Staphylococcus carnosus and application thereof in improving sour taste of soy sauce

Abstract

The application discloses a strain of Staphylococcus carnosus and application of the strain in improving sour taste of soy sauce, and belongs to the technical field of food fermentation. The Staphylococcus carnosus CS1.21 is first isolated from soy sauce mash, and has the characteristics of salt tolerance, strong acid production, ester production and bacteriostasis. The strain is used for fermenting high-salt dilute soy sauce, and can effectively produce lactic acid, acetic acid and succinic acid, and the production of lactic acid, acetic acid and succinic acid is respectively increased by 28.13%, 27.78% and 50.00% compared to the soy sauce without adding the CS1.21. Meanwhile, the accumulation of ester substances is promoted, and the content of the ester substances is increased by 86.23% compared to the soy sauce without adding the CS1.21, wherein the content of ethyl lactate and ethyl acetate is respectively increased by 70.11% and 85.76%. The addition of the Staphylococcus carnosus CS1.21 is beneficial to improving the sour taste of the soy sauce, and can improve the quality and flavor of the soy sauce without adding preservatives.

Description

Technical Field

[0001] This invention relates to the field of food fermentation technology, and more specifically to a strain of Staphylococcus aureus and its application in enhancing the acidity of soy sauce. Background Technology

[0002] Soy sauce is a traditional condiment originating in my country, made from soybeans and wheat through microbial fermentation. During the fermentation process, besides Aspergillus oryzae dominating protein hydrolysis, acid-producing bacteria (such as halophilic Staphylococcus aureus) produce organic acids (such as lactic acid and acetic acid) through high-salt fermentation. This enhances the soy sauce's acidity, contributing to its sweetness and richness. The accumulated organic acids also promote the formation of organic esters, improving the aroma. Furthermore, the accumulation of organic acids moderately lowers the pH of the mash, achieving a certain antibacterial effect, allowing for the production of preservative-free soy sauce.

[0003] Staphylococcus carnosus is an acid-producing bacterium commonly found in fermented foods. It has the ability to produce acid, promote the accumulation of free amino acids, and protect color and enhance flavor, thus improving the flavor and quality of fermented foods.

[0004] However, there are few reports on the research and application of Staphylococcus aureus screened in soy sauce mash. Its strain performance and its effect on soy sauce fermentation will be beneficial to the improvement of soy sauce fermentation technology and quality.

[0005] In summary, how to provide a Staphylococcus aureus suitable for enhancing acidity and flavor in soy sauce fermentation and how to develop its soy sauce fermentation process are problems that urgently need to be solved by those skilled in the art. Summary of the Invention

[0006] In view of this, the present invention provides a strain of Staphylococcus aureus and its application in enhancing the acidity of soy sauce.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] A strain of Staphylococcus carnosus, named CS1.21 and classified as Staphylococcus carnosus, was deposited on November 29, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 32851. The deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0009] Furthermore, Staphylococcus aureus CS1.21 can tolerate up to 16 g / 100 mL of NaCl.

[0010] The above-mentioned application of Staphylococcus aureus in increasing the organic acid content in the production of soy sauce through high-salt dilute fermentation.

[0011] Furthermore, the organic acids are lactic acid, acetic acid, and succinic acid. Compared with raw soy sauce without Staphylococcus aureus fermentation, the content of the above-mentioned organic acids is increased in raw soy sauce with added fermentation.

[0012] The main steps of applying Staphylococcus aureus in the fermentation production of soy sauce include:

[0013] S1. Inoculate the fermented sauce mash with the aforementioned Staphylococcus aureus CS1.21;

[0014] S2. Continue fermenting the mash obtained by adding Staphylococcus aureus CS1.21 to S1 for 90 days;

[0015] S3. Press, membrane filter, and sterilize the fermented soy sauce mash obtained in S2 to obtain crude fermented soy sauce with added Staphylococcus aureus CS1.21.

[0016] Furthermore, in step S1, the fermented sauce mash is a high-salt, dilute sauce mash with a salt concentration of 16-18 g / 100 mL, and its fermentation time is 10-25 days.

