Staphylococcus equorum p4-2 and use thereof

Abstract

The present application relates to the technical field of microbial inoculant, and particularly relates to equine gastric staphylococcus P4-2 and application thereof. Staphylococcus equorum ) P4-2, which is preserved in China Center for Type Culture Collection and has a preservation number of CCTCC NO: M 20261316. The equine gastric staphylococcus P4-2 can improve the physicochemical properties of fermented sausages: while effectively improving product color, antioxidant stability and biological safety and significantly shortening the production cycle, the characteristic flavor substances are targeted to systematically reshape the flavor fingerprint. The strain can comprehensively overcome the quality bottleneck of the traditional fermentation process, and provides key germplasm resource guarantee for developing high-quality, green and local flavor meat product starter.

Description

Technical Field

[0001] This invention relates to the field of microbial inoculants, and more particularly to Staphylococcus equi P4-2 and its applications. Background Technology

[0002] Fermented meat products (such as ham and sausages) are highly favored by consumers for their unique flavor, color, and texture. Traditional fermented meat products mostly rely on the spontaneous fermentation of microbial communities in the natural environment; however, this method has significant drawbacks, including long production cycles, susceptibility to seasonal climate changes, and unstable product quality. Therefore, artificial inoculation using exogenous microbial starter cultures has become an important technical means to improve the quality and safety of fermented meat products and ensure consistent quality.

[0003] Among various candidate starter cultures, coagulase-negative staphylococci (CNS) have attracted widespread attention due to their positive contributions to product color, flavor, and texture. Among them, *Staphylococcus equi* (…) Staphylococcus equorum It has been proven to be one of the dominant strains in the maturation process of various naturally fermented meat products, and has good environmental adaptability and application potential.

[0004] Chinese invention patent CN114507628B discloses a strain of Staphylococcus equi FS310 isolated from Xuanwei ham in Yunnan Province (accession number CGMCC No. 24492). This strain exhibits high salt, nitrate, and acid tolerance, and displays protease, nitrate reductase, and antioxidant enzyme activities. This patent indicates that applying FS310 to the fermentation of ham, pork sausages, and fish sausages can promote color development in meat products, reduce nitrite residues, inhibit lipid peroxidation, and improve the texture and flavor of meat products.

[0005] However, the Staphylococcus equi FS310 disclosed in CN114507628B still has the following shortcomings in practical applications: First, this strain has limited effect on improving the textural properties of fermented meat products. Although the application of FS310 can increase the product's redness value ( a While the value has improved, the effect on improving core texture indicators such as product hardness, chewiness, and cohesiveness is not significant, making it difficult to meet the higher requirements of modern consumers for the taste of fermented meat products.

[0006] Secondly, the efficacy of this strain in inhibiting lipid and protein oxidation needs further improvement. Although FS310 has certain antioxidant enzyme activity, existing technologies have not provided sufficient experimental data to support its effects on reducing thiobarbituric acid (TBA) values ​​and carbonyl content in fermented meat products, as well as its role in maintaining total sulfhydryl content. The comprehensiveness of its antioxidant system is still insufficient.

[0007] Third, the ability of this strain to directionally regulate the flavor composition of fermented meat products is unclear. CN114507628B mainly describes the overall flavor of the product through sensory evaluation, but lacks systematic analysis of changes in specific volatile flavor components, especially the impact on the formation and accumulation of irritating aldehydes and characteristic nitrogen-containing heterocyclic flavoring substances (such as pyrazine compounds).

[0008] Fourth, the functional characteristics of this strain are relatively simple, and it fails to give full play to the comprehensive potential of Staphylococcus equi as a multifunctional fermentation agent in fermented meat products. It is significantly insufficient in terms of multi-dimensional synergistic improvement of product color, texture, antioxidant stability and flavor quality.

[0009] Therefore, developing a novel Staphylococcus equi strain with more comprehensive functions, more significant texture improvement, stronger antioxidant capacity, and more precise flavor control is of great technical significance and market value for overcoming the application limitations of the FS310 strain in existing technologies (such as CN114507628B) and further improving the overall quality of fermented meat products. Summary of the Invention

[0010] The purpose of this invention is to address the aforementioned shortcomings of existing technologies by proposing a Staphylococcus equi P4-2 strain and its applications. This strain not only effectively improves the physicochemical quality of fermented sausages but also enhances their flavor and sensory properties.

[0011] The first objective of this invention is to provide Staphylococcus equi (equine gastric staphylococcus) Staphylococcus equorum P4-2, deposited on June 15, 2026 at the China Center for Type Culture Collection, located at Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province, with accession number CCTCC NO:M 20261316.

[0012] A second objective of this invention is to provide the application of Staphylococcus equi P4-2 in the preparation of fermented meat products.

[0013] Furthermore, the fermented meat products include fermented sausages.

[0014] Furthermore, at room temperature, strain P4-2 guided nitrogen-containing organic matter to undergo deep condensation transformation into nitrogen-containing heterocyclic compounds.

[0015] Furthermore, nitrogen-containing organic compounds include amino acids.

[0016] Furthermore, nitrogen-containing heterocyclic compounds include 2-vinyl-6-methylpyrazine, 2,5-dimethylpyrazine, and 2,6-diethylpyrazine.

