Streptococcus salivarius subsp. thermophilus LSE-G45 and application thereof

Abstract

The application belongs to the technical field of microorganisms, and particularly discloses a Streptococcus salivarius subsp. thermophilus LSE-G45 and application thereof. Streptococcus salivarius subsp .thermophilus The strain is preserved in the China General Microbiological Culture Collection Center on October 29, 2025, and has a preservation number of CGMCC No. 36396. The Streptococcus salivarius subsp. thermophilus LSE-G45 provided by the application can produce lipase at a high yield, and has good intestinal tolerance, hydrophobic self-aggregation and antioxidant and other probiotic properties. When the Streptococcus salivarius subsp. thermophilus LSE-G45 is used for fermenting whipped cream, the problem of weak aroma and single taste of the whipped cream can be obviously improved.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology and relates to a thermophilic subspecies of Streptococcus salivarius LSE-G45 and its applications. Background Technology

[0002] Lipases, as glycerol ester hydrolases, specifically hydrolyze the ester bonds of triglycerides in milk fat to generate glycerol and free fatty acids (FFA). These FFAs are not only the core contributors to the characteristic flavors of dairy products such as cream, butter, and cheese, but can also be further metabolized into volatile compounds such as lactones, methyl ketones, and esters, significantly enriching the diversity of dairy flavors. Therefore, they have extremely high application value in the dairy industry.

[0003] Whipping cream, a high-fat component obtained from whole milk, is rich in unsaturated fatty acids (such as linoleic acid and linolenic acid), protein, and trace elements, possessing both nutritional and flavor potential. However, traditional whipping cream relies on physical separation processes, lacking the complex milk fat aroma produced during fermentation, resulting in a monotonous taste and insufficient flavor complexity. While existing technologies can improve flavor by adding exogenous lipases or introducing starter cultures, chemical enzyme preparations can easily lead to flavor distortion, and the lipase activity of conventional fermentation strains such as lactic acid bacteria is limited, making it difficult to achieve precise control of FFA and the steady-state formation of flavor compounds.

[0004] Furthermore, existing cream fermentation strains generally suffer from weak aroma and monotonous flavor after fermentation, and cannot simultaneously meet the dual requirements of efficient enzyme production and flavor modification. Therefore, there is an urgent need to develop a microbial strain with high lipase activity, excellent fermentation adaptability, and flavor regulation capabilities to overcome the technical bottlenecks of weak aroma and monotonous flavor in traditional cream, and to provide a new approach for the development of fermented dairy products. Summary of the Invention

[0005] In view of this, the present invention provides a thermophilic subspecies of Streptococcus salivarius LSE-G45 and its application.

[0006] To achieve the above-mentioned objectives, the embodiments of the present invention employ the following technical solutions: This invention provides a thermophilic subspecies of Streptococcus salivarius, LSE-G45, whose Latin name is Streptococcus salivarius subsp .thermophilus The strain was deposited on October 29, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 36396.

[0007] Compared with existing technologies, the LSE-G45 subsp. thermophilic of Streptococcus salivarius provided by this invention not only has high lipase production, but also has good gastrointestinal tolerance, hydrophobic self-agglutination and antioxidant properties, and has no risk of hemolysis, making it safe and reliable.

[0008] This invention provides the application of the above-mentioned Streptococcus salivarius thermophilic subsp. LSE-G45 in the production of lipase.

[0009] The thermophilic subspecies of Streptococcus salivarius LSE-G45 provided by this invention can produce lipase at a high rate, with a lipase yield of up to 5.68±0.03 U / mL after fermentation.

[0010] The present invention provides a fermentation agent comprising Streptococcus salivarius thermophilic subsp. LSE-G45.

[0011] This invention provides a method for preparing the above-mentioned fermenting agent, comprising the following steps: The thermophilic subspecies of Streptococcus salivarius LSE-G45 was inoculated into a seed culture medium for fermentation to obtain a starter culture.

[0012] Preferably, the seed culture medium comprises: 19-21g yeast powder, 19-21g lactose, 1.9-2.1g glucose, 1.9-2.1g dipotassium hydrogen phosphate, 0.9-1.1g Tween 80, 4.9-5.1g sodium acetate, diluted with water to 1000mL, with a pH of 5.5.

[0013] For example, the seed culture medium also needs to be sterilized at 110~118℃ for 15min-20min.

[0014] Preferably, the fermentation conditions include a temperature of 40-42°C and a rotation speed of 100-120 r / min.

[0015] Preferably, the inoculum amount of the thermophilic subspecies of Streptococcus salivarius LSE-G45 is 2% to 3%.

