Enterococcus faecalis and use thereof

Abstract

The present application relates to a kind of enterococcus faecalis and its application.The enterococcus faecalis is preserved in China Microorganism Culture Collection Center on September 24, 2025, and the preservation number is CGMCC No.36054.The enterococcus faecalis CGMCC No.36054 has strong antioxidant capacity, and has strong tolerance to gastrointestinal fluid.In addition, the bacteria have strong diacetyl production capacity, and the diacetyl content in yogurt can be greatly improved by inoculating the bacteria after ultrasonic treatment, and other flavor substances in yogurt are also enriched.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a type of Enterococcus faecalis and its applications. Background Technology

[0002] Yogurt is a fermented dairy product made from pasteurized milk through fermentation with lactic acid bacteria. The metabolic differences among different strains of lactic acid bacteria can significantly regulate the formation and release of its flavor components. In pasteurized milk, sugars are metabolized by lactic acid bacteria to produce large amounts of lactic acid, acetaldehyde, and dimethylglyoxal; fats are hydrolyzed to form free fatty acids, precursors of methyl ketones, secondary alcohols, esters, and lactones; casein, through the action of proteolytic enzymes, produces a series of small molecule compounds, which are further converted into alcohols, aldehydes, and esters, thus forming the unique flavor of fermented milk.

[0003] Diacetyl is a major flavor compound in yogurt, imparting a creamy aroma. It can be obtained through natural extraction, chemical synthesis, and microbial fermentation. In recent years, driven by the "clean label" consumer trend in the food industry, research on reducing the use of exogenous food additives and enhancing the endogenous aroma production of lactic acid bacteria has become a key focus in the dairy sector. Currently, the academic community has used genomics, metabolomics, and other multi-omics technologies to elucidate the aromatic substance metabolic pathways of some lactic acid bacteria, clarifying the synthetic regulatory mechanisms of key flavor components such as diacetyl. However, research on how to "targetedly enhance" the efficiency of endogenous aroma production in lactic acid bacteria still lacks efficient, green, and scalable field intervention technologies.

[0004] Ultrasonic technology, with its advantages of being green, low-consumption, and sustainable, forms a thermo-ultrasonic synergistic system under temperature-controlled coupling. Through the synergistic effect of cavitation, mechanical vibration, and thermal effects, it can alter membrane permeability and activate key enzymes, significantly amplifying its potential to regulate microbial activity. Currently, research on the application of ultrasonic technology largely focuses on its impact on the physicochemical properties and functional activities of dairy products. However, systematic evidence regarding the effects of ultrasonic pretreatment on the flavor synthesis of lactic acid bacteria remains lacking. Summary of the Invention

[0005] The present invention aims to overcome the shortcomings of the prior art and provide Enterococcus faecalis and its applications.

[0006] To achieve the above objectives, the technical solution provided by this invention is as follows: The Enterococcus faecalis ( Enterococcus faecalisThis strain was deposited on September 24, 2025, at the China General Microbiological Culture Collection Center (CGMCC), accession number CGMCC No. 36054, located at Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China. Safety evaluation results showed that the strain is non-hemolytic, highly sensitive to antibiotics such as penicillin, ampicillin, and levofloxacin, and does not produce amines. This strain possesses excellent characteristics such as strong diacetyl production, strong antioxidant capacity, and strong tolerance to gastrointestinal fluids. The Enterococcus faecalis can be used to prepare diacetyl preparations.

[0007] The *Enterococcus faecalis* can be used to produce diacetyl by applying ultrasound treatment during the cultivation or fermentation of the *Enterococcus faecalis*. Preferably, the ultrasound parameters include 30-50°C, ultrasound power 100-350W, and ultrasound time 5-40 min. More preferably, the ultrasound parameters include ultrasound temperature 42°C, ultrasound power 150W, and ultrasound time 20 min.

[0008] The Enterococcus faecalis can be used to prepare high-yield diacetyl yogurt.