[0017] In the preparation of fermented sausages, the Staphylococcus equi P4-2 strain of this invention is inoculated into the meat matrix as a core fermenting agent. This strain exhibits extremely strong biochemical metabolic activity during fermentation, and its highly active nitrate reductase can significantly enhance the redness of the finished product. a Meanwhile, the extracellular proteases secreted by the organism can effectively disrupt the muscle's three-dimensional water network and accelerate water migration, causing the water activity (Aw) to drop rapidly to a safe level and prompting the texture indicators such as hardness and chewiness to reach the maturity standard ahead of time, thereby significantly shortening the fermentation cycle of traditional processes.

[0018] Furthermore, the *Staphylococcus equi* P4-2 strain of this invention plays a crucial role in enhancing product safety and antioxidant stability. Validated using a sterile fermented meat model, this strain is decarboxylase negative and possesses significant biogenic amine inhibition capabilities, substantially reducing the total accumulation of biogenic amines in the finished product. In the fermentation system, this strain exhibits excellent antioxidant activity, effectively inhibiting lipid and protein oxidative spoilage, significantly reducing thiobarbituric acid (TBA) values ​​and protein carbonyl content, and maintaining a high total sulfhydryl level, thereby reducing the risk of oxidative off-flavors at the source.

[0019] In terms of flavor reshaping, the *Staphylococcus equi* P4-2 strain of this invention, through specific proteolytic hydrolysis and catabolism, can significantly downregulate the abundance of aldehydes with pungent odors and (2R,3R)-(-)-2,3-butanediol, and target the consumption of precursors such as serine and lysine to convert them into characteristic volatile components. Furthermore, this strain can overcome the limitations of ambient temperature fermentation conditions and specifically synthesize trace amounts of nitrogen-containing heterocyclic characteristic aroma compounds, represented by 2-vinyl-6-methylpyrazine and 2,5-dimethylpyrazine, endowing the product with a unique "roasted meat" and "nutty" aroma.

[0020] In summary, *Staphylococcus equi* P4-2 can improve the physicochemical properties of fermented sausages: effectively enhancing product color, antioxidant stability, and biosafety while significantly shortening the production cycle, it also targets and enriches characteristic aroma compounds to systematically reshape the flavor fingerprint. This strain comprehensively overcomes the quality bottlenecks of traditional fermentation processes, providing crucial germplasm resource support for developing high-quality, green meat product starter cultures that are well-suited to local flavors. Attached Figure Description

[0021] Figure 1This is a colony morphology diagram of the Staphylococcus equi P4-2 strain of the present invention. Figure 2 A phylogenetic tree constructed for the Staphylococcus equi P4-2 strain of the present invention; Figure 3 The effect of Staphylococcus equi P4-2 fermentation on the textural properties of sausage, as described in this invention; Figure 4 The effect of Staphylococcus equi P4-2 fermentation on TBA value; Figure 5 The effect of Staphylococcus equi P4-2 fermentation on carbonyl content; Figure 6 The effect of Staphylococcus equi P4-2 fermentation on total sulfhydryl content. Detailed Implementation

[0022] The following are specific embodiments of the present invention, which are described in conjunction with the accompanying drawings. However, the present invention is not limited to these embodiments.

[0023] Example 1: Isolation, purification, culture and identification of strains 1. Sample collection and bacterial isolation and purification Sausages collected from farmers in Kaizhou District, Chongqing, were aseptically processed by removing the casings and crushing them. 25 g of the crushed sausages were placed in an Erlenmeyer flask pre-filled with 225 mL of sterile physiological saline and thoroughly mixed using a shaker to prepare a dilution of 10. -1 A meat paste suspension. 100 μL of the suspension was serially diluted in 900 μL of physiological saline to obtain 10... -3 ~ 10 -6 Dilute the suspension. Spread 100 μL of the suspension onto MSA medium and incubate at 37℃ for 48 h to obtain the purified strain *Staphylococcus equi* P4-2. Meanwhile, the purified strain was stored in glycerol and lyophilized in the microbial resource bank of the School of Food Science and Biology, Jingchu University of Technology for future use.

[0024] 2 Experimental Methods 2.1 Morphological identification Staphylococcus equi P4-2 was stained according to the instructions of the Gram staining kit, and then its morphology was observed under a binocular microscope at 100x oil immersion.

[0025] 2.2 PCR amplification of 16S rDNA sequence DNA was extracted from the bacterial strain using a bacterial genomic DNA extraction kit. PCR amplification was performed in a 25 μL reaction system, and the results were detected by agarose gel electrophoresis. Qualified samples were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing, and the sequencing results were analyzed for homology using the BLAST program in NCBI.

[0026] 2.3 Construction of Phylogenetic Tree Staphylococci with greater than 98% homology to the obtained Staphylococcus 16S rDNA gene sequence were selected from GeneBank as reference sequences, and then the Neighbor-Joining method in MEGA12.0 was used to construct the phylogenetic tree of the obtained strains.

[0027] 3 Results and Analysis 3.1 Strain Identification: Morphological Identification Figure 1 Observation shows that the colony diameter is between 0.5-2 mm, the front is round, the side is convex, the edges are neat, the color is milky white, opaque, and the surface is moist, smooth and glossy. Figure 1 ).

[0028] 3.2 16S rDNA sequence analysis of the strain The 16S rDNA sequence is shown in SEQ ID No:1, and the results show that the strain is Staphylococcus equi.