[0016] For example, the *Streptococcus salivarius* subsp. *thermophilus* LSE-G45 also needs to be activated. The activation culture medium is not limited here, and the operation can be carried out according to the conventional technical means known to those skilled in the art.

[0017] Preferably, the viable count of Streptococcus salivarius subsp. thermophilus LSE-G45 in the fermentation agent is 1.2 × 10⁻⁶. 8 CFU / mL ~1.4×10 10 CFU / mL.

[0018] Fermentation culture of Streptococcus salivarius subsp. thermophilus LSE-G45 yielded a starter culture with high concentration of live bacteria and high strain utilization, which significantly shortened the fermentation cycle and reduced fermentation costs.

[0019] This invention provides a probiotic freeze-dried powder, comprising the above-mentioned starter culture agent.

[0020] This invention provides a method for preparing probiotic freeze-dried powder, comprising the following steps: The fermentation agent is subjected to solid-liquid separation, a protective agent is added to the separated bacterial sludge, and the mixture is freeze-dried to obtain probiotic freeze-dried powder.

[0021] Preferably, the solid-liquid separation includes centrifugation.

[0022] More preferably, the centrifugation conditions include: a rotation speed of 4500 rpm to 4800 rpm and a time of 20 min to 30 min.

[0023] Preferably, the mass ratio of the bacterial sludge to the protective agent is (1~2):1.

[0024] More preferably, the protective agent comprises the following components in weight percentage: 0.6%~1% glycine, 5%~7% trehalose, 0.5%~1% L-cysteine, 10%~12% skim milk, and the balance being water.

[0025] Preferably, the freeze-drying conditions include: pre-freezing at a temperature of -80℃ to -60℃ for 1.5h to 3h under a vacuum of 1Pa to 2Pa; and then freezing at -80℃ to -50℃ for 24h to 36h.

[0026] This invention provides a method for preparing fermented light cream, comprising the following steps: The starter culture or the probiotic freeze-dried powder is inoculated into light cream for fermentation.

[0027] The method for preparing fermented light cream provided by this invention can not only use a starter containing Streptococcus salivarius thermophilus subsp. LSE-G45 to ferment light cream, but also use a probiotic freeze-dried powder containing Streptococcus salivarius thermophilus subsp. LSE-G45 to ferment light cream. It is flexible in application, can meet different production needs, and has a wider range of applications.

[0028] Preferably, the inoculum amount of the fermenting agent is 0.05% to 1%.

[0029] It should be noted that the inoculation amount mentioned above refers to a volume percentage.

[0030] More preferably, the viable count of *Streptococcus salivarius* subsp. *thermophilus* LSE-G45 in the fermentation agent is 1.2 × 10⁻⁶. 8CFU / mL ~1.4×10 10 CFU / mL.

[0031] Preferably, the inoculation amount of the probiotic freeze-dried powder is 50~150g / t.

[0032] Preferably, the viable count of Streptococcus salivarius subsp. thermophilus LSE-G45 in the probiotic freeze-dried powder is 5.0 × 10⁻⁶. 11 CFU / g ~1.0×10 13 CFU / g.

[0033] Preferably, the fermentation culture temperature is 38℃~42℃.

[0034] Preferably, the fermentation culture time is 8h~12h.

[0035] Compared with the prior art, the present invention has the following beneficial effects: (1) The survival rate of the thermophilic subspecies of Streptococcus salivarius LSE-G45 provided by the present invention reached 86.00±4.10% in gastric juice, 78.66±5.1% in intestinal juice, and 77.66±5% after sequential treatment with gastric and intestinal juices, showing good potential for colonization in the digestive tract.

[0036] (2) The hydrophobicity of Streptococcus salivarius subsp. thermophilicus LSE-G45 provided by the present invention can reach 76.17±0.33%, which has excellent intestinal mucosal adhesion ability.

[0037] (3) The autoagglutination rate of Streptococcus salivarius subsp. thermophilus LSE-G45 provided by the present invention is 87.00±0.10% in 24h, which shows excellent gastrointestinal adhesion and colonization ability.

[0038] (4) The *Streptococcus salivarius* subsp. *thermophilus* LSE-G45 provided by this invention can achieve a DPPH free radical scavenging rate of 69.33±0.58% and an ABTS free radical scavenging rate of... + With a free radical scavenging rate as high as 98.33±0.58%, it has excellent antioxidant activity. In addition, Streptococcus salivarius thermophilus subsp. LSE-G45 does not produce β-hemolysin, has good safety, and can be used in the food processing field.

