Staphylococcus sciuri LB1-2-6 and application of staphylococcus sciuri LB1-2-6 in meat product fermentation

Abstract

The invention relates to the technical field of microorganisms, in particular to staphylococcus sciuri LB1-2-6 and application thereof in meat product fermentation. The staphylococcus sciuri LB1-2-6 disclosed by the invention is preserved in the China General Microbiological Culture Collection Center on March 31, 2025, the preservation number is CGMCC No.34032, and the address of the preservation unit is No.3, Yard 1, Beichen West Road, Chaoyang District, Beijing. When the staphylococcus sciuri LB1-2-6 disclosed by the invention is applied to meat product fermentation, the meat product can be promoted to produce fragrance and develop color, the flavor of the meat product is favorably improved, the quality of the fermented meat product and the food safety are improved, and the industrialized and large-scale production of the fermented meat product can be realized.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a strain of Staphylococcus aureus LB1-2-6 and its application in meat product fermentation. Background Technology

[0002] Traditional meat products hold an important place in Chinese culinary culture, among which dried meat is particularly popular due to its long shelf life, unique flavor, and rich nutrition. However, traditional dried meat relies heavily on natural fermentation. This fermentation process involves not only its own naturally occurring bacteria but also contaminating bacteria from the environment. Consequently, the fermentation process may face unpredictable microbial contamination, leading to unstable product quality and even food safety risks such as excessive levels of spoilage microorganisms and nitrites.

[0003] Microbial fermentation technology can improve the functionality, sensory characteristics, and nutritional quality of meat products. The type of fermenting agent and fermentation process directly affect the quality of fermented meat products. Existing technologies mostly use lactic acid bacteria and yeast for fermentation, but fermentation is typically carried out at 25–30℃. Higher fermentation temperatures can easily lead to the growth of unwanted microorganisms, affecting product quality. Published literature, such as Chinese patent application number 202311409381.4, discloses a compound fermenting agent and its application for improving sausage fermentation quality. This agent uses *Staphylococcus squirrelii* S.18, *Lactobacillus plantarum* YR07, *Lactobacillus sakei* L.48, and *Staphylococcus xylose* S.14 to prepare a compound for sausage fermentation. Another Chinese patent application number 201310281948.4 uses *Staphylococcus squirrelii* SL4 and *Lactobacillus pentosaccharide* 31-1 to prepare a compound for fish paste fermentation sausages. However, both of these require the use of compound fermentation agents to achieve the desired effect.

[0004] Therefore, the present invention aims to screen a single strain of Staphylococcus aureus that is suitable for low-temperature fermentation and has excellent performance, in order to solve the problems of high dependence on chemical additives, imprecise flavor control, and high nitrite content in fermented meat products during industrialization, so as to further promote the standardization and industrialization of traditional fermented meat product production. Summary of the Invention

[0005] The technical problem solved by this invention is to provide a strain of Staphylococcus aureus LB1-2-6, which is Gram-positive, has protease and lipase activity, and is positive for nitrate reductase, catalase, and negative for amino acid decarboxylase. At the same time, it does not produce mucus, ammonia, gas, or hydrogen sulfide, and can be used for the safe fermentation of meat products and to improve the flavor of fermented meat products.

[0006] The second technical problem solved by this invention is to apply the aforementioned Staphylococcus aureus LB1-2-6 to the fermentation of meat products without the addition of nitrite, so as to improve the flavor of dried meat and enhance the safety of meat products.

[0007] The technical problem solved by this invention is achieved by the following technical solution:

[0008] A strain of Staphylococcus sciuri S.sciuri LB1-2-6 was deposited on March 31, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 34032. The address of the depository is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0009] The *Staphylococcus sciuri* S.sciuri LB1-2-6 is characterized by having an opaque, round colony morphology with a smooth surface, neat edges, and a lemon-yellow color.

[0010] The *Staphylococcus sciuri* S. sciuri LB1-2-6 is Gram-positive and contains one or more of the following characteristics: it has protease activity and lipase activity; it is positive for nitrate reductase, positive for catalase, and negative for amino acid decarboxylase; it does not produce mucus, ammonia, gas, or hydrogen sulfide gas.

[0011] The application of Staphylococcus sciuri S.sciuri LB1-2-6 in food fermentation.

[0012] Furthermore, the food product is a fermented meat product.

[0013] The preparation method of the fermented meat product includes: inoculating fresh meat with a suspension of Staphylococcus sciuri S.sciuri LB1-2-6 bacteria; fermenting and culturing the inoculated meat sample and then baking it to obtain fermented jerky.

