Lactobacillus plantarum h2-5 with cholesterol-lowering function and application thereof

Abstract

The application discloses a plant lactobacillus H2-5 with a cholesterol-lowering function and application thereof, the plant lactobacillus H2-5 has been preserved, and the preservation number is CGMCC No. 38949, and the strain genome NCBI number is SRR38400354. The application has excellent gastrointestinal tolerance (survival rate is greater than 60% after being treated by pH 3.0 simulated gastric juice for 3 h), good intestinal adhesion and colonization potential, high BSH enzyme activity (about 0.435 U / mg) and significant in-vitro cholesterol-lowering capacity (about 46 mg / L in the live bacteria group and about 40 mg / L in the dead bacteria group). After the strain is compounded with a commercial ferment YF-L904, a significant synergistic effect can be generated, so that the live bacteria number of fermented milk is stably maintained to be greater than 8.6 lg CFU / mL within 28 days of storage, the water holding capacity is increased by 6%-8%, and key texture indexes such as hardness, consistency, viscosity and cohesiveness are all better than those of a commercial control group, and the total score of comprehensive sensory is 81; the ferment composition has good broad-spectrum applicability to various characteristic milk sources such as cow milk, sheep milk, horse milk, camel milk and donkey milk, and can significantly improve the commodity quality and added value of products.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology and food engineering, specifically involving a strain of Lactiplantibacillus plantarum H2-5 with excellent gastrointestinal tolerance, high bile salt hydrolase (BSH) activity, and significant in vitro cholesterol-lowering ability. The strain has the preservation number CGMCC No. 38949 and the NCBI genome number SRR38400354. The invention also relates to the application of this strain in the preparation of various specialty milk sources such as cow's milk, goat's milk, mare's milk, camel's milk, and donkey's milk after being compounded with commercial starter cultures. Background Technology

[0002] Cardiovascular disease is one of the leading causes of death from chronic diseases worldwide, and its primary controllable risk factors are elevated levels of serum total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C). Long-term hypercholesterolemia is closely associated with adverse events such as coronary atherosclerosis, myocardial infarction, and stroke. In addition to dietary control and statin intervention, consuming cholesterol-lowering foods through daily diet has become an important way to assist in the prevention and improvement of hypercholesterolemia, with significant advantages of high safety and good adherence.

[0003] The core mechanism by which probiotics lower host serum cholesterol is currently recognized. Probiotics with high BSH activity can hydrolyze conjugated bile acids into free bile acids, promoting their excretion in feces. To compensate for the loss of bile acid reserves, the body converts more cholesterol into new bile acids, thereby lowering serum cholesterol levels. In addition, the functional structural components contained on the surface of live probiotics or their inactivated bacterial cells can also exert cholesterol-lowering effects synergistically through multiple pathways, such as cholesterol adsorption and co-precipitation, and inhibition of intestinal cholesterol absorption.

[0004] Lactiplantibacillus plantarum (reclassified from the genus *Lactiplantibacillus*) is a recognized safe probiotic widely used in fermented foods. Different strains of this species exhibit significant strain specificity in terms of BSH activity, acid production capacity, fermentation characteristics, and adaptability to milk matrices. Therefore, screening new strains with clear probiotic functions and excellent fermentation characteristics from natural sources has significant industrial value.

[0005] On the other hand, fermented milk is the most important carrier food for probiotics. However, commercially available fermented milk generally has the following problems: (1) severe post-acidification during storage, with the acidity of the product continuously rising and producing an excessive sour taste during the shelf life, leading to a decrease in consumer acceptance; (2) insufficient gel network strength, whey easily separating, resulting in damage to appearance and taste; (3) continuous deterioration of texture during storage, with a decrease in hardness, consistency and viscosity; (4) single flavor and lack of clear health function claims, resulting in serious product homogenization.

[0006] With the upgrading of consumption, the market demand for specialty milk sources such as goat milk, mare milk, camel milk, and donkey milk is increasing due to their high nutritional value, low allergenicity, and richness in characteristic active peptides and functional fatty acids. However, these specialty milk sources face unique bottlenecks in fermentation processing: (1) significant differences in protein composition (e.g., the ratio of κ-casein to αs1-casein, the whey protein ratio, and the casein micelle structure are all significantly different from those of cow milk), resulting in weak coagulation ability of traditional starter cultures, thin product texture, and severe whey separation; (2) strong inherent flavors (e.g., the goaty smell of goat milk and the salty and savory taste of camel milk), which are not easily accepted by the public, and the flavor coordination is poor after fermentation; (3) lack of dedicated functional fermentation strains, resulting in product homogenization and low added value. Therefore, there is an urgent need to develop a dedicated probiotic fermentation solution that can broadly adapt to various specialty milk protein systems and simultaneously achieve functional enhancement, texture improvement, and flavor coordination.

