 1, characterized in that The Staphylococcus equorum WY58 has at least one of the following characteristics: It has protease activity and lipase activity, and does not produce ammonia, viscosity, gas, or H2S. It does not have amino acid decarboxylase, but has catalase and nitrate reducing properties.

3. The Staphylococcus equorum WY58 according to claim 1, wherein: The protease activity of the strain was 10.51±0.64U / mL; the lipase activity was 7.14±0.11U / mL; the strain had good tolerance when the NaCl content was 8%; and the strain had the best acid resistance when the pH was 4.5-6.

5.

4. A product containing Staphylococcus equorum WY58 according to any one of claims 1 to 3, characterized in that: Therefore, the products include but are not limited to Staphylococcus equorum WY58 bacterial suspension and Staphylococcus equorum WY58 bacterial agent.

5. Use of Staphylococcus equorum WY58 according to any one of claims 1 to 3 in fermented meat products.

6. The use according to claim 4, characterized in that: The meat products include but are not limited to sausages and cold-cooked rabbit.

7. A sausage, characterized by: In the process of preparing sausages, the bacterial suspension containing Staphylococcus equorum WY58 as claimed in claim 4 is used during fermentation.

8. The sausage according to claim 7, characterized in that: The preparation method of the Staphylococcus equorum WY58 bacterial suspension comprises the following steps: activating Staphylococcus equorum, culturing, and fermenting to obtain Staphylococcus equorum WY58 fermentation liquid; centrifuging the fermentation liquid at 6000 rpm and 4° C. for 10 minutes, removing the supernatant, washing three times with physiological saline, and finally resuspending the suspension with an equal volume of physiological saline.

9. The sausage according to claim 8, characterized in that: The initial strain concentration in the Staphylococcus aureus WY58 fermentation broth was 2×10 8 CFU / mL—5×10 8 CFU / mL.

10. A method for preparing a sausage according to any one of claims 7 to 9, characterized in that The following steps are involved: 1) Pretreatment: Cut the lean meat into strips and mince the fat meat; centrifuge the Staphylococcus aureus WY58 fermentation broth, remove the supernatant, wash three times with saline, and finally resuspend in an equal volume of saline and pour into a beaker; 2) Fermentation: Weigh the required seasonings for the sausages in proportion, mix the lean meat and fat meat pretreated in step 1) with the seasonings, then add the Staphylococcus equorum WY58 suspension obtained in step 1), stir evenly, and fill and form; 3) Air drying: air-dry the filled sausages at 15° C., 5 days, and 80% humidity.

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