[0029] 3.3 Results of constructing the phylogenetic tree of the strains Strain phylogenetic tree such as Figure 2 As shown, this strain forms independent subclusters within its branches, suggesting that it possesses unique genetic characteristics that distinguish it from other strains of the same species.

[0030] Example 2: Fermentation enzyme activity test, safety test and pre-test of fermentation capacity of Staphylococcus equi P4-2 1. Fermentation enzyme activity test 1.1 Protease activity assay Staphylococcus equi P4-2 strain, activated on tryptic soy broth (TSB), was resuspended in 1×PBS buffer (pH 7.4 ± 0.1) to a concentration of approximately 10 log CFU / mL. Then, 10 μL of the bacterial suspension was inoculated onto tryptic soy agar (TSA, supplemented with 10% skim milk from the same genus) containing circular blank test strips (6 mm in diameter). After incubation at 37°C for 48 h, the diameter of the transparent zone around the test strip was measured.

[0031] 1.2 Lipase Activity Test Adjust the bacterial concentration as described in section 1.1, and inoculate 10 μL of the bacterial suspension onto lipase medium containing circular blank test strips (6 mm in diameter). (Add 2.5 mL of polyvinyl alcohol-ol-ol emulsion and 1 mL of rhodamine B solution (0.001% wt / vol) to every 100 mL of sterile TSA medium cooled to 55°C, mix well, and quickly pour onto sterile Petri dishes, allowing to solidify and dry.) Incubate at 37°C for 48–72 h. Observe under UV light whether an orange fluorescent ring appears around the bacterial colonies on the test strips, and determine the lipase activity of the Staphylococcus strain based on the intensity of the orange fluorescence and the size of the ring.

[0032] 1.3 Nitrate reductase activity test Adjust the bacterial suspension concentration as described in section 1.1, and inoculate 10 μL of the bacterial suspension onto nitrate-reducing agar medium containing circular blank test paper discs (6 mm in diameter) and incubate at 37°C for 24–48 h. Subsequently, add Griess reagent A and B to the colonies and observe and record whether red appears around the colonies. The intensity of the red color indicates the nitrate reductase activity of the Staphylococcus strain.

[0033] 2. Safety Inspection 2.1 Hemolytic test The activated bacterial strain was streaked onto TSA plates supplemented with 5% sterile defibrinated sheep blood and incubated at 37°C for 24 h. The presence of hemolytic zones around the colonies was observed. Simultaneously, known hemolytic Staphylococcus aureus strains were selected. Staphylococcus aureus (CCTCC AB 91093) was used as a positive control.

[0034] 2.2 Thermoresistant nuclease activity test Thermostable nuclease activity was tested using DNase agar. The activated strain was streaked onto a DNase agar plate and incubated at 37°C for 24 h. Then, 1 mol / L hydrochloric acid was added around the colonies. If the thermostable nuclease was positive, a clear zone would appear around the colony.

[0035] 2.3 Coagulase Activity Test 500 μL of the activated bacterial culture was added to 500 μL of lyophilized rabbit plasma dissolved in physiological saline and incubated at 37°C for 6 h. The plasma was observed for coagulation every 0.5 h; coagulation was considered positive, otherwise negative. Staphylococcus aureus CCTCC AB 91093 was used as a positive control.

[0036] 2.4 Decarboxylase Activity Test The activated strains were inoculated into test tubes containing decarboxylase medium containing L-lysine, L-arginine, L-ornithine, and L-cysteine, respectively. Test tubes without added L-amino acids served as blank controls. The cultures were incubated at 37°C for 4 days, and the color changes of the medium were recorded. Purple was considered positive, and yellow was considered negative.

[0037] 3. Preliminary test of fermentation capacity 3.1 Establishment of an aseptic fermented meat model Fresh pork hind leg and backfat were soaked whole in boiling water for 10 minutes to ensure microbial safety. Then, under aseptic conditions, the surface cooked meat was removed and discarded, and the remaining lean and fat meat was aseptically chopped in a ratio of 8:2. 2.5% NaCl, 0.015% NaNO2, and 0.05% ascorbic acid were added to the mixed chopped meat, and after thorough mixing, it was divided into portions and packaged. Activated Staphylococcus equi P4-2 strain was inoculated into the packaged mixture at a 5% inoculum (approximately 8 log CFU / g). Anaerobic fermentation was carried out at 10°C for 15 days, with samples taken every 3 days.

[0038] 3.2 Determination of biogenic amines The content of biogenic amines in fermented minced meat was determined by liquid chromatography. Sample preparation, extraction, and purification were performed according to GB5009.208-2016, "National Food Safety Standard - Determination of Biogenic Amines in Food". Pre-column derivatization of the sample extract and biogenic amine standard solution was then performed. 1 mL of the extract and standard solution were added to 200 μL of 2mol / L NaOH, 300 μL of saturated NaHCO3, and 2.0 mL of dansyl chloride solution (10 mg / mL, dissolved in acetone) under light-protected conditions. The mixture was reacted at 40℃ for 45 min in the dark, then 100 μL of ammonia was added and allowed to stand at room temperature for 30 min. The volume was then adjusted to 5 mL with acetonitrile. Both the sample solution and standard solution were filtered through a 0.22 μm filter membrane for analysis. Liquid chromatography was performed under the conditions specified in GB5009.208-2016, and a standard curve was plotted.