[0007] Given the above background, there is an urgent need in the field to provide a new strain of *Lactobacillus plantarum* that possesses excellent gastrointestinal tolerance, high BSH activity, significant cholesterol-lowering function, and can exert synergistic effects when combined with commercial fermentation agents in various specialty milk sources, significantly improve product texture, inhibit post-acidification and whey separation, and maintain a high viable count during storage. Summary of the Invention

[0008] To overcome the shortcomings of the prior art, the purpose of this invention is to provide a plant lactobacillus H2-5 with cholesterol-lowering function and its application.

[0009] To achieve the aforementioned objective, the technical solution of the present invention is as follows: a strain of *Lactobacillus plantarum* H2-5 and its related applications.

[0010] In a first aspect, the present invention provides a strain of Lactiplantibacillus plantarum H2-5, which is taxonomically named Lactiplantibacillus plantarum. This strain was deposited on June 1, 2026, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 38949 and NCBI genome number SRR38400354.

[0011] The *Lactobacillus plantarum* H2-5 described in this invention has the following biological characteristics: the colonies are milky white, smooth, with neat edges, and opaque; the cells are Gram-positive, short rod-shaped, and arranged singly, in pairs, or in chains; catalase-negative, non-spore-forming, non-flagellated, and non-motile; the optimal growth temperature is 37℃, and the optimal growth pH is 6.5.

[0012] The *Lactobacillus plantarum* H2-5 described in this invention has the following functional characteristics: (1) its survival rate is higher than 60% after treatment in simulated gastric juice at pH 3.0 for 3 h; (2) its cells can continue to proliferate after treatment in an environment containing 0.5%-1.0% bile salts for 6 h, showing excellent bile salt tolerance; (3) its cell surface hydrophobicity is about 34% and its self-aggregation is about 43%, indicating good potential for intestinal adhesion and colonization; (4) its BSH enzyme activity is as high as about 0.435 U / mg, significantly higher than that of the tested strains; (5) in a medium containing exogenous cholesterol, the cholesterol degradation of the live bacteria group reaches about 46.12 mg / L, while the cholesterol adsorption / degradation of the heat-killed bacteria group still reaches about 40.08 mg / L, indicating that its cholesterol-lowering function does not completely depend on live bacteria, and inactivated bacteria can also play a role, significantly broadening its application scenarios.

[0013] Secondly, the present invention provides the application of the aforementioned *Lactobacillus plantarum* H2-5 in the preparation of products with cholesterol-lowering functions. These products include, but are not limited to, fermented milk, yogurt, probiotic beverages, probiotic solid beverages, probiotic freeze-dried powders, probiotic capsules, and probiotic tablets.

[0014] Thirdly, the present invention provides a starter culture composition containing *Lactobacillus plantarum* H2-5 and a commercial starter culture. Preferably, the commercial starter culture is YF-L904 purchased from Chr. Hansen; the starter culture composition is suitable for fermenting one or more dairy raw materials selected from cow's milk, goat's milk, mare's milk, camel's milk, and donkey's milk.

[0015] Fourthly, the present invention provides a fermented milk, which is obtained by fermentation of Lactobacillus plantarum H2-5 alone, or by fermentation of the starter culture composition (i.e., Lactobacillus plantarum H2-5 combined with a commercial starter culture); the raw milk of the fermented milk is selected from one or more of cow's milk, goat's milk, mare's milk, camel's milk, and donkey's milk.

[0016] Fifthly, the present invention provides a method for improving the quality of fermented milk, the method comprising simultaneously inoculating a commercial starter culture and the *Lactobacillus plantarum* H2-5 into a milk raw material and fermenting it at a suitable temperature; the milk raw material is selected from one or more of cow's milk, goat's milk, mare's milk, camel's milk, and donkey's milk; preferably, the fermentation temperature is 40-43°C, for example 42°C; the inoculation amount of the commercial starter culture is 0.002‰-0.01‰ (w / w), for example 0.004‰; the inoculation amount of the *Lactobacillus plantarum* H2-5 starter culture is 0.5%-2.0% (v / v), for example 1.0%; and the fermentation endpoint is pH 4.5-4.6.