[0009] The present invention also provides a method for preparing high-diacetyl yogurt, the method comprising the following steps: (1) using reconstituted milk as a base material, inoculating with a suspension of the aforementioned Enterococcus faecalis pretreated by ultrasound; (2) fermenting at 30°C for 6 hours, followed by ripening at 4°C for 24 hours. Preferably, the inoculum amount of Enterococcus faecalis suspension is 4% (v / v).

[0010] Preferably, a commercial starter culture is also inoculated during fermentation, the commercial starter culture being composed of Streptococcus thermophilus and Lactobacillus bulgaricus in a mass ratio of 1:1.

[0011] The present invention also provides yogurt prepared by the above method, wherein the diacetyl content is not less than 22 μg / mL and the water holding capacity is not less than 65%. The present invention will be further described below.

[0012] This invention first screens high-diacetyl-producing lactic acid bacteria strains from a laboratory-preserved bacterial strain library. These strains are identified using 16S rDNA sequencing combined with Gram staining, and their safety and probiotic properties are systematically evaluated. Further, the influence of ultrasonic pretreatment on the diacetyl-producing ability of the target strains is investigated. Response surface methodology is used to optimize key parameters such as ultrasonic power, ultrasonic time, and ultrasonic temperature, aiming to clarify the feasibility of using ultrasonic technology to enhance endogenous flavor production in lactic acid bacteria. This provides a theoretical basis and technical support for the targeted flavor regulation and industrial production of clean-label yogurt. This invention innovatively combines ultrasonic pretreatment technology to establish a green, efficient, and chemical-residue-free method for high diacetyl production and its application system in yogurt, significantly improving the product's flavor and market acceptance. This invention applies ultrasound with specific parameters to Enterococcus faecalis in a fermentation system, enhancing the transcriptional activity of the diacetyl system genes, increasing cell membrane permeability, accelerating the flux of pyruvate to α-acetolactate, and thus promoting diacetyl synthesis. It also synergistically increases the activity of lactate dehydrogenase (LDH) and α-acetolactate synthase (ALS), promoting net diacetyl accumulation. Attached Figure Description

[0013] Figure 1 Screening and identification of diacetyl-producing strains: (a) shows the screening results of 40 diacetyl-producing strains; (b) shows the Gram microscopic image of Enterococcus faecalis L402; (c) shows the phylogenetic tree of Enterococcus faecalis L402 constructed based on the 16S rDNA sequence.

[0014] Figure 2 Characterization of Enterococcus faecalis L402: (a) shows the growth curve (OD) of strain L402 in M17 liquid medium. 600 (a) shows the acid production curve (pH value); (b) shows the survival rate of Enterococcus faecalis L402 in artificial gastric and intestinal fluids.

[0015] Figure 3 Safety evaluation of Enterococcus faecalis L402 of the present invention: hemolysis test results.

[0016] Figure 4 The effects of single-factor experiments on the synthesis of diacetyl by Enterococcus faecalis L402: (a) the effects of ultrasonic power, (b) ultrasonic time and (c) ultrasonic temperature on diacetyl yield.

[0017] Figure 5 Interaction analysis of the process for optimizing diacetyl yield using response surface methodology: Three-dimensional response surface plots of the interaction of various factors reflect the interactive effects of ultrasonic power, ultrasonic time, and ultrasonic temperature on diacetyl yield. Figure 6Rheological and water-holding properties of yogurt: (a), (b), and (c) show the curves of apparent viscosity as a function of shear rate; (d) shows the results of water-holding capacity measurement of yogurt.

[0018] Figure 7 Results of non-targeted GC-MS analysis: (a) Principal component analysis score plot; (b) Flavor classification plot.