[0039] 3.3 Determination of polypeptide content Weigh 5 g of fermented minced meat and homogenize it with 25 mL of PBS buffer (pH 7.0) at 10 × 3 s (10000 r / min) on ice. After standing at 4℃ for 2 h, centrifuge for 20 min (4℃, 10000 r / min), filter, and obtain the supernatant. Mix the supernatant with an equal volume of 15% trichloroacetic acid (v / w), let stand at room temperature for 30 min, and centrifuge for 15 min (4200 r / min) to obtain the supernatant for analysis. The peptide concentration in the supernatant was determined using the biuret method. A gradient standard solution was prepared using bovine serum albumin as the standard substance, and the absorbance of the standard solution and sample supernatant was measured at 540 nm. A standard curve was plotted with absorbance on the x-axis and standard solution concentration on the y-axis, and the peptide concentration in the sample was calculated.

[0040] 4 Results and Analysis 4.1 Results of fermentation enzyme activity and safety tests of Staphylococcus equi P4-2 Table 1 Performance evaluation of Staphylococcus aureus P4-2

[0041] Table 1 shows that *Staphylococcus equi* strain P4-2 possesses a good ability to produce extracellular proteases and nitrate reductases. Furthermore, *Staphylococcus equi* strain P4-2 is a coagulase-negative staphylococcal strain lacking decarboxylase activity, hemolysis, and thermostable nuclease-negative properties. This indicates that *Staphylococcus equi* strain P4-2 has the potential to safely ferment sausages.

[0042] 4.2 Results of detection of biogenic amines and peptides in Staphylococcus equi P4-2 Table 2. Changes in total biogenic amine content (μg / g) over time (d)

[0043] Table 3. Changes in polypeptide content (mg / g) over time (d)

[0044] Fermented meat products are rich in protein, which is easily degraded during maturation to produce free amino acids (FAAs). These FAs are then produced and accumulated by decarboxylation by microorganisms or endogenous enzymes. To eliminate the interference of complex environmental microorganisms and accurately assess the actual control ability of Staphylococcus equi P4-2 on FAs, this study inoculated Staphylococcus equi P4-2 into a sterile fermented meat model for dynamic monitoring of fermentation. As shown in Table 2, the total FAs content in the control group showed a trend of first increasing and then decreasing during fermentation, reaching a peak on day 12 (400.90 ± 68.71 μg / g). After inoculation with Staphylococcus equi P4-2, which showed decarboxylase-negative characteristics in the previous qualitative experiment, the total FAs content in this study on day 12 was only 132.58 ± 20.27 μg / g, significantly lower than the peak value of the CK group at the same time (400.90 ± 68.71, p<0.05). This indicates that Staphylococcus equi P4-2 has the potential to reduce BAs in fermented meat products.

[0045] In the maturation process of fermented meat products, protein hydrolysis is a key biochemical reaction that imparts characteristic flavor. Peptides, as important intermediate products in the conversion of proteins to FAAs, show changes in their content reflecting the combined effects of endogenous and microbial enzymes. As shown in Table 3, the peptide content in the control group initially decreased and then slightly increased during fermentation, mainly attributed to the hydrolytic action of endogenous proteases and peptidases in the sterile meat matrix. After inoculation with *Staphylococcus equi* P4-2, the peptide content showed a higher level (38.82 ± 1.37 mg / g) in the later stage of fermentation (day 12), indicating the production of more FAAs and other flavor precursors. This further demonstrates that strain *Staphylococcus equi* P4-2 has the potential to promote flavor development in fermented meat products.

[0046] Example 3: Effects of fermentation inoculated with Staphylococcus equi P4-2 on the physicochemical properties and sensory flavor of sausage 3.1 Preparation of Fermented Sausage The activated second-generation Staphylococcus equi P4-2 bacterial culture was inoculated into TSB medium at a 2% inoculum and cultured overnight at 37°C to obtain fresh bacterial culture. Subsequently, the fresh bacterial culture was centrifuged (4°C, 8000 × g, 4 min) and resuspended in 0.9% physiological saline, with OD600 adjusted to approximately 0.5. Then, lean pork from the hind leg and back fat were shredded (approximately 4 × 4 × 20 mm) and diced (approximately 4 × 4 × 4 mm), respectively. The lean and fat were mixed evenly at a mass ratio of 4:1, and 2.5% salt, 1% sucrose, 0.05% sodium isoascorbate, 0.008% nitrite (by mass of the mixed meat), and 10 mL / kg of white liquor were added and stirred thoroughly. The mixture was marinated in a refrigerator at 0–4°C for 24 h. The cured meat was divided into two groups. Except for the control group, the treatment groups were inoculated with *Staphylococcus equi* P4-2 at a 5% inoculum (approximately 8 Log CFU / g) and thoroughly mixed. The inoculated meat was stuffed into natural pig casings to prepare sausages. Each sausage section weighed approximately 150 g and was hung in an artificial climate incubator at 25℃ and 70-75% relative humidity for 3 days of fermentation, followed by maturation at 15-16℃ and 65-70% relative humidity for 12 days. Each group had 3-4 replicates. Sausage samples were collected on days 0, 3, 6, 9, 12, and 15 and stored at -80℃ for later use.

