Staphylococcus pasteuri for promoting hydrolysis of fat and phospholipid and application thereof

Abstract

The application relates to the technical field of food processing, and particularly relates to a pasteuria delphini for promoting hydrolysis of fat and phospholipid and application thereof. The preservation number of the pasteuria delphini (Staphylococcus pasteurii) Staphylococcus pasteuri ) is CCTCC NO. M 20261629; the preservation unit is China Center for Type Culture Collection, and the preservation time is July 13, 2026. The pasteuria delphini (Staphylococcus pasteurii) Staphylococcus pasteuri ) has high lipase activity and phospholipase activity, can significantly enhance fat aroma, milk aroma, fruit aroma and overall flavor levels of sausage meat products, and can improve flavor quality while considering oxidation stability and microbial safety.

Description

Technical Field

[0001] This application relates to the field of food processing technology, and in particular to a Staphylococcus pasteurellii strain that promotes the hydrolysis of fats and phospholipids and its application. Background Technology

[0002] Fermented meat products are meat products with unique flavor, color, and texture made through microbial fermentation. During fermentation, lipases such as lipases and phospholipases produced by microorganisms catalyze the hydrolysis of triglycerides and phospholipids in the meat, releasing free fatty acids. These free fatty acids further undergo oxidation and esterification reactions to generate volatile flavor compounds such as aldehydes, alcohols, esters, and acids. These volatile flavor compounds are the important material basis for the characteristic fatty aroma of fermented sausages. Therefore, screening fermentation strains with excellent fat and phospholipid hydrolysis capabilities is of great significance for the targeted regulation of the flavor of fermented sausages.

[0003] Coagulase-negative staphylococci (CNS) are among the dominant microbial communities in fermented meat products, with *Staphylococcus xylose* and *Staphylococcus carminatus* being widely used as starter cultures. However, existing commercial or research-grade staphylococcal starter cultures often focus on aroma, color development, or antibacterial properties, with generally limited lipase and phospholipase activity, resulting in insufficient hydrolysis of fats and phospholipids. This leads to inadequate release of free fatty acids and a weak, fatty flavor in fermented sausages, hindering product quality improvement. Furthermore, some strains pose safety risks due to hemolysis, production of plasma coagulase, or carrying thermostable nuclease genes. Additionally, some strains lack tolerance to fermentation environments (low pH, high salt, nitrite, low-temperature maturation), making them unsuitable for industrial production.

[0004] Staphylococcus pasteurellosis ( Staphylococcus pasteuri As a coagulase-negative staphylococcus, the functional characteristics of *Staphylococcus pasteurellii* in fermented meat products lack systematic research and development. Therefore, if *Staphylococcus pasteurellii* strains with good safety, strong production adaptability, and efficient hydrolysis of fats and phospholipids can be screened, it will be beneficial to improve the flavor and quality of fermented meat products (especially fermented sausages). Summary of the Invention

[0005] This application provides a Staphylococcus pasteurian strain that promotes the hydrolysis of fats and phospholipids and its application, in order to solve the following technical problem: how to improve the flavor quality of fermented meat products prepared by Staphylococcus pasteurian.

[0006] In a first aspect, embodiments of this application provide a Staphylococcus pasteurellium that promotes the hydrolysis of fats and phospholipids, wherein the Staphylococcus pasteurellium ( Staphylococcus pasteuri The preservation number of the *Staphylococcus pasteurellii* is CCTCC NO. M 20261629; Staphylococcus pasteuri The depositary institution for the *Staphylococcus pasteurellii* is: China Center for Type Culture Collection.Staphylococcus pasteuri The preservation period for this item is July 13, 2026.

[0007] Optionally, the *Staphylococcus pasteurellii* ( Staphylococcus pasteuri The 16S rRNA sequence of the rRNA is shown in SEQ ID NO.1.

[0008] Optionally, the *Staphylococcus pasteurellii* ( Staphylococcus pasteuri It has ≥99% homology with Staphylococcus pasteurellus S71MG680735.1.

[0009] Optionally, the *Staphylococcus pasteurellii* ( Staphylococcus pasteuri The phospholipase activity of *Staphylococcus pasteurellii* was 3.56 U / mL ± 0.67 U / mL. Staphylococcus pasteuri The lipase activity of the sample was 31.07 U / mL ± 0.70 U / mL.

[0010] Secondly, embodiments of this application provide a microbial inoculant, the microbial inoculant including the Staphylococcus pasteurellum described in the first aspect (…). Staphylococcus pasteuri The Staphylococcus pasteurellum ( ); Staphylococcus pasteuri The number of viable bacteria is ≥10. 9 CFU / g.

[0011] Thirdly, embodiments of this application provide an application of *Staphylococcus pasteurellii* that promotes the hydrolysis of fats and phospholipids, the application comprising using the *Staphylococcus pasteurellii* described in the first aspect ( Staphylococcus pasteuri It is used in sausage meat products.

[0012] Optionally, the *Staphylococcus pasteurellii* ( Staphylococcus pasteuri The inoculation amount is ≥1×10 8 CFU / g.

[0013] Optionally, the *Staphylococcus pasteurellii* ( Staphylococcus pasteuri The acid lipase activity of *Staphylococcus pasteurellii* was ≥6.83 U / mg after 14 days of inoculation into sausage meat products; Staphylococcus pasteuri Phospholipase activity ≥7.36 U / mg after 14 days of inoculation into sausage meat products.

[0014] Optionally, the *Staphylococcus pasteurellii* ( Staphylococcus pasteuri The saturated fatty acid content produced by *Staphylococcus pasteurellii* 14 days after inoculation into sausage meat products is ≥52 mg / g. Staphylococcus pasteuri The content of monounsaturated fatty acids produced by *Staphylococcus pasteurellii* ≥82 mg / g after 14 days of inoculation into sausage meat products is specified. Staphylococcus pasteuri The content of polyunsaturated fatty acids produced after 14 days of inoculation into sausage meat products is ≥30mg / g.

[0015] Optionally, the *Staphylococcus pasteurellii* ( Staphylococcus pasteuri The sensory evaluation score of the fat aroma in sausage meat products 14 days after inoculation was 1.17 to 1.52 times that of the blank control group. The Staphylococcus pasteurellum ( Staphylococcus pasteuri The total sensory evaluation score of the inoculated sausage meat products 14 days after inoculation was 1.02 to 1.52 times that of the blank control group.

[0016] The technical solutions provided in this application have the following advantages compared with the prior art: This application provides a Staphylococcus pasteurian strain that promotes the hydrolysis of fats and phospholipids. This strain possesses both strong lipase and phospholipase B activity, capable of hydrolyzing triglycerides and phospholipid sn-1 and sn-2 acyl groups, and activating acidic lipases and phospholipases in fermented meat product systems, thereby fully releasing free fatty acid flavor precursors such as oleic acid and linoleic acid. These precursors are then converted into characteristic flavor substances such as acetoin, 2,3-butanediol, 3-methylbutyric acid, isoamyl isovalerate, and (Z)-dec-2-enal through oxidation, esterification, and amino acid co-metabolism, significantly enhancing the product's fatty aroma, milky aroma, fruity aroma, and overall flavor profile. Simultaneously, this strain guides lipid oxidation towards benign flavor products and inhibits the increase of peroxide value and TBARS, suppressing the proliferation of Enterobacteriaceae and other miscellaneous bacteria, thus improving flavor quality while ensuring oxidative stability and microbial safety. Attached Figure Description

[0017] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments consistent with this application and, together with the description, serve to explain the principles of this application.

[0018] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, for those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 Gram staining image of Staphylococcus pasteurellus S71 provided in the embodiments of this application; Figure 2 This is a diagram showing the colony morphology of Staphylococcus pasteurellus S71 provided in an embodiment of this application. Figure 3 A phylogenetic tree diagram of 16S rRNA of Staphylococcus pasteurellus S71 provided in the embodiments of this application; Figure 4 This is a diagram showing the hemolysis results of Staphylococcus pasteurellus S71 provided in an embodiment of this application; wherein, Figure 4A represents Staphylococcus aureus ATCC 43300. β Diagram of hemolysis. Figure 4 B represents Staphylococcus pasteurellosis S71. γ Diagram of hemolysis; Figure 5 The image shows the plasma coagulase experiment of Staphylococcus pasteurellus S71 provided in the embodiments of this application. In the image, "-" represents sterile saline as a negative control, "intermediate group" represents Staphylococcus pasteurellus S71, and "+" represents the positive reference strain Staphylococcus aureus ATCC 43300. Figure 6 Electrophoresis diagram of the thermostable nuclease gene of Staphylococcus pasteurellus S71 provided in the embodiments of this application; Figure 7 The image shows the acid resistance test results of Staphylococcus pasteurellus S71 provided in the embodiments of this application; where "++" represents the positive control group, S71 represents the Staphylococcus pasteurellus S71 group, and "-" represents the negative control. Figure 8 The figure shows the results of the low-temperature resistance test of Staphylococcus pasteurellus S71 provided in the embodiments of this application; where "++" represents the positive control group, S71 represents the Staphylococcus pasteurellus S71 group, and "-" represents the negative control. Figure 9 The image shows the sodium chloride resistance test results of Staphylococcus pasteurellus S71 provided in the embodiments of this application; where "++" represents the positive control group, S71 represents the Staphylococcus pasteurellus S71 group, and "-" represents the negative control. Figure 10 The diagram shows the nitrite resistance test results of Staphylococcus pasteurellus S71 provided in the embodiments of this application; where "++" represents the positive control group, S71 represents the Staphylococcus pasteurellus S71 group, and "-" represents the negative control. Figure 11 This is a diagram showing the experimental results of nitrate reductase in Staphylococcus pasteurellus S71 provided in the embodiments of this application; Figure 12 The image shows the results of the acetoin production experiment of Staphylococcus pasteurellus S71 provided in the embodiments of this application; wherein, the left side is the positive control group, the middle is the Staphylococcus pasteurellus S71 group, and the right side is the negative control group; Figure 13 The figure shows the H2O2 enzyme experiment results of Staphylococcus pasteurellus S71 provided in the embodiments of this application; where ATCC43300 is the positive control, S71 is the Staphylococcus pasteurellus S71 group, and blank is the negative control group. Figure 14 The figure shows the lipase hydrolysis results of Staphylococcus pasteurellus S71 provided in the embodiments of this application; Figure 15 The diagram shows the phospholipase hydrolysis results of Staphylococcus pasteurellus S71 provided in the embodiments of this application. Figure 16 The chromatogram of the hydrolysis products of Staphylococcus pasteurellum S71 provided in the embodiments of this application, wherein, Figure 16 A is a thin-layer chromatogram of the hydrolysis products of Staphylococcus pasteurellii S71. In the figure, 1 represents phosphatidylcholine standard, 2 represents phosphatidic acid standard, 3 represents lysophosphatidylcholine standard, 4 represents palmitic acid standard, 5 represents oleic acid standard, and 6 and 7 both represent the lecithin hydrolysis products of Staphylococcus pasteurellii S71. Figure 16 B is the gas chromatography-mass spectrometry total ion chromatogram of oleic acid and palmitic acid standards of Staphylococcus pasteurellus S71. Figure 16 C is the total ion chromatogram of the hydrolysis products of Staphylococcus pasteurellis S71 by gas chromatography-mass spectrometry. Detailed Implementation

[0020] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0021] The range descriptions used in this application, such as numerical ranges and proportional ranges, include all possible sub-ranges and single numerical values ​​within that range. For example, the range descriptions of "1 to 6" or "1~6" cover all sub-ranges (such as 1 to 3, 2 to 5, etc.) and single numbers (such as 1, 2, 3, 4, 5, 6) between 1 and 6. Unless otherwise specified, the terms "comprising" and others used herein mean "including but not limited to"; relational terms such as "first" and "second" are used only to distinguish different entities or operations and do not imply an actual order or relationship; "and / or" indicates that multiple situations can exist individually or simultaneously; expressions such as "at least one," "multiple," and "at least one" refer to any combination of the corresponding objects, including combinations of single or multiple objects. The proportional relationships involved in this document, such as mass ratios and molar ratios, should be understood as the correspondence between the first and second terms of a proportional formula, according to the order of description. The raw materials, reagents, instruments, and equipment used herein can all be obtained by purchasing from the market or by existing methods.