[0017] Furthermore, in step S1, the inoculation amount of Staphylococcus aureus CS1.21 is 10 mg / L of fermented mash. 8 ~10 10 CFU.

[0018] Furthermore, the fermentation temperature of the soy sauce mash in steps S1 and S2 is 25–35°C.

[0019] The aforementioned application of Staphylococcus aureus in the fermentation production of soy sauce can further be used to increase the content of organic acids, amino acid nitrogen, and volatile flavor components in soy sauce.

[0020] The above-mentioned application of Staphylococcus aureus in enhancing the acidity of soy sauce.

[0021] As can be seen from the above technical solution, compared with the prior art, the beneficial effects achieved by the present invention are as follows:

[0022] The *Staphylococcus aureus* CS1.21 strain of this invention was isolated from soy sauce mash for the first time. It possesses characteristics such as salt tolerance, strong acid production, ester production, and antibacterial activity, making it valuable for enhancing the acidity and flavor of soy sauce during fermentation. Fermenting high-salt, dilute-state soy sauce using this strain can produce a certain concentration of organic acids and organic esters, thereby enhancing the quality and flavor of the soy sauce without the need for preservatives. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0024] Figure 1 This is a Gram staining image of strain CS1.21 in Example 2 of the present invention;

[0025] Figure 2 This is an electrophoresis image of the 16S rDNA sequence fragment of strain CS1.21 in Example 2 of the present invention;

[0026] Figure 3 This is the phylogenetic tree of strain CS1.21 in Example 2 of the present invention;

[0027] Figure 4 This is the organic acid chromatogram of Staphylococcus aureus CS1.21 in Example 4 of the present invention;

[0028] Figure 5 The antibacterial properties of Staphylococcus aureus CS1.21 in Example 4 of this invention;

[0029] Figure 6 This is a hemolytic safety diagram of Staphylococcus aureus CS1.21 in Example 4 of the present invention;

[0030] Figure 7 Metabolic characteristics of six organic acids in Staphylococcus aureus CS1.21 during soy sauce fermentation in Example 6 of this invention;

[0031] Figure 8 The sensory evaluation results of the fermented soy sauce with Staphylococcus aureus CS1.21 added in Example 6 of the present invention are shown. Detailed Implementation

[0032] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0033] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by those skilled in the art. The technical terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the scope of the invention.

[0034] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be purchased from the market or prepared by existing methods.

[0035] Example 1

[0036] strain screening

[0037] Strains were isolated and screened from high-salt, dilute fermented soybean paste using MRS medium (solid medium supplemented with 18g / 100mL agar powder) containing 0.2g / 100mL bromocresol purple and 8g / 100mL NaCl. Samples of fermented soybean paste fermented for 15 and 45 days were selected. 10g of the paste was placed in a sterile Erlenmeyer flask with glass beads, and 90mL of sterile physiological saline was added. The mixture was shaken at 100 rpm for 5 minutes to ensure thorough mixing and homogeneity. After serial dilution, 100μL of the bacterial suspension was spread onto NaCl-bromocresol purple-MRS plates and incubated at 37℃ for 2–5 days. Acid-producing strains with a high ratio of color change zone to colony diameter were screened, isolated, purified, and then subjected to secondary screening.

[0038] The isolated and purified salt-tolerant acid-producing strains with good growth were inoculated into MRS liquid medium containing 80 g / L NaCl and cultured at 37°C and 180 rpm for 48 h. 5 mL of fermentation broth was centrifuged and the supernatant was measured to determine the pH. Strains with a fermentation broth pH < 4.5 were screened out.

[0039] Two mL of the fermentation broth of the selected strain was filtered through a 0.22 μm aqueous membrane and analyzed for lactic acid content by liquid chromatography (LC conditions: mobile phase 0.1% phosphoric acid-methanol (95:5), flow rate 0.6 mL / min, injection volume 10 μL, UV detection wavelength 210 nm, column temperature 30 ℃). The strain with the highest lactic acid production was selected and named CS1.21.

[0040] Example 2

[0041] Strain identification

[0042] 1. Morphological characteristics

[0043] (1) The fermentation broth of strain CS1.21 was serially diluted and spread on MRS plates, and incubated at 37℃ for 48 h. The colony morphology was observed. The results of the plate culture showed that strain CS1.21 was a smooth, opaque, milky white, round colony with a raised center and neat edges.