[0019] Figure 8 The following are the metabolic analysis diagrams for ultrasound and non-ultrasound: (a) cluster heatmap; (b) flavor comparison radar chart; (c) metabolic difference volcano chart; (d) inter-group differential metabolite cluster statistical chart; (e) comprehensive sensory evaluation score of yogurt. Detailed Implementation

[0020] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. These embodiments are based on the technical solution of the present invention and provide detailed implementation methods and specific operating procedures. However, the scope of protection of the present invention is not limited to the following embodiments. Example 1

[0021] This invention screened and identified a high-diacetyl-producing strain L402, which was classified and named Enterococcus faecalis ( Enterococcus faecalis ). (1) Screening and isolation of lactic acid bacteria Take 10g of milk sample, add 90mL of 0.9% physiological saline, homogenize, and then perform 10-fold serial dilutions. Select appropriate dilutions and spread them on M17 solid medium (tryptone 2.5g / L, beef extract 5.0g / L, yeast extract 2.5g / L, MgSO4 0.25g / L, meat peptone 2.5g / L, soybean peptone 5.0g / L, lactose 5.0g / L, sodium β-glycerophosphate 19.0g / L, ascorbic acid 0.5g / L, agar 15.0g / L, pH 6.9±0.2), and incubate at 37℃ for 36-48h. Select large and fast-growing strains for streaking purification, subculture three times to obtain single colonies, inoculate them on M17 liquid medium, incubate at 37℃ for 16-24h, and store at 4℃ for later use. (2) Initial screening of diacetyl-producing strains The above-mentioned strains were inoculated into M17 liquid medium and cultured at 37°C for 12 h. Then, they were inoculated into milk at a rate of 4% (v / v) and cultured at 30°C for 6 h, followed by refrigeration for 24 h. The sample was mixed with an equal volume of 8% (w / v) trichloroacetic acid, centrifuged, and the supernatant was collected. 0.5 mL of 1% (w / v) o-phenylenediamine solution was added, and the absorbance at 335 nm was measured.

[0022] (3) Secondary screening of diacetyl-producing strains An ultrasound-assisted method was used. The diacetyl-producing bacterial suspension from the initial screening was placed in a 30℃ constant temperature bath and ultrasonically treated at 200W for 10 min, then removed and set aside. It was inoculated into cow's milk at a 4% (v / v) inoculum, incubated at 30℃ for 6 h, and then refrigerated for 24 h. The sample was mixed with an equal volume of 8% (w / v) trichloroacetic acid, centrifuged, and the supernatant was collected. 0.5 mL of 1% (w / v) o-phenylenediamine solution was added, and the absorbance at 335 nm was measured. The results are shown below. Figure 1 -a.

[0023] (4) Identification of strains Through primary and secondary screening, a strain was obtained and named L402. The colonies were white, round, with regular edges, and appeared spherical under a microscope. Figure 1 -b, Gram-positive, non-flagellated, cells arranged singly, in pairs or short chains, see Table 1.

[0024] Table 1. Colony morphology of the strains

[0025] Sequencing was performed on the PCR amplification product of 16S rDNA, and the sequencing results are shown in the sequence listing. Blast sequence alignment was performed on the NCBI website, and the results showed that strain L402 had over 99% homology with *Enterococcus faecalis* 16S rDNA, confirming that this bacterium is *Enterococcus faecalis*. A phylogenetic tree of *Enterococcus faecalis* L402 was constructed using MEGA11 software. Figure 1 -c).

[0026] (5) Growth characteristics of the strain Activated Enterococcus faecalis L402 was inoculated into M17 liquid medium at a 4% inoculum and cultured at 37°C for 16 hours. The absorbance of the bacterial solution at 600 nm was measured every 2 hours, and the pH of the bacterial solution was also measured. The resulting growth curve is shown below. Figure 2 As shown in -a. The results indicate that Enterococcus faecalis L402 grows rapidly in M17 medium, entering the logarithmic phase at around 4 hours and the stationary phase at 8-10 hours.

[0027] (6) Safety evaluation of the strain a) Hemolysis test The bacterial suspension to be tested was streaked onto agar plates containing 5% (v / v) sheep blood and incubated at 37°C for 48 h. Staphylococcus aureus ATCC25923 was used as a positive control, and Lactococcus lactis L500 as a negative control. After incubation, clear hemolytic zones were observed around the colonies. The presence of a complete clear hemolytic zone indicated β-hemolysis, while the absence of a zone indicated no hemolysis. Results are shown below. Figure 3 L402 did not exhibit hemolytic activity.