[0022] This application embodiment provides a Staphylococcus pasteurellii strain that promotes the hydrolysis of fats and phospholipids. The Staphylococcus pasteurellii strain (… Staphylococcus pasteuri The preservation number of the *Staphylococcus pasteurellii* is CCTCC NO. M 20261629; Staphylococcus pasteuri The depositary institution for the *Staphylococcus pasteurellii* is: China Center for Type Culture Collection.Staphylococcus pasteuri The preservation period for this item is July 13, 2026.

[0023] It should be noted that the Staphylococcus pasteurellium provided in this application promotes the hydrolysis of fats and phospholipids, and the mechanism by which this Staphylococcus pasteurellium improves the flavor and quality of fermented meat products is as follows: 1. Strong lipolysis ability directly releases flavor precursors (fat → free fatty acids).

[0024] The strain exhibits lipase activity of 31.07 U / mL ± 0.70 U / mL and phospholipase activity of 3.56 U / mL ± 0.67 U / mL. The phospholipase was identified as phospholipase B, which can simultaneously hydrolyze the acyl groups at the sn-1 and sn-2 positions of phospholipids (the hydrolysis products contain approximately a 1:1 ratio of oleic acid to palmitic acid, with no accumulation of lysophosphatidylcholine). Furthermore, it forms short-chain fatty acids (precursors to milky and characteristic fatty aromas) through lipase, providing ample substrates for the flavor formation of fermented meat products.

[0025] 2. Continuously activate endogenous lipases in the sausage system.

[0026] The acid lipase activity in the inoculated group continuously increased throughout the fermentation process, while the phospholipase activity was higher than that in the control group at all time points. This indicates that the strain not only produces its own enzymes but also regulates the fermentation microenvironment and activates the acid lipases in the system, forming a synergistic lipolysis of "strain enzymes + endogenous enzymes".

[0027] 3. Free fatty acids are directionally converted into characteristic flavor compounds.

[0028] (1) Aromatic aldehydes: The characteristic product of oleic acid oxidation, (Z)-dec-2-enal, increased from 56.91 μg / kg to 172.71 μg / kg (an increase of 203%, which was not detected in the blank group after 3 days); (E)-2-undecenal increased by 280% from 0 days after 3 days. (2) Milk ketones / alcohols: acetoin reached 6010.36 μg / kg at 3 days, which was 4.78 times that of the blank group; 2,3-butanediol was 2.69 times that of the blank group at 14 days, and the two showed redox equilibrium transformation; (3) Cheese and nut flavor acids: 3-methylbutyric acid reached 942.60 μg / kg at 14 days, which was 4.47 times that of the blank group; (4) Fruit flavor esters: Isoamyl isovalerate increased from 6.92 μg / kg to 231.80 μg / kg (an increase of 33.5 times); Ethyl linoleate was only detected in the inoculated group; (5) Others: phenylacetaldehyde (389.05 μg / kg, floral scent), benzaldehyde (122.47 μg / kg, almond scent), and 2-pentylfuran (84.95 μg / kg, oily scent) were only detected in the bacterial group.

[0029] 4. Regulates the direction of lipid oxidation, inhibiting deterioration and enhancing flavor.

[0030] The peroxide value and TBARS of the inoculated group were lower than those of the control group at 14 days, and there was no difference in the rancidity score. The early deterioration aldehydes such as nonanal continued to decrease with fermentation, while the oxidation of linoleic acid was directed to benign flavor products such as 2-pentylfuran rather than rancid small molecule aldehydes. The strain was catalase positive and produced the antioxidant 2,4-di-tert-butylphenol, both of which reduced the risk of oxidative deterioration.

[0031] 5. Optimize the fermentation microecology and physicochemical processes.

[0032] This strain is resistant to acid, salt, nitrite, and low temperature, and can be established in a dominant manner: the number of Staphylococcus aureus in the inoculated group is dominant throughout the process, and it works in conjunction with lactic acid bacteria to maintain a high level, while the number of Enterobacteriaceae is inhibited; the acid production is mild and the dehydration and maturation are accelerated, ensuring the stable formation of flavor from both the perspective of microbial community and physicochemical properties.

[0033] Sensory endpoint verification: The inoculated group showed significantly higher lipid aroma, overall flavor, and overall acceptability than the control group.

[0034] In some alternative embodiments, the *Staphylococcus pasteurellii* ( Staphylococcus pasteuri The 16S rRNA sequence is shown in SEQ ID NO.1.

[0035] In these embodiments, *Staphylococcus pasteurellii* having the 16S rRNA sequence shown in SEQ ID NO.1 ( Staphylococcus pasteuri This indicates that the Staphylococcus pasteurellum ( Staphylococcus pasteuri This species belongs to the genus Staphylococcus pasteurellum. 。

[0036] In some alternative embodiments, the *Staphylococcus pasteurellii* ( Staphylococcus pasteuri It has ≥99% homology with Staphylococcus pasteurellus S71MG680735.1.

[0037] In these embodiments, *Staphylococcus pasteurellum* with ≥99% homology to *Staphylococcus pasteurellum* S71MG680735.1 ( Staphylococcus pasteuri This indicates that the Staphylococcus pasteurellum ( Staphylococcus pasteuri It belongs to this genus of Staphylococcus pasteurellum.

[0038] In some alternative embodiments, the *Staphylococcus pasteurellii* ( Staphylococcus pasteuri The phospholipase activity of *Staphylococcus pasteurellii* was 3.56 U / mL ± 0.67 U / mL. Staphylococcus pasteuri The lipase activity of the sample was 31.07 U / mL ± 0.70 U / mL.

[0039] In these embodiments, *Staphylococcus pasteurellii* with a phospholipase activity of 3.56 U / mL ± 0.67 U / mL ( Staphylococcus pasteuri The bacteria exhibit good phospholipase activity. Based on the identification result that the phospholipase type is phospholipase B, it indicates that this *Staphylococcus pasteurellii* (…) Staphylococcus pasteuri This process can simultaneously hydrolyze the acyl groups at the sn-1 and sn-2 positions of phospholipids, resulting in an approximately 1:1 ratio of oleic acid to palmitic acid in fermented meat products, without the accumulation of lysophosphatidylcholine. Additionally, *Staphylococcus pasteurellii* with a lipase activity of 31.07 U / mL ± 0.70 U / mL... Staphylococcus pasteuri It can form a large amount of short-chain fatty acids, which provides sufficient substrate for the flavor formation of fermented meat products.

[0040] Staphylococcus pasteurellosis ( Staphylococcus pasteuri The phospholipase activity can be 3.56 U / mL, 3.56 U / mL ± 0.1 U / mL, 3.56 U / mL ± 0.2 U / mL, 3.56 U / mL ± 0.3 U / mL, 3.56 U / mL ± 0.4 U / mL, 3.56 U / mL ± 0.5 U / mL, 3.56 U / mL ± 0.6 U / mL, or 3.56 U / mL ± 0.67 U / mL.

[0041] Staphylococcus pasteurellosis ( Staphylococcus pasteuri The lipase activity can be 31.07 U / mL, 31.07 U / mL ± 0.1 U / mL, 31.07 U / mL ± 0.2 U / mL, 31.07 U / mL ± 0.3 U / mL, 31.07 U / mL ± 0.4 U / mL, 31.07 U / mL ± 0.5 U / mL, 31.07 U / mL ± 0.6 U / mL, or 31.07 U / mL ± 0.7 U / mL.

[0042] Based on a general inventive concept, embodiments of this application provide a microbial inoculant, the microbial inoculant comprising Staphylococcus pasteurellii (… Staphylococcus pasteuri The Staphylococcus pasteurellum ( ); Staphylococcus pasteuri The number of viable bacteria is ≥10. 9 CFU / g.

[0043] This microbial agent is based on the aforementioned Staphylococcus pasteurellum ( Staphylococcus pasteuri This is achieved by using the Staphylococcus pasteurellum ( Staphylococcus pasteuri For specific information, please refer to the above embodiments. Since the microbial agent adopts some or all of the technical solutions of the above embodiments, it has at least all the beneficial effects brought about by the technical solutions of the above embodiments, which will not be repeated here.

[0044] Based on a general inventive concept, embodiments of this application provide an application of *Staphylococcus pasteurellii* that promotes the hydrolysis of fats and phospholipids, the application comprising using the *Staphylococcus pasteurellii* (… Staphylococcus pasteuri It is used in sausage meat products.

[0045] This application is based on the aforementioned Staphylococcus pasteurellum ( Staphylococcus pasteuri This is achieved by using the Staphylococcus pasteurellum ( Staphylococcus pasteuri For specific information, please refer to the above embodiments. Since the application adopts some or all of the technical solutions of the above embodiments, it has at least all the beneficial effects brought about by the technical solutions of the above embodiments, which will not be repeated here.

[0046] In some alternative embodiments, the *Staphylococcus pasteurellii* ( Staphylococcus pasteuri The inoculation amount is ≥1×10 8 CFU / g.

[0047] In these embodiments, the inoculation amount is ≥1×10 8 CFU / g of Staphylococcus pasteurellosis ( Staphylococcus pasteuri This allows sausage meat products to ferment fully, and during the fermentation process, a large number of characteristic flavor substances (such as lipases, aldehydes, cheese and nut acids, and fruit esters) are produced through lipases and phospholipases.