[0044] (2) Gram staining of strain CS1.21 and observation of its morphological characteristics under an oil microscope (100x) revealed that the strain was arranged in single cells, pairs, or short chains, with some strains showing irregular clusters, and was a Gram-positive bacterium. Figure 1 ).

[0045] 2. Molecular biological characteristics

[0046] Genomic DNA was extracted from bacterial strain CS1.21 using a bacterial kit from Sangon Biotech (Wuhan) Co., Ltd. PCR amplification of 16S rDNA was performed using universal bacterial primers 27F and 1492R.

[0047] Primer design: Forwardprimer: 5'-AGAGTTTGATCCTGGCTCAG-3', SEQ ID No. 1; Reverseprimer: 5'-AAGGAGGTGATCCAGCCGCA-3', SEQ ID No. 2.

[0048] PCR reaction mixture (50 μL): 2 μL template DNA, 2 μL upstream primer (10 μmol / mL), 2 μL downstream primer (10 μmol / mL), 5 μL 10×PCR Buffer, 3 μL Mg 2+ (25mol / mL), 4μL dNTP (2.5mol / mL), 1μL Taq DNA polymerase (5U / μL), 31μL double-distilled water.

[0049] PCR reaction conditions: 95℃ template DNA pre-denaturation for 5 min; 95℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 1 min, 30 cycles; 72℃ extension for 10 min, storage at 4℃.

[0050] The PCR products were detected by agarose gel electrophoresis. The results are as follows: Figure 2 As shown.

[0051] The PCR product was sent to the Changsha branch of Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The sequencing results are shown in SEQ ID No. 3. The obtained 16S rDNA results were compared with the Genbank database of NCBI (National Center for Biotechnology Information). The result identified it as Staphylococcus carnosus. Figure 3 ).

[0052]

[0053]

[0054] Example 3

[0055] Preservation of strains

[0056] Staphylococcus carnosus CS1.21 was deposited on November 29, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 32851, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0057] Example 4

[0058] Performance determination of Staphylococcus aureus CS1.21 strain

[0059] 1. Salt resistance:

[0060] To be cultivated to 10 7 Staphylococcus aureus CS1.21 at CFU / mL was inoculated onto MRS plates containing 8, 10, 12, 14, 16, 18 and 20 g / 100 mL NaCl, respectively, and incubated at 37°C until visible colonies were observed. The salt tolerance of the strain was determined based on its growth on the salt plates.

[0061] Table 1. Salt tolerance results of Staphylococcus aureus CS1.21

[0062]

[0063] Note: "++" indicates very good growth, "++" indicates good growth, "+" indicates average growth, and "-" indicates no growth.

[0064] As shown in Table 1, Staphylococcus aureus CS1.21 can grow on MRS medium with 8–16 g / 100 mL NaCl, and can tolerate up to 16 g / 100 mL NaCl, demonstrating a good ability to tolerate high concentrations of salt.

[0065] 2. Acid production performance:

[0066] To be cultivated to 10 7 Staphylococcus aureus CS1.21 (CFU / mL) was inoculated into MRS liquid medium containing 16 g / 100 mL NaCl and fermented at 37 °C for 48 h. 2 mL of the fermentation broth was taken, mixed with 5 mL of zinc sulfate solution (3 g / 100 mL) and 5 mL of potassium ferrocyanide solution (10.6 g / 100 mL), and the mixture was brought to a final volume of 100 mL. After standing for 30 min, the mixture was centrifuged and the supernatant was collected. The mixture was then filtered through a 0.22 μm aqueous membrane, and the organic acid content of the filtrate was analyzed by liquid chromatography.