[0039] (5) The thermophilic subspecies of Streptococcus salivarius LSE-G45 provided by the present invention can produce lipase in large quantities, and the yield of lipase produced by fermentation is as high as 5.68±0.03U / mL.

[0040] (6) When using the starter containing Streptococcus thermophilus subsp. LSE-G45 or probiotic freeze-dried powder provided by the present invention to ferment light cream, it can effectively solve the problems of weak aroma and single flavor of existing light cream. The fermented light cream has a prominent milky aroma, delicate taste, and enhanced flavor layers. Attached Figure Description

[0041] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the embodiments 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.

[0042] Figure 1 The cell morphology of Streptococcus salivarius subsp. thermophilus LSE-G45 provided in Example 1 of this invention; Figure 2 This is a morphological image of the cell culture of Streptococcus salivarius subsp. thermophilus LSE-G45 provided in Example 1 of the present invention; Figure 3 This is a phylogenetic tree of the thermophilic subspecies of Streptococcus salivarius LSE-G45 provided in Example 2 of the present invention; Figure 4 The growth curve of Streptococcus salivarius thermophilic subsp. LSE-G45 strain provided in Example 3 of the present invention; Figure 5 This is a growth diagram of the thermophilic subspecies of Streptococcus salivarius LSE-G45 provided in Example 4 of the present invention on glycerol tartrate agar plates. Figure 6 This is a p-NP standard curve diagram of the *Streptococcus salivarius* subsp. *thermophilicus* LSE-G45 strain provided in Example 4 of the present invention; Figure 7 This is a statistical chart of the lipase activity assay results of different strains provided in Example 4 of the present invention; Figure 8 This is a hemolytic test diagram of the Lactobacillus rhamnosus LSE-H023-A-07 strain provided in Example 10 of the present invention; Figure 9 This is a hemolytic test diagram of the *Streptococcus salivarius* subsp. *thermophilicus* LSE-G45 strain provided in Example 10 of the present invention; Figure 10 Radar chart for sensory evaluation of light cream fermented using the starter culture provided in Example 10 of this invention; Figure 11 Radar chart for sensory evaluation of light cream fermented with probiotic freeze-dried powder provided in Example 10 of the present invention. Detailed Implementation

[0043] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0044] Unless otherwise specified, the raw materials and reagents used in this invention are all conventional commercially available products; unless otherwise specified, the methods used in this invention are all conventional methods in the field.

[0045] 1. The culture medium used in this invention is as follows: MRS liquid culture medium: 10g beef extract, 10g casein peptone, 5g yeast extract, 6.6g K2HPO4, 2g diammonium citrate, 0.3g MnSO4·H2O, 0.5g anhydrous sodium acetate, 0.5g MgSO4·7H2O, 20g glucose and 1mL Tween 80, add water to a final volume of 1000mL; sterilize at 118℃ for 20min.

[0046] Tributyric acid glyceride plate medium: Dissolve 2.5g meat peptone, 2.5g casein peptone, 3g yeast extract powder, and 12g agar in 1000mL of water, sterilize at 121℃ for 15min, and add 10mL of tributyric acid glyceride while hot after sterilization, and mix thoroughly.

[0047] TJA liquid culture medium: 50 mL tomato juice, 10 g beef extract, 5 g yeast extract, 2 g glucose, 20 g lactose, 2 g dipotassium hydrogen phosphate, 5 g anhydrous sodium acetate, 1 g Tween 80, add water to make up to 950 mL, and sterilize at 118 °C for 20 min.

[0048] Columbia blood agar medium: 10g casein trypsin digest, 5g meat stomach enzyme digest, 3g cardiac trypsin digest, 5g yeast extract, 1g corn starch, 5g sodium chloride, 13.5g agar, add water to make up to 950mL, sterilize at 118℃ for 20min, cool to 45-50℃ after sterilization, then add 50mL of sterile defibrinated sheep blood, gently shake and pour into plates, cool to obtain Columbia blood agar medium.

[0049] High-density culture medium: 20g yeast powder, 20g lactose, 2g glucose, 2g dipotassium hydrogen phosphate, 1g Tween 80, 5g anhydrous sodium acetate, add water to make up to 1000mL, sterilize at 118℃ for 20min.

[0050] Example 1 This embodiment provides the isolation and purification of Streptococcus salivarius thermophilic subspecies LSE-G45.

[0051] Weigh 1g of milk fan and quickly place it into a sterile mortar. Use a sterile pipette to add 9mL of sterile physiological saline to the mortar. Grind the milk fan thoroughly and evenly until the sample tissue is uniformly broken down, preparing 10 -1 The initial bacterial suspension was serially diluted 10-fold, followed by multiple streak plate separations to ultimately select pure strains. These strains exhibited a milky-white, smooth colony morphology with regular edges and were Gram-positive, staining purple. (Specific details are as follows...) Figures 1-2 As shown.

