Method for improving expression of nitric oxide synthase of staphylococcus saprophyticus by low-frequency ultrasound and application thereof in improving color of fermented meat products

Abstract

The application belongs to the technical field of food science and technology, and particularly relates to a method for improving expression of saprophytic Staphylococcus nitric oxide synthase by low-frequency ultrasound and application of the method in improving color and luster of fermented meat products. The ultrasonic process of the application increases the enzyme activity of strain HLMY980 NOS and does not cause logarithmic death of the strain, prolongs the logarithmic growth phase, and enhances the NiR and MRA activities. After the strain HLMY980 is treated by low-frequency ultrasound and inoculated into meat paste for fermentation, the NOS enzyme activity is significantly enhanced, nos the gene is up-regulated, nitroso-myoglobin is generated, the redness value and yellowness value are significantly improved, excellent pink color is exhibited, and the effect is better than that of a commercial starter, and the application has important practical significance and industrial value for realizing nitrite reduction and substitution, improving product safety, and promoting green upgrading of the industry.

Description

Technical Field

[0001] This invention belongs to the field of food science and technology, and specifically relates to a method for improving the expression of nitric oxide synthase in Staphylococcus saprophyticus. Background Technology

[0002] Fermented meat products are a mainstream and distinctive product in the meat processing industry. In industrialized production, nitrites are commonly used as a core auxiliary agent. Nitrites can combine with myoglobin in meat to form bright red nitrosomyoglobin, which is crucial for the stable color development of meat products. However, nitrites pose serious food safety risks. Secondary amines naturally present in meat can react with residual nitrites to form N-nitrosamines, which have strong mutagenic and carcinogenic risks, seriously endangering human health and posing food safety hazards. With the popularization of green and clean production concepts in the food industry and increasingly stringent food safety standards in various countries, low-nitrite and nitrate-free fermented meat products have become the mainstream trend in the industry. Developing safe and efficient nitrite-based color development technologies is a key research topic in the current field of fermented meat processing.

[0003] Existing research confirms that microbial endogenous nitric oxide color development technology is one of the optimal technical pathways to replace chemical nitrites. Coagulase-negative staphylococci, as dominant microorganisms in fermented meat products, carry nitric oxide synthase (NOS), which catalyzes the conversion of L-arginine to nitric oxide (NO) and L-citrulline, thereby promoting the production of nitrosomyoglobin (NO-Mb) and improving the color of meat products. Although the nos gene is ubiquitous in coagulase-negative staphylococci, NOS derived from coagulase-negative staphylococci only contains an oxygenase domain, requiring an external reductase system to provide electrons to complete the catalytic process. This structural defect results in low NO production efficiency mediated by coagulase-negative staphylococci's NOS enzyme, making it difficult to completely replace nitrites in terms of color development. To address the aforementioned issue of insufficient color development efficiency, invention patent CN110800913A discloses a color-developing agent composed of coagulase-negative staphylococci and L-arginine, confirming the feasibility of nitrate-free color development mediated by staphylococcal NOS, but failing to solve the core problem of insufficient NOS activity in the strain. Invention patent CN114568644A improves the expression level of NOS in coagulase-negative staphylococci by treating fermented sausages inoculated with low-intensity high hydrostatic pressure, but this physical method has limited effect on improving enzyme activity, requires large equipment investment, is costly, and the batch processing mode is difficult to efficiently integrate with continuous sausage filling production lines.

[0004] Therefore, developing a green, safe, easy-to-operate, and industrially adaptable technology to efficiently upregulate the expression of the nos gene in coagulase-negative staphylococci, enhance NOS enzyme activity and nitric oxide synthesis efficiency, and break through the bottleneck of existing microbial color development technology is of great practical significance and industrial value for reducing and replacing nitrite in fermented meat products, improving product safety, and promoting the green upgrading of the industry. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention proposes a method for enhancing the expression of nitric oxide synthase in Staphylococcus saprophyticus using low-frequency ultrasound and its application in improving the color of fermented meat products.

[0006] The technical solution of this invention is implemented as follows:

[0007] This invention utilizes low-frequency ultrasound technology to enhance the activity of Staphylococcus saprophyticus HLMY980 NOS through its cavitation effect and mechanical mass transfer. The strains were then screened for use in fermented meat matrices based on their a* value and sensory evaluation. Finally, the strains were applied to duck breast mince, and the color-developing effect of low-frequency ultrasound treatment was verified through color-related physicochemical indicators (nitrosohemoglobin content, UV-Vis spectrum, nitrite content, etc.) and quality indicators. Through this application, we aim to clarify the effect of low-frequency ultrasound treatment on enhancing the enzyme activity of Staphylococcus saprophyticus HLMY980 NOS and understand the influence of the strains on the color and quality of duck meat. This will provide scientific evidence and theoretical support for the application of Staphylococcus aureus.

[0008] On the one hand, the present invention provides a method for improving the expression of nitric oxide synthase in Staphylococcus saprophyticus using low-frequency ultrasound, the steps of which are: treating the bacterial culture of Staphylococcus saprophyticus HLMY980 with low-frequency ultrasound, thereby improving the expression of nitric oxide synthase in Staphylococcus saprophyticus.

[0009] Preferably, the frequency of the ultrasonic treatment is 30-50 kHz, the power is 180-220 W, the total time of the ultrasonic treatment is 5-12.5 min, the number of ultrasonic treatments is 2-3, the interval time is 7-9 h, and the temperature of the ultrasonic treatment is 35-38℃.

[0010] Preferably, the preservation number of the above-mentioned *Staphylococcus saprophyticus* HLMY980 is CCTCC M 2026518; the concentration of the bacterial solution used is 10. 5 -10 7 CFU·mL -1 .

[0011] Preferably, the aforementioned low-frequency ultrasound can prolong the logarithmic growth phase of Staphylococcus saprophyticus and increase the activity of nitrite reductase and myoglobin reductase in the cells of Staphylococcus saprophyticus.

[0012] Before ultrasound, the logarithmic growth phase of *Staphylococcus saprophyticus* HLMY980 ranged from 10 h to 28 h; after ultrasound, the logarithmic growth phase ranged from 4 h to 48 h. λbefore = 4.48796 > λafter = -1.5928, indicating that the lag phase of *Staphylococcus saprophyticus* HLMY980 was significantly shortened by 6 h after ultrasound.

[0013] At 0 h, no nitrite was detected in any group, indicating that there was no nitrite in the initial culture medium. The nitrite content in the ultrasonic treatment group increased sharply in the first 32 h, reaching a peak at 32 h, where the NiR enzyme activity was 0.069 (μmol·h⁻¹). -1 ·10 4 cell); NiR activity in the non-ultrasound group increased in the first 28 h, with a peak value of 0.0411 (μmol·h). -1 ·10 4 (cell). NiR enzyme activity was enhanced by 67.88% by ultrasound. The MRA activities of both the ultrasound-treated and untreated groups showed an increasing trend in the early stages of culture, reaching a maximum of 25 U / mL at 24 h. -1 15.74 U·mL -1 Ultrasound enhanced the MRA activity of Staphylococcus aureus by 58.83%.

[0014] The highest NOS enzyme activity of Staphylococcus was observed after 12.5 min of sonication, with an enzyme activity of 1.480. The highest enzyme activity was observed 8 h after two sonication treatments, with an enzyme activity of 1.803 under this condition, which was 80.34% higher than that of the control group and 21.87% higher than that of sonication treatment without interval.

[0015] After ultrasound treatment, differentially expressed genes were mainly enriched in metabolic pathways, with the highest enrichment levels in carbohydrate metabolism and amino acid metabolism, suggesting their core role in the regulation of basic substances and energy metabolism. Highly enriched BP entries were mainly concentrated in the positive regulation of RNA metabolism and transcription (Rich Factor close to 1.0), including positive regulation of RNA metabolism, DNA template transcription, and nitrogenous base compound metabolism. Under low-frequency ultrasound conditions, the nos gene associated with NOS enzyme in *Staphylococcus saprophyticus* HLMY980 showed a significant upregulation, with nos gene expression increasing by 1.14-fold. This indicates that the increased NOS enzyme activity in *Staphylococcus saprophyticus* HLMY980 was not due to nos gene overexpression alone, but rather the result of multiple mechanisms, including enhanced transcriptional regulation, systemic metabolic remodeling, optimization of the cellular microenvironment, and synergistic quorum sensing.