[0067] Using common organic acids found in soy sauce as standard references, the organic acids produced by Staphylococcus aureus CS1.2 fermentation were qualitatively analyzed using high performance liquid chromatography (HPLC). The chromatograms of the organic acids are shown below. Figure 4The standard curves for organic acids are shown in Table 2. The results of organic acid production by Staphylococcus aureus CS1.21 fermentation are shown in Table 3. The total content of the six organic acids was 1.43 g / 100 mL. Among them, lactic acid and acetic acid accounted for more than 75% of the total content of the six organic acids. Acetic acid, lactic acid, and citric acid accounted for 0.91 g / 100 mL, 0.19 g / 100 mL, and 0.30 g / 100 mL, respectively, and were the main organic acids.

[0068] Table 2 Standard curves of organic acids

[0069]

[0070]

[0071] Note: x is the concentration of organic acid; y is the peak area.

[0072] Table 3. Content of organic acids produced by Staphylococcus aureus CS1.21 metabolism

[0073]

[0074] Note: * indicates that the unit of fumaric acid is "mg / 100mL".

[0075] 3. Antibacterial properties:

[0076] Staphylococcus aureus CS1.21 strain was streaked onto the lower layer of an MRS plate and incubated at 37°C for 24 hours. Then, a mixture of 10... 5 CFU / mL Bacillus megatherium BB15 (isolated and preserved from spoiled soy sauce by Hunan Provincial Condiment Fermentation Engineering Center) was poured onto the top layer of beef extract peptone medium and cultured for 8–24 hours. Results were then observed. The formation of a clear zone around the streaked area indicates that the strain can inhibit the growth of Bacillus megatherium; conversely, the absence of a clear zone indicates that it cannot inhibit growth.

[0077] The results of the antibacterial test of Staphylococcus aureus CS1.21 against Bacillus megaterium BB15 are shown in [the table below]. Figure 5 Staphylococcus aureus CS1.21 formed a transparent inhibition zone significantly larger than the streak area on agar plates containing Bacillus megaterium, indicating that this strain can significantly inhibit the growth of Bacillus megaterium, which causes soy sauce spoilage.

[0078] 4. Hemolytic safety performance:

[0079] Staphylococcus CS1.21 was inoculated onto defibrinated sheep blood agar plates, with Staphylococcus aureus ATCC 6538 as a positive control and Escherichia coli DH-5α as a negative control. The culture conditions for Staphylococcus CS1.21, the positive control, and the negative control bacteria were 37℃ for 24 hours. The presence of a hemolytic zone was marked as "positive," and the absence of a hemolytic zone was marked as "negative."

[0080] Defibrinated sheep blood culture medium: 33g of 7% sheep blood agar base, 1000mL of water, sterilized at 121℃ for 20min; after sterilization, when cooled to 50℃, add 5% sterile defibrinated sheep blood, shake well, pour into sterile plates and cool for later use.

[0081] Comparing the positive and negative controls, the CS1.21 hemolysis test results showed no hemolysis. Figure 6 According to research, Staphylococcus carnosus is a species listed in the "List of Microbial Strains for Traditional Fermented Foods in China". It is a food-grade strain of the Staphylococcus genus that has been completely confirmed to be safe. Therefore, Staphylococcus carnosus CS1.21 can be used as a safe fermentation strain for food.

[0082] Example 5

[0083] Application of Staphylococcus aureus CS1.21 in the fermentation production of soy sauce

[0084] The following steps are adopted:

[0085] (1) Inoculate Staphylococcus aureus CS1.21 into high-salt, dilute fermented soy sauce mash with a salt concentration of 16–18 g / 100 mL and fermentation for 15 days. The inoculation amount is 1 × 10 g / L of fermented soy sauce mash. 9 CFU;

[0086] (2) The fermented mash inoculated with Staphylococcus aureus CS1.21 continued to ferment for 90 days, with the temperature controlled at 30℃ throughout the process;

[0087] (3) Press, filter and sterilize the fermented mash that has been fermented for 90 days in the above steps to obtain fermented soy sauce with added Staphylococcus aureus CS1.21.

[0088] Example 6

[0089] Quality indicators of Staphylococcus aureus CS1.21 fermented soy sauce

[0090] Fermented soy sauce containing Staphylococcus aureus CS1.21 was prepared according to the method in Example 5, with a control group not containing Staphylococcus aureus CS1.21.