[0017] The beneficial effects of this invention are reflected in: (1) The plant lactobacillus H2-5 has significant gastrointestinal tolerance, high BSH activity and excellent in vitro cholesterol-lowering ability. Both live bacteria and heat-killed bacteria can produce cholesterol-lowering effects, and can be used as an excellent industrial strain for developing functional fermented foods. (2) The combination of *Lactobacillus plantarum* H2-5 and commercial starter YF-L904 showed a significant synergistic effect: the viable count remained stable at above 8.6 lg CFU / mL after 28 days of storage, significantly higher than the single-strain group and the commercial control group; water holding capacity increased by about 6%-8% and whey separation was significantly reduced; key textural indicators such as hardness, consistency, viscosity and cohesion were better than the commercial control group throughout the process; the overall sensory score of the product reached about 81 points, significantly higher than the commercial control group (about 64 points) and the H2-5 single-strain group (about 72.7 points). (3) The fermentation agent composition exhibits good broad-spectrum applicability to various specialty milk sources such as cow's milk, goat's milk, mare's milk, camel's milk, and donkey's milk. It can overcome the inherent defects of the protein system differences of specialty milk sources, weak coagulation ability, easy whey separation, and poor flavor coordination. The resulting product has dense coagulation, uniform texture, and mild and harmonious flavor. The number of viable bacteria remains stable at a high level within 21 days of storage at 4℃, and the pH and titratable acidity change steadily. It can significantly improve the commercial quality and added value of specialty milk source fermented products. Attached Figure Description

[0018] In the attached diagram: Figure 1 A complete genome circle map of Lactobacillus plantarum H2-5; Figure 2 A bar chart comparing the survival rates of Lactobacillus plantarum H2-5 and the control strain under simulated gastric juice conditions at pH 3.0 and 10 g / L bile salts. Figure 3 A bar chart comparing the BSH enzyme activity and in vitro cholesterol-lowering ability (live group and lethal group) of Bacterium plantarum H2-5 and control strains. Figure 4The graph shows the changes in viable cell count, pH, and titratable acidity after storage at 4℃ for 0, 7, 14, 21, and 28 days in the commercial control group, the H2-5 single-strain group, and the H2-5 combined with commercial starter culture group. Figure 5 This is a comparative curve showing the changes in hardness, consistency, viscosity, and cohesiveness of fermented milk in the commercial control group and the H2-5 compound group during storage. Figure 6 The images show actual photos of fermented milk products obtained by fermenting cow's milk, goat's milk, mare's milk, camel's milk, and donkey's milk with the fermentation agent composition of the present invention, respectively, demonstrating that the coagulated milk from each milk source has a good state, fine texture, and no serious whey separation. Detailed Implementation

[0019] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments. Obviously, the described embodiments are only a part of the embodiments of the invention, and not all of them. Unless otherwise specified, the embodiments and features described in this application can be combined with each other. All other embodiments obtained by those skilled in the art based on the embodiments of the invention without inventive effort are within the scope of protection of the invention.

[0020] Please refer to the instruction manual appendix. Figures 1-6 This invention provides a *Lactobacillus plantarum* H2-5 with cholesterol-lowering function and its application. The commercial fermentation agent YF-L904 used in this invention was purchased from Chr. Hansen. MRS liquid culture medium and agar were purchased from Beijing Luqiao Technology Co., Ltd. Bile salts (bovine bile salts), cholesterol standards, o-phthalaldehyde (OPA), sodium taurine deoxycholate (TDCA) and other reagents were purchased from Sigma-Aldrich. Fresh cow's milk, goat's milk, mare's milk, camel's milk and donkey's milk were purchased from quality-assured pastures in Inner Mongolia.

[0021] Example 1: Isolation, identification and preservation of Lactobacillus plantarum H2-5 1.1 Strains Isolation: Traditional fermented milk samples collected from Inner Mongolia were anaerobically cultured on MRS agar at 37°C for 48 h using a serial dilution method. Single colonies with typical morphology were selected and purified for three generations. The activated and frozen strains were preserved and numbered H2-5.