[0047] 3.2 Determination of sausage color The color of fermented sausage was determined with reference to the research of Zhao Jingru

[81] . The sausage sample was cut into 1 cm thick cylinders, and the brightness value was measured after blank plate correction using an LS170 colorimeter. L ), redness value ( a ) and yellowness value ( b ).

[0048] 3.3 Determination of Sausage Water Activity (Aw) Take 5 g of chopped fermented sausage and place it in a water activity meter test dish. Use the water activity meter to measure the Aw of the sausage for 10 min.

[0049] 3.4 Determination of moisture content in sausages The moisture content of fermented sausages was determined according to the method in GB 5009.3-2016 "Determination of Moisture in Food".

[0050] 3.5 Determination of pH value of sausage The pH value of fermented sausage was determined according to the method in GB 5009.237-2016 "Determination of pH value of food".

[0051] 3.6 Determination of sausage texture Sausage samples with uniform fat and lean meat and the casings removed were cut into 2 cm thick cylinders, and their hardness, elasticity, cohesiveness and chewiness were measured using a texture analyzer.

[0052] 3.7 Determination of Thiobarbituric Acid (TBA) Content in Sausage Weigh 5.00 g of fermented sausage sample (after removing the casing) and chop it. Add 25 mL of 20% trichloroacetic acid and 20 mL of deionized water, homogenize, and let stand at room temperature for 1 h. Then filter and collect the supernatant, and make up to 50 mL with deionized water to obtain the test solution. Take 5.00 mL of the test solution, add 5.00 mL of thiobarbituric acid (0.02 mol / L), mix well, incubate at 90℃ for 30 min, cool to room temperature, and measure the absorbance of the reaction solution at 532 nm using an ELISA reader. Use distilled water as a blank control. Each group has three replicates.

[0053] 3.8 Determination of carbonyl content in sausage The carbonyl content of fermented sausages was determined using the Solarbio Protein Carbonyl Content Detection Kit.

[0054] 3.9 Determination of total sulfhydryl content in sausages The carbonyl content of fermented sausages was determined using the Solarbio Total Sulfhydryl Content Detection Kit.

[0055] 3.10 Determination of FAA content in sausages Weigh 0.5 ~ 1.0 g of sample into a 10 mL volumetric flask, dissolve it completely, add water to the mark, mix well, and let stand at 4℃ for 24 h. Take 1 mL of the supernatant into a 10 mL centrifuge tube, add 1 mL of 5% sulfosalicylic acid solution, mix well, centrifuge (6000 × g, 10 min), take 1 mL of the supernatant, filter it through a 0.22 μm filter membrane, and then use a fully automated amino acid analyzer for determination.

[0056] 3.11 Sensory evaluation Fourteen healthy, sensory-normal evaluators (5 men and 9 women, aged 23-30 years) with experience in food sensory evaluation conducted a sensory evaluation of the fermented sausages. The sensory evaluation criteria are shown in Table 4, and the evaluators evaluated the sausages from five dimensions: appearance, color, aroma, texture, and taste.

[0057] Table 4 Sensory Evaluation Table for Fermented Sausage

[0058] 3.12 VOCs analysis of sausage Accurately weigh 5.0 g of chopped fermented sausage sample, then add 2 μL of 0.816 mg / mL (dissolved in hexane) 2-methyl-3-heptanone solution as an internal standard before headspace solid-phase microextraction (SPME). After equilibration at 60℃ for 15 min, insert the aged extraction head into the headspace extraction bottle and place it in a 60℃ constant temperature water bath for 30 min for adsorption. After adsorption, remove the extraction head and then quickly insert it into the sample inlet for desorption at 250℃ for 5 min, while simultaneously starting the instrument to acquire data. The extraction fiber head used was 50 / 30 μm DVB / CAR / PDMS fiber. The capillary used for separation was a DB-5MS (15 m × 0.25 mm × 0.25 μm, Agilent). The sample inlet temperature was 250℃, and helium was used as the carrier gas at a flow rate of 1 mL / min. VOCs were separated according to the following procedure: first, the temperature was held at 50 °C for 1 min, then increased to 110 °C at a rate of 4 °C / min and held for 1 min; then increased to 250 °C at a rate of 7 °C / min and held for 5 min. The mass spectrometer was operated in total ion current mode at an ionization energy of 70 eV. The scan range was 30–500 m / z, and full scan mode was used. Compounds were identified based on mass spectra and retention indices in the mass spectral library (NIST17-1.lib). Compounds with a match ≥ 75 were selected to determine the volatile composition of the sample, and semi-quantitative concentrations were obtained by calculating the relative peak areas of each compound.

[0059] 4 Results and Analysis 4.1 Effects of Staphylococcus equi P4-2 fermentation on sausage color, Aw, moisture content and pH As shown in Table 5, at the end of fermentation on day 15, the redness of the control group sausages ( a The value was 5.04 ± 0.44, while the sausage inoculated with Staphylococcus equi P4-2 strain... a The value significantly increased to 6.96 ± 0.05. This fully demonstrates that the *Staphylococcus equi* P4-2 strain of this invention possesses extremely excellent nitrate reductase activity. This strain can efficiently reduce nitrate in the fermentation system to nitrite, thereby generating nitric oxide, which combines with myoglobin in the meat matrix to form a highly stable bright red pigment (nitrosomyoglobin), giving sausages the typical reddish color of cured meat.