[0052] Example 2 This embodiment provides the identification and preservation of Streptococcus salivarius thermophilic subspecies LSE-G45.

[0053] DNA was extracted from the strain isolated in Example 1 and PCR amplification was performed using universal primers (upstream primer 27F: 5'-AGAGTTTGATCMTGGCTCAG-3'; downstream primer 1492R: 5'-GGTTACCTTGTTACGACTT-3').

[0054] The PCR reaction system consisted of: 25 μL of 1×Taq PCR Master Mix, 1 μL of upstream primer 27F, 1 μL of downstream primer 1492R, 2 μL of DNA template, and 21 μL of ddH2O.

[0055] The PCR amplification program was as follows: 95℃ pre-denaturation for 5 min, 95℃ denaturation for 1 min, 55℃ annealing for 1 min, 72℃ extension for 1 min 30 s, denaturation to extension for 30 cycles, and finally 72℃ extension for 10 min.

[0056] The PCR products were sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. The quality-controlled paired-end sequencing results were assembled to obtain the 16S rRNA sequence, as shown below:

[0057] The assembled 16S rRNA sequence was compared with the NCBI database. The species of the sample was determined to be *Streptococcus thermophilus*, and it was named *Streptococcus thermophilus* (subspecies *Salicornica*). Streptococcus salivarius subsp .thermophilus LSE-G45, phylogenetic tree as follows Figure 3 As shown. This strain is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 36396, located at Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing, on October 29, 2025.

[0058] Example 3 This embodiment provides the determination of the growth performance of Streptococcus salivarius thermophilic subspecies LSE-G45.

[0059] 2% of *Streptococcus salivarius* subsp. *thermophilus* LSE-G45 was inoculated into MRS liquid medium and thoroughly mixed. 200 μL of the bacterial solution was transferred into honeycomb plates, which were then placed in an automated growth curve analyzer and fermented at 38°C for 24 hours. The OD (octane rating) of the bacterial solution was automatically measured every 1 hour during the fermentation process. 600nm The values ​​were then plotted as a curve, and the specific results are as follows: Figure 4 As shown, 0-2h is the lag phase, 3-8h is the logarithmic growth phase, and 9-24h is the stationary phase.

[0060] Example 4 This embodiment provides a screening test for high-lipase-producing strains of Streptococcus salivarius subspecies LSE-G45.

[0061] 1. Screening of high-lipase-producing strains 1.1 Initial screening of lipase-producing strains Make small wells (0.8 cm in diameter) on tributylate agar plates and add 1.2 × 10⁻⁶ viable bacteria. 8 ~1.6×10 12 The cFU / mL *Streptococcus salivarius* subsp. *thermophilus* LSE-G45 strain was inoculated into TJA liquid medium at a volume fraction of 2% and incubated at 38°C for 16 hours. 50–100 μL of the inoculum was then precisely measured and inoculated into small wells, and incubated at 38°C for 48 hours. The presence of a clear zone on the plate indicated that the strain produced lipase.

[0062] like Figure 5 As shown in Table 1, the thermophilic subspecies of Streptococcus salivarius LSE-G45 can produce lipase with a diameter of 20±1 mm.

[0063] Table 1

[0064] 1.2 Secondary screening of lipase-producing strains Preparation of crude enzyme solution: The bacterial culture of Streptococcus salivarius subsp. thermophilus LSE-G45 was centrifuged at 8000 rpm for 10 min at 4℃, and the resulting supernatant was the crude enzyme solution.

[0065] Substrate buffer preparation: Dissolve 2.3g sodium deoxycholate and 1.1g gum arabic in water, adjust the pH to 8, and bring the volume to 1000mL.

[0066] Preparation of p-nitrophenol (p-NP) standard curve: Weigh 0.01391 g of p-NP and dissolve it in 50 mL of isopropanol to obtain a 2 mol / L p-NP working solution. Add the reagents according to Table 2.

[0067] Table 2

[0068] After mixing the above solutions in a test tube, react in a 30°C water bath for 15 minutes. Then, remove the tube and add 2 mL of 95% ethanol to terminate the reaction. Centrifuge at 12000 rpm for 2 minutes. Take the supernatant and measure the absorbance at 410 nm, zeroing the microscope with distilled water. Plot a standard curve based on the measurement results, as follows: Figure 6 As shown, the relevant linear formula is Y = 1.5318X + 0.0093, R0 2 =0.999, where Y is the p-NP concentration, X is the absorbance value, and a and b are coefficients.