[0048] In some alternative embodiments, the *Staphylococcus pasteurellii* ( Staphylococcus pasteuri The acid lipase activity of *Staphylococcus pasteurellii* was ≥6.83 U / mg after 14 days of inoculation into sausage meat products; Staphylococcus pasteuri Phospholipase activity ≥7.36 U / mg after 14 days of inoculation into sausage meat products.

[0049] In these embodiments, Staphylococcus pasteurellum ( Staphylococcus pasteuri The presence of acid lipase activity ≥6.83 U / mg and phospholipase activity ≥7.36 U / mg in Staphylococcus pasteurellii 14 days after inoculation into sausage meat products indicates that the *Staphylococcus pasteurellii* strain is positive for the virus. Staphylococcus pasteuri The acid lipase and phospholipase activities of the α-lipase remained relatively active even after several days of fermentation, indicating that a large number of characteristic flavor substances (such as lipases, cheese and nut acids, and fruit esters) could still be produced during the fermentation process.

[0050] In some alternative embodiments, the *Staphylococcus pasteurellii* ( Staphylococcus pasteuri The saturated fatty acid content produced by *Staphylococcus pasteurellii* 14 days after inoculation into sausage meat products is ≥52 mg / g. Staphylococcus pasteuri The content of monounsaturated fatty acids produced by *Staphylococcus pasteurellii* ≥82 mg / g after 14 days of inoculation into sausage meat products is specified. Staphylococcus pasteuri The content of polyunsaturated fatty acids produced after 14 days of inoculation into sausage meat products is ≥30mg / g.

[0051] In these embodiments, Staphylococcus pasteurellum ( Staphylococcus pasteuri The presence of saturated fatty acid content ≥52 mg / g, monounsaturated fatty acid content ≥82 mg / g, and polyunsaturated fatty acid content ≥30 mg / g in sausage meat products after 14 days of inoculation indicates that the *Staphylococcus pasteurellii* strain is infected. Staphylococcus pasteuri Lipase hydrolysis of lipase can produce a large amount of short-chain saturated fatty acids. These short-chain saturated fatty acids are the core flavor precursors of the milky aroma and characteristic fatty aroma of fermented sausages, thereby enhancing the flavor of sausage meat products.

[0052] In some alternative embodiments, the *Staphylococcus pasteurellii* ( Staphylococcus pasteuri The sensory evaluation score of the fat aroma in sausage meat products 14 days after inoculation was 1.17 to 1.52 times that of the blank control group. The Staphylococcus pasteurellum ( Staphylococcus pasteuri The total sensory evaluation score of the inoculated sausage meat products 14 days after inoculation was 1.02 to 1.52 times that of the blank control group.

[0053] In these embodiments, Staphylococcus pasteurellum ( Staphylococcus pasteuri The sensory evaluation score of the fat aroma in sausage meat products inoculated 14 days after inoculation was 1.17 to 1.52 times that of the blank control group, and the fat aroma of Staphylococcus pasteurellis (S. pasteurellis) was also significantly higher. Staphylococcus pasteuri The total sensory evaluation score of *Staphylococcus pasteurellii* 14 days after inoculation into sausage meat products was 1.02 to 1.52 times that of the blank control group, indicating that this *Staphylococcus pasteurellii* (*Staphylococcus pasteurellii*) was present. Staphylococcus pasteuri When inoculated into sausage meat products, it can produce a rich flavor, especially a fatty aroma, thereby improving the sensory quality of sausage meat products.

[0054] The present application is further illustrated below with reference to specific embodiments. Experimental methods in the following embodiments that do not specify specific conditions are generally determined according to national / industry standards; if there is no corresponding national / industry standard, they are performed according to general international standards, conventional conditions, or conditions recommended by the manufacturer.

[0055] Example 1 Experiments were conducted with different bacterial strains: I. Experimental Materials 1. Experimental strain: Staphylococcus aureus ( Staphylococcus aureus ATCC 43300), Staphylococcus xylose ( Staphylococcus xylosusCICC 22112) and preserved Staphylococcus pasteurella S71 (Staphylococcus) pasteuri (The accession number is CCTCC NO. M 20261629).

[0056] 2. Experimental reagents: as shown in Table 1.

[0057] Table 1 Experimental Reagents 3. Experimental instruments and equipment: as shown in Table 2.

[0058] Table 2 Instruments and Equipment III. Experimental Methods 1. Activation of bacterial strains and preparation of fresh bacterial culture.

[0059] (1) Activation of strains: Take out Staphylococcus pasteurellus S71, Staphylococcus xylose (preservation number CICC 22112), and Staphylococcus aureus ATCC 43300 strains that were stored at -80℃, thaw them at room temperature, take 100μL of the stored bacterial solution, and then inoculate it into TSB broth medium at an inoculation rate of 2%. Incubate at 37℃ with shaking at 150rpm for 18h. Then take fresh bacterial solution and streak it onto mannitol sodium chloride agar medium. Incubate at 37℃ for 24h until single colonies grow.

[0060] (2) Preparation of fresh bacterial culture: single colonies were picked and inoculated into TSB broth medium and cultured at 37°C with shaking at 150 rpm for 18 h to obtain fresh bacterial culture in the logarithmic growth phase.

[0061] 2. Preparation of culture medium.

[0062] (1) Acid resistance test medium: Adjust the pH of the TSB medium to 4.8–5.0 using 0.1% HCl, and then autoclave it at 121°C for 15 minutes.

[0063] (2) Low-temperature resistance testing medium: The TSB medium was autoclaved at 121°C for 15 minutes.

[0064] (3) Sodium chloride tolerant culture medium: Add 6% NaCl to TSB medium and autoclave at 121°C for 15 min.

[0065] (4) Nitrite-tolerant culture medium: Add 150 mg / kg NaNO2 to TSB medium and autoclave at 121°C for 15 min.

[0066] (5) Acetonitrile-producing (VP) medium: Dissolve 5.0g of peptone, 5.0g of glucose, and 5.0g of NaCl in 1000mL of distilled water, adjust the pH to 7.0-7.2, and autoclave at 115℃ for 20min.

[0067] (6) Nitrate reductase medium: Dissolve 1g of tryptone, 0.5g of yeast extract, and 0.1g of potassium nitrate in 100mL of distilled water, adjust the pH to 7.0-7.2, and autoclave at 121℃ for 15min.

[0068] (7) Fat hydrolysis plate culture medium: Weigh 4g of glyceryl tartrate agar basal medium and add it to 200mL of distilled water. Heat to boiling to dissolve and autoclave at 121℃ for 15min. Since glyceryl tartrate is insoluble in water, polyvinyl alcohol (PVA) is used as an emulsifier. Homogenize and emulsify the mixture at a glyceryl tartrate to PVA mass ratio of 1:3. When the glyceryl tartrate basal medium cools to 60℃, add 2mL of the sterilized glyceryl tartrate PVA emulsion per 200mL of medium, shake well, and then pour into plates for later use.

[0069] (8) Egg yolk hydrolysis plate medium: 1) Preparation of 50% egg yolk emulsion: Take fresh eggs, disinfect them by soaking in 75% alcohol for 30 minutes, and after the surface alcohol evaporates, break the shell and remove the egg white. Use a sterile pipette to draw the egg yolk into a sterile beaker, add sterile physiological saline at a 1:1 volume ratio, shake to mix evenly, and store briefly at 4℃~8℃.

[0070] 2) Preparation of egg yolk agar plates: Weigh the required amount of egg yolk agar basal medium, sterilize it at 121℃ for 15 minutes, cool it to about 50℃, and mix it evenly with 10 mL of 50% sterile egg yolk saline suspension per 100 mL of medium. Quickly pour the mixture into petri dishes and let it solidify before use.

[0071] 3. Morphology and identification of the strain.

[0072] (1) Gram staining and cell morphology observation experiments: Gram staining experiments were performed, and the cellular morphology of Staphylococcus pasteurellosis S71 was observed and recorded under an optical microscope.

[0073] (2) Colony morphology observation experiment: Staphylococcus pasteurellus S71 culture, which has been cultured in TSB medium for 18 hours, was streaked onto mannitol sodium chloride agar medium and cultured at 37°C for 24 hours. Single colonies were grown, and the morphological characteristics of the colonies were observed and recorded.

[0074] (3) Homology analysis of 16S rRNA: 1) DNA extraction: Add 500 μL of DNA extraction buffer (final concentration of 20 mM Tris; 2 mM EDTA; 1% Triton X-100; 0.1 mM NaOH) to a 1.5 mL centrifuge tube. Pick a single colony and resuspend it in the extraction buffer. Boil in a water bath for 10 min, then cool rapidly. Centrifuge at 10000 × g for 3 min. The supernatant is the DNA solution.

[0075] 2) PCR reaction system and conditions: Amplification of the 16S rRNA gene was performed using universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492 R (5'-GGTTACCTTGTTACGACTT-3'). The amplified fragments were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing (Shanghai, China). The sequenced gene sequences were input into the NCBI (National Center for Biotechnology Information) nucleic acid database for BLAST analysis. Homology analysis was performed based on similarity and coverage. Phylogenetic tree analysis was conducted using MEGA6 software to construct a phylogenetic tree.

[0076] 4. Safety testing methods.

[0077] (1) Hemolysis test: Single colonies were streaked onto blood agar plates and incubated upside down at 37°C for 24 hours. Hemolysis was observed, with β-hemolytic Staphylococcus aureus ATCC 43300 serving as a positive control. If a clear hemolytic zone appeared around the colony, it was considered hemolytic. β Hemolysis is a positive result. If no hemolysis zone appears, it is considered negative. γ Hemolysis (i.e., no hemolysis) is a negative result.

[0078] (2) Plasma coagulase test: Add 500 µL of sterile physiological saline to a vial containing lyophilized rabbit plasma, shake until dissolved, then add 300 µL of fresh bacterial culture, mix thoroughly, and incubate at 37°C. Observe every 30 minutes. If coagulation occurs, the result is considered positive; otherwise, it is negative. A vial inoculated with Staphylococcus aureus ATCC 43300 serves as a positive control, and a vial inoculated with sterile water serves as a negative control.

[0079] (3) Thermoresistant nuclease gene detection experiment: DNA was extracted from the samples using a boiling method. The positive control was *Staphylococcus aureus* ATCC 43300, and the negative control was a sample lacking the thermostable nuclease gene. nuc The strain was selected, with sterile physiological saline as a blank control. PCR primers, reaction system, and reaction procedure are shown in Tables 3, 4, and 5.