[0060] Water activity (Aw) and moisture content are important indicators for evaluating the drying and dehydration efficiency and storage safety of fermented sausages. The *Staphylococcus equi* strain P4-2 of this invention exhibits excellent dehydration-promoting properties. As fermentation and drying proceeded, the moisture loss rate of sausages inoculated with P4-2 was significantly faster than that of the control group. By day 15 of fermentation, the moisture content of the P4-2 group had dropped sharply to 31.60% ± 6.86%, significantly lower than the 37.11% ± 7.77% of the control group. This is mainly because the P4-2 strain secretes abundant extracellular proteases in the early stages of fermentation, which degrade large muscle proteins, disrupting the original three-dimensional water maintenance network and promoting the conversion of bound water into free water, thereby significantly improving the efficiency of moisture migration and evaporation from the sausage interior.

[0061] As moisture rapidly evaporated, the final Aw level in group P4-2 stabilized at 0.86 ± 0.01 (compared to 0.87 ± 0.01 in the control group). This extremely low Aw level effectively cut off the moisture conditions necessary for the survival of foodborne pathogens and spoilage bacteria, providing a reliable safety guarantee for the long-term storage of fermented sausages.

[0062] Inoculation with strain P4-2 resulted in a safer and more stable dynamic succession of pH in the sausage system. In the early stages of fermentation (0–3 days), the pH of the P4-2 group rapidly decreased from an initial 5.90 ± 0.05 to 5.71 ± 0.08, quickly establishing an acidic environment that inhibited contaminating microorganisms. In the later stages of fermentation, endogenous enzymes and proteases secreted by P4-2 hydrolyzed proteins, releasing peptides and amino acids, and deamination produced alkaline substances, leading to a moderate pH rise. By the end of day 15 of fermentation, the final pH of the P4-2 group was 6.21 ± 0.12, effectively buffering excessive acidity and astringency, and the overall pH level was lower than the control group (6.32 ± 0.09). This more stable final pH indicates that *Staphylococcus equi* P4-2 can more effectively ensure the microbial safety of fermented sausages from an acid-base control perspective.

[0063] In summary, by comparing the fermentation process of Staphylococcus equi P4-2 inoculated with the present invention with that of the natural fermentation control group, it was confirmed that this strain has significant technical advantages in the preparation of fermented sausages.

[0064] Table 5. Effects of Staphylococcus aureus P4-2 fermentation on sausage color, Aw, moisture content, and pH.

[0065] 4.2 Effects of Staphylococcus equi P4-2 fermentation on sausage textural properties like Figure 3As shown in (A), (C), and (D), the overall firmness, chewiness, and cohesiveness of the sausages in each group showed a significant upward trend with the extension of fermentation time. However, inoculation with Staphylococcus equine P4-2 significantly altered and optimized this texture formation process, exhibiting a remarkably significant "rapid maturation" characteristic. Experimental results showed that the fermented sausages inoculated with strain P4-2 reached their peak firmness, chewiness, and cohesiveness on day 12 of fermentation, at 995.01 ± 13.08 g, 550.72 ± 10.45 g, and 0.63 ± 0.01 g, respectively, significantly higher than the naturally fermented control group (CK group) at the same time. Compared to the slow physical evolution of natural fermentation, strain P4-2 enabled the sausages to achieve the density and chewiness required for high-quality dry-fermented sausages in a shorter time. More importantly, the Staphylococcus equine P4-2 strain of this invention can secrete a large amount of highly active extracellular proteases during fermentation. These enzymes effectively promote the moderate degradation and matrix remodeling of macromolecular myofibril proteins in meat, thereby accelerating the cross-linking and solidification of the internal dense three-dimensional gel network. This provides direct technical support for shortening the fermentation cycle and reducing storage and energy costs in actual industrial production.

[0066] In addition, unlike the trends in hardness, chewiness, and cohesion, the elasticity of each group of samples showed a trend of first increasing and then leveling off or slightly decreasing during fermentation. Figure 3 (B) The physical basis of elasticity is highly dependent on the structured myofibril protein network in the muscle. The P4-2 inoculated group showed a slight decrease in elasticity in the later stage of fermentation, which further confirms the strong extracellular protease secretion activity of this strain. This moderate protein degradation not only does not destroy the overall sliceability of the sausage, but also cuts through the excessively cross-linked tough fibers, giving the final product a better melt-in-your-mouth texture and avoiding the unpleasant chewing experience of the product being dry and hard due to excessive drying.

[0067] Therefore, the Staphylococcus equi P4-2 strain of this invention exhibits superior fermentation efficiency, significantly driving the biochemical evolution and physical dehydration of the meat matrix. Compared to the naturally fermented control group (CK), this strain enables sausages to achieve excellent firmness, chewiness, and cohesiveness by day 12, improving the physical sensory quality of the product while significantly shortening the fermentation cycle of traditional processes, demonstrating extremely high potential for industrial application.

[0068] 4.3 Effects of Staphylococcus equi P4-2 fermentation on lipid and protein oxidation in sausage The appropriate biochemical transformation of lipids and proteins is fundamental to the unique flavor of fermented meat products. However, excessive oxidation not only leads to the loss of nutritional value but also produces off-flavor substances such as aldehydes and ketones (e.g., rancidity), severely affecting the sensory quality and food safety of the product. Experimental results show that the Staphylococcus equi P4-2 strain of this invention exhibits excellent antioxidant effects in the fermentation system.