[0069] 2. Sample enzyme activity assay Weigh 0.03 g of p-nitrophenol palmitate (p-NPP) and dissolve it in 10 mL of isopropanol to prepare a p-NPP solution. Take 2 mL of the p-NPP solution and add 18 mL of pH 8.0 buffer solution, mix well, and obtain the working solution. Take 4 test tubes and add 2.4 mL of the above working solution to each tube, and incubate them in a water bath at 37 °C for 3 min. Add 100 μL of crude enzyme solution to 3 test tubes as samples, and add 100 μL of crude enzyme solution that has been denatured by boiling (95 °C, 10 min) to the other tube as a control. Mix well, and place all 4 test tubes in a water bath at 37 °C for 15 min. After removing the tubes, add 2 mL of 95% ethanol to terminate the reaction, centrifuge at 12000 rpm for 2 min, and retain the supernatant to measure the absorbance value. The spectrophotometer was zeroed with distilled water, and the absorbance of the supernatant of Streptococcus salivarius subsp. thermophilus LSE-G45 was measured at 410 nm. A is the absorbance of the sample, A0 is the absorbance of the reference standard, and A-A0 is the absorbance of the actual sample. The lipase activity was calculated using a regression equation.

[0070] Based on the above standard curve, the formula for calculating lipase activity is: Lipase activity (U / mL) = (a×△OD+b)×n×1×10 / 15; In the formula, △OD: A-A0 (absorbance value at 410nm); a and b: coefficients in the p-NP standard curve formula; The enzyme activity in a 10:100 μL enzyme solution is calculated as the enzyme activity per 1 mL. 15: The reaction time is 15 minutes; n: The dilution factor of the enzyme solution.

[0071] like Figure 7 As shown, the enzyme activity of Streptococcus salivarius subsp. thermophilus LSE-G45 (denoted as LSE-G45) is 5.68±0.03 U / mL.

[0072] Example 5 This embodiment provides tests on the resistance of Streptococcus salivarius subsp. thermophilicus LSE-G45 to intestinal and gastric fluids.

[0073] Gastric juice: Dissolve 10g pepsin and 16.4mL of 0.1mol / L hydrochloric acid in 950mL of water, adjust the pH to 2.0, and then bring the volume to 1000mL.

[0074] Intestinal fluid: Dissolve 6.8g potassium dihydrogen phosphate and 10g trypsin in 950mL of water, adjust the pH to 6.8, and then bring the volume to 1000mL.

[0075] The thermophilic subsp. *Salinomyces* LSE-G45 strain was activated three times in TJA liquid medium to obtain a viable count of 1.2 × 10⁻⁶. 8 CFU / mL of activated bacterial culture was inoculated into TJA liquid medium at a 2% (v / v) inoculum and incubated at 38°C for 16 h. After centrifugation at 8000 rpm for 20 min, the culture was washed twice with PBS buffer and resuspended in gastric juice (pH 2), intestinal juice (pH 6.8), or physiological saline, respectively. Four groups were established: gastric juice group, intestinal juice group, and a group that incubated in gastric juice first and then intestinal juice. Viable bacterial counts were measured after incubation at room temperature for 3 h (for the group incubated in gastric juice first, then intestinal juice for 3 h).

[0076] Survival rate (%) = N0 / N t ×100%; In the formula, N t viable bacteria count over 3 hours, N o : 0h viable bacteria count.

[0077] After 3 hours of culture in gastric juice, the viable bacterial survival rate of Streptococcus thermophilus subsp. LSE-G45 was 86±4.1%, and after 3 hours of culture in intestinal juice, the viable bacterial survival rate was 78.66±5.1%. After 3 hours of culture in gastric juice and then 3 hours in intestinal juice, the viable bacterial survival rate was 77.66±5.00%, which proves that a large number of Streptococcus thermophilus subsp. LSE-G45 survives in the gastrointestinal tract and has good tolerance to gastric and intestinal juices.

[0078] Example 6 This embodiment provides a test of the hydrophobic properties of the thermophilic subspecies of Streptococcus salivarius LSE-G45.

[0079] Phosphate buffer (PBS buffer): Weigh 0.24 g potassium dihydrogen phosphate, 1.44 g disodium hydrogen phosphate, 0.8 g sodium chloride, and 0.2 g potassium chloride, dissolve in 500 mL of pure water, bring the volumetric flask to 1000 mL, and sterilize at 118 °C for 20 min.