[0091] 1. Physicochemical indicators of Staphylococcus aureus CS1.21 fermented soy sauce:

[0092] Physicochemical properties of soy sauce fermented with Staphylococcus aureus CS1.21 were analyzed. The method for determining reducing sugar content was in accordance with the People's Republic of China National Standard GB / T 5009.7-2016, the method for determining total acid content was in accordance with the People's Republic of China National Standard GB / T 12456-2021, and the method for determining amino acid nitrogen was in accordance with the People's Republic of China National Standard GB / T 5009.39-2003.

[0093] The effect of Staphylococcus aureus CS1.21 in soy sauce fermentation was investigated. Soy sauce fermented without CS1.21 was used as a control group. Physicochemical indicators of the soy sauce after 90 days of fermentation were measured. Table 4 shows that the reducing sugar content decreased by 4.40% compared to the control group, while the total acid and amino acid nitrogen contents were higher. The total acid content was 1.76 g / 100 mL, an increase of 15.79% compared to the control group; the amino acid nitrogen content was 0.96 g / 100 mL, an increase of 4.35% compared to the control group, exceeding the 20% limit for premium soy sauce (amino acid nitrogen ≥ 0.80 g / 100 mL). The total acid index met the national standard (total acid ≤ 2.5 g / 100 mL). Adding CS1.21 to fermented soy sauce increased the total acid content and enhanced the sourness without exceeding the total acid content limit. Therefore, the addition of Staphylococcus aureus CS1.21 helps to increase the total acid and amino acid nitrogen content of soy sauce, enhancing its sourness and umami flavor. Overall, compared to the control group, the addition of Staphylococcus aureus CS1.21 during fermentation can accelerate the utilization of reducing sugars, promote the accumulation of total acid and ammonia nitrogen in soy sauce, and thus improve the quality of soy sauce.

[0094] Table 4 Physicochemical Indicators of Staphylococcus aureus CS1.21 Fermented Raw Soy Sauce

[0095]

[0096] 2. Composition and content of organic acids in soy sauce fermented with Staphylococcus aureus CS1.21:

[0097] Referring to the organic acid analysis method in Example 4, the contents of six organic acids (L-malic acid, lactic acid, acetic acid, citric acid, succinic acid and fumaric acid) in Staphylococcus carcinoma CS1.21 fermented soy sauce were determined, and the results are shown in Table 5.

[0098] Table 5. Organic acid content in fermented soy sauce with added Staphylococcus aureus CS1.21

[0099]

[0100] Note: * indicates that the unit of fumaric acid is "mg / 100mL".

[0101] The results are shown in Table 5. The total content of six organic acids in the soy sauce fermented with Staphylococcus aureus CS1.21 was 1.24 g / 100 mL, which was 24.00% higher than that of the control group. Fermentation with Staphylococcus aureus CS1.21 mainly affected the content of lactic acid, acetic acid, and succinic acid among the six organic acids, which were 0.41, 0.46, and 0.18 g / 100 mL, respectively, representing increases of 28.13%, 27.78%, and 50.00% compared to the control group.

[0102] Figure 7 In the study, the net molar increases of lactic acid, acetic acid, and succinic acid in soy sauce fermented with Staphylococcus aureus CS1.21 were all upward; while the net molar increases of L-malic acid, citric acid, and fumaric acid were downward. The most significant increases were in lactic acid and acetic acid, which is beneficial for the formation of important lactic and acetate esters in soy sauce. Lactic acid and acetic acid are important organic acids contributing to the sourness of soy sauce, while succinic acid has a typical meaty umami flavor. The upward adjustment of these three organic acids had a significant effect on the formation of palatable sourness and the enhancement of umami in soy sauce.

[0103] 3. Volatile flavor compounds and their content in Staphylococcus aureus CS1.21 fermented soy sauce:

[0104] Take 2 mL of soy sauce sample fermented with Staphylococcus aureus CS1.21 for 90 days and place it in a 20 mL GC-MS vial. Add 2 μL of 2-methyl-3-heptanone solution (0.816 μg / μL) as an internal standard. SPME conditions: shaker temperature 50℃, shaking time 20 min, extraction time 20 min; GC conditions: injection port temperature 250℃, resolution time 5 min, temperature program: 40℃ held for 1 min, increase to 120℃ at 5℃ / min, hold for 2 min, increase to 250℃ at 10℃ / min, hold for 5 min, carrier gas is high-purity helium, carrier gas flow rate is 1.0 mL / min, splitless injection; MS conditions: EI ion source, electron energy 70 eV, emission current 200 μA, transfer line and ion source temperature 250℃, ion fragment mass scan range 40–500 m / z.