[0028] b) Antibiotic susceptibility testing Following the CLSI 45-A3 guidelines, antibiotic susceptibility was determined using the Kirby-Bauer disk diffusion method. The bacterial suspension to be tested was evenly spread onto M17 agar plates, dried, and then three disks of the same antibiotic susceptibility testing agent were placed at equal intervals. The plates were incubated at 37°C for 48 hours. The diameter of the inhibition zone was measured using a digital caliper (0.1 mm accuracy), and the average of the three measurements was taken as the final result. The results are shown in Table 2. L402 showed high sensitivity to antibiotics such as penicillin, ampicillin, and levofloxacin (the diameter of the inhibition zone met the CLSI definition of "sensitive").

[0029] Table 2 Results of drug susceptibility test and determination of drug resistance

[0030] Note: R indicates drug resistance, I indicates moderate sensitivity, and S indicates sensitivity. c) Biogenic amine test The biogenic amine formation potential was determined using a colorimetric reaction with a decarboxylase kit, using *Staphylococcus aureus* ATCC25923 as a positive control. The biogenic amine content was determined using high-performance liquid chromatography-fluorescence detection (HPLC-FLD). 5 mL of logarithmic-phase bacterial culture was centrifuged at 4℃ and 9777×g for 15 min. The supernatant was mixed with 25 mL of 0.1 nmol / L HCl, centrifuged again, and the supernatant was collected. 1 mL of the supernatant was added sequentially to 0.1 mL of 0.01% (w / v) 1,7-diaminoheptane, 0.5 mL of saturated Na₂CO₃, and 1 mL of 1% (w / v) 5-(dimethylamino)naphthalene-1-sulfonyl chloride-acetone solution. The mixture was derivatized at 45℃ in the dark for 60 min. The derivatization was terminated with 0.5 mL of 10% L-proline, followed by ether extraction, vacuum concentration, and redissolved in acetonitrile. The results are shown in Table 3. L402 does not have amino acid decarboxylase activity and does not generate biogenic amines during the culture process, indicating that strain L402 has good safety in terms of biogenic amine production.

[0031] Table 3. Growth status and biogenic amine count in decarboxylase.

[0032] (7) Evaluation of the beneficial properties of the strain a) Survival rate of bacteria in simulated gastric and intestinal fluids Artificial saliva, gastric juice, and intestinal juice were prepared. Overnight culture of the bacterial strain was centrifuged for 5 min, and the cells were resuspended in 0.9% NaCl. The cells were inoculated into artificial saliva at 10% (v / v) and incubated at 37°C for 10 min. Samples were taken at 0 and 10 min for plate counting. The cells were collected by centrifugation, washed three times with PBS, and resuspended in artificial gastric juice. The cells were incubated at 37°C for 2 h, and samples were taken for counting. The cells were then transferred to artificial intestinal juice by centrifugation and incubated at 37°C for 3 h. Plate counting was performed at the endpoint. Results are shown below. Figure 2-b indicates that L402 has a strong tolerance to digestive stress factors such as gastric acid and bile salts, and has better adaptability to the gastrointestinal environment.

[0033]

[0034] In the formula: N0 is the number of viable bacteria sampled at 0h, N t The number of viable bacteria of the strain when exposed to simulated saliva, gastric juice, and intestinal juice.

[0035] b) Antioxidant Test (ABTS) + (Superoxide anion, DPPH free radical scavenging rate) Centrifuge the bacterial suspension at 10000×g for 10 min at 4℃, discard the supernatant, wash the bacterial cells three times with 0.01mol / L PBS (pH 7.4), and resuspend to obtain an intact cell (IC) suspension. Sonicate at 800W for 30 min on ice, 15 s on / 15 s off. Centrifuge the lysate for 15 min as above; the supernatant is the cell-free extract (CFE).