[0080] The thermophilic subsp. *Salinomyces* LSE-G45 strain was activated three times in TJA liquid medium to obtain a viable count of 1.2 × 10⁻⁶. 8 The activated bacterial culture was prepared at CFU / mL and then inoculated into TJA liquid medium at a volume fraction of 2%. The culture was incubated at 38°C for 16 hours. After incubation, the bacterial culture was mixed and centrifuged at 4500 rpm for 10 minutes. The bacterial cells were collected, washed twice with PBS buffer, and then the OD value was adjusted with PBS buffer. 600nm The value was adjusted to 0.6 ± 0.05, recorded as A0. 1 mL of xylene was added to the adjusted bacterial cell concentration, mixed well, and incubated at room temperature for 10 min. Then, the mixture was vortexed for 2 min, and reacted at room temperature for 30 min. The absorbance of the aqueous phase at 600 nm was measured and recorded as A1. Three parallel samples were used for each measurement. The hydrophobicity was calculated using the following formula: Hydrophobicity (%) = (1 - A1 / A0) × 100%.

[0081] Strain hydrophobicity refers to the degree of hydrophobicity on the surface of a strain cell, reflecting the ability of the bacteria to bind to hydrophobic substances (such as the hydrophobic region on the surface of intestinal epithelial cells). It is an important prerequisite for strains to adhere to the intestinal mucosa. When the hydrophobicity of a strain is ≥40%, its adhesion and colonization potential is excellent.

[0082] The hydrophobicity of the Streptococcus salivarius thermophilus subsp. LSE-G45 strain provided by this invention is 76.17±0.33%, indicating that this strain has good hydrophobic properties.

[0083] Example 7 This embodiment provides a test of the self-agglutination ability of Streptococcus salivarius thermophilus subspecies LSE-G45.

[0084] The thermophilic subsp. *Salinomyces* LSE-G45 strain was activated three times in TJA medium to obtain a viable count of 1.2 × 10⁻⁶ cells / year. 8 The activated bacterial culture was prepared at CFU / mL and then inoculated into TJA liquid medium at a volume fraction of 2%. The culture was incubated at 38°C for 16 hours. After incubation, the bacterial culture was mixed and centrifuged at 4500 rpm for 10 minutes. The bacterial cells were then washed twice with PBS (0.01 mol / L) buffer, and the OD value was adjusted with PBS buffer. 600nm The absorbance was recorded as A0 when the concentration reached 0.6 ± 0.05. After incubation at 38℃ for 24 h, the absorbance of the supernatant at 600 nm was measured and recorded as A1. Three parallel samples were used for each measurement. The self-agglutination rate was calculated using the following formula: Self-agglomeration rate (%) = (1-A1 / A0)×100%.

[0085] The self-agglutination ability of a strain refers to the ability of the strain's own cells to aggregate and form a colony. It is an important characteristic for the strain to form a biofilm in the intestine and enhance its colonization stability. The 24-hour self-agglutination rate of the strain is ≥70%, indicating excellent colonization stability.

[0086] The autoagglutination rate of the thermophilic subspecies of Streptococcus salivarius LSE-G45 strain provided by this invention is 87.00±0.10% in 24 hours, indicating that this strain has good autoagglutination ability.

[0087] Example 8 This embodiment provides an antioxidant activity test of Streptococcus salivarius subsp. thermophilicus LSE-G45 strain.

[0088] 1. Determination of DPPH free radical scavenging ability The thermophilic subsp. *Salinomyces* LSE-G45 strain was activated three times in TJA liquid medium to obtain a viable count of 1.2 × 10⁻⁶. 8 The activated bacterial culture was prepared at CFU / mL and then inoculated into TJA liquid medium at a volume fraction of 2%. The culture was incubated at 38℃ for 16 hours. After incubation, the bacterial culture was mixed and centrifuged at 10000 rpm for 10 minutes. The supernatant was collected and stored at 4℃ for later use. 0.0079 g of 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH) was dissolved in 100 mL of anhydrous ethanol to prepare the working solution. 2 mL of the supernatant was mixed with the working solution to prepare the bacterial culture. The culture was reacted at room temperature in the dark for 30 minutes, then centrifuged (4℃, 6000 rpm, 10 minutes). The supernatant was collected for later use. The solutions were: blank group: 2 mL supernatant + 2 mL anhydrous ethanol; control group: 2 mL DPPH + 2 mL anhydrous ethanol; sample group: 2 mL DPPH + 2 mL supernatant. The DPPH free radical scavenging rate was calculated using the following formula: DPPH free radical scavenging rate (%) = [1 - (Ai -A0) / A j ×100%; In the formula, A i : The absorbance of the sample group at 517 nm; A0: Absorbance of the blank group at 517nm; A j : Absorbance of the control group at 517nm.