[0080] Table 3. Information on thermostable nuclease primers Table 4 PCR reaction system Table 5 PCR reaction procedure 5. Production adaptability test methods.

[0081] (1) Acid resistance test: Single colonies were picked and inoculated into acid-resistant medium (pH 4.9–5.0) and incubated at 37°C and 150 rpm for 24–48 hours. The acid resistance of the strains was defined by the degree of turbidity of the medium, with positive labels "+", "++", and "++++" and negative labels "-". A bacterial culture cultured at an initial pH of 6.5 served as a positive control, and an acid-resistant medium inoculated with sterile water served as a negative control.

[0082] (2) Low temperature resistance test: Single colonies were picked and inoculated into corresponding cryotolerant media, and cultured at 10°C and 150 rpm for 24-48 hours. The cryotolerantness of the strains was defined by the degree of turbidity of the media, with positive labels marked as "+", "++", and "++++", and negative labels marked as "-". A culture cultured at 37°C served as a positive control, and a culture inoculated with sterile water at 10°C served as a negative control.

[0083] (3) Salt tolerance test: Single colonies were picked and inoculated onto salt-tolerant medium, and cultured at 37°C and 150 rpm for 24-48 hours. The salt tolerance of the strains was defined by the degree of turbidity of the medium, with positive labels marked as "+", "++", and "++++", and negative labels marked as "-". A bacterial culture with added NaCl at 37°C served as a positive control, and a salt-tolerant medium inoculated with sterile water served as a negative control.

[0084] (4) Nitrite tolerance test: Single colonies were picked and inoculated into nitrite-tolerant medium, and cultured at 37°C and 150 rpm for 24-48 hours. The nitrite tolerance of the strains was defined by the degree of turbidity of the medium, with positive labels "+", "++", and "++++" and negative labels "-". A bacterial culture without NaNO2 at 37°C served as a positive control, and a nitrite-tolerant medium inoculated with sterile water served as a negative control.

[0085] (5) Nitrate reductase experiment: Single colonies of Staphylococcus aureus were picked and inoculated onto nitrate broth medium and incubated at 37°C for 1-3 days. Two to three drops each of solution A and solution B from the nitrate reduction kit were added, mixed thoroughly, and the results were observed immediately. A positive reaction is indicated by a red color, while a negative reaction shows no color change (no red appearance). If a negative result is obtained, a small amount of zinc powder can be added. If the color turns red, the result is confirmed as negative; if the color remains unchanged, the result is positive. The intensity of the red coloration defines the nitrate reductase activity of the strain: pink is marked as "+", magenta as "++", blood red as "++++", and reddish-brown as "++++". No color change without zinc powder and a red coloration after adding zinc powder are marked as "-".

[0086] (6) Ethinosome test: Single colonies of Staphylococcus aureus were picked and inoculated onto VP medium. Incubation was carried out at 37°C and 150 rpm for 24–48 hours. Then, 10 drops of VP reagent A and 4 drops of VP reagent B were added to the medium, and the mixture was shaken thoroughly and incubated at 37°C for 10–20 minutes. A red color indicates a positive result, while no color change indicates a negative result. The intensity of the red color indicates the strength of the strain's ability to produce acetoin. VP medium inoculated with sterile water served as a negative control.

[0087] (7) Catalase experiment: Take 100 μL of Staphylococcus bacterial suspension and drop it onto a glass slide. Add H2O2 solution and determine the catalase production capacity based on the amount of bubbles produced: no bubbles indicate negative (-), bubbles indicate positive, and the more bubbles there are, the stronger the enzyme activity of the strain. Record the number of bubbles as +, ++, +++.

[0088] 6. Experimental methods for detecting the lipid and phospholipase activities of the strain.

[0089] (1) Fat hydrolysis plate culture medium: Single colonies of Staphylococcus pasteurellis S71 were picked and inoculated on glycerol tretinoin basal medium. They were incubated at 37℃ for 3-4 days. The formation of clear zones was observed. The lipase activity was determined by the difference between the radius of the clear zone and the radius of the colony (in cm). 0 < difference ≤ 0.1 was marked as "+", 0.1 < difference ≤ 0.2 as "++", 0.2 < difference ≤ 0.3 as "+++", 0.3 < difference ≤ 0.4 as "++++", difference > 0.4 as "+++++", and no clear zone was marked as "-".

[0090] (2) Phospholipid hydrolysis plate culture medium: Single colonies of Staphylococcus pasteurellus S71 were selected and inoculated onto egg yolk agar plates for initial screening. The colonies were incubated at 37°C for 36-48 hours. The presence or absence of a milky white halo was observed. The difference between the diameter of the milky white halo and the diameter of the colony was used to determine the phospholipase activity (unit: cm). A value of 0 < difference ≤ 0.1 was marked as "+", 0.1 < difference ≤ 0.2 as "++", 0.2 < difference ≤ 0.3 as "+++", 0.3 < difference ≤ 0.4 as "++++", and a value > 0.4 as "+++++". No milky white halo was marked as "-".

[0091] (3) Determination of lipase activity: Lipase activity was determined using the p-nitrophenol method. Lipase catalyzes the hydrolysis of the substrate p-nitrophenol palmitate (p-NPP) to produce p-nitrophenol. p-Nitrophenol exhibits maximum absorbance at 410 nm under alkaline conditions, and the amount produced can be reflected by measuring the absorbance value.

[0092] Enzyme activity is defined as the amount of enzyme required to generate 1 μmol of p-nitrophenol per minute under the specified assay conditions. One unit of enzyme activity (U) is defined as the amount of enzyme required to generate 1 μmol of p-nitrophenol per minute.

[0093] 1) Fermentation medium: The composition (w / v) of fermentation medium A is: 3.0% tryptone, 2.0% glucose, 0.1% Tween-80, and 1.0% olive oil.

[0094] 2) Preparation of crude enzyme solution: Add Staphylococcus pasteurellus S71 colonies to fermentation medium A and incubate at 37℃ and 150 r / min for 3-5 days. Then, take 1 mL of fermentation broth into a 1.5 mL centrifuge tube, centrifuge at 9570×g for 5 min, and take the supernatant as crude enzyme solution and determine enzyme activity.

[0095] 3) Preparation of substrate solution: Solution A is an 8.94 mmol / L p-nitrophenol palmitate (p-NPP) isopropanol solution, stored at 4°C; Solution B is a 20 mmol / L Tris-HCl buffer (pH 8.0) containing 0.1% gum arabic, stored at 4°C. When using, mix Solution A and Solution B at a volume ratio of 1:9 to prepare the substrate working solution, and use immediately.

[0096] 4) Determination of enzyme activity: Add 600 μL of substrate solution to a 1.5 mL centrifuge tube, followed by 25 μL of appropriately diluted crude enzyme solution; the blank control was replaced with Tris-HCl buffer at pH 7.0. After reacting at 40 °C for 15 min, 500 μL of 95% ethanol was added to terminate the reaction. Centrifuge at 9570 × g for 3 min, and measure the absorbance of the supernatant at 410 nm. When plotting the p-nitrophenol standard curve, the reaction volume and conditions were kept consistent with the enzyme activity assay conditions; distilled water was used as a blank control, and the absorbance was measured at 410 nm. A standard curve was then plotted based on the results.

[0097] (4) Determination of phospholipase activity: Phospholipase can catalyze the hydrolysis of lecithin to generate free fatty acids. The content of free fatty acids in the system is determined by acid-base titration, and the titration consumption of standard NaOH solution is used to indirectly characterize phospholipase activity.

[0098] Enzyme activity is defined as the amount of enzyme required to generate 1 μmol of free fatty acid per minute under the specified conditions. One unit of enzyme activity (U) is defined as the amount of enzyme required to generate 1 μmol of free fatty acid per minute.

[0099] 1) Fermentation medium: The composition (w / v) of fermentation medium B is: 1.0% glucose, 1.5% peptone, 0.5% sodium chloride, 1.0% phospholipids, 0.4% K2HPO4·3H2O, and 0.06% MgSO4·7H2O.

[0100] 2) Preparation of crude enzyme solution: Strains with phospholipase hydrolysis ability were selected, and single colonies were added to fermentation medium B. After culturing at 37℃ and 150 r / min for 2 days, the supernatant was taken as crude enzyme solution and the enzyme activity was measured.

[0101] 3) Preparation of substrate solution: Solution C is a 200 mmol / L acetic acid solution; solution D is a 200 mmol / L sodium acetate solution. Mix these two solutions in a suitable ratio to prepare an acetic acid-sodium acetate buffer solution with the desired pH.

[0102] 4) Determination of enzyme activity: Add 10g of lecithin to 300mL of pH 5.0 buffer solution and homogenize for 10min to prepare a substrate solution. Place 10mL of the substrate solution in a 50mL Erlenmeyer flask and preheat in a water bath at 37℃ for 10min. Then add 1mL of enzyme solution and mix immediately. Set up a blank control without enzyme solution. After reacting at 37℃ with shaking at 150rpm for 15min, immediately add 10mL of 95% ethanol to terminate the reaction. Titrate with 100mmol / L NaOH standard solution and record the amount of alkali consumed. Repeat the experiment three times and calculate the average value.

[0103] 7. Identification of phospholipase types.

[0104] (1) Thin-layer chromatography of the strain hydrolysis products: 1) Preparation of crude enzyme solution: The enzyme-producing strain was inoculated into the fermentation medium and cultured at 37℃ for 48h at 150r / min. The fermentation broth was centrifuged at 7104×g for 20min, and the supernatant was collected and stored at 4℃ for later use.

[0105] 2) Preparation and analysis of hydrolysis products: 1 mL of 0.5% lecithin solution was added to 4 mL of crude enzyme solution, and the mixture was incubated in a constant temperature shaker at 37℃ and 150 r / min for 48 h. The reaction was then terminated by adding 2 mL of 0.05 mol / L ethylenediaminetetraacetic acid solution. 20 mL of a chloroform-methanol mixture (2:1, V / V) was added for extraction, and the mixture was thoroughly shaken and allowed to stand for phase separation. This process was repeated three times. Finally, the extracted organic phase was dried under nitrogen to prevent oxidation. The dried lipid residue was redissolved in 400 μL of a chloroform and methanol mixture (2:1, V / V), and developed using chloroform-methanol-acetic acid (40:15:6, V / V) as the developing solvent and iodine vapor fumigation.