[0036] DPPH free radical scavenging rate: Mix 0.1 mL of sample solution with 0.1 mL of 0.2 mmol / L DPPH-ethanol solution, incubate at room temperature in the dark for 40 min, and measure the absorbance at 517 nm. Each sample was tested in triplicate. The blank group used an equal volume of ethanol instead of the sample solution, and the control group used an equal volume of ethanol instead of the DPPH solution. The scavenging rate was calculated using the following formula:

[0037] In the formula: A1 is the absorbance of the control group, A2 is the absorbance of the sample group, and A3 is the absorbance of the blank group.

[0038] Superoxide anion scavenging rate: 3 mL Tris-HCl (pH 8.2) was preheated at 25℃ for 20 min, 1 mL pyrogallol (25℃) was added to initiate the reaction, and 1 mL sample solution was immediately added. After mixing, the reaction was carried out at 25℃ for 10 min, and the absorbance was measured at 320 nm. Each sample was repeated 3 times. The blank group was replaced with 1 mL physiological saline instead of the sample solution, and the sample blank group was replaced with 1 mL sample solution plus 4 mL physiological saline. The absorbance was read at 325 nm to correct for background. The scavenging rate was calculated according to the formula:

[0039] In the formula: A1 is the absorbance of the sample, A2 is the absorbance of the blank group, and A0 is the absorbance of the blank group.

[0040] ABTS radical scavenging rate: An equal volume of 7 mmol / L LABTS and 2.45 mmol / L K₂S₂O₈ was mixed and reacted at room temperature in the dark for 14 h to obtain an ABTS⁺ stock solution. The stock solution was diluted with anhydrous ethanol to a final absorbance of 0.70 ± 0.02 at 734 nm to obtain the working solution. The fermentation broth was diluted 5-fold with distilled water, and 0.4 mL of the solution was added to 4.6 mL of the working solution. The mixture was incubated at 30 °C in the dark for 3 min, centrifuged at 4500 × g for 3 min, and the absorbance of the supernatant was measured at 734 nm. The blank was used as the working solution, and the scavenging rate was calculated using the formula:

[0041] In the formula: A1 is the absorbance value of the sample after reacting with the ABTS⁺ free radical solution, A2 is the background absorbance value of the sample solution, and A0 is the absorbance value of the blank control.

[0042] Table 4 Antioxidant Capacity Analysis

[0043] The complete cell suspension (IC) of L402 showed a superoxide anion scavenging rate and ABTS cationic free radical scavenging capacity of over 90%, indicating excellent activity. The DPPH free radical scavenging rate of cell-free extract (CFE) of strain L402 was significantly higher than that of L405 (P<0.05), indicating that L402 has good antioxidant properties.

[0044] (8) Preparation of bacterial suspension Enterococcus faecalis L402 was inoculated into M17 liquid medium and activated at 37°C for 12 h. This activation was repeated three times. The activated bacterial solution was then inoculated into M17 liquid medium at a 3% inoculum and incubated at 37°C for 12 h. The colony count was determined using the dilution plate method. Based on the colony count, the cultured solution was centrifuged, the supernatant was discarded, and sterile physiological saline was added to adjust the cell count to 102. 6 ～8 CFU / mL, store at 4℃.

[0045] In conclusion, the selected Enterococcus faecalis L402 exhibits good probiotic properties and can be used as the starting strain for the next stage of experiments. Example 2

[0046] The method of the present invention for high diacetyl production by Enterococcus faecalis L402 under ultrasound stress includes the following steps: (1) Effects of ultrasonic power, time and processing temperature A bacterial suspension was prepared and co-inoculated with a commercial starter culture into sterilized skim milk. A single-factor experimental design was used to investigate the effects of ultrasonic power (0–350 W), time (0–40 min), and temperature (0–50 °C) on diacetyl yield. Each factor had 5–8 gradients, with each gradient replicated three times. The optimal parameter combination was determined based on the highest diacetyl yield. Figure 4 As can be seen from the data, the optimal ultrasonic conditions are an ultrasonic temperature of 37℃, an ultrasonic power of 150W, and an ultrasonic time of 10min. (2) Box-Behnken response surface optimization Based on the single-factor results, a three-factor, three-level Box-Behnken model was established using Design-Expert 8.0.6. Ultrasonic time (A), ultrasonic temperature (B), and ultrasonic power (C) were used as independent variables, and diacetyl concentration was used as the response value to examine the interaction. The factor levels were set according to the optimal intervals for single-factor experiments, as shown in Table 5. Each experimental point was repeated three times, and the mean diacetyl value was input into the model. The optimal parameter combination was determined through model fitting and analysis of variance.

