Compound lactic acid bacteria agent for mare milk fermentation, fermented mare milk and preparation method of fermented mare milk

Abstract

The invention discloses a compound lactic acid bacteria agent for mare milk fermentation, fermented mare milk and a preparation method of the fermented mare milk, and belongs to the technical field of mare milk biological fermentation. The compound lactic acid bacteria agent comprises five viable bacteria of lactobacillus bulgaricus, streptococcus thermophilus, lactobacillus helveticus, lactobacillus plantarum and lactobacillus casei, the optimal ratio of lactobacillus bulgaricus to streptococcus thermophilus to lactobacillus helveticus to lactobacillus plantarum to lactobacillus casei is 1: 1: 0.5: 0.5: 0.5 during mare milk fermentation, the optimal inoculum size is 3%, the optimal fermentation temperature is 40.5 DEG C, and the optimal fermentation time is 12 hours. The prepared fermented mare milk (mare milk yoghurt) is excellent in flavor, pure and elegant, the bad flavor is successfully inhibited, the elegant fragrance is created, and the flavor level is improved; the functions are enhanced, the health value is high, high-value metabolites are enriched, bioactive peptides are released, and an anti-oxidation system is enhanced; the process is stable, and the quality is controllable: by optimizing process parameters, the viable count reaches an extremely high level, stable and efficient expression of a synergistic metabolic network is ensured, and uniform and reproducible product quality is ensured.

Description

Technical Field

[0001] This invention belongs to the field of mare's milk bio-fermentation technology, specifically relating to a compound lactic acid bacteria agent for mare's milk fermentation, fermented mare's milk and its preparation method. Background Technology

[0002] Mare's milk is a unique milk resource that is rich in nutrients and has low allergenicity. Its protein composition is special (high whey protein content and loose casein structure), high in unsaturated fatty acids, and rich in bioactive substances such as vitamin C and lactoferrin, making it a "natural functional milk" with great development potential. However, compared to the huge market size of cow's milk yogurt, the industrial development of mare's milk is severely lagging behind, and there are almost no standardized, commercialized mare's milk fermented products similar to yogurt on the market.

[0003] Currently, fermented mare's milk products mainly focus on traditional mare's milk wine (such as Koumiss). These products rely on the mixed fermentation of yeast and lactic acid bacteria, typically contain alcohol, and have a strong sour and astringent flavor and a foamy texture. This inherent characteristic limits consumption by children, pregnant women, and groups with specific dietary needs, and also fails to meet the mainstream market's expectation of a mild, smooth, and pleasantly sweet and sour yogurt. Therefore, developing a new type of non-alcoholic mare's milk yogurt product with an elegant flavor and pleasant texture is of great significance for tapping into the value of this unique resource and expanding the healthy dairy product portfolio.

[0004] However, achieving this goal faces two major technological bottlenecks: the lack of specialized starter cultures and the challenge of flavor control. Directly applying traditional starter cultures used in the preparation of ordinary cow's milk yogurt (usually containing only Lactobacillus bulgaricus and Streptococcus thermophilus) to mare's milk often fails to achieve the desired flavor profile. Existing research indicates that traditional fermentation easily produces an unpleasant "fecal odor" (mainly related to substances such as indole) and lacks a rich and complex aroma, resulting in poor product flavor quality.

[0005] To address the aforementioned issues, existing technologies offer some partial solutions. For example, various functional lactic acid bacteria have been isolated from traditional mare's milk wine, such as *Lactobacillus plantarum* (with the potential to produce extracellular polysaccharides, improving texture), *Lactobacillus helveticus* (with strong proteolytic ability, releasing flavor precursors and deeply hydrolyzing proteins), and *Lactobacillus casei* (with probiotic properties and mild acid-producing ability). These strains are considered to have potential in improving the texture, flavor, and function of fermented milk. However, most existing research remains at the stage of evaluating the function of single strains or simple mixing. There is a lack of in-depth research and feasible technical solutions on how to scientifically and quantitatively combine these functional strains with traditional basic strains (*Lactobacillus bulgaricus*, *Streptococcus thermophilus*) to construct a synergistic metabolic network in the special matrix of mare's milk, thereby systematically solving flavor defects (suppressing unpleasant odors and creating elegant aromas) and simultaneously enriching functional components. In particular, the metabolic flux regulation mechanism of compound microbial agents during mare's milk fermentation and their global impact on the overall quality of the final product (especially the profile of volatile flavor compounds and non-volatile functional metabolites) remain a blank.