[0069] During fermentation, sausages inoculated with Staphylococcus equi P4-2 exhibited good lipid oxidation intervention capabilities. Experimental results are as follows: Figure 4 As shown, during the core fermentation stage (0-12 days), the TBA value of the P4-2 group remained at a low level overall, and the accumulation rate of its oxidation products was significantly slower than that of the natural fermentation control group (CK group). This indicates that inoculating the P4-2 strain of the present invention can effectively delay the lipid oxidation chain reaction in fermented sausages, inhibit spoilage caused by excessive fat oxidation, and thus ensure the flavor purity of the final product.

[0070] Protein oxidation also degrades meat texture and reduces its nutritional properties, and an increase in carbonyl content is a key indicator for assessing the severity of this oxidation. Figure 5 As shown, with the progress of fermentation, the CK group, which underwent natural fermentation, experienced a rapid and continuous increase in protein carbonyl content due to the lack of antioxidant microecological protection, reaching a high level by day 6. In contrast, inoculation with strain P4-2 significantly inhibited the accumulation of carbonyl groups within the system. Especially in the early to mid-stages of fermentation (days 3-6), the carbonyl content in the P4-2 group not only did not increase but also showed a significant decrease (dropping to 0.07 ± 0.01 μmol / g by day 3 of fermentation), and its overall protein oxidation level remained lower than that of the CK group until the end of the fermentation period. This trend demonstrates that *Staphylococcus equi* P4-2 can effectively block free radical attacks on amino acid side chains or peptide bonds in the fermentation system, reducing the risk of carbonyl derivative formation.

[0071] Total sulfhydryl content is another core physicochemical indicator characterizing the degree of protein oxidation. For example... Figure 6 As shown, the total sulfhydryl content of the P4-2 inoculated group was consistently significantly higher than that of the CK group. Especially at the end of fermentation (day 15), the total sulfhydryl content of the P4-2 group stabilized at 1.17 ± 0.12 μmol / g, significantly superior to that of naturally fermented sausages at the same stage. The efficient maintenance of total sulfhydryl content and the significant inhibition of carbonyl content highly corroborate each other, further confirming that the *Staphylococcus equi* P4-2 of this invention possesses excellent protein antioxidant efficacy, capable of slowing down the oxidative degradation process of proteins at the microscopic level, providing a solid antioxidant biological barrier for the industrial production of high-quality fermented meat products.

[0072] 4.4 Effects of Staphylococcus equi P4-2 fermentation on sausage FAAs Free amino acids (FAAs) are not only crucial flavor compounds in fermented sausages, but also core precursors for the formation of higher volatile flavor compounds such as aldehydes, ketones, and alcohols. Precise quantitative analysis of the FAA composition of each sausage group at the end of fermentation confirmed that the *Staphylococcus equi* P4-2 strain of this invention possesses remarkable targeted regulatory capabilities in flavor substance metabolism and flavor profile reshaping.

[0073] Table 6 shows that inoculation with Staphylococcus equi P4-2 did not destroy the inherent base flavor of the sausage. There was no significant difference in total amino acid content between the P4-2 inoculated group and the naturally fermented control group (CK group) (7.612 ± 0.755 mg / g and 7.368 ± 0.353 mg / g, respectively, p>0.05). Furthermore, glutamic acid (Glu), which imparts umami flavor, and alanine (Ala), which imparts sweetness, were the two most abundant amino acids and remained highly consistent between the two groups. This fully demonstrates that inoculation with the P4-2 strain of this invention can excellently preserve and stabilize the basic umami and sweet flavor profile of fermented sausage, laying an excellent taste foundation for the subsequent development of complex aromas. However, the P4-2 strain exhibited extremely significant metabolic specificity for some specific flavor amino acids. Compared with the control group, the contents of valine (Val) and leucine (Leu) in sausages inoculated with P4-2 were significantly increased (p<0.05), with Val increasing from 0.399 ± 0.024 mg / g to 0.456 ± 0.036 mg / g and Leu increasing from 0.387 ± 0.036 mg / g to 0.457 ± 0.033 mg / g. Meanwhile, fermentation with strain P4-2 significantly reduced the contents of serine (Ser) and lysine (Lys) (p<0.05), with Ser decreasing dramatically from 0.374 ± 0.060 mg / g to 0.233 ± 0.025 mg / g and Lys decreasing from 0.967 ± 0.103 mg / g to 0.740 ± 0.071 mg / g. In the fermented meat microecology, FAAs can be converted into a variety of characteristic volatile flavor compounds through Strecker degradation, lipid oxidation pathways, and microbial enzymatic action. The significant consumption of specific amino acids such as Ser and Lys is due to the high metabolic activity of the P4-2 strain in the later stages of fermentation, which utilizes them as key substrates and efficiently converts them into specific volatile aroma components.

[0074] In summary, the Staphylococcus equi P4-2 of this invention not only perfectly preserves the stable sweet and fresh base flavor of fermented sausage, but also precisely consumes and reshapes the microscopic ratio of specific free amino acids through specific proteolytic and decompositional metabolism, providing an excellent precursor conversion mechanism for the formation and enrichment of complex and high-grade aroma in fermented sausage.