[0047] Table 5 Factors and Levels in Response Surface Experiment

[0048] The experimental design and results are shown in Table 6. Multiple regression fitting was performed on the experimental results to construct a quadratic regression equation between the diacetyl concentration (Y) and its independent variables: Y = 19.68 + 0.64A + 0.48B + 0.016C + 0.25AB − 0.49AC − 0.069BC + 1.47A 2 +0.48B 2 +0.28C 2 .

[0049] Table 6 Response Surface Experiment Scheme and Results

[0050] Based on the variance analysis results of the regression equations in Table 7, the following findings are obtained: Model significance: The P-value of this regression model is <0.001, indicating extremely significant statistical significance, demonstrating that the model effectively reflects the relationship between ultrasound time, ultrasound temperature, ultrasound power, and diacetyl concentration; Model fit: The model's coefficient of determination R² = 0.9625, indicating that 96.25% of the response value variation can be explained by the selected independent variables (A, B, C); Lack of fit P = 0.3803 (>0.05), indicating a small model fit error and good consistency between experimental data and model predictions; Model reliability: The adjusted coefficient of determination R²adj = 0.9143 after correction for degrees of freedom further confirms the model's high reliability, effectively explaining 91.43% of the response value variation. Through the significance analysis of the regression coefficients, the order of influence of each factor on diacetyl concentration is determined to be: A (ultrasound time) > B (ultrasound temperature) > C (ultrasound power). In summary, the response surface model established in this study has high fitting degree and strong reliability, and can be used for the analysis and prediction of diacetyl concentration optimization process conditions, providing a theoretical basis for subsequent determination of the optimal combination of ultrasonic parameters.

[0051] Table 7. Analysis of Variance for Regression Equations

[0052] The interaction effects of the response surface optimization experimental data were analyzed using Design-Expert 8.0.6 software, and response surface plots and contour plots were generated. The results are as follows: Figure 5 As shown in the figure. Interaction effect analysis based on response surface methodology revealed significant differences in the interaction effects of different factor combinations (AB, BC, AC) on diacetyl concentration: The interaction between AB (ultrasound time-ultrasound temperature) and BC (ultrasound temperature-ultrasound power) showed that both had relatively flat response surfaces with small curvatures; the contour plots showed sparse and nearly circular lines, and statistical results showed P>0.05, indicating that the interaction between these two factors had no significant effect on diacetyl concentration. The interaction between AC (ultrasound time-ultrasound power) showed a significantly nonlinear response surface (P<0.05), with large curvatures and a pronounced slope; the contour plots showed dense lines in a typical elliptical shape, suggesting that changes in ultrasound time and ultrasound power synergistically affect diacetyl concentration, and their interaction effect was significantly stronger than other combinations. Based on the combined response surface characteristics and statistical analysis results, the order of influence of the interaction of various factors on diacetyl concentration is: AC (ultrasound time - ultrasound power) > AB (ultrasound time - ultrasound temperature) > BC (ultrasound temperature - ultrasound power). This conclusion is consistent with the F-value judgment results of each interaction term in the analysis of variance, further verifying the reliability of the interaction effect analysis.

[0053] Example 3 This invention relates to a method for efficiently producing diacetyl-rich yogurt using Enterococcus faecalis L402 through ultrasonic treatment. (1) Preparation of bacterial suspension and ultrasonic pretreatment Take 50 mL of Enterococcus faecalis L402 bacterial culture into a centrifuge tube, centrifuge (4℃, 8000 r / min, 10 min), discard the supernatant, wash the bacterial cells with twice the volume of physiological saline, repeat the centrifugation twice, and resuspend the bacterial cells with an equal volume of physiological saline for later use. The bacterial suspension was treated with ultrasound at 150 W and 42 °C for 20 min, and then removed for later use.