[0014] Furthermore, the initial count of Staphylococcus sciuri S. sciuri LB1-2-6 in fresh meat was not less than 10. 7 CFU / g.

[0015] Furthermore, the fermentation culture temperature is 6–10℃, and the culture time is 6–8 days.

[0016] Furthermore, after inoculation, the inoculated meat is placed in a vacuum bag, vacuumed, and then fermented.

[0017] Furthermore, the baking temperature is 40–60℃, and the baking time is 3–10 hours.

[0018] Beneficial effects:

[0019] The *Staphylococcus sciuri* S. sciuri LB1-2-6 described in this invention is Gram-positive, possessing protease and lipase activity. It can hydrolyze fats and proteins, promoting the formation of specific aromatic substances. It can produce volatile compounds with a special aroma, such as 3-isopropylbenzaldehyde. Therefore, its application in meat product fermentation can promote aroma and color development, improve the flavor of meat products, shorten fermentation time, and increase production efficiency. Furthermore, the *Staphylococcus sciuri* S. sciuri LB1-2-6 described in this invention is characterized by being nitrate reductase-positive, catalase-positive, and amino acid decarboxylase-negative. It also does not produce mucus, ammonia, gas, or hydrogen sulfide. Multiple tests, including plasma coagulation tests, hemolysis tests, and DNase activity tests, all yielded negative results, making it safe and reliable for use in food fermentation.

[0020] The *Staphylococcus sciuri* S. sciuri LB1-2-6 described in this invention, when used in meat product fermentation, not only produces aroma and color, enhances umami flavor, and ensures the stability of meat product quality, but also effectively avoids the accumulation of harmful metabolites such as nitrites. It can be used for the standardized production of fermented meat products and has good application prospects. Attached Figure Description

[0021] Figure 1 This is a schematic diagram of the colony morphology of Staphylococcus sciuri S. sciuri LB1-2-6 on LB medium as described in Example 1 of the present invention.

[0022] Figure 2 This is a schematic diagram of the bacterial morphology of Staphylococcus sciuri S. sciuri LB1-2-6 described in Example 1 under a microscope.

[0023] Figure 3 This is the phylogenetic tree of Staphylococcus sciuriS.sciuri LB1-2-6 described in Example 1 of this invention.

[0024] Figure 4 The results of the plasma coagulation experiment of Staphylococcus sciuri S. sciuri LB1-2-6 described in Example 2 of this invention are as follows.

[0025] Figure 5 The results of the hemolysis experiment of Staphylococcus sciuri S. sciuri LB1-2-6 described in Example 2 of this invention are shown.

[0026] Figure 6 The results of the amino acid decarboxylase experiment of Staphylococcus sciuri S. sciuri LB1-2-6 described in Example 2 of this invention are shown.

[0027] Figure 7 The results of the toluidine blue-DNase experiment of Staphylococcus sciuri S. sciuri LB1-2-6 described in Example 2 of this invention are shown.

[0028] Figure 8 This is a schematic diagram showing the lipase detection results of Staphylococcus sciuri S. sciuri LB1-2-6 as described in Example 2 of this invention.

[0029] Figure 9 This is a schematic diagram showing the detection results of protease production by Staphylococcus sciuri S. sciuri LB1-2-6 as described in Example 2 of this invention.

[0030] Figure 10 This is a schematic diagram of the antibiotic susceptibility test results of Staphylococcus sciuri S. sciuri LB1-2-6 as described in Example 2 of the present invention. Detailed Implementation

[0031] To make the technical means, creative features, objectives and effects of this invention easier to understand, the invention will be further described below with reference to specific embodiments.

[0032] Example 1

[0033] Samples were taken from the central part of the leg of dried mutton from Lazi County, Tibet. The samples were cut into several small pieces, added to sterile 0.9% physiological saline, homogenized, diluted with 0.9% physiological saline, and then spread on LB solid medium and incubated at 37°C for 48 hours. Single colonies were picked and streaked on another LB solid medium for isolation, and then incubated at 37°C. The purification and isolation were repeated three times to obtain Staphylococcus sciuri S. sciuri LB1-2-6.

[0034] (I) Morphological identification

[0035] Observe the colony morphology of *Staphylococcus sciuri* S. sciuri strain LB1-2-6 on LB solid medium, such as... Figure 1 As shown, the colonies are opaque, round, with smooth surfaces and intact edges. The bacteria are Gram-positive. Figure 2 As shown, they are arranged in pairs, chains, or irregular clusters.