[0016] Secondly, this invention applies to protect the fermentation broth of ultrasonically treated Staphylococcus saprophyticus HLMY980 prepared by the above method.

[0017] Thirdly, this invention also applies for protection of the application of the above-mentioned fermentation liquid in improving the color of fermented meat products. The application steps are as follows: after processing the meat products, cut them into pieces, mince them using a meat grinder, add the fermentation liquid to the minced meat, stir evenly, and ferment.

[0018] Preferably, the amount of the above-mentioned fermentation broth added is 0.5-1.5 wt% of the minced meat, and the concentration of the fermentation broth is 10. 5 -10 7 CFU·mL -1 .

[0019] Preferably, the screen plate of the above-mentioned meat grinder has a hole diameter of 6-10 mm, the temperature of the minced meat does not exceed 6-8℃, the fermentation temperature is 4℃, and the fermentation time is 6-24 h.

[0020] Preferably, the above-mentioned minced meat is any one of pork minced meat, duck minced meat, and beef minced meat.

[0021] Preferably, the above-mentioned fermentation broth can increase the redness and yellowness values ​​of meat products, promote NO-Mb formation, and thus improve the color of fermented meat products. There was no significant difference in the ability of nitrite and the S900 strain to increase the redness (a*) and yellowness (b*) of duck meat.

[0022] In the beef matrix, the redness values ​​of all strain groups were significantly higher than those of the control group at the beginning, with the S900 group and the BS group showing the most outstanding effects. During fermentation, the redness decreased, but the decrease was slower for all inoculated strain groups. At 24 h, the S900 group had the highest redness and the best color development ability.

[0023] In the duck meat matrix, at 0 h, the redness values ​​of all inoculated strain groups were slightly higher than those of the control group, but the difference was not significant; the redness values ​​increased significantly from 6 to 12 h, and the S900 group and the BS group reached their peak values ​​at 12 h, with no significant difference between them. With the increase of fermentation time, there was no significant difference in the color development effect between the S900 group and the N900 group, and both were better than the BS group.

[0024] In the pork matrix, the S900 group of minced meat had the best redness value at the beginning, and the color development effect was the same as that of the duck meat group. The redness value continued to rise from 6 to 12 hours and reached the peak at 12 hours. When fermented to 18 hours, the S900 group still maintained the highest redness value and the color development effect was better than the BS group and the N900 group.

[0025] The critical period for reducing the redness value of the minced meat was 0-6 hours, during which the a* values ​​of each group showed significant differences. The color development advantage of the S900 group was evident in the early stage of fermentation. From 6 to 24 hours, the rate of decrease in a* values ​​in each group tended to level off, but the S900 group still maintained a significantly higher redness value than the other groups. This indicates that the ultrasonically treated Staphylococcus saprophyticus HLMY980 exhibited better color development than the BS and N900 groups.

[0026] Strain S900 can significantly increase the content of nitrosoheme in fermented meat without increasing nitrite residue, while ultrasonic treatment of Staphylococcus saprophyticus HLMY980 is more effective than commercial starter cultures.

[0027] The nitrite group showed a strong characteristic absorption peak at 400 nm and exhibited a typical double peak in the visible region of nitrosomyoglobin (NO-Mb) at 540 nm and 575 nm, indicating the presence of a high concentration of NO-Mb in this group. The spectral morphology of the BS and S900 groups was consistent with that of the nitrite group, but their overall absorbance was lower than that of the nitrite group, indicating that the chromogenic substances formed in the BS and S900 groups were the same as those in the nitrite group. This verified at the molecular level that the S900 and BS strains promoted the formation of NO-Mb, and that the NO-Mb concentration in the S900 group was significantly higher than that in the BS group.

[0028] The present invention has the following beneficial effects:

[0029] 1. This invention employs low-frequency ultrasound technology to treat the NOS activity of *Staphylococcus saprophyticus* HLMY980. After optimizing the ultrasound process (total time 5-12.5 min, 2-3 ultrasound cycles, with an interval of 8 h), the NOS enzyme activity of the strain was significantly increased by 80.34% without causing order-of-magnitude lethality. Ultrasonic treatment prolonged the logarithmic growth phase of strain HLMY980 and enhanced NiR and MRA activities.

[0030] 2. This invention uses ultrasonically treated strains for the production of fermented meat products. Different varieties, parts and forms of meat substrates are inoculated and fermented. Based on the redness value and sensory evaluation results, duck breast mince was selected as the final fermentation substrate. After fermentation, the duck mince has an excellent pink color and the effect is better than commercial fermentation agents. Nitrosomoglobin is generated, which confirms that this application achieves the biocolorization of meat products through the endogenous NO pathway.

[0031] 3. This invention elucidates the molecular mechanism by which low-frequency ultrasound enhances NOS enzyme activity through transcriptome sequencing. GO enrichment analysis results show that differentially expressed genes are mainly enriched in the positive regulation of RNA metabolism and transcription, including positive regulation of RNA metabolism, DNA template transcription, and the metabolism of nitrogenous base compounds. KEGG enrichment analysis results show that the endoplasmic reticulum protein processing pathway has the highest enrichment factor and significance, making it the core enrichment pathway for differentially expressed genes. Furthermore, the NOS gene shows significant upregulation, with an upregulation of 1.14-fold. In summary, this application has significant practical and industrial value for achieving nitrite reduction and substitution in fermented meat products, improving product safety, and promoting the green upgrading of the industry. Attached Figure Description

[0032] 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 some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0033] Figure 1 The process for treating Staphylococcus saprophyticus HLMY980 using low-frequency ultrasound is shown in Figure a. Figure a represents the determination of ultrasound duration, figure b represents the determination of ultrasound interval duration, and figure c represents the determination of the number of ultrasound cycles. Different lowercase letters indicate significant differences between the two groups. P <0.05), the same applies below.

[0034] Figure 2 The survival of Staphylococcus saprophyticus HLMY980 after low-frequency ultrasound treatment is shown in Figure a, where ultrasound duration is shown in Figure a, ultrasound interval is shown in Figure b, and number of ultrasound cycles is shown in Figure c.

[0035] Figure 3 The growth curves of Staphylococcus saprophyticus HLMY980 48 h before and after ultrasound are shown.

[0036] Figure 4 The changes in enzyme activity of Staphylococcus saprophyticus HLMY980 before and after ultrasound were shown; Figure a shows the activity of metmyoglobin reductase (MRA) and Figure b shows the activity of nitrite reductase (NIR).

[0037] Figure 5 The effect of fermentation strains on the redness value of minced meat within 24 h is shown in Figure a, where the redness value of beef is shown in Figure b, the redness value of duck meat is shown in Figure c, and the redness value of pork is shown in Figure c.

[0038] Figure 6 The values ​​represent the redness values ​​of fermented meat matrices; Figures a and e show the changes in redness values ​​of beef, b and f show the changes in redness values ​​of duck, and c and g show the changes in redness values ​​of pork.

[0039] Figure 7 Box plot for sensory evaluation of fermented meat substrate.

[0040] Figure 8 Fermented duck breast mince L*, a*, b* result.

[0041] Figure 9 The values ​​are nitrosoheme (a) and nitrite content (b).

[0042] Figure 10 The UV-Vis spectrum of nitrosomyoglobin.

[0043] Figure 11The sensory evaluation results of fermented duck breast mince before and after cooking are shown in Figure a. Figure a shows the sensory evaluation results before cooking, Figure b shows the sensory evaluation results after cooking, and Figure c shows the actual product.