[0106] (2) Gas chromatography-mass spectrometry analysis: To determine the composition of free fatty acids in the hydrolysis products of the strain, 0.25 mL of a 5 mg / mL solution of 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphatidylcholine was mixed with 1 mL of crude enzyme solution and incubated at 37 °C with shaking at 150 rpm for 12 h. 1 mL of the above hydrolysis solution, along with 2 mg each of palmitic acid and oleic acid standards, were placed in stoppered test tubes for fatty acid methyl esterification. 1 mL of a 1% sodium hydroxide-methanol solution was added to each test tube, vortexed to mix thoroughly, and then purged with nitrogen for 1 min. The mixture was then saponified in a water bath at 75 °C for 15 min, and cooled to room temperature. Subsequently, 2 mL of a 5% hydrochloric acid-methanol solution was added, vortexed to mix thoroughly, and purged with nitrogen for 1 min. The mixture was then methylated in a water bath at 75 °C for 15 min. After cooling, 2 mL of n-hexane was added to extract the fatty acid methyl esters. The mixture was allowed to stand and separate into layers, and the upper n-hexane extract was analyzed by gas chromatography-mass spectrometry. Mixed standard solutions of oleic acid and palmitic acid with concentrations of 21 μg / mL, 42 μg / mL, 84 μg / mL, 168 μg / mL, 337 μg / mL, and 675 μg / mL were prepared and determined using the above-mentioned methylation, acidification, and gas chromatography-mass spectrometry analysis methods, and standard curves were plotted.

[0107] IV. Fermented Sausage 1. Preparation of fermented sausage.

[0108] (1) Sausage recipe: mince pork into meat filling with a fat-to-lean ratio of 4:1, and add 2.5% salt, 1% white sugar, 0.05% ascorbic acid, 0.007% sodium nitrate and 0.007% sodium nitrite by mass.

[0109] (2) Process: The lean and fatty meat was diced and minced, marinated overnight at 4℃, inoculated with the corresponding bacterial strains, thoroughly mixed, stuffed into sausages, and degassed. Then, it was placed in a constant temperature and humidity incubator and fermented for 3 days at 25℃ and 90% humidity. It was then dried for 7 days at 15℃ and 85% humidity, and finally matured for 4 days at 15℃ and 75% humidity, for a total of 14 days. Samples were taken from each group at 0d, 3d, and 14d for subsequent index determination. The fermented sausages were divided into two groups: a blank group (no inoculation) and a single-strain group (Staphylococcus pasteurellis S71 inoculation amount of ~10). 8 CFU / g).

[0110] 2. Sensory evaluation.

[0111] Referencing and slightly modifying national standards GB / T 22210-2008 "Sensory Evaluation Standard for Meat and Meat Products", GB 2730-2015 "National Food Safety Standard" for cured meat products, and GB / T 23493-2022 "General Quality Rules for Chinese Sausages", Table 6, the sensory evaluation scoring table for sausages, was developed. Twelve well-trained food science students were selected to score the sensory quality of the sausages.

[0112] Table 6. Sensory Evaluation Scoring Table for Sausages 3. Routine physicochemical property determination.

[0113] (1) pH value determination: The pH value was determined according to the method for determining pH value in GB 5009.237-2016 "National Food Safety Standard for Food".

[0114] (2) Determination of moisture content: The moisture content was determined according to the method for determining moisture in GB 5009.3-2016 "National Food Safety Standard for Food".

[0115] (3) Determination of colorimetric values: After homogenization, the sample was evenly spread on the test plate and pressed down to completely block light transmission. The colorimetry of the sample at six points was measured using a CR-400 colorimeter, and the arithmetic mean of the results was used for statistical analysis.

[0116] (4) Determination of nitrite content: The determination was carried out in accordance with GB 5009.33-2016 "National Food Safety Standard - Determination of Nitrite and Nitrate in Food".

[0117] (5) Determination of microbial indicators: The determination of total bacterial count was performed in accordance with GB 4789.2-2022 "National Food Safety Standard - Microbiological Examination of Food - Determination of Total Bacterial Count"; the determination of suspected lactic acid bacteria was performed in accordance with GB 4789.35-2023 "National Food Safety Standard - Microbiological Examination of Food - Lactic Acid Bacteria Examination"; the determination of suspected staphylococci was performed in accordance with the detection method for Staphylococcus aureus: the sample was serially diluted and spread on mannitol sodium chloride agar medium, incubated at 37℃ for 48 hours, and then the number of suspected staphylococci was counted (due to the poor selectivity of mannitol sodium chloride agar medium, the sample may contain species similar to staphylococci); the determination of Escherichia coli was performed in accordance with GB 4789.41-2016 "National Food Safety Standard - Microbiological Examination of Food - Enterobacteriaceae Examination".

[0118] (6) Determination of POV: The peroxide value of the samples was determined using the first method, indicator titration, in the determination of peroxide value in food according to the national standard GB 5009.227-2023 "National Food Safety Standard".

[0119] (7) Determination of TBARS value: The determination was carried out in accordance with the national standard GB 5009.181-2016 "National Food Safety Standard for Malondialdehyde in Food".

[0120] 4. Determination of lipase activity in fermented sausages.

[0121] (1) Preparation of buffer solution: 1) Buffer for neutral lipase: Prepare a buffer containing 0.22 mol / L tris(hydroxymethyl)aminomethane hydrochloride (Tris / HCl) and 0.05% (w / v) Triton X-100, and adjust the pH to 7.5.

[0122] 2) Buffer solution for acid lipase: Prepare a buffer solution containing 0.1 mol / L disodium hydrogen phosphate, 0.05% Triton X-100, 0.8 mg / mL bovine serum albumin (BSA), and 0.05 mol / L citric acid, and adjust the pH to 5.0.

[0123] 3) Buffer for phospholipase: Prepare a buffer containing 0.1 mol / L disodium hydrogen phosphate, 0.05 mol / L citric acid, 150 mmol / L sodium fluoride, 0.05% (w / v) Triton X-100 and 0.8 mg / mL BSA, and adjust the pH to 5.0.

[0124] (2) Extraction of crude enzyme solution: Weigh 5.0g of sausage sample and add 25mL of phosphate buffer (50mmol / L, containing 5mmol / L EGTA, adjusted to pH 7.5). Homogenize under ice bath conditions. Centrifuge the homogenate at 10000×g for 20min at 4℃. After filtering to remove the upper fat layer, bring the volume to 25mL with the appropriate buffer. The filtrate is the crude enzyme solution.

[0125] (3) Enzyme activity assay: 1) Determination of neutral lipase activity: Add 2.8 mL of buffer solution (pH=7.5) and 0.1 mL of 1.0 mmol / L 4-methylumbelliferyl oleate to 0.1 mL of enzyme extraction solution, incubate at 37 °C for 30 min, then cool on ice. Measure fluorescence at λex=328 and λem=443. For the blank control, use the same volume of the enzyme extraction buffer instead of the enzyme extraction solution.

[0126] 2) Determination of acid lipase activity: Add 2.8 mL of buffer solution (pH=5.0) and 0.1 mL of 1.0 mmol / L 4-methylumbelliferyl oleate to 0.1 mL of crude enzyme extract. Incubate in a water bath at 37 °C for 30 min. Immediately terminate the reaction with 0.5 mL of 1 mol / L hydrochloric acid. Measure fluorescence at λex=328 and λem=470. For the blank control, use the same volume of the enzyme extract buffer instead of the enzyme extract solution.

[0127] 3) Determination of phospholipase activity: Add 2.8 mL of buffer solution (pH=5.0) and 0.1 mL of 1 mmol / L 4-methylumbelliferyl oleate to 0.1 mL of enzyme extraction solution, and react at 37 °C for 30 min. Immediately after the reaction, terminate the reaction with 0.5 mL of 1 mol / L hydrochloric acid. Measure the fluorescence at λex=328 and λem=443. The blank control is prepared using the same volume of the enzyme extraction buffer instead of the enzyme extraction solution.

[0128] Prepare a series of 4-methylumbelliferone solutions of different concentrations using the buffer solution used for the above enzyme assays, and construct a standard curve. Define 1 nmol of methylumbelliferone produced by 1 g of protein within 1 min as 1 unit of enzyme activity (U / g pro.).

[0129] 5. Determination of free fatty acids in fermented sausages.

[0130] Samples were pretreated according to the first method in the determination of fatty acids in food in GB 5009.168-2016 National Food Safety Standard. Qualitative analysis was performed using full scan mode (50~650 m / z) of gas chromatography-mass spectrometry (GC-MS) combined with the NIST14 mass spectrometry database. Fatty acid content was calculated using the quantitative ion and external standard method in Table 7.

[0131] Gas chromatography-mass spectrometry (GC-MS): Trace ultra DSQ II (Thermo Fisher Scientific, USA); Column: 70% poly(TR-FAME) cyanopropyl polysiloxane capillary column (100 m × 0.25 mm × 0.20 μm); Injector temperature: 270℃; Detector temperature: 280℃; Temperature program: Initial temperature 100℃, hold for 13 min; 100℃~180℃, ramp rate 10℃ / min, hold for 6 min; 180℃~200℃, ramp rate 1℃ / min, hold for 20 min; 200℃~230℃, ramp rate 4℃ / min, hold for 10.5 min. Carrier gas: Nitrogen; Split ratio 100:1, injection volume 1.0 μL.

[0132] Table 7 Quantitative Ions of Fatty Acids 6. Determination of volatile flavor compounds.

[0133] (1) HS-SPME analysis conditions: Weigh 5g of fermented sausage sample and place it in a 20mL headspace vial. Add 10μL of the internal standard cyclohexanone (0.475μg / μL acetone) and seal the vial. After equilibration at 80℃ for 10min, extract for 30min (80℃) using a 50μm / 30μm DVB / CAR / PEMS (Supelco, USA) extraction head. Then, desorb the extraction head at the injection port (temperature 230℃, time 2min).

[0134] (2) GC conditions: Detection was performed using a TRACE 1600 GasChromatograph manufactured by Thermo Fisher Scientific. A TR-FFAP column (30m × 0.25mm, 0.25μm) was used, with splitless mode selected, helium (purity >99.999%) as the carrier gas, a flow rate of 1.0 mL / min, and an injection port temperature of 230℃. The initial column temperature was 40℃, held for 3 min, and then increased to 210℃ at a rate of 5℃ / min, held for 5 min.

[0135] (3)MS conditions: Detection was performed using an ISQ 7610 Single Quadrupole Mass Spectrometer manufactured by Thermo Fisher Scientific. The interface temperature was 210°C, the transmission line temperature was 230°C, the voltage was 1.2 kV, full scan mode was used, the mass scan range was 40 m / z to 500 m / z, the scan time was consistent with the gas chromatography temperature rise time, an EI ion source was used, the ion source temperature was 250°C, and the electron energy was 70 eV.

[0136] (4) Qualitative analysis: The measurement results were automatically retrieved from the NIST 20 spectral library, with both the SI and RSI scores required to be greater than 800.