[0036] (II) Molecular Identification

[0037] Genomic DNA was extracted from *Staphylococcus occussciuri* S. sciuri LB1-2-6 using a commercially available rapid bacterial genomic DNA extraction kit. Using the extracted genome as a template, 16S rDNA fragments were amplified using universal bacterial primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCT TGTTACGACTT-3'). The amplified products were sequenced, and the sequencing results were analyzed using BLAST on NCBI. A phylogenetic tree was constructed using MEGA 7.0 software. Figure 3 As shown, based on morphological characteristics and molecular identification results, strain LB1-2-6 was identified as Staphylococcus sciuri.

[0038] (III) Safety Evaluation

[0039] Plasma coagulation test: Add 0.5 mL of sterile physiological saline to a vial containing lyophilized rabbit plasma until completely dissolved. Then add 0.3 mL of LB liquid culture of Staphylococcus sciuri S. sciuri LB1-2-6, mix thoroughly, and incubate at 30°C. Observe every half hour for 6 hours. Figure 4 As shown, no solidification occurred.

[0040] Hemolysis test: Staphylococcus sciuri S. sciuri LB1-2-6 cultured in LB broth was streaked onto blood agar plates and incubated at 30°C for 24 hours. No hemolytic zone was observed. Figure 5 As shown, this invention demonstrates that the *Staphylococcus sciuri* S. sciuri LB1-2-6 strain described herein has no hemolytic activity.

[0041] Amino acid decarboxylase test: Staphylococcus sciuri S. sciuri LB1-2-6 was inoculated into culture media with and without added amino acids, and incubated at 28°C for 24 hours. If both the control and experimental tubes were yellow, the result was negative; if the control tube was yellow and the experimental tube was purple, the result was positive. Figure 6 As shown, the results indicate that the *Staphylococcus sciuri* S. sciuri LB1-2-6 described in this invention is an amino acid decarboxylase-negative strain.

[0042] Toluidine blue-DNase assay: Staphylococcus cussciuri S. sciuri LB1-2-6 cultured in LB medium was inoculated onto toluidine blue-DNA agar medium and incubated at 30°C for 24 h. Results are as follows: Figure 7 As shown, the colony did not turn rose red, indicating that the *Staphylococcus sciuri* LB1-2-6 described in this invention is a DNase-negative strain.

[0043] Lipase activity assay: Staphylococcus sciuri S. sciuri LB1-2-6 was inoculated into LB liquid medium and cultured at 37℃ for 30 h. 10 μL of the bacterial culture was then spotted onto porcine backfat (PF) medium to observe the clear zone around the colonies. After 5 days of culture, if... Figure 8 As shown, a clear zone appears around the colonies in the plate, indicating that the *Staphylococcus sciuri* described in this invention is present.

[0044] S. sciuri)LB1-2-6 has lipase activity.

[0045] Protease activity assay: Staphylococcus sciuri S. sciuri LB1-2-6 was inoculated into LB liquid medium and cultured at 37℃ for 30 h. 10 μL of the bacterial culture was then spotted onto casein medium (CA) and cultured for 5 days. 10% trichloroacetic acid was then added around the colonies, and the clear zone around the colonies was observed. Figure 9 As shown, the presence of a clear zone around the colony in the plate indicates that the *Staphylococcus sciuri* S. sciuri LB1-2-6 described in this invention possesses protease activity.

[0046] Antibiotic susceptibility testing: Following the guidelines of the Clinical and Laboratory Standards Institute, the susceptibility of *Staphylococcus sciuri* S. sciuri LB1-2-6, as described in this invention, to gentamicin (GEN), tetracycline (TET), amikacin (AMK), ciprofloxacin (CIP), chloramphenicol (CHL), amoxicillin (AMC), cefoxitin (FOX), rifampin (RIF), penicillin (PEN), vancomycin (VAN), erythromycin (ERY), and clindamycin (CLI) was determined using the disk diffusion method. The bacterial suspension was spread onto a solid culture medium, and the drug susceptibility test discs were placed on the medium. The culture was incubated at 37°C for 24 hours, and the antibiotic resistance of the strain was observed. Figure 10 As shown, the *Staphylococcus sciuri* S. sciuri LB1-2-6 described in this invention is relatively sensitive to antibiotics.

[0047] Example 2

[0048] The *Staphylococcus sciuri* S. sciuri LB1-2-6 described in this invention is applied to the production of fermented mutton jerky.