[0044] Figure 12 The quality analysis of fermented duck breast mince is shown in Figure a, where pH value of fermented duck breast mince is shown in Figure a, total bacterial count of fermented duck breast mince is shown in Figure b, and thiobarbituric acid content of fermented duck breast mince is shown in Figure c.

[0045] Figure 13 Venn analysis of genes expressed between two samples (a), correlation coefficient heatmap (b), and PCA analysis plot (c).

[0046] Figure 14 A volcano plot visualizes differentially regulated genes among samples; up: indicates upregulated genes (red); down: indicates downregulated genes (blue); gray indicates indifferential genes.

[0047] Figure 15 GO annotation for differentially expressed genes.

[0048] Figure 16 A bar chart annotating the KEGG Pathway for differentially expressed genes across transcriptomes.

[0049] Figure 17 Bubble charts for GO enrichment analysis (a) and KEGG enrichment analysis of sample DEGs (b). Note: Richfactor: enrichment factor; P adjust: confidence level. The size of the vertical axis is directly proportional to the significance of the pathway on the horizontal axis, and the redder the color, the deeper the enrichment. Detailed Implementation

[0050] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. 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 of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0051] Unless otherwise specified, the experimental methods used in the following experimental examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0052] This invention utilizes low-frequency ultrasound technology to enhance the activity of Staphylococcus saprophyticus HLMY980 NOS through cavitation and mechanical mass transfer. The strains were then screened for use in fermented meat matrices based on a* values ​​and sensory evaluation. Finally, the strains were applied to duck breast mince, and the color development effect of low-frequency ultrasound treatment was verified through color-related physicochemical indicators (nitrosohemoglobin content, UV-Vis spectrum, nitrite content, etc.) and quality indicators. Through this study, we aim to clarify the effect of low-frequency ultrasound treatment on enhancing the enzyme activity of Staphylococcus saprophyticus HLMY980 NOS and understand the influence of the strains on the color and quality of duck meat. This will provide scientific evidence and theoretical support for the application of Staphylococcus aureus.

[0053] 1. Main reagents used in this application

[0054] Nutrient agar (NA), Luria-Bertani (LB) broth, Qingdao Haibo Biotechnology Co., Ltd.; S0025 Nitric Oxide Synthase Assay Kit, Shanghai Beyotime Biotechnology Co., Ltd.; BL1070B / 48T Nitrite Reductase Activity Assay Kit, Anhui Baisha Biotechnology Co., Ltd.; Commercial starter BactoFlavor ScarLet NO.3745020 (BS), mixed culture of Staphylococcus aureus and Staphylococcus calf, Novonesis, a multinational biotechnology company.

[0055] 2. The main equipment and instruments used in this application are shown in Table 1.

[0056] Table 1 Main Instruments and Equipment

[0057]

[0058] 3. Strains and culture conditions

[0059] Staphylococcus saprophyticus HLMY980 was streaked from a -80°C glycerol cryovial onto NA agar medium and incubated at 37°C for 24 hours. Single colonies were picked and incubated overnight (24 hours) in 100 mL LB broth.

[0060] Example 1: Determination of Low-Frequency Ultrasonic Processing Technology

[0061] The *Staphylococcus saprophyticus* HLMY980 bacterial suspension was ultrasonically stimulated using a SYU-10-200DT ultrasonic cleaner with a fixed ultrasonic frequency of 40 kHz and a fixed ultrasonic power of 200 W. Test tubes were placed in the center of the ultrasonic cleaner, 3 cm away from the bottom. The ultrasonic water bath temperature was controlled at 37℃ using cooling water circulation. The bacterial suspension without ultrasonic treatment served as a control group to investigate the effects of different ultrasonic stimulation parameters on the NOS activity of *Staphylococcus saprophyticus* HLMY980.

[0062] (1) Effect of ultrasonic treatment time on NOS activity of Staphylococcus saprophyticus HLMY980

[0063] The HLMY980 bacterial suspension of Staphylococcus saprophyticus, which had been cultured for 24 hours, was sonicated for specific time periods of 5, 7.5, 10, 12.5, 15, 17.5, and 20 minutes.

[0064] (2) Effect of ultrasonic treatment time interval on NOS activity of Staphylococcus saprophyticus HLMY980

[0065] The HLMY980 culture of Staphylococcus saprophyticus, which had been cultured for 24 hours, was sonicated for a total of 12.5 minutes. Two sonication sessions were performed, with the intervals between the sonication sessions being 0 h, 2 h, 4 h, 6 h, 8 h, 10 h, and 12 h.

[0066] (3) Effect of the number of ultrasonic treatments on the NOS activity of Staphylococcus saprophyticus HLMY980

[0067] The total duration of ultrasonic fixation of the 24-hour cultured Staphylococcus saprophyticus HLMY980 bacterial suspension was 12.5 min, with ultrasonic sessions spaced 8 h apart, and the number of ultrasonic sessions selected was 1, 2, 3, or 4.

[0068] (4) Determination of relative enzyme activity of Staphylococcus saprophyticus HLMY980 NOS after ultrasonic treatment

[0069] The relative NOS activity of Staphylococcus saprophyticus 900 was determined according to the instructions of the S0025 nitric oxide synthase assay kit. The DAF-FM DA probe bound to NO produced by NOS, and fluorescence detection was performed using a microplate reader. The fluorescence intensity was evaluated and calculated, and the amount of NO produced reflected the relative NOS activity before and after ultrasonic stimulation.

[0070] (1)

[0071] The ultrasonic treatment process was determined based on the activity of nitric oxide synthase (NOS), the major chromogenic enzyme of *Staphylococcus saprophyticus* HLMY980. Compared with the control group, the NOS enzyme activity of *Staphylococcus* increased by 9.67%, 22.15%, 25.33%, and 47.98% after ultrasonic treatment for 5 min, 7.5 min, 10 min, and 12.5 min, respectively. The highest NOS enzyme activity was observed after 12.5 min of ultrasonic treatment, with an activity of 1.480 (…). Figure 1a) This may be because low-frequency ultrasound increases cell permeability by creating transient pores in the cell membrane, which enhance transmembrane transport and the secretion of metabolites such as enzymes. However, with increasing ultrasound duration, the NOS enzyme activity of *Staphylococcus saprophyticus* HLMY980 drops sharply. Excessive exposure of *Staphylococcus* to ultrasound can cause severe shock waves and mechanical stress to microbial cells, leading to negative or destructive modifications that affect enzyme production.

[0072] Based on the ultrasound time results, staphylococcal cultures that had reached the logarithmic growth phase were subjected to two ultrasound sessions, with a total ultrasound duration of 12.5 min. The ultrasound interval results are as follows: Figure 1 As shown in b, the enzyme activity was highest at an interval of 8 hours between the two ultrasound treatments, reaching 1.803, an increase of 80.34% compared to the control group and 21.87% compared to ultrasound treatment without interval. Further extending the interval reduced the NOS enzyme activity of Staphylococcus aureus, possibly due to the weakening effect of ultrasound stimulation caused by the extended interval, leading to a decrease in enzyme activity. This is because low-intensity ultrasound generates stable cavitation and provides repairable damage to cells, altering the survival state of microbial cells and leading to accelerated bacterial proliferation and increased metabolic products.

[0073] Based on the established ultrasonic treatment duration of 12.5 min and interval of 8 h, the number of ultrasonic treatments was determined again. For example... Figure 1 As shown in c, there was no significant difference in NOS enzyme activity of Staphylococcus aureus under conditions of 2 and 3 ultrasound stimulations. However, the enzyme activity growth effect decreased with the increase of stimulation times, which is because repeated stimulation can damage the cell membrane.

[0074] (5) Survival analysis of Staphylococcus saprophyticus HLMY980 after ultrasonic treatment

[0075] The bacterial colonies in the ultrasonically treated bacterial suspension samples were counted using the spread method.