[0137] (5) Quantitative analysis: A semi-quantitative analysis method combining full scan and internal standard was used: Cyclohexanone was selected as the internal standard, and acetone was used as the internal standard solvent. An internal standard solution with a mass concentration of 0.475 μg / μL was prepared for spiking. The concentration of each volatile component was calculated using the following formula: , In the formula, C is the mass concentration of the analyte, in μg / kg; Ax is the peak area of ​​the analyte, in AU·min; C0 is the concentration of the internal standard, in 0.475 μg / μL; V is the injection volume of the internal standard, in 10 μL; A0 is the peak area of ​​the added internal standard, in AU·min; and m is the mass of the sample, in g. The mass of the internal standard C0×V is 4.75 μg, and the mass of the fermented sausage sample is 5 g.

[0138] Relevant experimental and effect data: 1. Results of strain identification.

[0139] (1) Gram staining and cell morphology experiments: The experimental results are as follows Figure 1 As shown, Staphylococcus pasteurellus S71 was identified as a Gram-positive coccus. The Gram-stained microscopic image of Staphylococcus pasteurellus S71 showed a purple color, and the bacteria were distributed in a spherical aggregate.

[0140] (2) Results of colony morphology experiment: The experimental results are as follows Figure 2 As shown, the colonies of Staphylococcus pasteurellus S71 are of moderate size, generally round, and golden in color. The colonies of Staphylococcus pasteurellus S71 are raised and full, with regular outlines and edges, smooth and shiny surfaces, and opaque texture with obvious luster.

[0141] (3) Results of 16S rRNA homology analysis: The PCR product of Staphylococcus pasteurellis S71 was sequenced, and the resulting sequences were compared for homology in the NCBI BLAST database to identify similar strains and construct a phylogenetic tree. The results are as follows: Figure 3 As shown. The 16S rRNA sequencing results of Staphylococcus pasteurellosis S71 are as follows:

[0142] The 16S rRNA sequencing results showed 100% homology with Staphylococcus pasteurellus S71MG680735.1 known in the Gene Bank database.

[0143] 2. Results of the safety performance test of the strain.

[0144] (1) Results of hemolysis test: The results are as follows Figure 4 As shown, compared to the β-hemolysis observed in Staphylococcus aureus ATCC 43300 ( Figure 4 As shown in A), Staphylococcus pasteurellus S71 is... γ hemolysis ( Figure 4 As shown in B), no hemolysis occurred, indicating that the hemolysis result of Staphylococcus pasteurellis S71 was negative.

[0145] (2) Results of plasma coagulase test: The results are as follows Figure 5 As shown, no plasma coagulation was observed in Staphylococcus pasteurellus S71, indicating that Staphylococcus pasteurellus S71 does not produce plasma coagulase.

[0146] (3) Results of thermostable nuclease gene detection: The results are as follows Figure 6 As shown, the thermostable nuclease gene of Staphylococcus aureus ( nuc PCR amplification was performed, and no 279bp product band was amplified in Staphylococcus pasteurellus S71, indicating that it was negative for the thermostable nuclease gene.

[0147] 3. Results of thermostable nuclease gene detection.

[0148] (1) Acid resistance test, the results are as follows Figure 7 As shown, based on the degree of turbidity of the culture, Staphylococcus pasteurellus S71 has strong acid resistance and is marked as +++.

[0149] (2) Low temperature resistance test: The results are as follows Figure 8 As shown, based on the turbidity of the culture, Staphylococcus pasteurellus S71 has a certain degree of low-temperature resistance, which is marked as ++.

[0150] (3) Salt tolerance test: The results are as follows Figure 9 As shown, based on the turbidity of the culture, Staphylococcus pasteurellus S71 has a strong salt tolerance, marked as +++.

[0151] (4) Nitrite tolerance test: The results are as follows Figure 10 As shown, based on the turbidity of the culture medium, Staphylococcus pasteurellus S71 has a strong tolerance to nitrite, marked as +++.

[0152] (5) Nitrate reductase experiment: Results are as follows Figure 11As shown, Staphylococcus pasteurellus S71 lacks nitrate reductase activity and is marked as -.

[0153] (6) Ethylania test: Results are as follows Figure 12 As shown, Staphylococcus pasteurellus S71 appears pale red, indicating that Staphylococcus pasteurellus S71 can produce acetoin, which is marked as ++.

[0154] (7) H2O2 enzyme experiment: The results are as follows Figure 13 As shown, obvious air bubbles are visible in Staphylococcus pasteurellus S71, indicating that Staphylococcus pasteurellus S71 can secrete catalase, which is marked as +.

[0155] 4. Experimental results of lipid and phospholipase activity of the strain.

[0156] (1) Results of lipase hydrolysis of the strain: The results are as follows Figure 14 It can be seen that Staphylococcus pasteurellus S71 has a strong lipase hydrolysis ability, marked as +++++.

[0157] (2) Phospholipid hydrolysis plate culture medium: Results are as follows Figure 15 It can be seen that Staphylococcus pasteurellus S71 has a strong phospholipase hydrolytic ability, marked as +++++.

[0158] (3) Results of lipase activity and phospholipase activity: The results are shown in Table 8.

[0159] Table 8. Results of lipase and phospholipase activity assays in the strains. As shown in Table 8, the phospholipase activity of Staphylococcus pasteurellus S71 was 3.56 U / mL ± 0.67 U / mL, and the lipase activity reached 31.07 U / mL ± 0.70 U / mL. This indicates that Staphylococcus pasteurellus S71 can not only degrade fats, but also has a better ability to decompose triglycerides.

[0160] 5. Types of phospholipase in Staphylococcus pasteurellis S71.

[0161] Thin-layer chromatography was used for preliminary detection of phospholipid hydrolysis products of Staphylococcus pasteurellosis S71, and the results are as follows: Figure 16 As shown. By Figure 16 As shown in A, thin-layer chromatography only detected palmitic acid and oleic acid, preliminarily indicating that *Staphylococcus pasteurellii* S71 can produce phospholipase B; further qualitative analysis was then performed by gas chromatography-mass spectrometry (chromatogram of standard substances is shown in Figure A). Figure 16 As shown in B, palmitic acid RT = 14 min, oleic acid RT = 15.57 min), the results are as follows: Figure 16 As shown in C, this indicates that phospholipase B can simultaneously hydrolyze the sn-1 and sn-2 acyl groups of phospholipids, confirming that Staphylococcus pasteurellis S71 can produce phospholipase B.

[0162] Then, based on the oleic acid standard curve y = 0.6156x - 5.6355 (R... 2 =0.998), palmitic acid standard curve y=1.2462x-4.4985 (R 2 Quantitative analysis using a concentration of 0.999 g / mL revealed that the hydrolysis products contained 107.09 mg / 100g of oleic acid and 108.05 mg / 100g of palmitic acid, with a mass ratio of approximately 1:1. This indicates that the hydrolysis products of Staphylococcus pasteurellosis S71 do not contain lysophosphatidylcholine, which is consistent with the results of thin-layer chromatography.

[0163] 6. Experimental results of the application of fermented sausage.

[0164] (1) Sensory evaluation results: The results are shown in Table 9.

[0165] Table 9 Sensory evaluation results of fermented sausages Table 9 shows that, compared with the control group, the fermented sausage inoculated with Staphylococcus pasteurellium S71 had superior overall sensory quality. The overall flavor score and fat aroma score of the Staphylococcus pasteurellium S71 group were significantly higher than those of the control group, with the fat aroma score showing the most significant improvement. This indicates that Staphylococcus pasteurellium S71 can promote fat hydrolysis in the sausage system, enrich lipid-based flavor compounds, and effectively enhance the product's oil aroma and overall flavor profile. There was no significant difference in rancidity scores between the two groups, indicating that Staphylococcus pasteurellium S71 does not exacerbate excessive fat oxidation and produce rancid off-flavors, posing no risk of flavor degradation. Regarding appearance, the fermentation strain had no negative impact on the sausage's color and shape, and even slightly improved its appearance. In conclusion, Staphylococcus pasteurellium S71, as a fermentation strain, significantly enhanced the product's fat aroma and overall flavor intensity, achieving the highest overall acceptability.

[0166] (2) Results of conventional physicochemical indicators: The results are shown in Table 10.

[0167] Table 10 Results of Basic Physicochemical and Microbiological Indicators of Fermented Sausage Table 10 shows that the various indicators of the control group and the *Staphylococcus pasteurellii* S71 group exhibited different trends throughout the fermentation process. At fermentation day 0, there was no significant difference in pH between the two groups. With increasing fermentation time, the pH first decreased and then increased. At day 14, the pH of the *Staphylococcus pasteurellii* S71 group was slightly higher than that of the control group, indicating that *Staphylococcus pasteurellii* S71 has a mild acid-producing capacity and will not cause excessive acidification. The moisture content continuously decreased with fermentation. After day 14, the moisture content of the *Staphylococcus pasteurellii* S71 group (22.63% ± 3.12%) was lower than that of the control group (30.66% ± 1.66%), indicating that the metabolism of *Staphylococcus pasteurellii* S71 can accelerate the dehydration and maturation of sausages, further enhancing their storage quality. Regarding color… L * The value decreases with fermentation time. a * Value rises, b * The value gradually decreased, and after 14 days of fermentation, the Staphylococcus pasteurella S71 group... L * , b * All were higher than the blank group. a * The similar values ​​indicate that Staphylococcus pasteurellii S71 can maintain higher brightness and yellowness in fermented meat products, while the redness remains largely unchanged. Nitrite content shows significant degradation with prolonged fermentation time; the nitrite levels in both groups were similar throughout the entire process, decreasing to below 3 mg / kg after 14 days.

[0168] The lipid oxidation indicators TBARS and POV reflected the degree of lipid oxidation: with the change of fermentation time, the TBARS of both the control group and the Staphylococcus pasteurellis S71 group increased. After 14 days of fermentation, the TBARS of the Staphylococcus pasteurellis S71 group was slightly lower than that of the control group. After 14 days of fermentation, the POV of the Staphylococcus pasteurellis S71 group (23.05 mg / 100g ± 0.79 mg / 100g) was lower than that of the control group (25.80 mg / 100g ± 1.08 mg / 100g). Combined with the data in Table 9, it can be seen that Staphylococcus pasteurellis S71 can effectively inhibit the lipid oxidation process and reduce the risk of spoilage of fermented meat products.

[0169] At the microbiological level, the number of lactic acid bacteria in the *Staphylococcus pasteurellii* S71 group remained consistently high throughout the fermentation process, with significantly higher *Staphylococcus* counts compared to the control group. The number of *Enterobacter* was consistently lower in the control group, indicating that *Staphylococcus pasteurellii* S71 can preferentially colonize, synergistically increase lactic acid bacteria abundance, and inhibit the proliferation of harmful *Enterobacter*, thus optimizing the sausage microbial community structure. Therefore, inoculating with *Staphylococcus pasteurellii* S71 can regulate the acidification and dehydration process of fermented sausages, optimize the color of fermented meat products, slow down fat oxidation, enrich beneficial microorganisms, inhibit the growth of unwanted microorganisms, and balance physicochemical properties with microbial safety, making it an excellent fermentation strain suitable for fermented sausage processing.