[0022] 1.2 Morphological identification: Colonies are milky white, round, smooth, with regular edges, and opaque; Gram-positive, short rod-shaped, single or paired, or arranged in chains; catalase-negative, non-spore-forming, non-flagellated, and non-motile.

[0023] 1.3 Molecular biological identification: Genomic DNA was extracted using the CTAB method; the 16S rRNA gene was amplified by PCR using universal primers 27F / 1492R and bidirectionally sequenced. The sequencing results (approximately 1450 bp) were compared with the GenBank database using BLAST, and the results showed that the similarity to the type strain of *Lactiplantibacillus plantarum* was over 99.9%; further sequencing of recA, pheS, and tuf housekeeping genes and analysis of the average nucleotide identity (ANI) of the whole genome confirmed that the strain was *Lactiplantibacillus plantarum* H2-5.

[0024] 1.4 Preservation of strain: The *Lactobacillus plantarum* H2-5 described in this invention has been preserved with the preservation number CGMCCNo.38949 and the NCBI genome number of the strain is SRR38400354.

[0025] Example 2: In vitro probiotic properties and cholesterol-lowering function characterization of Lactobacillus plantarum H2-5 To evaluate the probiotic potential of *Lactobacillus plantarum* H2-5, it was compared in parallel with two other *Lactobacillus plantarum* strains (H3-2 and H3-12) isolated from conventional fermented milk during the same period. All strains were activated under the same conditions and used the same detection methods, with three replicates per group.

[0026] 2.1 Simulated Gastric Fluid Tolerance: The activated bacterial culture was centrifuged (8000×g, 4℃, 10 min) to collect bacterial cells. The cells were washed twice with sterile PBS and resuspended to the same OD600. The cells were then inoculated at 10% (v / v) into simulated gastric fluid containing 3 mg / mL pepsin at pH 3.0 and incubated at 37℃ for 3 h. Samples were taken at 0 h and 3 h, and the viable cell count was determined using the MRS agar pour plate method. The results are shown in Table 1.

[0027] Table 1. Changes in viable bacterial counts of each strain in simulated gastric fluid at pH 3.0 (×10⁻¹⁰) 9 (CFU / mL, n=3) As shown in Table 1, the survival rate of *Lactobacillus plantarum* H2-5 after treatment in simulated gastric juice at pH 3.0 for 3 h reached approximately 64.71%, which was significantly higher than that of the control strain tested, indicating that it has excellent gastric juice tolerance.

[0028] 2.2 Bile salt tolerance: The activated bacterial culture was inoculated at 10% (v / v) into MRS liquid medium containing 0.3%, 0.5% and 1.0% bovine bile salts, and incubated at 37℃ for 0 h and 6 h. The viable bacterial count was then counted on pour plates. The results are shown in Table 2.

[0029] Table 2. Changes in viable bacterial counts of various strains in different concentrations of bile salts (×10⁻¹⁰) 9 (CFU / mL, n=3) As shown in Table 2, the *Lactobacillus plantarum* H2-5 strain can still maintain stable proliferation under bile salt stress of 0.3%, 0.5%, and up to 1.0%. After 6 h of treatment, the number of viable bacteria increased by about 30%-58% compared with 0 h, and the survival rate was ≥80%. This indicates that the strain has excellent bile salt tolerance and can tolerate the intestinal environment of human small intestine physiological bile salt concentration (typical value 0.3%-0.5%).

[0030] 2.3 Cell surface hydrophobicity and self-aggregation: The hydrophobicity of the bacterial cell surface was determined according to the xylene extraction method of Rosenberg et al.; the self-aggregation was determined by allowing the bacterial suspension to settle naturally. The results showed that the cell surface hydrophobicity of *Lactobacillus plantarum* H2-5 was approximately 34.27%, and the self-aggregation was approximately 43.47%, suggesting that this strain has good potential for intestinal adhesion and colonization, which is beneficial for its probiotic effects in the intestinal environment.

[0031] 2.4 BSH enzyme activity assay: After the bacterial cells were lysed, intracellular proteins were collected by centrifugation. Using sodium taurodeoxycholate (TDCA) as a substrate, the reaction was carried out at 37℃ for 30 min. The amount of released amino acids was determined at 570 nm using the ninhydrin colorimetric method, and the BSH activity was calculated. The results are shown in Table 3.