[0076] Low-frequency ultrasound can cause the dispersion of growing cell aggregates, thereby increasing the surface-to-volume ratio. The highest Staphylococcus colony count was observed after 7.5 min of ultrasound treatment, reaching 7.80 lg (CFU·mL⁻¹). -1 The level increased by approximately 7.59 × 10⁻⁶ compared to the control group. 7 lg(CFU·mL) -1 With prolonged sonication, the total bacterial count decreased slightly, reaching 7.53 × 10⁻⁶ after 20 minutes of sonication. 7 lg(CFU·mL) -1 There was no significant difference compared to the control group. Figure 2 a).

[0077] Within the interval from 0 h to 4 h, as the ultrasound interval increased, Staphylococcus aureus was inactivated due to the cavitation and mechanical effects of ultrasound, and the total colony count showed a decreasing trend, reaching a minimum of 7.48 × 10⁻⁶ at an ultrasound interval of 4 h. 7 lg(CFU·mL) -1 There was no significant difference between the control group and the control group; however, with further extension of the interval, the acoustic cavitation caused by sonochemical and acoustic physical effects caused repairable damage to bacterial cells, such as increased cell membrane permeability, thereby accelerating the excretion of cellular waste and the membrane transport of necessary substrates, and thus promoting cell growth. At a 12-hour interval, the total colony count increased to 7.75 lg (CFU·mL⁻¹). -1 The level increased by 7.52 lg (CFU·mL) compared to the non-ultrasound group. -1 () Figure 2 b).

[0078] Furthermore, compared with the control group, both two and three ultrasound sessions significantly increased the number of Staphylococcus colonies, reaching 7.74 lg (CFU·mL⁻¹). -1 ) and 7.80 lg (CFU·mL) -1 The increase was 7.50 lg (CFU·mL). -1 ) and 7.60 lg (CFU·mL) -1 The decrease in colony count after four sonications may be related to the fact that prolonged sonication time caused physical damage to Staphylococcus cells, leading to leakage of cell contents and ultimately cell death. Figure 2 c).

[0079] (6) Effect of ultrasonic treatment on the growth of Staphylococcus saprophyticus HLMY980

[0080] The growth curves of *Staphylococcus saprophyticus* HLMY980 before and after ultrasonic treatment were determined using turbidimetric assay. Seed culture was inoculated into LB broth at a volume fraction of 1% (φ) and cultured at 37℃ for 48 h. The OD of the bacterial culture was measured using a microplate reader. 600 .

[0081] From Table 2 and the growth curve ( Figure 3 The determination coefficient R of the growth curve fitting equation before and after ultrasound can be obtained. 2The values ​​were all above 0.95, indicating that the Gompertz model can well describe the growth of *Staphylococcus saprophyticus* HLMY980 before and after ultrasound. Before ultrasound, the logarithmic growth phase of *Staphylococcus saprophyticus* HLMY980 was 10 h–28 h; after ultrasound, the logarithmic growth phase was 4 h–48 h. λbefore = 4.48796 > λafter = -1.5928, indicating that the lag phase of *Staphylococcus saprophyticus* HLMY980 was significantly shortened by 6 h after ultrasound. The increase in Pm after ultrasound enhanced the growth capacity of the strain. Low-intensity ultrasound produces stable cavitation and causes repairable damage to the cells, thereby altering the bacterial state, promoting the growth and replication of *Staphylococcus saprophyticus*, and accelerating its proliferation. However, Rm after ultrasound (0.04323) < Rm before ultrasound (0.07968), indicating a decrease in the growth rate and an increase in the logarithmic growth phase.

[0082] Table 2. Growth curve fitting of Staphylococcus saprophyticus HLMY980 before and after ultrasound.

[0083]

[0084] (7) Effect of ultrasonic treatment on enzyme activity of Staphylococcus saprophyticus HLMY980

[0085] ① Methemoglobin reductase (MRA) activity

[0086] The reaction system consisted of 0.3 mL of 5.0 mM EDTA, 0.3 mL of 50 mM phosphate buffer (pH 7.0), 0.3 mL of 3.0 mM K4Fe(CN)6, 0.3 mL of deionized water, 0.6 mL of 0.75 mM MetMb, 0.9 mL of crude enzyme extract, and 0.3 mL of 2 mM NADH. NADH was added to initiate the reaction. The control group did not receive NADH. Absorbance was measured at 580 nm, recorded every 5 seconds for a total duration of 1 min. MRA activity units were defined as the amount of enzyme required to convert 1 nmol of MetMb per minute, calculated using the following formula:

[0087] (2)

[0088] In the formula: ΔA represents the absorbance change at 580 nm, V1 represents the volume of the reaction system (ML), and ε represents the difference in molar extinction coefficients, 12 × 10⁻⁶. 3 (L·(mol·cm)) -1 V2 represents the sample volume (mL), L represents the optical path of the cuvette (Cm), and m represents the mass of myoglobin (mg).

[0089] ② Nitrite reductase (NIR) activity

[0090] Nitrite reductase activity (NIR) was determined using a nitrite reductase activity assay kit on crude bacterial enzyme solutions. The unit of NIR activity was defined as a reduction of 1 μmol NO per milliliter of bacteria per hour. 2− The amount.

[0091] Based on the above results, Staphylococcus saprophyticus HLMY980 samples were collected at 0, 8, 10, 16, 24, 28, 32, 40, and 48 hours before ultrasound; and Staphylococcus saprophyticus HLMY980 samples were collected at 0, 4, 8, 16, 24, 32, 40, and 48 hours after ultrasound to measure the enzyme activity of Staphylococcus saprophyticus HLMY980 before and after ultrasound.

[0092] Nitric oxide (NO) produced by bacteria readily penetrates the cell membrane and is oxidized to nitrate or nitrite in the culture medium. Since the culture medium originally does not contain nitrite, the detected nitrite can only originate from NO produced by bacteria through the catalysis of nitric oxide synthase activity. Therefore, measuring nitrite concentration can indirectly reflect the level of NO production within the bacteria. However, nitrite reductase (NiR) is widely present in Staphylococcus aureus, which can reduce nitrite in the presence of a substrate and regenerate NO. Figure 4 As shown in Figure a. At 0 h, no nitrite was detected in any group, indicating that there was no nitrite in the initial culture medium. The nitrite content in the ultrasonically treated group increased sharply in the first 32 h, reaching a peak at 32 h, where the NiR enzyme activity was 0.069 (μmol·h⁻¹). -1 ·10 4 cell); NiR activity in the non-ultrasound group increased in the first 28 h, with a peak value of 0.0411 (μmol·h). -1 ·10 4 (cell). NiR enzyme activity was enhanced by 67.88% by ultrasound.

[0093] Nitric oxide should bind to the ferrous iron in hemoglobin, not the ferric iron, to form NO-Hb, thus giving it a red color. Therefore, before further binding with nitric oxide, the ferric iron in methemoglobin (Met-Hb) must be reduced to ferrous iron. Methemoglobin reductase (MRA) is an enzyme capable of reducing Met-Mb, composed of NADH-dependent cytochrome b5 reductase, cytochrome b5, and mitochondrial outer membrane cytochrome b. This enzyme promotes the transfer of two electrons from the cofactor NADH to the intermediate cytochrome b, which is then further transferred to Met-Mb, thereby initiating the reduction process of Met-Mb. Methemoglobin reductase may be synthesized during the stable growth phase of bacteria, suggesting a link between decreased enzyme activity and changes in electron transfer during substrate redox processes. Figure 4As shown in b, the MRA activity in both the ultrasonic-treated and untreated groups showed an increasing trend in the early stage of culture, reaching a maximum value of 25 U·mL at 24 h. -1 15.74 U·mL -1 Ultrasound enhanced the MRA activity of Staphylococcus aureus by 58.83%.

[0094] In summary, the activities of the two Staphylococcus aureus color-related enzymes showed an initial increase followed by a decrease during the growth of the strains, and ultrasound enhanced the activity of both enzymes. Therefore, when using ultrasound-treated Staphylococcus saprophyticus HLMY980 in a fermented meat paste system, it is necessary to appropriately control the fermentation time to maximize the strain's color development ability.