[0105] Table 6. Volatile flavor compounds of Staphylococcus aureus CS1.21 fermented soy sauce

[0106]

[0107] Table 6 shows the results of the types and contents of volatile substances in soy sauce fermented with Staphylococcus aureus CS1.21 for 90 days. The soy sauce fermented with Staphylococcus aureus CS1.21 contained 54 types of volatile flavor compounds, an increase of 9 compared to the control group. The total content of volatile flavor compounds in the soy sauce fermented with Staphylococcus aureus CS1.21 was 5424.52 μg / L, an increase of 50.04% compared to the control group. Furthermore, ethyl acetate and ethyl lactate are esters related to the organic acid metabolism of Staphylococcus aureus CS1.21, and their contents were significantly higher than those in the control group. The ethyl acetate content for fruity aroma increased by 85.76% compared to the control group, and the ethyl lactate content for creamy aroma increased by 70.11% compared to the control group. Therefore, the addition of Staphylococcus aureus CS1.21 during fermentation is beneficial for increasing the content and richness of volatile flavor compounds in soy sauce.

[0108] In summary, fermented soy sauce with added Staphylococcus aureus CS1.21 is significantly superior to fermented soy sauce without added CS1.21 in terms of amino acid nitrogen, total acid, important organic acids, and volatile flavor compounds. Adding Staphylococcus aureus CS1.21 is beneficial for improving the quality and flavor of soy sauce.

[0109] Example 7

[0110] Acidity and overall sensory evaluation of Staphylococcus aureus CS1.21 fermented soy sauce

[0111] 1. Acidity characteristics of Staphylococcus aureus CS1.21 fermented soy sauce

[0112] Based on the lactic acid content of different brands of soy sauce, it was found that the lactic acid content in premium soy sauce was generally low. In order to determine the influence of lactic acid content on the taste of soy sauce, a sensory evaluation method for the acidity characteristics of soy sauce fermented by Staphylococcus aureus CS1.21 was formulated in reference (Wu Moucheng. Food Analysis and Sensory Evaluation [M]. Beijing: China Agriculture Press; 2002.) and the basic principles of sensory evaluation of soy sauce (GB18186-2025). Before evaluation, the RANDBETWEEN function in Excel 2023 was used to randomly number soy sauce samples with different lactic acid contents from 100 to 999. The personnel who numbered the samples did not participate in the evaluation. Eleven individuals (5 men and 6 women) from different age groups and regions were invited to conduct sensory evaluations of the soy sauces. Lactic acid was used as the main organic acid affecting the flavor of soy sauce. The flavor of soy sauces with different lactic acid contents was evaluated on a 10-point scale. The evaluation rules were as follows: scores were given based on acidity, saltiness, and bitterness. Excessive acidity, saltiness, or bitterness resulted in corresponding deductions, with a score range of 0-5 points; moderate acidity, a balanced acidity and saltiness, and a savory flavor were scored from 5-8 points; and a moderate acidity and saltiness with a delicious flavor were scored from 8-10 points. The average score of all evaluators was the final score. SPSS 18.0 data analysis software was used for statistical analysis of the sensory evaluation data.

[0113] Using a premium-grade soy sauce brewed using a specific brewing process from a certain soy sauce company as a base, its lactic acid content was determined to be 0.30±0.00 g / 100 mL. Based on this, soy sauces with different lactic acid concentrations were blended, with concentration gradients set at 0.30, 0.35, 0.40, 0.45, 0.50, 0.55, 0.60, and 0.70 g / 100 mL. The evaluation results of the soy sauce's acidity and flavor at different lactic acid concentrations using the above evaluation method are shown in Table 7. Table 7 shows that when the lactic acid content is 0.4–0.5 g / 100 mL, the sensory score of the sample is 9.14–9.56, indicating that the acidity and flavor harmony are optimal within this lactic acid content range. The lactic acid content of the Staphylococcus aureus CS1.21 fermented soy sauce is 0.41 g / 100 mL (Table 5). Taking lactic acid as an example, it can be seen that the acidity and flavor harmony of the Staphylococcus aureus CS1.21 fermented soy sauce are within a good range.