[0006] Therefore, existing technologies lack a specific compound lactic acid bacteria agent and its supporting process for mare's milk fermentation. This agent should effectively overcome the fermentation difficulties inherent in mare's milk substrate, working synergistically to simultaneously achieve: 1) significantly inhibiting the formation of undesirable flavor compounds (such as indole); 2) specifically creating abundant positive flavor compounds (such as vanillin); and 3) directionally enriching functional metabolites with health potential (such as specific bioactive peptides and neurosteroid precursors). This would result in the production of high-quality fermented mare's milk products with elegant flavor, enhanced functionality, and acceptable texture. Summary of the Invention

[0007] This invention is proposed based on the aforementioned background. The invention aims to provide a specific compound lactic acid bacteria agent, fermented mare's milk products derived therefrom, and a method for preparing the same, through rational design and optimization, in order to solve the technical problems existing in the prior art.

[0008] To achieve the above-mentioned technical objectives, the present invention adopts the following technical solution: One objective of this invention is to provide a bacterial strain combination for fermenting mare's milk, which consists of Lactobacillus bulgaricus (Lactobacillus bulgaricus). Lactobacillus delbrueckii subsp. bulgaricus ), Streptococcus thermophilus ( Streptococcus thermophilus Lactobacillus helveticus ( Lactobacillus helveticus Lactobacillus plantarum ( Lactobacillus plantarum ) and Lactobacillus casei ( Lactobacillus casei It consists of five kinds of bacteria.

[0009] Preferably, the ratio of the five bacteria used is 1:1:(0.1-0.9):(0.1-0.9):(0.1-0.9).

[0010] More preferably, the ratio of the five bacteria used is 1:1:0.5:0.5:0.5.

[0011] The second objective of this invention is to provide the application of the aforementioned bacterial strain combination in the preparation of a compound lactic acid bacteria agent for mare's milk fermentation.

[0012] The third objective of this invention is to provide a compound lactic acid bacteria agent for fermenting mare's milk, which contains live bacteria of the aforementioned bacterial strain combination.

[0013] Preferably, the number of viable bacteria is not less than 1×10⁻⁶. 10 CFU / g.

[0014] The fourth objective of this invention is to provide the application of the aforementioned compound lactic acid bacteria agent in the preparation of fermented mare's milk.

[0015] The fifth objective of this invention is to provide a fermented mare's milk, which is produced by fermenting mare's milk with the aforementioned compound lactic acid bacteria agent.

[0016] The sixth objective of this invention is to provide a method for preparing the fermented mare's milk, wherein the fermentation temperature is 36-44℃, the fermentation time is 8-16 hours, and the inoculation amount of compound lactic acid bacteria is 1-5%.

[0017] Preferably, the fermentation temperature is 40-41℃, the fermentation time is 11-13 hours, and the inoculation amount of the compound lactic acid bacteria agent is 2.5-3.5%.

[0018] Compared with the prior art, the present invention has the following significant advantages: 1. Successfully developed a fermented mare's milk (mare's milk yogurt) product with an elegant flavor.

[0019] Currently, there are no mainstream products on the market that use mare's milk as a base, have a stable gel texture, and possess a typical yogurt flavor. This invention, through specific bacterial agent compounding and process optimization, has for the first time achieved pure lactic acid bacteria fermentation of mare's milk, successfully producing a non-alcoholic, smooth-tasting fermented dairy product (mare's milk yogurt) with a flavor similar to traditional yogurt but with more distinctive characteristics, providing consumers with a brand-new, high-quality, and unique dairy product option.

[0020] 2. The product flavor has been revolutionaryly optimized, forming a unique and elegant flavor profile.

[0021] (1) Precise suppression of undesirable flavors: Compared to the traditional fermented mare's milk (FS group), which produces a strong "fecal odor" with an indole flavor activity value (OAV) as high as 24.18, the product of this invention (FM group) successfully suppressed the indole OAV to an extremely low 0.49, greatly improving the purity and acceptability of the product. This effect is directly attributed to the specific combination of Lactobacillus plantarum, Lactobacillus casei, and Lactobacillus helveticus and their synergistic effect at the preferred ratio. Metabolic pathway analysis confirmed that this combination can more efficiently direct tryptophan metabolism to the kynurenine pathway (the content of the key intermediate 2-aminobenzoic acid in the FM group is 3.6 times that in the FS group) and enhance the oxidative conversion of indole, thereby suppressing undesirable flavors from both the source and end.