[0095] Example 2: Determination of Fermentation Process

[0096] A fermentation broth was prepared by mixing commercial starter culture BactoFlavor Scarlet NO.3745020 with water at a mass ratio of starter culture to water of 6.26:500, resulting in a cell concentration of 10. 9 CFU·mL -1 Staphylococcus saprophyticus HLMY980 was purified and inoculated into LB broth, then cultured at 37°C for 24 h. The experimental group of bacterial cultures was centrifuged, and the bacterial pellet was washed with physiological saline, resulting in a cell concentration of 102. 7 CFU·mL -1 Additives: 0.5% salt, 0.5% white sugar.

[0097] Blank control group A: 100 g minced meat, no exogenous additives;

[0098] Experimental group B (BS): Commercial starter culture (BS) was added;

[0099] Experimental group C (N900): Added unultrasonicated Staphylococcus saprophyticus HLMY980;

[0100] Experimental group D (S900): Added with ultrasonically treated Staphylococcus saprophyticus HLMY980.

[0101] Because chicken meat has a low myoglobin content, duck, pork, and beef were used as the meat substrates for Staphylococcus saprophyticus HLMY980 fermentation. The fermentation process was determined based on the redness value (a*) and color sensory evaluation of the meat substrates.

[0102] (1) Effect of fermentation strains on minced meat a* within 24 h

[0103] Skinless duck breast, pork hind leg, and beef shank were processed and cut into chunks, then passed through a meat grinder with an 8 mm mesh. After mixing with auxiliary materials, 1 wt.% of fermentation liquid was added to the minced meat, and the mixture was stirred for 5 minutes while maintaining a center temperature not exceeding 8°C. The minced meat was then placed in sterile sampling bags and marinated in a 4°C cold storage. Pork minced meat was marinated for 18 hours according to industrial processes, while duck and beef minced meat were marinated for 24 hours. The redness value of the minced meat during the marinating process was measured using a colorimeter.

[0104] The color development ability of different fermentation strains (commercial starter cultures BS, N900, and S900) varies significantly, and their effectiveness is influenced by the type of meat substrate. Beef substrate ( Figure 5 In (a), due to its high myoglobin content, the methemoglobin content increases with prolonged fermentation time, leading to decreased meat color stability and intensified browning. Initially, the redness values ​​of all strain groups were significantly higher than the control group, with the S900 and BS groups showing the most significant effects. During fermentation, the redness decreased in all groups, but the decrease was slower in all inoculated strain groups. At 24 h, the S900 group had the highest redness and the best color development ability. Duck meat substrate ( Figure 5 In (b), at 0 h, the redness values ​​of all inoculated strain groups were slightly higher than those of the control group, but the difference was not significant; the redness values ​​increased significantly from 6 to 12 h, and the S900 group and the BS group reached their peak values ​​at 12 h, with no significant difference between them. With increasing fermentation time, there was no significant difference in color development between the S900 group and the N900 group, and both were superior to the BS group. Pork substrate ( Figure 5 In c), the S900 group of minced meat had the best redness value at the beginning, and the color development effect was the same as that of the duck meat group. The redness value continued to rise from 6 to 12 hours and reached its peak at 12 hours. When fermented to 18 hours, the S900 group still maintained the highest redness value and the color development effect was better than that of the BS group and the N900 group.

[0105] The critical period from 0 to 6 hours was the reduction of redness value in the minced meat. Significant differences were observed in a* values ​​among the groups, with the S900 group showing a color development advantage in the early stages of fermentation. From 6 to 24 hours, the rate of decrease in a* values ​​in each group tended to level off, but the S900 group still maintained a significantly higher redness value than the other groups, indicating that the ultrasonically treated Staphylococcus saprophyticus HLMY980 exhibited better color development than the BS and N900 groups.

[0106] (2) Determination of fermented meat substrate

[0107] Five types of meat—duck breast, duck leg, pork hind leg, pork tenderloin, and beef shank—were selected and prepared into cubes and minced meat, respectively. They were then inoculated with bacterial strains for fermentation to investigate the effects of meat shape, variety, and part on the color development characteristics of the strains.

[0108] Commercial starter culture (BS) and ultrasound-treated Staphylococcus saprophyticus HLMY980 (S900) were inoculated into minced and block meat matrices from duck breast, duck leg, pork tenderloin, pork hind leg, and beef shank, and then compared with a blank control. The overall redness value of the meat matrices ( Figure 6 ) and sensory evaluation results ( Figure 7 It can be seen that the variety, part and temperature fluctuation all have a significant impact on the color of the meat matrix. Moreover, the BS group and the S900 group showed stable color development under different conditions, that is, the S900 group was better than the BS group and both were significantly better than the control group.

[0109] From the perspective of meat matrix varieties, pork and duck have muscle fiber patterns similar to beef, but due to differences in myoglobin content and basic characteristics, there are significant differences in redness values ​​and sensory performance among various meats. Beef has a high basic redness, and after fermentation with the inoculated strain S900, the color stability of the group was significantly improved, and its sensory quality was superior to the control group under all conditions. Pork has a moderate basic redness, and after fermentation with the inoculated strain, the redness of the pork matrix increased most significantly, resulting in a significant improvement in the overall sensory score. Duck has a low basic redness, but after fermentation with the strain, the redness of the duck meat was significantly improved, and it maintained good sensory stability after cooking. Overall, ultrasonic fermentation with Staphylococcus saprophyticus HLMY980 has a more direct effect on improving the color of pork and duck, while for beef, it mainly maintains color stability and optimizes the overall sensory performance.

[0110] From the perspective of part and morphology, different parts of the same meat exhibit variations in color after fermentation due to differences in myoglobin content. The most significant improvement in redness was observed in pork tenderloin and duck breast, resulting in higher sensory scores. Pork hind leg and beef shank showed more stable color and sensory characteristics after fermentation with bacterial strains. In terms of meat morphology, the changes in meat chunks and minced meat showed a similar trend. Minced meat, after processing, has a more uniform tissue structure, which is more conducive to the even distribution of bacterial strains compared to meat chunks, promoting the color-developing effect of the strains and resulting in a more uniform and consistent color distribution. Meat chunks, on the other hand, have an advantage in maintaining their color.

[0111] Based on the above two points, duck breast meat has a relatively low base redness and uniform meat paste shape and color. After ultrasonic fermentation with Staphylococcus saprophyticus HLMY980, the redness is significantly improved, and the sensory quality improvement effect is intuitive and stable. Therefore, duck breast meat paste was ultimately selected as the fermentation substrate.

[0112] (3) The effect of fermentation temperature fluctuation on the color of meat substrate

[0113] The experimental group of meat substrate was placed at 4℃ for 18 h and then at 32℃ for 1 h to investigate the effect of temperature fluctuation on the color development characteristics of the strain.

[0114] Temperature changes significantly affect the color and sensory quality of meat matrix. Under low-temperature conditions, the control group showed browning and a decline in sensory quality, while fermentation with the strain effectively enhanced the redness of the meat. At 32℃, the increased temperature accelerated the biochemical reactions and bacterial growth of the meat matrix, leading to a significant decline in the redness and sensory quality of the control group. However, the fermented groups, especially the S900 group, maintained stable color and sensory performance. Sensory evaluation results ( Figure 7 As can be seen, compared with the control group, the BS group and the S900 group maintained higher scores after 15 minutes of high-temperature cooking. Because the fermentation of the color-developing strains can increase the heat-stable nitrosochromogen in the meat matrix, the sensory results of the meat fermented by both strains after heat processing were superior to the blank control group. Among them, the S900 group showed the most outstanding effect in retaining the redness of the meat matrix, significantly better than the BS group and the control group.

[0115] Example 3: Color Analysis of Fermented Duck Breast Mince

[0116] (1) Sample preparation

[0117] Skinless duck breast meat was processed using an 8 mm perforated plate meat grinder, with the addition of 0.5% salt and 0.5% white sugar, and then inoculated with a fermentation strain for fermentation. The fermentation process groups are shown in Table 3.