[0075] Table 6. Effects of Staphylococcus aureus P4-2 fermentation on sausage FAAs

[0076] 4.5 Effects of Staphylococcus equi P4-2 fermentation on sensory properties of sausage As shown in Table 7, fermentation with Staphylococcus equine P4-2 improved the sensory properties of sausages in five dimensions: appearance, color, texture, aroma, and taste. This indicates that the Staphylococcus equine P4-2 of this invention has a positive effect on improving the sensory quality of sausages.

[0077] Table 7 Effects of Staphylococcus aureus P4-2 fermentation on sensory properties of sausage

[0078] 4.6 Effect of Staphylococcus equi P4-2 fermentation on sausage flavor As shown in Tables 8 and 9, inoculation with Staphylococcus equi strain P4-2 effectively enriched the composition of volatile compounds in sausages. Compared with the natural fermentation control group (CK group), the number of aldehyde components in the P4-2 treatment group increased from 4 to 7, the number of hydrocarbons increased from 13 to 19, and the number of other substances increased from 5 to 8.

[0079] In terms of flavor balance and regulation of oxidation byproducts, *Staphylococcus equi* P4-2 exhibited a significant deodorizing effect. Natural fermentation systems, lacking effective guidance for lipid and amino acid metabolism, are prone to excessive accumulation of linear and branched aldehydes, leading to unpleasant putrid or oxidative odors. Experimental data showed that after inoculation with strain P4-2, the total aldehyde content in sausages decreased from 1031.08 ± 113.80 μg / 100g to 577.40 ± 60.90 μg / 100g. Among them, the content of 3-methylbutyraldehyde, which has a pungent odor, decreased from 499.09 ± 79.76 μg / 100g to 210.68 ± 46.19 μg / 100g, while the content of (2R,3R)-(-)-2,3-butanediol, which easily produces an overly sweet taste and masks the meaty aroma, was also reduced from 107.40 ± 8.04 μg / 100g to 36.28 ± 15.59 μg / 100g.

[0080] Furthermore, *Staphylococcus equi* P4-2 exhibits high specificity in the targeted enrichment of specific flavor compounds. Several key flavor markers undetectable in the control group were synthesized and accumulated in high abundance in the P4-2 treatment group. Most notably, nitrogen-containing heterocyclic compounds (pyrazines) were generated. Although pyrazines are generally considered products of high-temperature Maillard reactions, under ambient fermentation conditions, strain P4-2 can guide the deep condensation transformation of nitrogen-containing precursors such as amino acids through specific microbial metabolic pathways. Experimental results confirmed the specific generation of 2-vinyl-6-methylpyrazine (14.85 ± 7.44 μg / 100g), 2,5-dimethylpyrazine (5.65 ± 3.37 μg / 100g), and 2,6-diethylpyrazine (0.31 ± 0.14 μg / 100g) in the P4-2 treatment group. These substances have extremely low thresholds and significantly impart unique nutty and roasted aromas to the product. Meanwhile, P4-2 also induced the formation of volatile components such as benzaldehyde (9.07 ± 3.17 μg / 100g) and hexanal (3.78 ± 3.55 μg / 100g), further enhancing the aroma richness of the product.

[0081] In summary, *Staphylococcus equi* P4-2 systematically optimizes the flavor composition of fermented sausages. This strain, while inhibiting the accumulation of naturally occurring fermentation byproducts, achieves the targeted accumulation of high-grade flavor compounds, primarily pyrazines. Based on this unique metabolic characteristic, *Staphylococcus equi* P4-2 can serve as an excellent reserve of native microbial resources, and can be used to develop novel meat product fermentation agents that highly suit the traditional regional flavors of my country.

[0082] Table 8. Types and contents of volatile flavor compounds in naturally fermented sausages and sausages fermented with Staphylococcus equi P4-2.

[0083] Table 9. Differences in flavor compounds and content between naturally fermented sausage and Staphylococcus equi P4-2 fermented sausage. p <0.05)

[0084] Note: ND indicates that the relevant flavor compounds were not detected in this group.

[0085] For any points not covered above, existing technologies shall apply.

[0086] Although specific embodiments of the present invention have been described in detail by way of examples, those skilled in the art should understand that the above examples are for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make various modifications or additions to the described specific embodiments or use similar methods to replace them, without departing from the direction of the invention or exceeding the scope defined by the appended claims. Those skilled in the art should understand that any modifications, equivalent substitutions, improvements, etc., made to the above embodiments based on the technical essence of the present invention should be included within the protection scope of the present invention.

Claims

1. Staphylococcus equi ( Staphylococcus equorum P4-2, which is deposited at the China Center for Type Culture Collection, accession number CCTCC NO:M 20261316.

2. The application of Staphylococcus equi P4-2 as described in claim 1 in the preparation of fermented meat products.

3. The application as described in claim 2, characterized in that, The fermented meat products include fermented sausages.

4. The application as described in claim 3, characterized in that, At room temperature, strain P4-2 guides nitrogen-containing organic matter to undergo deep condensation transformation into nitrogen-containing heterocyclic compounds.

5. The application as described in claim 4, characterized in that, Nitrogen-containing organic compounds include amino acids.

6. The application as described in claim 4, characterized in that, Nitrogen-containing heterocyclic compounds include 2-vinyl-6-methylpyrazine, 2,5-dimethylpyrazine, and 2,6-diethylpyrazine.

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