[0022] (2) The elegant aroma is specifically created: The product of this invention can generate vanillin (OAV>1), which has an elegant vanilla sweet aroma that is almost undetectable in traditional fermentation. At the same time, the bioconversion efficiency of terpene precursors in raw materials is higher, resulting in a more significant enrichment of terpene alcohols such as borneol (which impart a fresh woody aroma and a cooling sensation). The generation of these positive aromas is closely related to the unique enzyme activity of Lactobacillus helveticus and the complementary metabolic network provided by Lactobacillus plantarum and Lactobacillus casei, and is a metabolic characteristic achieved by a specific combination of strains.

[0023] (3) Enhanced basic flavor intensity and layering: The product of this invention has a better ability to accumulate key substances (such as 2-undecone, n-decanoic acid, and 1-undecyl alcohol) that contribute to fruit aroma, fatty aroma and floral aroma than traditional fermentation, and more thoroughly removes medium-chain fatty acid esters that may produce waxy flavor, making the overall flavor profile richer and more layered.

[0024] 3. The product's functionality is targeted and enhanced, and it is enriched with high-value health-related metabolites.

[0025] (1) Specific enrichment of high-value functional factors: The content of metabolites such as pregnanelone (neuroactive precursor) and tauroursodeoxycholic acid (potential hepatobiliary protective effect) in the product of this invention is significantly higher than that in traditional fermentation products. This is because in the FM compound microbial agent, Lactobacillus helveticus may contribute the enzyme system for cholesterol backbone modification, while the bile salt hydrolase activity and synergistic effect of Lactobacillus plantarum and Lactobacillus casei jointly drive these high-value metabolic pathways.

[0026] (2) Abundant release of bioactive peptides: The protein hydrolysis profile of the product of this invention is more in-depth and specific, and can release a variety of characteristic short peptides that are almost undetectable in traditional fermentation (such as Tyr-Ile-Val, with an FC as high as 208.99; and Ile-Ser-Thr, etc.). This may be directly due to the synergistic hydrolysis effect formed by the powerful protein hydrolysis system of Lactobacillus helveticus and the complementary peptidase profile of Lactobacillus plantarum and Lactobacillus casei in the compound microbial agent.

[0027] (3) Systemic enhancement of the antioxidant system: This invention can not only universally enhance antioxidant phenolic substances such as rhodioloside through tyrosine metabolism, but its unique metabolic network may also construct a more complete multi-component antioxidant system by enriching substances such as allantoin.

[0028] 4. The fermentation process is stable and efficient, ensuring that the synergistic effect is fully expressed.

[0029] Through system optimization, the maximum number of viable bacteria in the compound microbial agent was achieved (approximately 2.08 × 10⁻⁶). 11 The optimal process parameters (temperature 40-41℃, time 11-13 hours, inoculum size 2.5-3.5%) were achieved. This optimized process provides a stable and efficient expression environment for the synergistic metabolic network determined by the specific bacterial ratio (1:1:0.5:0.5:0.5), ensuring the uniformity and reproducibility of product quality.

[0030] The root of all the above advantages lies in the core of this invention: the "combination and ratio of microbial strains." Specifically, *Lactobacillus bulgaricus* and *Streptococcus thermophilus* are used as the fermentation base, synergistically introducing *Lactobacillus helveticus*, *Lactobacillus plantarum*, and *Lactobacillus casei*, with the viable cell ratio controlled at 1:1:(0.1-0.9):(0.1-0.9):(0.1-0.9), preferably 1:1:0.5:0.5:0.5. This specific architecture constructs a unique metabolic network different from traditional two-strain systems, enabling precise regulation of key metabolic fluxes such as tryptophan and tyrosine, activating specific enzyme systems for synthesizing high-value functional substances, thereby achieving a synergistic enhancement of flavor and function. Attached Figure Description

[0031] Figure 1 This shows the fermentation results of different fermentation temperatures for the dominant strain combination in this invention.

[0032] Figure 2 This shows the fermentation results of different fermentation times for the dominant strain combinations in this invention.

[0033] Figure 3 This is the fermentation result of different strain ratios in the combination of dominant strains in this invention.

[0034] Figure 4 The fermentation results are shown for different amounts of the dominant strain combination added in this invention.

[0035] Figure 5 This refers to the interaction between fermentation temperature and ultrasonic extraction time in this invention.

[0036] Figure 6 This refers to the interaction between fermentation temperature and the amount added in this invention.