[0032] Table 3. BSH enzyme activity of each strain (U / mg protein, n=3) As shown in Table 3, the BSH enzyme activity of *Lactobacillus plantarum* H2-5 reached 0.435±0.008 U / mg protein, which was significantly higher than that of the tested control strain (P<0.05). This was the highest level among the strains screened in this study, suggesting that it has a strong bile acid debinding ability, which is the core mechanism of its cholesterol-lowering function.

[0033] 2.5 In vitro cholesterol-lowering ability: MRS liquid culture medium containing 0.1 mg / mL water-soluble cholesterol was prepared, and live bacteria (live bacteria group) or bacteria inactivated at 80℃ for 30 min (lethal group) were inoculated separately. After incubation at 37℃ for 24 h, the culture was centrifuged, and the residual cholesterol concentration of the supernatant was determined at 550 nm using the phthalaldehyde method. The cholesterol degradation / adsorption amount was calculated. The results are shown in Table 4.

[0034] Table 4. In vitro cholesterol-lowering capacity of each strain (mg / L, n=3) As shown in Table 4, the in vitro cholesterol degradation of the live *Lactobacillus plantarum* H2-5 group reached approximately 46.12 mg / L, significantly higher than that of the tested control strain. The cholesterol degradation / adsorption of the heat-killed bacterial group still reached 40.08 mg / L, indicating that the cholesterol scavenging effect of this strain does not entirely depend on live bacterial metabolism; the inactivated functional structural components on the bacterial surface can also play a role, significantly broadening the application of this strain in functional foods. Based on the comprehensive in vitro indicators in Example 2, H2-5 is indeed the most superior potential probiotic strain among the screened homologous strains.

[0035] Example 3: Compound application of Lactobacillus plantarum H2-5 in fermented milk and its product advantages 3.1 Preparation process of fermented milk: (1) Heat fresh milk to 55-60℃, add 7% (w / w) white sugar and stir until completely dissolved; (2) Preheat to 65°C and homogenize under high pressure at 20 MPa; (3) Sterilize at 95℃ for 5 min, then cool to 42℃; (4) Inoculation was performed in the following groups: ① Commercial control group: only 0.004‰ (w / w) of commercial starter culture YF-L904 was inoculated; ② H2-5 single-strain group: only 1.0% (v / v) of Bacillus plantarum H2-5 liquid starter culture (live count ≥1×10⁻⁶) was inoculated. 9 CFU / mL); ③ H2-5 compound group: simultaneously inoculated with 0.004‰ YF-L904 and 1.0% Lactobacillus plantarum H2-5; (5) Ferment at a constant temperature of 42℃ until pH 4.5-4.6 (about 5-6 h), then cool to below 10℃; (6) After ripening at 4℃ for 12 h, it is then divided into portions for use.

[0036] 3.2 Viable bacteria count during storage: The three groups of fermented milk were stored at 4℃ for 28 days. Samples were taken on days 1, 7, 14, 21 and 28, respectively. The viable bacteria count was determined by MRS agar pour plate counting method. The results are shown in Table 5.

[0037] Table 5. Changes in viable bacterial counts during storage of fermented milk in each group (lg CFU / mL, n=3) As shown in Table 5, the viable count of the H2-5 compound fermented milk remained stable at above 8.6 lg CFU / mL for 28 days after storage at 4℃, which was significantly higher than that of the commercial control group and the H2-5 single-strain group (P<0.05). This indicates that the fermentation agent composition provided by the present invention can stably maintain a high viable count during storage, which is beneficial to meeting the requirements of probiotic products for viable count during shelf life.

[0038] 3.3 Changes in pH and titratable acidity during storage: The determination methods were in accordance with GB 5009.239-2016 and GB 5413.34. The results are shown in Tables 6 and 7.

[0039] Table 6. pH changes during storage of fermented milk in each group (n=3) Table 7. Changes in titratable acidity of fermented milk in each group during storage (°T, n=3) As shown in Tables 6 and 7, the pH and titratable acidity of the H2-5 compound fermented milk during storage were reasonable and the overall trend was stable. After 28 days of storage, the pH remained above 4.11 and the titratable acidity did not exceed 115°T. There was no obvious excessive post-acidification, and the overall taste of the product was more harmonious.