[0049] The preparation method is as follows: Fresh, commercially available lamb hind leg meat is used as raw material. The tendons and membranes are removed, and the meat is boiled and then cut into pieces for later use. A fermentation group and a control group are set up. The fermentation group is inoculated with a Staphylococcus sciuri S. sciuri LB1-2-6 bacterial suspension, ensuring that the total viable bacterial count in the lamb reaches 1×10⁻⁶. 7 CFU / g; the control groups included no bacterial suspension inoculation (CK0 group), inoculation with Staphylococcus aureus S.18 (CK1 group), and inoculation with Staphylococcus aureus SL4 (CK2 group). Lamb hind leg meat from each group was placed in vacuum bags, vacuum-sealed, and incubated at 8℃ for 7 days. Then, it was removed and baked at 60℃ for 3.5 hours, and after natural cooling, fermented lamb jerky was obtained.

[0050] The fermented mutton jerky obtained from each group was evaluated for basic physicochemical properties, textural characteristics, volatile flavor compounds, and sensory characteristics.

[0051] (I) Determination of basic physicochemical indicators and textural properties of dried mutton.

[0052] Moisture content determination: The determination was carried out according to the direct drying method in GB5009.3—2016.

[0053] pH determination: The determination shall be performed in accordance with the method in GB5009.237—2016.

[0054] Determination of total thiol content: The total thiol content was measured using the Solarbio Total Thiol Reagent Kit, which utilizes the reaction of thiol with (DTNB).

[0055] Malondialdehyde (MDA) content determination: The content was measured using the Beyotime MDA kit with 2-thiobarbituric acid extraction (TBA) method.

[0056] Total bacterial count determination: The method for determining the total bacterial count is based on GB4789.2-2016.

[0057] Rehydration rate determination: Place 1g of sample in a beaker and bathe it in a 90℃ water bath for 15 minutes. After the water bath treatment, transfer the sample to a sieve and drain it for 8 minutes to remove surface moisture. Then, remove the treated sample and weigh it. Calculate the rehydration rate using the following formula: Rehydration rate = (m1 - m2) / m1. Where, m1: weight after rehydration, g; m2: weight before rehydration, g.

[0058] Determination of free amino acid content: Take 5g of pulverized and dried sample, place it in a beaker, then add 50mL of distilled water and approximately 5g of activated carbon. Heat to boiling and filter. Next, wash the activated carbon with approximately 50mL of hot water, collect the filtrate in a 100mL volumetric flask, add an appropriate amount of water to the mark, and mix thoroughly for subsequent analysis. The content of free amino acids is calculated using the following formula:

[0059] The calculation formula is: Free amino acid content (mg / 100g) = 100 x C / (m x 100). Where, C: the number of amino acids in μg obtained from the standard curve; m: the mass of the sample solution being measured, equivalent to the sample mass, in g.

[0060] The results are shown in Table 1.

[0061] Table 1. Statistical analysis of physicochemical indicators of fermented mutton jerky in each group

[0062] Note: Data in the table are mean ± standard deviation; different letters in "ad" in the same row indicate significant differences (p<0.05).

[0063] As shown in Table 1, the content of key indicators such as MDA, nitrite, and free amino acids in this invention are significantly superior to those in groups CK0, CK1, and CK2. Groups CK1 and CK2, which are control groups, were fermented using Staphylococcus sciuri S.18 and Staphylococcus sciuri SL4, respectively, which have a certain effect on improving the physicochemical properties of fermented mutton. However, compared to these, the Staphylococcus sciuri S.sciuri LB1-2-6 described in this invention achieved unexpectedly superior results in improving the physicochemical properties of fermented mutton.

[0064] (II) The volatile flavor compounds of the mutton jerky obtained in each group were detected by gas chromatography-mass spectrometry (GC-MS), and the results are shown in Table 2.

[0065] Table 2. Relative content of volatile flavor compounds in each group of fermented mutton jerky.

[0066]

[0067] Note: - indicates not detected.

[0068] Table 2 shows that the fermented mutton jerky prepared according to this invention has the best flavor. The content of decanal and 3-isopropylbenzaldehyde in this group is significantly higher than that in the blank group. The high concentration of decanal and 3-isopropylbenzaldehyde can also have a significant impact on the overall flavor of the mutton jerky. The content of irritating and unpleasant flavor substances such as (Z)-9-hexadecenal was not detected or was low in the experimental group, indicating that fermentation with *Staphylococcus sciuri* S.sciuri LB1-2-6 as described in this invention can reduce the irritating odor in fermented meat. In summary, the flavor substances produced by *Staphylococcus sciuri* S.sciuri LB1-2-6 during the fermentation of meat products can significantly improve the flavor of the product, optimize the texture of the mutton jerky, and thus enhance the overall flavor performance of the meat product.

[0069] (III) Sensory evaluation of dried mutton.

[0070] Fifteen sensory evaluators were selected to conduct sensory evaluations on the color, aroma, taste, and texture of each group of fermented mutton jerky. The sensory evaluation criteria and results are shown in Table 3.