[0170] 7. The determination of lipase activity in fermented sausages, and the results are shown in Table 11.

[0171] Table 11 Results of lipase activity determination in fermented sausages Table 11 shows that at fermentation day 0, there were no significant differences in the activities of neutral lipase, acid lipase, and phospholipase between the control group and *Staphylococcus pasteurellii* S71. However, with the extension of fermentation time, the activity of neutral lipase in both groups showed a continuous decreasing trend. Since neutral lipase can hydrolyze triglycerides and release a large amount of free fatty acids, its decreased activity may be due to the lower pH leading to lower neutral lipase activity. In contrast, the activity of acid lipase showed significant differences between the two groups. The acid lipase activity in the control group fluctuated little throughout the fermentation process, decreasing slightly after 14 days; while the *Staphylococcus pasteurellii* S71 group showed a continuous increasing trend, reaching 7.48±0.65 U / mg pro. at 14 days of fermentation, significantly higher than the 5.99±0.34 U / mg pro. in the control group at the same time point. P The value was <0.05, suggesting that Staphylococcus pasteurellii S71 can activate acidic lipase by regulating the sausage fermentation microenvironment. Acidic lipase can directionally catalyze the production of key flavor precursors such as short-chain and medium-chain volatile fatty acids. These precursors are important enzymes that constitute the typical fatty aroma in fermented sausages. In addition, this result provides direct evidence from an enzymatic perspective for the significant improvement in the fatty aroma sensory score of the Staphylococcus pasteurellii S71 group.

[0172] Furthermore, phospholipase activity in both groups exhibited a dynamic change of first increasing and then decreasing, reaching a peak at day 3 of fermentation. Throughout the fermentation process, the phospholipase activity in the Staphylococcus pasteurella S71 group was higher than that in the control group at all time points, reaching 7.46±0.10 U / mg pro. and 6.96±0.09 U / mg pro. at day 14 of fermentation, respectively. Since phospholipase catalyzes the hydrolysis of phospholipids, it can generate small molecule compounds with flavor activity, such as aldehydes and alcohols, which contribute to enriching the flavor profile of the product.

[0173] 8. Results of free fatty acids in fermented sausages are shown in Table 12.

[0174] Table 12 Changes in fatty acid content during sausage fermentation in different groups As shown in Table 12, with the extension of fermentation time, the levels of saturated fatty acids (SFA), monounsaturated fatty acids (MUFA), and polyunsaturated fatty acids (PUFA) in both the control group and the *Staphylococcus pasteurellum* S71 group showed an increasing trend, but the increase in the *Staphylococcus pasteurellum* S71 group was significantly greater than that in the control group. After 14 days of fermentation of the meat products, the total SFA, MUFA, and PUFA levels in the *Staphylococcus pasteurellum* S71 group reached 52.724 mg / g, 82.369 mg / g, and 30.498 mg / g, respectively, all of which were higher than those in the control group (40.118 mg / g, 58.078 mg / g, and 22.328 mg / g).

[0175] Further analysis of the fatty acid components revealed that the total amount of saturated fatty acids in both groups gradually accumulated during fermentation. At 14 days of fermentation, the total saturated fatty acid content in the *Staphylococcus pasteurellii* S71 group reached 52.724 mg / g, significantly higher than the 40.118 mg / g in the control group. Short-chain saturated fatty acids, such as C8:0, C10:0, and C11:0, are the core flavor precursors for the milky and characteristic fatty aromas of fermented sausages. At 14 days of fermentation, the content of short-chain fatty acids in the *Staphylococcus pasteurellii* S71 group was higher than that in the control group. Palmitic acid (C16:0) and stearic acid (C18:0) are the main components of saturated fatty acids in sausages. After 14 days of fermentation, the contents of these two components in the *Staphylococcus pasteurellii* S71 group were 22.381 mg / g and 24.123 mg / g, respectively, higher than those in the control group at the same time point, thus providing a rich, fatty flavor base for the sausage. Long-chain saturated fatty acids (C20-C24) decreased slightly in the later stages of fermentation, suggesting that these long-chain saturated fatty acids participated in the conversion of aldehydes and ketones as secondary flavor compounds or underwent weak oxidation and consumption. The significant increase in MUFA was mainly attributed to the substantial accumulation of oleic acid (C18:1n9c), reaching 78.181 mg / g at 14 days of fermentation, a 42.1% increase compared to the control group. Oleic acid imparts a mellow and full-bodied fatty aroma to meat products, directly enhancing the flavor profile. Among PUFAs, linoleic acid (C18:2n6c) showed the most significant increase, with the Staphylococcus pasteurellis S71 group reaching 25.005 mg / g, a 36.8% increase compared to the control group. Combined with the lipase activity assay results in sausages, the continuous increase in acidic lipase and phospholipase activities in the Staphylococcus pasteurellis S71 group (7.48 U / mg pro. and 7.46 U / mg pro., respectively, at 14 days of fermentation) corresponded well to the enrichment of these unsaturated fatty acids. This indicates that Staphylococcus pasteurellis S71 can directionally promote the release of flavor precursor fatty acids such as oleic acid and linoleic acid by activating key lipolytic enzymes.

[0176] 9. Results of volatile flavor compounds in fermented sausages are shown in Table 13.

[0177] Table 13 Results of determination of volatile flavor compounds in fermented sausages Note: "-" in Table 13 indicates that it was not detected.

[0178] Table 13 shows that the alcohol content results indicate that *Staphylococcus pasteurellii* S71 can significantly enrich branched-chain alcohols and unsaturated alcohols. Among them, 2,3-butanediol, a core component of creamy sweetness, continuously accumulated in the *Staphylococcus pasteurellii* S71 group with fermentation time. It was undetectable at day 0 of fermentation, reached 947.42 μg / kg after 3 days, and reached 1618.49 μg / kg after 14 days. In the control group, 2,3-butanediol reached its peak after 3 days of fermentation and then decreased, with a content of only 601.03 μg / kg after 14 days. The 2,3-butanediol content in the *Staphylococcus pasteurellii* S71 group after 14 days was 2.69 times that of the control group. Since 2,3-butanediol is generated from sugar metabolism and acetoin reduction, its high content in the later stages of fermentation indicates that *Staphylococcus pasteurellii* S71 still possesses vigorous carbon metabolism capacity during the maturation stage.

[0179] The dynamic changes of phenylethanol were basically consistent in both groups, with a mass content of approximately 177 μg / kg after 3 days of fermentation, decreasing to 28 μg / kg after 14 days. Phenyleneethanol mainly contributes to the rose aroma. 4-Methyl-3-heptanol and 3-methylheptane-2-ol were only detected in the Staphylococcus pasteurellis S71 group after 14 days of fermentation, with mass contents of 77.74 μg / kg and 30.37 μg / kg, respectively. These branched alcohols originate from the deamination and decarboxylation of amino acids and can enrich the complex aroma of meat products. Meanwhile, 2-octen-1-ol, 4-methylheptol, and 2-propanol were only detected in the Staphylococcus pasteurellis S71 group at day 0, while no corresponding substances were found in the control group at the same time. This indicates that Staphylococcus pasteurellis S71 can specifically activate the alcohol synthesis pathway in the early stage of fermentation, and store precursors for esterification reactions in advance.

[0180] The aldehyde composition results showed that *Staphylococcus pasteurellii* S71 could simultaneously promote the formation of lipophilic unsaturated aldehydes and Strecker aromatic aldehydes, significantly improving the flavor maturity of fermented sausages. (Z)-dec-2-enal is a characteristic lipophilic substance of oleic acid oxidation. The mass content of (Z)-dec-2-enal in the *Staphylococcus pasteurellii* S71 group continuously increased with fermentation, from 56.91 μg / kg at 0d to 172.71 μg / kg at 14d, an increase of 203%. In the control group, only 28.90 μg / kg was detected at the beginning of fermentation at 0d, and it was not detected at 3d and 14d. (Z)-dec-2-enal is a characteristic substance of the rich lipophilic aroma in the *Staphylococcus pasteurellii* S71 group. Combined with the free fatty acid results, it was found that *Staphylococcus pasteurellii* S71 promotes the accumulation of oleic acid through metabolic pathways, and the oleic acid is further oxidized to generate unsaturated aldehydes, forming a complete lipophilic aroma enhancement metabolic chain. Phenylacetaldehyde and benzaldehyde were detected only after 14 days of fermentation in the *Staphylococcus pasteurellium* S71 group, with mass contents of 389.05 μg / kg and 122.47 μg / kg, respectively. They impart floral and almond-nut aromas to the product, respectively. Both are products of phenylalanine decomposition metabolism, indicating that *Staphylococcus pasteurellium* S71 exhibits vigorous amino acid catabolism activity at the end of fermentation. 2-Heptenoal and 3-methylbutyraldehyde were also detected only after 14 days of fermentation in the *Staphylococcus pasteurellium* S71 group, with mass contents of 120.37 μg / kg and 20.68 μg / kg, respectively. 3-Methylbutyraldehyde is a product of leucine transamination, decarboxylation / dehydrogenation. Nonanal and octanal are early markers of lipid oxidation; the contents of both groups decreased continuously with fermentation. Nonanal completely disappeared at the end of fermentation in the control group, a change consistent with the TBARS lipid oxidation detection results, indicating that oxidative deterioration of the sausage was effectively controlled in the later stages of fermentation. (E)-2-Undecenal reached 75.36 μg / kg after 3 days of fermentation in the Staphylococcus pasteurellus S71 group, which is 280% higher than the 19.82 μg / kg at the start of fermentation 0 days. In the blank group, this substance was only detected at the start of fermentation 0 days, which further indicates that Staphylococcus pasteurellus S71 can promote the generation of unsaturated aldehyde flavor substances.