[0040] 3.4 Water-holding capacity and whey separation: The water-holding capacity of the samples at the fermentation endpoint and after 12 h of post-ripening was determined by centrifugation (4℃, 5000×g, 30 min). The results are shown in Table 8.

[0041] Table 8. Water-holding capacity of fermented milk (%, n=3) As shown in Table 8, the water-holding capacity of the fermented milk in the H2-5 compound group was increased by 6%-7% compared with the commercial control group, and the amount of whey separation was significantly reduced. This indicates that the combination of H2-5 and YF-L904 can significantly enhance the water-binding capacity of the gel network and improve the appearance and taste of the product.

[0042] 3.5 Texture characteristics during storage: The hardness, consistency, viscosity and cohesion of samples stored for different times were measured using a texture analyzer (TA.XT Plus, Stable Micro Systems) equipped with an A / BE probe. The results are shown in Tables 9-12.

[0043] Table 9. Changes in hardness during storage (g, n=3) Table 10 Consistency changes during storage (g·s, n=3) Table 11 Viscosity changes during storage (cP, n=3) Table 12 Changes in cohesion during storage (g·s, n=3) As shown in Tables 9-12, compared with the commercial control group, the H2-5 compound fermented milk group had significantly higher absolute values ​​of hardness, consistency, viscosity and cohesion throughout the storage process, especially maintaining a significant advantage in the middle and late stages of storage (14-28 days). This indicates that the addition of H2-5 can work synergistically with YF-L904 to build a more robust gel network structure and effectively resist network collapse and texture decay during storage.

[0044] 3.6 Sensory evaluation: A group of 10 trained sensory evaluators were formed to conduct blind evaluations of each group of fermented milk from five dimensions: color (10 points), aroma (25 points), texture (15 points), taste (25 points), and mouthfeel (25 points). The total score was 100 points. The results are shown in Table 13.

[0045] Table 13 Sensory evaluation of fermented milk in each group (scores, n=10) As shown in Table 13, the H2-5 compound fermented milk achieved a total sensory score of 81.0±1.26, significantly higher than the commercial control group (64.0±1.84) and the H2-5 single-strain group (72.7±1.26). Specifically, the H2-5 compound group scored significantly better than the other two groups in aroma, texture, taste, and mouthfeel, indicating that this starter culture composition has outstanding sensory quality advantages. Based on the results of viable cell count, pH, titratable acidity, water-holding capacity, texture, and sensory properties during storage in Example 3, it can be concluded that the combination of *Lactobacillus plantarum* H2-5 described in this invention with the commercial starter culture YF-L904 produces a significant synergistic effect, endowing the product with higher potential health benefits and substantially improving the product's commercial quality and shelf life, achieving a "1+1>2" synergistic effect.

[0046] Example 4: Applicability of Lactobacillus plantarum H2-5 in various specialty milk sources 4.1 Preparation process: Fresh goat milk, mare milk, camel milk, and donkey milk were used as raw materials. The raw materials were heated, homogenized, sterilized (95℃, 5 min), and cooled to 42℃ according to the process in Example 3. Then, 0.004‰ YF-L904 and 1.0% Lactobacillus plantarum H2-5 starter culture were inoculated according to the H2-5 compound group scheme. The fermentation was carried out at a constant temperature of 42℃ until the pH reached 4.5-4.6 (about 5 h for goat milk, about 6.5 h for mare milk, about 6 h for camel milk, and about 7 h for donkey milk). After that, the fermentation was cooled and stored at 4℃.

[0047] 4.2 Observation of Product Appearance and Texture: All fermented milks from various milk sources exhibited good curdling state, uniform and dense texture, smooth surface, and no obvious whey separation. Goat milk fermented milk had a typical fermented milk flavor with significantly reduced goaty odor and a delicate texture; mare milk fermented milk fermented smoothly, with a refreshing taste and mellow flavor; camel milk fermented milk formed a stable gel system, with a mellow flavor and a balanced salty and savory taste; donkey milk fermented milk had a relatively soft and delicate texture and a mild and refreshing flavor.

[0048] 4.3 Physicochemical indicators during storage: Samples were taken on days 0, 7, 14 and 21 to determine the viable count (1g CFU / mL), pH and titratable acidity (°T) of fermented milk from each milk source. Each group was in triplicate. The results are shown in Tables 14-16.