[0071] Table 3 Sensory evaluation criteria and results for each group of fermented mutton jerky.

[0072]

[0073] As shown in Table 3, compared with the control group, the mutton jerky prepared by fermentation with Staphylococcus sciuri S. sciuri LB1-2-6 as described in this invention scored higher than the control group in terms of color, aroma, taste and texture, and the differences were significant.

[0074] Example 3

[0075] In Example 2, fresh beef hind leg meat was used instead of the raw material, and the experimental and control groups were set up identically. The basic physicochemical properties, textural characteristics, and sensory evaluations of the fermented beef jerky prepared in each group were then performed. The control groups included a group without any bacterial suspension (CK3), a group inoculated with *Staphylococcus aureus* S.18 (CK4), and a group inoculated with *Staphylococcus aureus* SL4 (CK5). The inoculated beef hind leg meat from each group was placed in vacuum bags, vacuum-sealed, and incubated at 6°C for 8 days. It was then removed and baked at 40°C for 10 hours, and after natural cooling, fermented beef jerky was obtained. The rest was the same as in Example 2.

[0076] (I) Determination of basic physicochemical indicators and textural properties of beef jerky.

[0077] The results are shown in Table 4.

[0078] Table 4. Statistical analysis of physicochemical indicators of fermented beef jerky for each group

[0079]

[0080] Note: Data in the table are mean ± standard deviation; different letters in "ad" in the same row indicate significant differences (p<0.05).

[0081] As shown in Table 4, the present invention significantly outperforms groups CK3, CK4, and CK5 in terms of key indicators such as MDA, nitrite, and free amino acids. Groups CK4 and CK5, which were fermented using Staphylococcus sciuri S.18 and SL4 respectively, disclosed in existing technologies, showed no significant effect on improving the physicochemical properties of fermented beef. In contrast, the Staphylococcus sciuri S.sciuri LB1-2-6 described in this invention achieved unexpectedly excellent results in improving the physicochemical properties of fermented beef.

[0082] (ii) Fifteen sensory evaluators were selected to conduct sensory evaluations on the color, aroma, taste and texture of each group of fermented beef jerky. The sensory evaluation criteria and results are shown in Table 5.

[0083] Table 5. Sensory evaluation criteria and results for each group of fermented beef jerky.

[0084]

[0085] As shown in Table 5, compared with the control group, the beef jerky prepared by fermentation with Staphylococcus sciuri S. sciuri LB1-2-6 as described in this invention scored higher than the control group in terms of color, aroma, taste and texture, and the differences were significant.

[0086] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of this invention is defined by the appended claims and their equivalents.

Claims

1. A strain of Staphylococcus sciuri S.sciuri LB1-2-6 was deposited on March 31, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 34032. The address of the depository is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

2. The *Staphylococcus sciuri* S. sciuri LB1-2-6 as described in claim 1, characterized in that, The colony morphology of *Staphylococcus sciuri* S. sciuri LB1-2-6 is as follows: opaque, round colonies with smooth surfaces, regular edges, and a lemon-yellow color.

3. The Staphylococcus sciuri S. sciuri LB1-2-6 as described in claim 1, characterized in that, The *Staphylococcus sciuri* S. sciuri LB1-2-6 is a Gram-positive bacterium containing one or more of the following characteristics: protease activity, lipase activity; nitrate reductase positivity, catalase positivity, and amino acid decarboxylase negative; and does not produce mucus, ammonia, gas, or hydrogen sulfide.

4. The application of Staphylococcus sciuri S. sciuri LB1-2-6 as described in any one of claims 1 to 3 in food fermentation.

5. The application as described in claim 4, characterized in that, The food product in question is a fermented meat product.

6. The application as described in claim 5, characterized in that, The method for preparing the fermented meat product includes: inoculating fresh meat with a suspension of Staphylococcus sciuri S.sciuri LB1-2-6 bacteria; fermenting and culturing the inoculated fresh meat and then baking it to obtain fermented jerky.

7. The application as described in claim 6, characterized in that, The initial count of Staphylococcus sciuri S. sciuri LB1-2-6 in fresh meat after inoculation should not be less than 1 × 10⁻⁶. 7 CFU / g.

8. The application as described in claim 7, characterized in that, The fermentation culture temperature is 6–10℃, and the culture time is 6–8 days.

9. The application as described in claim 8, characterized in that, After inoculation, the inoculated meat sample is placed in a vacuum bag, vacuumed, and then fermented.

10. The application as described in claim 6, characterized in that, The baking temperature is 40-60℃, and the baking time is 3-10 hours.

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