[0118] Table 3 Fermentation process grouping

[0119]

[0120] (2) Determination of color difference in fermented minced meat

[0121] The L*, a*, and b* values ​​were measured using a colorimeter. Five points were taken from each sample group, and the average value was recorded.

[0122] The effects of different treatments on the color of fermented duck meat are as follows: Figure 8 As shown, there was no significant difference in L* values ​​among the treatment groups ( Figure 8 a, P>0.05), indicating that the treatment method had a weak effect on the brightness of duck meat. The a* value was significantly highest in the nitrite group, followed by the S900 group; both were significantly higher than the control group and the BS group ( Figure 8 b, P<0.05, indicating that there was no significant difference in the ability of nitrite and S900 strain to increase the redness of duck meat. Regarding b* values, there was no significant difference between the nitrite group and the S900 group, but the values ​​were significantly higher than those of the control group and the BS group. Figure 8 (c, P<0.05), indicating that both can increase the yellowness of duck meat.

[0123] (3) Fermented minced meat nitrosoheme

[0124] Weigh approximately 4.0 g of minced meat sample, add 18 mL of 75% acetone solution, mix thoroughly, and let stand for 10 minutes in the dark. Measure its absorbance (A) at a wavelength of 540 nm. 540 ), and calculate the content of nitroso pigments according to formula (3). The content of nitrosoheme is expressed in ppm.

[0125] (3)

[0126] (4) Nitrite content in fermented minced meat

[0127] Mix the chopped sample (1 g) with distilled water (30 mL) and heat in a boiling water bath for 20 minutes. Then filter through filter paper and dilute with distilled water to a final volume of 50 mL. The filtrate (20 mL) is then mixed with 1 mL of sulfonic acid (30 mmol·L⁻¹). -1 ) and 1 mL of N-(1-naphthyl)ethylenediamine dihydrochloride (5 mmol·L) -1 Mix thoroughly. After thorough mixing, dilute with deionized water to 25 mL. Allow the reaction mixture to stand at room temperature (25 ± 1 °C) for 20 minutes. The absorbance of the sample solution was measured at 540 nm, and the nitrite content in the sample was determined using a standard curve prepared with sodium nitrite, y = 0.0221x + 0.02, R0. 2 =0.9996, calculate. The result is expressed in mg·kg⁻¹. -1 express.

[0128] Nitrosamine and nitrite content in fermented duck breast mince: The effects of different treatments on the residual amounts of nitrosamine and nitrite in fermented duck meat. Figure 9 As shown, the nitrosoheme content was significantly highest in the nitroso group, followed by the S900 group, which was significantly higher than both the BS group and the control group. Figure 9 The results indicate that strain S900 can promote the formation of nitrosoheme, thereby improving the color of meat products. Regarding nitrite residue, the nitrite group was significantly higher than the other three groups (P<0.05), while there was no significant difference between the control group, the BS group, and the S900 group. Figure 9 (b, P>0.05). In conclusion, Staphylococcus can significantly increase the content of nitrosohematopoietics in fermented meat without increasing nitrite residues, and the effect of ultrasonic treatment of Staphylococcus saprophyticus HLMY980 is better than that of commercial starter cultures.

[0129] (5) Ultraviolet-visible spectrum of fermented minced meat nitrosomyoglobin

[0130] Absorption scans were performed at 1 nm intervals in the 350-700 nm range using UV-Vis spectrophotometry. Approximately 5.0 g of minced meat sample was weighed and added to 25 mL of pre-cooled 75% acetone solution. The mixture was homogenized for 1 minute using a homogenizer. The mixture was then placed on ice in the dark for 30 min, followed by centrifugation at 8,000 g for 10 min at 4 °C. The supernatant was filtered through a 0.45 μm filter membrane to obtain the pigment extract.

[0131] UV-Vis spectra of fermented duck meat from different treatment groups are as follows: Figure 10 As shown in the figure, the nitrite group exhibited a strong characteristic absorption peak at 400 nm and showed typical double peaks in the visible region of nitrosomyoglobin (NO-Mb) at 540 nm and 575 nm, indicating the presence of a high concentration of NO-Mb in this group. This result is consistent with the determination results of nitrosomyoglobin content and a* value. The spectral morphology of the BS and S900 groups was consistent with that of the nitrite group, but the overall absorbance was lower than that of the nitrite group, indicating that the chromogenic substances formed were the same as those in the nitrite group. This verifies at the molecular level that the S900 and BS strains promote the formation of NO-Mb, and the NO-Mb concentration in the S900 group was significantly higher than that in the BS group. The control group showed no obvious characteristic absorption peak and almost no NO-Mb formation was observed.

[0132] (6) Sensory evaluation of the color of fermented minced meat before and after cooking

[0133] Sensory evaluation of the fermented minced meat samples was conducted according to Table 4:

[0134] Table 4 Sensory Evaluation Table of Color

[0135]

[0136] The color uniformity and sensory evaluation results of fermented duck meat in different treatment groups are as follows: Figure 11 As shown in ab, Figure 11 a and b represent the sensory evaluation results. The Range value represents the color uniformity; the smaller the value, the more uniform the color.

[0137] The control group had the lowest Range value, while the nitrite group had the highest. The large data dispersion indicates that although the traditional nitrite process produces excellent color development, it has the worst color uniformity and is prone to localized uneven coloring. The Range values ​​of the S900 and BS groups were significantly lower than those of the nitrite group (P < 0.05), showing improved uniformity, but the sensory score of the BS group was lower than that of the S900 group. The Range value of the S900 group was significantly lower than that of the nitrite group (P < 0.05), with a concentrated data distribution and low dispersion, indicating that the S900 strain was evenly distributed in the duck mince, improving the uniformity and consistency of the meat color. (Image of actual product) Figure 11 c) This further validates the conclusions drawn from the aforementioned quantitative indicators.

[0138] (7) Quality analysis of fermented duck breast mince

[0139] ① pH value determination of fermented meat paste

[0140] pH value was determined according to the national standard GB 5009.237-2016 "National Food Safety Standard: Determination of pH Value in Food".

[0141] pH is a core indicator of the quality formation of fermented meat products, and its changes directly reflect the level of acid production by microbial metabolism. Results are as follows: Figure 12 As shown in Figure a, inoculation with fermentation strains (commercial starter culture and S900 strain) effectively promoted the acidification process of fermented meat paste and significantly reduced the pH value of the system. Furthermore, the acid-producing capacity of the commercial starter culture strain was slightly better than that of the S900 strain. The addition of nitrite could also reduce the pH value of the meat paste to some extent, but its acidification effect was weaker than that of the strain-inoculated group.

[0142] ② Determination of total bacterial count in fermented meat paste

[0143] The total bacterial count was determined according to GB 4789.2-2022, "National Food Safety Standard - Microbiological Examination of Food - Determination of Total Bacterial Count".

[0144] The total bacterial count results of fermented minced meat are as follows: Figure 12 As shown in b, inoculation with both commercial starter culture and S900 strain significantly increased the total bacterial count of fermented meat paste. The S900 strain exhibited superior proliferation compared to the strains in the commercial starter culture; however, the addition of nitrite had no significant effect on the total bacterial count of the fermented meat paste, showing no obvious antibacterial effect. Based on the above results, it can be concluded that after inoculation with the fermentation strain, the massive proliferation of the fermentation strain not only lowered the pH of the meat paste through metabolic acid production but also inhibited the growth of other microorganisms by competing for nutrients and occupying ecological niches, ultimately leading to a significant increase in the total bacterial count. This is a crucial characteristic of the successful colonization and dominance of the fermentation strain in the fermentation process.

[0145] ③ Determination of thiobarbituric acid value of fermented meat paste

[0146] The experiment was conducted in accordance with GB / T 35252-2017, "Direct Method for Determination of 2-Thiobarbituric Acid Value in Animal and Vegetable Oils".