[0049] Table 14 Changes in viable bacteria count during storage of fermented milk from different milk sources (lg CFU / mL, n=3) Table 15 pH changes during storage of fermented milk from different dairy sources (n=3) Table 16 Changes in titratable acidity of fermented milk from different dairy sources during storage (°T, n=3) As shown in Tables 14-16, the starter culture composition (H2-5 and YF-L904 compounded) of this invention exhibits good fermentation performance and storage stability in four characteristic milk sources: goat milk, mare milk, camel milk, and donkey milk. The viable cell count remained above 8.5 1 g CFU / mL throughout 21 days of storage; the pH decreased steadily without drastic post-acidification (pH ≥ 4.25 at 21 days); and the titratable acidity changed gradually with a reasonable overall trend, not exceeding 115°T. Based on the above indicators and sensory performance, the starter culture composition of this invention has good broad-spectrum adaptability to various characteristic milk protein systems. It overcomes the inherent defects of weak coagulation ability, easy whey separation, and poor flavor harmony in characteristic milk sources. The resulting fermented milk products from characteristic milk sources exhibit good coagulation state, harmonious flavor, and stable storage, demonstrating significant industrial and commercial application value.

[0050] 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.

[0051] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, all variations falling within the meaning and scope of equivalents of the claims are intended to be included within the present invention. No reference numerals in the claims should be construed as limiting the scope of the claims.

[0052] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A type of *Lactobacillus plantarum* H2-5, characterized in that, The taxonomic name of the Lactiplantibacillus plantarum strain H2-5 is Lactiplantibacillus plantarum, with accession number CGMCC No. 38949 and NCBI genome number SRR38400354.

2. The application of *Lactobacillus plantarum* H2-5 according to claim 1 in the preparation of products with cholesterol-lowering function; preferably, the product is selected from one or more of fermented milk, yogurt, probiotic beverages, probiotic solid beverages, probiotic freeze-dried powder, probiotic capsules, and probiotic tablets.

3. A fermentation agent composition, characterized in that, The fermentation agent composition comprises *Lactobacillus plantarum* H2-5 as described in claim 1 and a commercial fermentation agent.

4. The fermentation agent composition according to claim 3, characterized in that, The commercial starter culture is YF-L904; the inoculum amount of the commercial starter culture in the starter culture composition is 0.002‰-0.01‰ (w / w), and the inoculum amount of Lactobacillus plantarum H2-5 is 0.5%-2.0% (v / v); the starter culture composition is suitable for fermenting one or more milk raw materials selected from cow's milk, goat's milk, mare's milk, camel's milk, and donkey's milk.

5. A fermented milk, characterized in that, The fermented milk is prepared by fermentation of Lactobacillus plantarum H2-5 as described in claim 1, or by fermentation of the starter culture composition as described in claim 3 or 4.

6. The fermented milk according to claim 5, characterized in that, The raw milk for the fermented milk is selected from one or more of cow's milk, goat's milk, mare's milk, camel's milk, and donkey's milk; the number of live bacteria in the fermented milk is maintained above 8.6 1 g CFU / mL after being stored at 4°C for 28 days.

7. A method for improving the quality of fermented milk, characterized in that, The method includes simultaneously inoculating a commercial starter culture agent and the *Lactobacillus plantarum* H2-5 of claim 1 into a milk raw material, and then fermenting it.

8. The method according to claim 7, characterized in that, The commercial starter culture is YF-L904, with an inoculum size of 0.002‰-0.01‰ (w / w); the inoculum size of *Lactobacillus plantarum* H2-5 is 0.5%-2.0% (v / v); the milk raw material is selected from one or more of cow's milk, goat's milk, mare's milk, camel's milk, and donkey's milk; the fermentation temperature is 40-43℃; and the fermentation endpoint is pH 4.5-4.

6.

9. The method according to claim 7 or 8, characterized in that, The method also includes a step of post-fermentation at 0-6°C for 8-15 h.

10. The use of *Lactobacillus plantarum* H2-5 according to claim 1 or the starter culture composition according to claim 3 or 4 in the preparation of fermented milk, characterized in that, The fermented milk has one or more of the following characteristics: (a) Viable bacterial count ≥ 8.6 lg CFU / mL after 28 days of storage; (b) The water-holding capacity is increased by more than 6% compared to fermented milk made with commercial starter culture alone; (c) The hardness, consistency, viscosity and cohesiveness are superior to those of fermented milk made with commercial starter culture alone.

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