[0114] Table 7 Sensory evaluation of different lactic acid contents in brewed soy sauce

[0115]

[0116] Note: In the sensory rating column, letters a, b, c, d, etc. indicate the significance of differences in sensory ratings among samples (p < 0.05).

[0117] 2. Analysis of total acid and lactic acid content in different brands of premium soy sauce

[0118] To determine the lactic acid and total acid content of commercially available soy sauces and to ascertain the impact of lactic acid content on sourness, ten different brands of premium soy sauce (amino acid nitrogen content ≥0.8g / 100mL) were selected for testing. The results are shown in Table 8. Table 8 reveals that the highest lactic acid content in premium soy sauce was only 0.33g / 100mL, with seven brands falling below 0.30g / 100mL. This falls short of the optimal lactic acid content corresponding to the sourness score in Table 7. Therefore, utilizing endogenous Staphylococcus aureus CS1.21 selected during soy sauce fermentation to produce lactic acid and other organic acids is an important and safe method to enhance the sourness of soy sauce without the need for exogenous food acidulants.

[0119] Table 8. Lactic acid and total acid content of various premium soy sauces

[0120]

[0121]

[0122] 3. Overall sensory evaluation of Staphylococcus aureus CS1.21 fermented soy sauce

[0123] The sensory evaluation of soy sauce adopted a 100-point system based on four indicators: color, taste, aroma, and appearance (Table 9). The average of all scores for each individual indicator was used as the final score for that indicator. The sensory evaluation panel consisted of 11 healthy and professionally trained evaluators (5 men and 6 women, aged between 20 and 45 years old).

[0124] Table 9 Sensory Evaluation Standards for Soy Sauce

[0125]

[0126] Sensory differences between soy sauce fermented with CS1.21 and the control group were compared and analyzed using sensory scoring. The results are as follows: Figure 8 As shown, the total sensory score of the CS1.21 group was 86.0, higher than the total sensory score of the control group (73.6). The flavor of the fermented soy sauce with added CS1.21 was significantly improved compared to the control group, with a flavor score increase of 20.69%, exhibiting a pleasantly sour and fresh taste. These results indicate that Staphylococcus aureus CS1.21 has a significant enhancing effect on the aroma, flavor, and overall taste of soy sauce, demonstrating good potential for application in soy sauce brewing.

[0127] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0128] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. 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 the invention. Therefore, the invention 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 disclosed herein.

Claims

1. A strain of Staphylococcus aureus ( Staphylococcus carnosus ), characterized in that, Named CS1.21, its taxonomic name is Staphylococcus aureus. Staphylococcus carnosus It was deposited on November 29, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 32851, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

2. The application of Staphylococcus aureus in the fermentation production of soy sauce, characterized in that, Includes the following steps: S1. Inoculate the fermented sauce mash with Staphylococcus aureus CS1.21; S2. Continue fermenting the mash obtained in S1 for 90 days; S3. Press, membrane filter, and sterilize the fermented soy sauce mash obtained in S2 to obtain crude soy sauce. The aforementioned Staphylococcus aureus, named CS1.21, is classified as Staphylococcus aureus. Staphylococcus carnosus It was deposited on November 29, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 32851, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. In step S1, the fermented sauce mash is a high-salt, dilute sauce mash with a salt concentration of 16-18 g / mL, and its fermentation time is 10-25 days. In step S1, the inoculation amount of Staphylococcus aureus CS1.21 is 10 mg / L of fermented mash. 8 ~10 10 CFU; The fermentation temperature of the sauce mash in step S1 is 25~35℃. Used to increase the content of organic acids, amino acid nitrogen, and volatile flavor components in soy sauce; The organic acids are lactic acid, acetic acid, and succinic acid.

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