[0089] The DPPH free radical scavenging rate of the LSE-G45 strain of Streptococcus salivarius of this invention is 69.33±0.58%, indicating that this strain has good DPPH free radical scavenging ability.

[0090] Example 9 This embodiment provides ABTS of Streptococcus salivarius subsp. thermophilus LSE-G45. + Tests to determine free radical scavenging capacity.

[0091] ABTS + Preparation of the diluent working solution (absorbance at 734 nm is 0.7): Accurately weigh 0.192 g of 3-ethyl-benzothiazole-6-sulfonic acid (ABTS). + Dissolve the standard sample in water and bring the volume to 50 mL. At the same time, accurately weigh 0.067 g of potassium persulfate, dissolve it in water and bring the volume to 50 mL. Mix the two solutions prepared above in a 1:1 volume ratio and store them in a dark environment for 12 h. Dilute them 35 times with PBS buffer (0.01 mol / L, pH=7.4) before use.

[0092] Take 250 μL of sample; the viable bacterial count is 1.2 × 10⁻⁶. 8 A solution of Streptococcus salivarius subsp. thermophilus LSE-G45 at CFU / mL was added to a centrifuge tube, followed by the addition of 1900 μL of ABTS. + Dilute the working solution and react at room temperature for 6 min. After the reaction is complete, use the sample and measure the absorbance at 734 nm. Transfer 1900 μL of ABTS. + The working solution was diluted and mixed with 250 μL of PBS buffer (0.01 mol / L, pH=7.4) as a blank control. ABTS was then calculated. + The free radical scavenging rate is calculated using the following formula: ABTS + Free radical scavenging rate (%) = [(A0-A1) / A0] × 100%; In the formula, A0 is the absorbance of the blank control; A1 is the absorbance of the sample.

[0093] The ABTS of Streptococcus salivarius subsp. thermophilus LSE-G45 strain of this invention +The free radical scavenging rate was 98.33±0.58%, indicating that this strain exhibited good ABTS (Alternative Targeted Therapy) performance. + Free radical scavenging ability.

[0094] Example 10 This embodiment provides a safety evaluation of the LSE-G45 strain of Streptococcus salivarius thermophilus.

[0095] Hemolytic test: The thermophilic subsp. *Salinomyces* LSE-G45 strain was activated three times in TJA liquid medium, yielding a viable count of 1.2 × 10⁻⁶. 8 The activated bacterial solution at CFU / mL was streaked onto Columbia blood agar medium and incubated at 38°C for 48 h. After incubation, the presence of hemolytic zones around the colonies was observed. During this process, *Lactobacillus rhamnosus* LSE-H023-A-07 (deposited at the China General Microbiological Culture Collection Center, accession number CGMCC No. 26668, address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Zhaoyang District, Beijing, deposited on February 21, 2023) was used as a negative control strain to assess and compare whether the test strains exhibited hemolytic activity.

[0096] This invention tested the hemolytic activity of *Streptococcus thermophilus* subsp. LSE-G45 on blood agar plates, using *Lactobacillus rhamnosus* LSE-H023-A-07 as a negative control. Figures 8-9 As shown, no obvious clear zone was observed around the two strains, indicating that the strains are γ-hemolytic and do not produce β-hemolysin.

[0097] Example 11 This embodiment provides the application of Streptococcus salivarius thermophilic subspecies LSE-G45 in the fermentation of light cream.

[0098] 1. Preparation of fermentation broth for Streptococcus salivarius subsp. thermophilus LSE-G45 strain The thermophilic subsp. salivarius LSE-G45 strain was inoculated into TJA liquid medium at an inoculation rate of 2% and cultured at 38°C for 16 hours to prepare a high-density yeast culture. The activated bacterial solution was inoculated into a high-density culture medium at an inoculum volume of 3%. The fermentation temperature was set at 42℃, pH at 5.5, and the rotation speed at 100 r / min. Fermentation was carried out for 4 h 30 min ~ 6 h, yielding a viable cell count of 1.4 × 10⁻⁶. 10 Fermentation agent at CFU / mL.

[0099] 2. Preparation of Fermented Whipping Cream Weigh a certain amount of light cream and raw milk to make the base material have a fat content of 28%~38.0% and a protein content of 1.5%~2.5%. Homogenize the base material under a pressure of 20~30MPa, sterilize it in a water bath at 98℃ for 30 minutes, and then let it stand and cool to obtain the light cream base material. After the temperature of the cream base material drops to 40-42℃, add the above-mentioned starter culture at inoculation rates of 0.05%, 0.2%, 0.5%, and 1.0%, respectively. Ferment at a constant temperature of 40℃ for 8-9 hours. The overall fermentation acidity is between 69-72°T, and the finished product acidity is between 70-78°T. Sensory evaluation of the finished product is shown in Table 3. The fermented cream prepared with an inoculation rate of 0.2% has a delicate texture, a rich buttery aroma, and a good taste. Specific sensory evaluations are as follows: Figure 10 As shown.