[0147] Free radicals generated by lipid oxidation attack nitrosomyoglobin, causing it to oxidize and discolor, turning meat products from bright red to brown. TBA values ​​are shown below. Figure 12 As shown in c, the addition of nitrite can inhibit lipid oxidation in fermented meat paste. Commercial starter culture and ultrasonically treated Staphylococcus saprophyticus strain HLMY980 can also reduce lipid oxidation and alleviate oxidative deterioration in meat paste to some extent, but their oxidative inhibition effect is weaker than that of the nitrite-treated group.

[0148] Example 4: Preliminary investigation by transcriptomics into the mechanism by which low-frequency ultrasound enhances the activity of nitric oxide synthase in Staphylococcus saprophyticus HLMY980

[0149] Transcriptomic analysis of Staphylococcus saprophyticus HLMY980 before and after ultrasound:

[0150] After collecting the sonicated Staphylococcus saprophyticus HLMY980 cells in the logarithmic growth phase and the control group HLMY980 cells in the same logarithmic growth phase, all samples were transported to the designated testing center using dry ice. The unsonicated group served as the control group (Control group), and the sonicated group served as the experimental group (EG group).

[0151] This study used the Illumina HiSeq sequencing platform to sequence and analyze the constructed Staphylococcus saprophyticus HLMY980 library. The experiment included a control group (untreated strain) and an ultrasonic treatment group, with three biological replicates in each group. The sequencing data quality statistics are shown in Table 5. The raw data volume (Raw Bases) of the control group were 317,702,3994, 326,207,442, and 305,496,6768, respectively, while the raw bases of the experimental group were 362,821,7128, 365,901,3880, and 356,995,0758, respectively. The raw sequence count (Raw Reads) of the control group were 21,039,894, 21,603,142, and 0,231,568, respectively, while the raw read counts of the experimental group were 24,027,928, 24,231,880, and 23,642,058, respectively. After quality filtering, the control group obtained 20,998,474, 21,541,568, and 20,172,100 clean reads, respectively, while the experimental group obtained 23,957,936, 24,158,174, and 23,579,416 clean reads, respectively. Sequencing data quality control results showed that the proportions of Q20 and Q30 sequences were above 99% and 96%, respectively, and the original base error rate and the base error rate after quality control were both 0.01%, indicating that the sequencing data obtained in this study were of acceptable quality and reliable, meeting the requirements for subsequent bioinformatics analysis.

[0152] Table 5. Statistical comparison of sequencing data quality control before and after.

[0153]

[0154] Reference genome alignment was performed using the transcriptome analysis software Bowtie2. High-quality reads, after quality control filtering, were aligned using the Burrows-Wheeler algorithm. The alignment statistics for the two groups are shown in Table 6. The total reads for the control group were 20,998,474, 20,998,474, and 20,998,474, respectively, while those for the experimental group were 23,957,936, 24,158,174, and 23,579,416, respectively. The number of reads aligned to unique positions on the reference genome were 20,060,140, ​​20,060,140, ​​and 19,419,200 (control) and 23,289,399, 23,500,564, and 22,884,159 (experiment), respectively, with unique alignment rates of 95.53%, 95.71%, and 96.27% and 97.21%, 97.28%, and 97.05%, respectively. The number of unique alignment reads for all samples was greater than 1.7 × 10⁻⁶. 7 Furthermore, the overall unique alignment rate was relatively high, indicating that there was no obvious exogenous contamination in the sample, and the sequencing depth could meet the needs of subsequent gene expression quantitative analysis.

[0155] Table 6. Statistical Table of Comparison Results

[0156]

[0157] 1. Inter-sample expression level analysis:

[0158] Gene expression analysis among different samples is a crucial method for assessing the reliability of transcriptome data and the rationality of sample selection. Quantitative analysis of gene expression levels can reveal the changing trends of differential gene expression among different samples, further elucidating gene regulatory mechanisms. This study used Venn analysis to identify common and specific genes expressed between two samples; untreated *Staphylococcus saprophyticus* HLMY980 was designated as the control group (Control group), while *Staphylococcus saprophyticus* HLMY980 treated with low-frequency ultrasound was designated as the experimental group (EG group). Figure 13 As can be seen, the number of expressed genes in the Control group and the EG group were 2513 and 2591, respectively, with a total of 2495 co-expressed genes between the two groups; in addition, there were 18 genes specifically expressed in the Control group and 96 genes specifically expressed in the EG group.

[0159] Pearson correlation coefficient (R) was used for analysis. This serves two purposes: firstly, it verifies whether the samples meet the pre-designed experimental requirements; secondly, it provides a reference for subsequent differential gene analysis, thus clearly presenting the differential characteristics between samples. A Pearson correlation coefficient closer to 1 indicates a higher similarity in gene expression among the samples. A correlation heatmap is shown below. Figure 13 Results b showed a strong correlation in gene expression levels among replicates of the same sample, R 2 The values ​​were all greater than 0.97, indicating good intra-group reproducibility; however, there were significant differences in gene expression between the control group and the experimental group (EG), with the lowest value being 0.906 (A2-B1) and the highest being 0.956 (A1-B3), which were all lower than the intra-group correlation, indicating that there were significant differences in gene expression patterns between the two groups.

[0160] To further verify the rationality of sample grouping and the differential gene expression patterns, principal component analysis (PCA) was performed on all samples. The results are as follows: Figure 13 As shown in Figure c, the three-dimensional PCA results show that the first three principal components explained a total of 90.12% of the total gene expression variation, with PC1 contributing the most (70.97%) and serving as the main dimension distinguishing the two groups. The control and experimental (EG) groups showed a clear separation trend in three-dimensional space, with closely spaced repetitive samples within each group, indicating a significant difference in gene expression patterns between the two groups and good sample reproducibility.

[0161] In summary, the PCA analysis results further confirm the reliability of the transcriptome data and the rationality of the experimental design in this study. The selected data are reasonable and reliable and can be used for subsequent related analyses.

[0162] 2. Differentially expressed gene analysis among samples

[0163] In transcriptome sequencing analysis, RSEM software was used to calculate the TPM expression values ​​of all genes in the samples, and differentially expressed genes (DEGs) were identified using a P < 0.05 and |log2FC| ≥ 1 as the screening threshold. A total of 308 differentially expressed genes were identified in the control group (Control group) and the experimental group (EG group), of which 280 genes were upregulated (90.91%) and 28 genes were downregulated (9.09%). These differentially expressed genes may be involved in the core processes of *Staphylococcus saprophyticus* HLMY980 responding to low-frequency ultrasound stress and increased nitric oxide synthase activity. Volcano plots are a commonly used method for visualizing the distribution of differentially expressed genes in the transcriptome, and can intuitively present the overall characteristics of gene expression changes between the two groups of samples. Figure 14In the graph, the horizontal axis represents the log2 fold change (log2FC) after log2 transformation, and the vertical axis represents the statistical significance test value (-log10 (p value)). Each point in the graph represents a gene; the points on the left are downregulated genes, and the points on the right are upregulated genes. The farther the gene is from the origin and the closer it is to the upper end of the vertical axis, the more significant its expression difference. The volcano plot results show that the experimental group (EG group) had differentially expressed genes, and the number of upregulated genes was greater than the number of downregulated genes. This phenomenon may be due to the fact that ultrasound stimulation can significantly increase the gene expression of *Staphylococcus saprophyticus* HLMY980 under ultrasound environmental stress.

[0164] 3. Analysis of differentially expressed gene functions among samples

[0165] (1) GO annotation

[0166] Differentially expressed genes (DEGs) obtained from the GO database were functionally classified into three main categories: biological processes, cellular components, and molecular functions. Figure 15 See Table 7 for classification details. Genes are involved in six categories during biological processes, with the most abundant function being cellular processes (63 genes). These functional pathways are related to intracellular chemical reactions and various cellular functions and activities, through which cells respond to different physiological needs and environmental challenges. Among cellular components, only cellular anatomical entities and protein-containing complexes stand out; molecular functions involve eight categories, with binding and catalytic activity genes being the most numerous (57 genes each), indicating that various activities within *Staphylococcus saprophyticus* HLMY980 facilitate its adaptation to low-frequency ultrasound stress.