[0181] The results of acid component analysis showed that *Staphylococcus pasteurellii* S71 synergistically increased the content of branched-chain acids and medium-chain saturated acids, imparting a typical fermented flavor to the product. 3-Methylbutyric acid (3-Methylbutyric acid) is a characteristic branched-chain acid from leucine metabolism, possessing a rich cheese and nutty aroma. The mass content of 3-Methylbutyric acid in the *Staphylococcus pasteurellii* S71 group increased continuously with fermentation, being undetectable at day 0, reaching 615.93 μg / kg at day 3, and as high as 942.60 μg / kg at day 14. In contrast, the peak mass content of the control group at day 3 was 287.25 μg / kg, decreasing to 211.03 μg / kg after day 14. The mass content of the *Staphylococcus pasteurellii* S71 group after 14 days of fermentation was 4.47 times that of the control group. The significant accumulation of 3-Methylbutyric acid reflects the remodeling effect of *Staphylococcus pasteurellii* S71 on the amino acid metabolic network. In addition, acetic acid, an end product of glycolysis, showed a continuous increase in content in both groups during fermentation. After 14 days of fermentation, the content of acetic acid in the *Staphylococcus pasteurellium* S71 group was 1080.58 μg / kg, approximately 15.3% higher than the 937.48 μg / kg content in the control group. This indicates that the *Staphylococcus pasteurellium* S71 group has a stronger carbohydrate metabolism capacity, and acetic acid can provide acyl donors for the esterification reaction in the system. Furthermore, nonanoic acid, derived from the oxidative cracking of oleic acid, had a content of 172.57 μg / kg in the *Staphylococcus pasteurellium* S71 group after 14 days of fermentation, 2.62 times that of the control group (65.76 μg / kg), corresponding to the fatty acid enrichment data of oleic acid in the *Staphylococcus pasteurellium* S71 group. Furthermore, caprylic acid, capric acid, and 3-methylvaleric acid were only detected in the *Staphylococcus pasteurellium* S71 group after 14 days of fermentation, with mass contents of 67.64 μg / kg, 37.25 μg / kg, and 192.28 μg / kg, respectively. The synergistic effect of medium-chain saturated acids and branched-chain acids enriched the aroma layers of cheese and fatty acid fermentation. The variation trend of (E)-3-hexenoic acid, which has a grassy, ​​oily aroma, showed significant differences between the groups, reflecting a clear difference in the oxidative metabolic pathways of unsaturated fatty acids between the two groups.

[0182] The changes in ester composition demonstrate that *Staphylococcus pasteurellii* S71 significantly increases the content of fruity flavor compounds. Isoamyl isovalerate was the most significantly different volatile component among the groups, exhibiting a prominent fruity aroma. In the *Staphylococcus pasteurellii* S71 group, the content increased from 6.92 μg / kg at day 0 of fermentation to 231.80 μg / kg after 14 days, an increase of 33.5 times. In contrast, the isovalerate content in the control group increased from 58.38 μg / kg to 107.48 μg / kg, an increase of only 1.84 times. After 14 days of fermentation, the content in the *Staphylococcus pasteurellii* S71 group was 2.16 times that of the control group. The synthesis of isovalerate depends on isovaleric acid and alcohol substrates. While 3-methylbutanol was not detected in the *Staphylococcus pasteurellii* S71 group, 3-methylbutyric acid accumulated in large quantities, suggesting that isovaleric acid is rapidly consumed in esterification reactions after formation, or that the strain synthesizes it efficiently via the isovaleryl-CoA pathway. Furthermore, ethyl isovalerate has a pineapple aroma. After 14 days of fermentation, the ethyl isovalerate content in the *Staphylococcus pasteurellium* S71 group was 43.02 μg / kg, higher than the 29.15 μg / kg in the control group. This is attributed to the abundant isovaleric acid and ethanol substrate in the *Staphylococcus pasteurellium* S71 group. In addition, ethyl linoleate was only detected in the *Staphylococcus pasteurellium* S71 group after 14 days of fermentation, with a content of 27.52 μg / kg. It is an esterification product of linoleic acid and ethanol, directly confirming that the *Staphylococcus pasteurellium* S71 group had a higher free linoleic acid content and stronger esterification reactivity.

[0183] The ketone component results showed that *Staphylococcus pasteurellii* S71 significantly activated acetoin synthesis, imparting a rich buttery aroma to the product. 3-Hydroxy-2-butanone (acetoin) is the core substance of the buttery flavor and also the component with the largest inter-group variation among all volatile substances. The mass content of acetoin in the *Staphylococcus pasteurellii* S71 group after 3 days of fermentation was 6010.36 μg / kg, which was 4.78 times that of the control group (1258.43 μg / kg) at the same time point. In the control group, acetoin was not detected after 0 days of fermentation, while the *Staphylococcus pasteurellii* S71 group still retained 961.29 μg / kg after 14 days of fermentation. Acetonitrile is converted from pyruvate to α-acetolactate. Its large accumulation indicates that the glucose metabolism flux of the *Staphylococcus pasteurellium* S71 group is stronger. Acetonitrile and 2,3-butanediol exhibit a reversible redox equilibrium. In the *Staphylococcus pasteurellium* S71 group, the mass content of acetonitrile was high and the mass content of 2,3-butanediol was low after 3 days of fermentation. After 14 days of fermentation, the mass content of acetonitrile decreased while the mass content of 2,3-butanediol increased significantly, showing a clear reductive conversion pattern. 2-Nonone was only detected in the control group after 3 days of fermentation, with a mass content of 224.31 μg / kg. It was not detected in the *Staphylococcus pasteurellium* S71 group throughout the entire fermentation process. 2-Nonone is generated from fatty acid β-oxidation or decarboxylation, indicating a significant difference in the fatty acid catabolism pathways between the two groups.

[0184] Significant differences in characteristics were also observed among the minor volatile components, such as phenols, ethers, and furans. 2,4-Di-tert-butylphenol, possessing antioxidant activity, was detected only in the *Staphylococcus pasteurellium* S71 group after 3 days of fermentation, with a content of 10.64 μg / kg, which can help improve the oxidative stability of the fermentation system. 2-Pentylfuran, a characteristic product of linoleic acid oxidation with an oily aroma, was detected only in the *Staphylococcus pasteurellium* S71 group. Its content rapidly increased from 5.14 μg / kg at 3 days to 84.95 μg / kg at 14 days of fermentation, an increase of approximately 16.5 times. 2-Pentylfuran is a benign flavor oxidation product; this increase indicates that *Staphylococcus pasteurellium* S71 can regulate the oxidation of linoleic acid towards flavor compound formation, reduce the accumulation of rancid small molecule aldehydes such as hexanal, and has the function of targeted regulation of lipid oxidation and improvement of product flavor quality.

[0185] In summary, the embodiments of this application provide a Staphylococcus pasteurian strain that promotes the hydrolysis of fats and phospholipids. Staphylococcus pasteuri ), the Staphylococcus pasteurellis ( Staphylococcus pasteuri This *Staphylococcus pasteurella* species exhibits high lipase and phospholipase activity, enabling it to produce abundant fatty aromas and unique volatile flavor compounds (such as branched-chain alcohols, unsaturated alcohols, unsaturated aldehydes, Strecker aromatic aldehydes, branched-chain acids, medium-chain saturated acids, acetoin, and minor volatile components like phenols, ethers, and furans) in sausage products, significantly enhancing their fatty, milky, and fruity aromas and overall flavor profile. Furthermore, this *Staphylococcus pasteurella* species... Staphylococcus pasteuri It can guide lipid oxidation to benign flavor products and inhibit the increase of peroxide value and TBARS, inhibit the proliferation of bacteria such as Enterobacteriaceae, and improve flavor quality while taking into account oxidative stability and microbial safety.

[0186] The above description is merely a specific embodiment of this application, enabling those skilled in the art to understand or implement this application. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of this application. Therefore, this application is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features claimed in this application.

Claims

1. A Staphylococcus pasteurellii strain that promotes the hydrolysis of fats and phospholipids, characterized in that, The preservation number of the Staphylococcus pasteuri (A) Staphylococcus pasteuri is CCTCC NO. M 20261629; the preservation unit of the Staphylococcus pasteuri (A) Staphylococcus pasteuri is China Center for Type Culture Collection, and the preservation time of the Staphylococcus pasteuri (A) Staphylococcus pasteuri is July 13, 2026.

2. The Staphylococcus pasteurellium according to claim 1 ( Staphylococcus pasteuri ), characterized in that, The Staphylococcus pasteurellum ( Staphylococcus pasteuri The 16S rRNA sequence of the rRNA is shown in SEQ ID NO.

1.

3. The Staphylococcus pasteurellium according to claim 1 ( Staphylococcus pasteuri ), characterized in that, The Staphylococcus pasteurellum ( Staphylococcus pasteuri It has ≥99% homology with Staphylococcus pasteurellus S71MG680735.

1.

4. The Staphylococcus pasteurellium according to claim 1 ( Staphylococcus pasteuri ), characterized in that, The Staphylococcus pasteurellum ( Staphylococcus pasteuri The phospholipase activity of *Staphylococcus pasteurellii* was 3.56 U / mL ± 0.67 U / mL. Staphylococcus pasteuri The lipase activity of the sample was 31.07 U / mL ± 0.70 U / mL.

5. A microbial inoculant, characterized in that, The microbial agent includes Staphylococcus pasteurellum as described in any one of claims 1 to 4. Staphylococcus pasteuri The Staphylococcus pasteurellum ( ); Staphylococcus pasteuri The number of viable bacteria is ≥10. 9 CFU / g.

6. The application of a Staphylococcus pasteurellii strain that promotes the hydrolysis of fats and phospholipids, characterized in that, The application includes using the Staphylococcus pasteurian as described in any one of claims 1 to 4. Staphylococcus pasteuri It is used in sausage meat products.

7. The application according to claim 6, characterized in that, The Staphylococcus pasteurellum ( Staphylococcus pasteuri The inoculation amount is ≥1×10 8 CFU / g.

8. The application according to claim 6, characterized in that, The Staphylococcus pasteurellum ( Staphylococcus pasteuri The acid lipase activity of *Staphylococcus pasteurellii* was ≥6.83 U / mg after 14 days of inoculation into sausage meat products; Staphylococcus pasteuri Phospholipase activity ≥7.36 U / mg after 14 days of inoculation into sausage meat products.

9. The application according to claim 6, characterized in that, The Staphylococcus pasteurellum ( Staphylococcus pasteuri The saturated fatty acid content produced by *Staphylococcus pasteurellii* 14 days after inoculation into sausage meat products is ≥52 mg / g. Staphylococcus pasteuri The content of monounsaturated fatty acids produced by *Staphylococcus pasteurellii* ≥82 mg / g after 14 days of inoculation into sausage meat products is specified. Staphylococcus pasteuri The content of polyunsaturated fatty acids produced after 14 days of inoculation into sausage meat products is ≥30mg / g.

10. The application according to claim 6, characterized in that, The Staphylococcus pasteurellum ( Staphylococcus pasteuri The sensory evaluation score of the fat aroma in sausage meat products 14 days after inoculation was 1.17 to 1.52 times that of the blank control group. The Staphylococcus pasteurellum ( Staphylococcus pasteuri The total sensory evaluation score of the inoculated sausage meat products 14 days after inoculation was 1.02 to 1.52 times that of the blank control group.

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