[0100] Table 3

[0101] 3. Preparation of Probiotic Freeze-Dried Powder Mix 1g glycine, 7g trehalose, 1g L-cysteine ​​and 10g skim milk evenly, add water to make 100mL, sterilize at 115℃ for 15min to obtain the protectant; The above-mentioned starter culture was centrifuged at 4800 rpm for 20 min. The centrifuged bacterial sludge and protective substrate were mixed evenly at a mass ratio of 1:1. The mixture was pre-frozen at -80°C for 2 h under a vacuum of 1 Pa. Then it was frozen at -65°C for 32 h to obtain probiotic freeze-dried powder. Weigh a certain amount of light cream and raw milk to make the base material have a fat content of 28%~38.0% and a protein content of 1.5%~2.5%. Homogenize the base material under a pressure of 20~30MPa, sterilize it in a 98℃ water bath for 30 minutes, and then let it stand and cool to obtain the light cream base material. The above-mentioned freeze-dried probiotic powder was added to the cream base at inoculation rates of 50g / t, 100g / t, and 150g / t, and fermented at a constant temperature of 40℃ for 8-12 hours. The overall fermentation acidity was between 67-73°T, and the finished product acidity was between 70-76°T. Sensory evaluation of the finished product was conducted, and the sensory scoring table is shown in Table 3. The fermented cream prepared with an inoculation rate of 100g / t had a delicate texture, a very rich fat aroma, and the best taste. Specific sensory evaluation results are as follows: Figure 11 As shown.

[0102] 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 or improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A thermophilic subspecies of Streptococcus salivarius ( Streptococcus salivarius subsp .thermophilus LSE-G45, characterized in that, Its accession number is CGMCC No.36396.

2. The application of the thermophilic subspecies of Streptococcus salivarius LSE-G45 as described in claim 1 in the production of lipase.

3. A fermenting agent, characterized in that, The fermentation agent includes LSE-G45 of Streptococcus salivarius as described in claim 1.

4. A method for preparing the fermenting agent according to claim 3, characterized in that, Includes the following steps: The thermophilic subspecies of Streptococcus salivarius LSE-G45 was inoculated into a seed culture medium for fermentation to obtain a starter culture.

5. The method for preparing the fermenting agent as described in claim 4, characterized in that, The fermentation conditions include: a temperature of 40℃~42℃ and a rotation speed of 100r / min~120r / min; The inoculation amount of the thermophilic subsp. LSE-G45 of Streptococcus salivarius was 2% to 3%. The viable count of *Streptococcus thermophilus* subsp. LSE-G45 in the fermentation spawn was 1.2 × 10⁻⁶. 8 CFU / mL ~1.4×10 10 CFU / mL.

6. A probiotic freeze-dried powder, characterized in that, Includes the fermenting agent as described in claim 3.

7. A method for preparing the probiotic freeze-dried powder according to claim 6, characterized in that, Includes the following steps: The fermentation agent is subjected to solid-liquid separation, a protective agent is added to the separated bacterial sludge, and the mixture is freeze-dried to obtain probiotic freeze-dried powder.

8. The method for preparing probiotic freeze-dried powder as described in claim 7, characterized in that, The mass ratio of the bacterial sludge to the protective agent is (1~2):1; The protective agent comprises the following components in weight percentage: 0.6%~1% glycine, 5%~7% trehalose, 0.5%~1% L-cysteine, 10%~12% skim milk, and the balance being water; The freeze-drying conditions include: pre-freezing at -80℃ to -60℃ for 1.5h to 3h under a vacuum of 1Pa to 2Pa; and then freezing at -80℃ to -50℃ for 24h to 36h.

9. A method for preparing fermented light cream, characterized in that, Includes the following steps: The fermenting agent of claim 3 or the lyophilized probiotic powder of claim 6 is inoculated into light cream for fermentation culture.

10. The method for preparing fermented light cream as described in claim 9, characterized in that, The inoculum amount of the fermenting agent is 0.05%~1%; The inoculation amount of the probiotic freeze-dried powder is 50~150g / t; The fermentation culture temperature is 38℃~42℃; The fermentation culture time is 8h~12h.

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