[0167] Table 7 Statistical Results of GO Annotations

[0168]

[0169] (2) KEGG annotation

[0170] KEGG metabolic pathway annotation results show that ( Figure 16Differentially expressed genes were primarily enriched in metabolic pathways, with the highest enrichment levels in carbohydrate and amino acid metabolism, suggesting their central role in the regulation of basic substance and energy metabolism. In the genetic information processing category, translation pathways were enriched with 9 genes, indicating that transcriptome differences mainly affect protein translation; environmental information processing was mainly focused on membrane transport and signal transduction, reflecting their function in substance exchange and environmental signal response. Under the cellular processes category, the prokaryotic pathway in cell communities was enriched with 7 genes, indicating their involvement in the regulation of prokaryotic population behavior; human disease and organismal system pathways showed only a small enrichment, suggesting that the functions of differentially expressed genes are biased towards basal metabolism and environmental adaptation.

[0171] 4. GO functional enrichment analysis of differentially expressed genes

[0172] To investigate the biological functions of differentially expressed genes in *Staphylococcus saprophyticus* HLMY980 under low-frequency ultrasound conditions, GO functional enrichment analysis was performed on all differentially expressed genes. Using P < 0.05 as the selection threshold, the top 20 enriched results were selected for visualization analysis. The results are shown below. Figure 17 Figure a shows the graph. The vertical axis represents GO functional entries, and the horizontal axis represents enrichment factors. Differentially expressed genes were significantly enriched at the biological process (BP), cellular component (CC), and molecular function (MF) levels. Using enrichment factors as the core indicator, highly enriched BP entries were mainly concentrated in the positive regulation of RNA metabolism and transcription (Rich Factor close to 1.0), including positive regulation of RNA metabolism, DNA template transcription, and nitrogenous base compound metabolism. At the CC level, there was only weak enrichment in membrane components. At the MF level, shikimate kinase activity, homologous protein binding, and phosphate transmembrane transport activity were the main enriched functions, suggesting that low-frequency ultrasound treatment may exert its effects by regulating transcription, interfering with cell wall homeostasis, and aromatic metabolic pathways.

[0173] 5. Differential gene KEGG pathway enrichment analysis

[0174] To reveal the metabolic pathway characteristics of differentially expressed genes in *Staphylococcus saprophyticus* HLMY980 between the control and experimental groups (EG) under nitrite stress, KEGG enrichment analysis was performed on the differentially expressed genes in the two groups, such as... Figure 17As shown in b. Pathway enrichment was performed based on the KEGG database. Using P≤0.05 as the screening criterion, the top 20 metabolic pathways with the highest significance were selected, and bubble charts were generated for visualization analysis. The results showed that the endoplasmic reticulum protein processing pathway had the highest enrichment factor and significance, making it the core enrichment pathway for differentially expressed genes. The pentose-glucuronide interconversion and carotenoid biosynthesis pathways also showed high enrichment significance. The quorum sensing pathway had the largest number of differentially expressed genes, indicating that intercellular signal regulation plays an important role in the response process. In addition, basic metabolic pathways such as purine metabolism and pyruvate metabolism, as well as genetic information processing pathways such as protein export and RNA degradation, also showed varying degrees of enrichment, collectively forming a functional regulatory network of differentially expressed genes. Most of these pathways are closely related to bacterial tolerance mechanisms to stress and mainly belong to the three categories of metabolism, genetic information processing, and environmental information processing in the KEGG classification.

[0175] 6. Analysis of nitric oxide synthase activity in Staphylococcus saprophyticus HLMY980

[0176] Transcriptomic analysis was used to preliminarily investigate the mechanism of enhanced nitric oxide synthase (NOS) activity in *Staphylococcus saprophyticus* HLMY980 under low-frequency ultrasound conditions. Specifically, the nos gene, associated with NOS enzyme, was significantly upregulated in *Staphylococcus saprophyticus* HLMY980 under low-frequency ultrasound conditions, with an upregulation of nos gene expression by 1.14-fold, indicating that NOS enzyme synthesis was promoted under these conditions. GO enrichment analysis showed that the positive transcriptional regulatory pathway was significantly activated; simultaneously, KEGG annotation results indicated an increase in gene abundance in genetic information processing pathways such as translation and protein folding. These changes collectively promoted efficient transcription and translation of the NOS gene and ensured the correct folding of newly synthesized proteins, thus providing sufficient conditions for enhanced enzyme activity. KEGG pathway annotation and enrichment analysis of differentially expressed genes revealed that pathways related to carbohydrate, amino acid, and coenzyme metabolism in *Staphylococcus saprophyticus* HLMY980 were significantly activated, while energy supply was enhanced. This provides a continuous and efficient supply of essential substrates, energy (ATP), and various coenzyme factors for the NOS-catalyzed reaction of L-arginine to nitric oxide, ensuring the efficient operation of the enzymatic reaction from a material and energy foundation. Furthermore, GO enrichment analysis showed that membrane component remodeling-related functions of *Staphylococcus saprophyticus* HLMY980 were activated, and KEGG enrichment analysis also highlighted antioxidant responses and protein processing pathways. These changes optimized the membrane localization and spatial conformation of NOS and effectively improved the intracellular antioxidant environment. The significant enrichment of the quorum sensing pathway indicates a synergistic mechanism based on quorum signaling between *Staphylococcus saprophyticus* HLMY980 cells. This pathway enhances the overall capacity of the strain at the quorum level, amplifying the biological effects of increased enzyme activity.

[0177] In conclusion, the increased NOS enzyme activity in Staphylococcus saprophyticus HLMY980 is not due to the single factor of nos gene overexpression, but rather to the combined effect of multiple mechanisms, including enhanced transcriptional regulation, systemic metabolic remodeling, optimization of the cellular microenvironment, and synergistic effects of quorum sensing.

[0178] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for enhancing the expression of nitric oxide synthase in Staphylococcus saprophyticus using low-frequency ultrasound, characterized in that, The steps are as follows: Low-frequency ultrasound treatment of the bacterial culture of Staphylococcus saprophyticus HLMY980 to increase the expression of nitric oxide synthase in Staphylococcus saprophyticus.

2. The method according to claim 1, characterized in that: The ultrasonic treatment frequency is 30-50 kHz, the power is 180-220W, the total ultrasonic treatment time is 5-12.5 min, the number of ultrasonic treatments is 2-3, the interval time is 7-9 h, and the ultrasonic treatment temperature is 35-38℃.

3. The method according to claim 2, characterized in that: The preservation number of the *Staphylococcus saprophyticus* HLMY980 is CCTCC M 2026518; the concentration of the bacterial solution used is 10. 5 -10 7 CFU·mL -1 .

4. The method according to claim 3, characterized in that: The low-frequency ultrasound can prolong the logarithmic growth phase of Staphylococcus saprophyticus and increase the activity of nitrite reductase and myoglobin reductase in the bacteria.

5. The fermentation broth of *Staphylococcus saprophyticus* prepared by the method according to any one of claims 1-4.

6. The application of the fermentation broth according to claim 5 in improving the color of fermented meat products, characterized in that, The steps are as follows: After processing the meat products, cut them into pieces, grind them into minced meat using a meat grinder, add fermentation liquid to the minced meat, stir well, and ferment.

7. The application according to claim 6, characterized in that: The amount of fermentation broth added is 0.5-1.5 wt% of the minced meat, and the concentration of the fermentation broth is 10. 5 -10 7 CFU·mL -1 .

8. The application according to claim 7, characterized in that: The screen plate of the meat grinder has a aperture of 6-10 mm, and the stirring temperature of the minced meat does not exceed 6-8℃; the fermentation temperature is 2-6℃, and the fermentation time is 6-24 h.

9. The application according to claim 7, characterized in that: The minced meat can be any one of pork mince, duck mince, or beef mince.

10. The application according to claim 9, characterized in that: The fermentation liquid can increase the redness and yellowness of meat products, promote the production of nitrosomyoglobin, and thus enhance the color of fermented meat products.

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