[0054] (2) Yogurt preparation process Preparation of reconstituted milk: Dissolve 12% (w / v) whole milk powder and 7% (w / v) white sugar in distilled water at 55℃, stir at 250r / min for 5min, then homogenize and emulsify at 300r / min for 10min; sterilize in a water bath at 95℃ for 10min, and cool to 30℃ for later use.

[0055] Fermentation process: The reconstituted milk was inoculated with a 4% (v / v) *Enterococcus faecalis* L402 suspension and 0.2% (w / v) commercial starter culture (*Streptococcus thermophilus*: *Lactobacillus bulgaricus* = 1:1), and incubated at 30°C until curdled, followed by ripening at 4°C for 24 hours. Experimental groups: The LIN group was inoculated with only the commercial starter culture; the CON group was inoculated with both the commercial starter culture and L402; the ULT group was inoculated with both the commercial starter culture and L402 pretreated with ultrasound.

[0056] (3) Yogurt quality determination a) Measurement of rheological properties Steady-state shear scans of yogurt were performed using a Kinexuspro+ rotational rheometer (PP50 tray, 40mm flat clamp, 1mm gap, 25℃): shear rate 0.01–100s. -1 Record the apparent viscosity. The results are as follows: Figure 6 As shown, the viscosity was significantly increased after ultrasonic treatment compared to the control, indicating that inoculation with ultrasonically treated L402 can increase the viscosity of fermented yogurt. b) Water-holding capacity measurement Take approximately 10g of fermented milk sample (weigh), centrifuge at 10000 rpm for 30 minutes at 4°C, discard the supernatant, invert the centrifuge for 10 minutes to allow the supernatant to flow out completely, and then immediately weigh the precipitate. Water-holding capacity is expressed as:

[0057] In the formula: the mass of the centrifuge tube is recorded as m0; the total mass of the centrifuge tube plus the fermented milk is recorded as m1; the total mass of the precipitate after centrifugation and the centrifuge tube is recorded as m2; Water Holding Capacity (WHC) test results are as follows Figure 6 As shown in -d. Inoculation with ultrasonically treated L402 can improve the water-holding capacity of yogurt.

[0058] c) Texture determination The determination was performed using a food physical property tester with the following parameters: probe diameter 25mm, trigger force 5g, initial speed 2mm / s, testing speed 1mm / s, and post-test speed 2mm / s. As shown in Table 8, inoculation with L402 after ultrasound increased the hardness and viscosity of yogurt, while showing no significant changes in cohesion and viscoelasticity.

[0059] Table 8. Determination of Yogurt Texture

[0060] (4) Yogurt flavor determination Accurately weigh 3g of sample into a 20mL headspace vial, add 2.0µL of internal standard (n-pentadecane-d3 250µg / mL), seal immediately, and proceed with HC-SPME-GC-MS analysis. The sample was analyzed by HS-SPME-GC-MS using an Agilent 7697A-8890-5977B headspace gas chromatography-mass spectrometer.

[0061] Principal component analysis score plot (7-a) shows that the cumulative contribution rate of the first principal component (PC1) and the second principal component (PC2) reaches 72.00%, with PC1 accounting for 61.40%, which is the main dimension distinguishing the samples. The figure shows that the ULT, CON, and LIN groups exhibit obvious intra-group clustering trends, indicating high consistency of replicate samples within each treatment group and good reproducibility of the experiment. Meanwhile, the distribution areas of samples from different groups differ in the two-dimensional space formed by PC1 and PC2, especially the ULT, CON, and LIN groups, which show significant separation along the PC1 direction. This indicates that different treatment conditions caused global and systematic differences in the metabolic profiles of the samples, reflecting the significance of the treatment effects. The volatile components in the samples were classified and their relative contents were statistically analyzed. From the perspective of compound category distribution (7-b), esters (18.54%) and hydrocarbons (18.28%) constitute the most important components, with similar proportions and significantly higher than other categories; ketones (10.44%), alcohols (10.18%), and organic heterocyclic compounds (10.18%) are the second most important components, each accounting for more than 10%. Other categories such as terpenes, aldehydes, acids, and phenols account for less than 6%, belonging to trace components. Overall, volatile components in the sample are dominated by esters and hydrocarbons, and contain a rich variety of medium and trace categories, exhibiting high chemical diversity, which may collectively constitute the unique odor or metabolic characteristics of this sample. The odor characteristics of the ULT and CON groups were visualized and compared using odor radar maps, and the results are shown in Figure (8-b), indicating significant differences between the different treatment groups in multiple odor dimensions. The ULT group showed a stronger response to odors such as "fatty," "buttery," and "creamy," with relatively convex outlines; the CON group showed a stronger response to odors such as "oily," "medicinal," and "sweet." Overall, the odor outlines of the two groups were clearly separated in the radar image, indicating that ultrasound significantly affected the odor properties of volatile components. Based on the heatmap analysis results (8-a), the different treatment groups showed clear clustering and differential patterns in the expression profiles of volatile compounds. Overall, most compounds showed significant differences in expression levels, indicating that the treatment significantly affected the accumulation of metabolites. Specifically, aldehydes and ketones (such as hexanal, 2,3-butanedione, and 2,3-pentanedione) were highly expressed in the ULT group (red area). The volcano plot results (8-c) showed that under ultrasound treatment conditions, a total of 383 differentially expressed metabolites were identified between the ULT and CON groups, of which 35 were upregulated and 17 were downregulated. The number of substances that were significantly upregulated was greater than the number that were downregulated, and some of the substances that were significantly upregulated (such as 2,3-pentanedione and acetoin) also had high VIP values.

[0062] (5) Sensory evaluation of yogurt A sensory evaluation panel of 10 food professionals was selected to score the fermented milk in terms of color, taste, aroma and texture according to the national standard GB19302-2010. The specific evaluation criteria are shown in the table.

[0063] Table 9 Sensory Evaluation Table for Yogurt

[0064] Sensory evaluation results showed that ultrasound synergistically enhanced the aroma and flavor of strain L402, improving overall acceptability. Figure 8 -e).

Claims

1. A type of Enterococcus faecalis, characterized in that, The Enterococcus faecalis was deposited at the China General Microbiological Culture Collection Center on September 24, 2025, with accession number CGMCC No. 36054.

2. The use of Enterococcus faecalis as described in claim 1 in the preparation of diacetyl-producing preparations.

3. The use of Enterococcus faecalis as described in claim 1 in the production of diacetyl.

4. The application as described in claim 3, characterized in that, The application involves applying ultrasound during the cultivation or fermentation of the Enterococcus faecalis.

5. The application as described in claim 4, characterized in that, The parameters of the ultrasound include ultrasound temperature 30-50℃, ultrasound power 100-350W, and ultrasound time 5-40min.

6. The application as described in claim 5, characterized in that, The parameters of the ultrasound include an ultrasound temperature of 42°C, an ultrasound power of 150W, and an ultrasound time of 20 minutes.

7. The application of Enterococcus faecalis as described in claim 1 in the preparation of high-yield diacetyl yogurt.

8. A method for preparing yogurt with high diacetyl yield, characterized in that, The method includes the following steps: (1) using reconstituted milk as a base material, inoculating the bacterial suspension of Enterococcus faecalis as described in claim 1, which has been pretreated by ultrasound; (2) fermenting at 30°C for 6 hours, followed by ripening at 4°C for 24 hours.

9. The method as described in claim 8, characterized in that, During fermentation, a commercial starter culture is also inoculated, which consists of Streptococcus thermophilus and Lactobacillus bulgaricus in a 1:1 mass ratio.

10. Yogurt prepared by the method of claim 8 or 9, characterized in that, The yogurt contains no less than 22 μg / mL of diacetyl and has a water-holding capacity of no less than 65%.

Images