[0037] Figure 7 This refers to the interaction between fermentation time and the amount added in this invention.

[0038] Figure 8 The following are multivariate statistical analysis charts of volatile flavor compounds in this invention: (a) PCA score chart, (b) PLS-DA score chart, and (c) PLS-DA permutation test at 200 times.

[0039] Figure 9 This is a Venn diagram of the key differentially expressed volatile metabolites in this invention.

[0040] Figure 10 This is a heatmap of differentially expressed nonvolatile metabolites in this invention.

[0041] Figure 11 This invention presents a hypothetical metabolic pathway for the biosynthesis of rhodioloside and the conversion of indole derivatives during the fermentation of mare's milk by lactic acid bacteria. (a) Rhodioloside biosynthesis pathway, (b) Indole derivative metabolic network. Detailed Implementation

[0042] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the invention. The reagents, products, and instruments used in the following examples are all commercially available, and the methods used in the examples, unless otherwise specified, are consistent with conventionally used methods.

[0043] In this invention: OAV (Odor Activity Value): This refers to the ratio of the concentration of a volatile substance to its olfactory threshold. An OAV ≥ 1 is generally considered to indicate that the substance contributes to the overall flavor; the higher the OAV, the stronger the contribution.

[0044] FC (Fold Change): refers to the ratio of the content of a certain metabolite in the experimental group to that in the control group.

[0045] Untargeted metabolomics: a technique that performs global analysis of all small molecule metabolites in a biological system without pre-setting a target.

[0046] PLS-DA (Partial Least Squares Discriminant Analysis): A multivariate statistical analysis method for identifying differentially expressed metabolites between groups.

[0047] Live bacteria ratio: In this invention, unless otherwise specified, it refers to the ratio of the number of live bacteria (CFU) of each strain at the start of the compound inoculant or fermentation.

[0048] The main contents of this invention are as follows: A. Product Option 1: Compound Lactic Acid Bacteria Agent The compound lactic acid bacteria agent is used for fermenting mare's milk, and its active ingredient includes Lactobacillus bulgaricus (Lactobacillus bulgaricus). Lactobacillus delbrueckii subsp. bulgaricus ), Streptococcus thermophilus ( Streptococcus thermophilus Lactobacillus helveticus ( Lactobacillus helveticus Lactobacillus plantarum ( Lactobacillus plantarum ) and Lactobacillus casei ( Lactobacillus casei The five types of bacteria are live bacteria. The ratio of live bacteria counts of the five types of bacteria is 1:1:(0.1-0.9):(0.1-0.9):(0.1-0.9), preferably 1:1:0.5:0.5:0.5. The bacterial agent can be in the form of lyophilized powder or liquid fermentation agent, wherein the live bacteria count of the lyophilized powder is not less than 1×10⁻⁶. 10 CFU / g.

[0049] B. Product Option Two: Fermented Mare's Milk The fermented mare's milk is produced by fermenting mare's milk with the aforementioned compound lactic acid bacteria agent. It contains vanillin with an flavor activity value (OAV) greater than 1; indole has an OAV value less than 1; and the content of the characteristic differential metabolites pregnanelone and / or tauroursodeoxycholic acid is significantly higher than that of mare's milk fermented under the same conditions with a starter culture composed of Lactobacillus bulgaricus and Streptococcus thermophilus.

[0050] C. Method Scheme 3: Preparation of Compound Lactic Acid Bacteria Agent The preparation method of the compound lactic acid bacteria agent includes: inoculating freeze-dried bacterial powders of *Lactobacillus bulgaricus*, *Streptococcus thermophilus*, *Lactobacillus helveticus*, *Lactobacillus plantarum*, and *Lactobacillus casei* into MRS liquid medium, activating and expanding them to the late logarithmic growth phase at a suitable temperature. The bacterial cells are collected by centrifugation, resuspended in sterile physiological saline, and the viable cell concentration of each suspension is determined using the plate count method. Finally, according to the ratio described in this invention (viable cell ratio of 1:1:0.5:0.5:0.5), the bacterial suspensions are quantitatively mixed to obtain the compound lactic acid bacteria agent. This agent can be used directly or further processed by centrifugation, addition of a freeze-drying protectant, and freeze-drying to prepare a freeze-dried powder.

[0051] D. Method Scheme Four: Preparation of Fermented Mare's Milk To determine the optimal fermentation process, single-factor experiments were first conducted. Figure 1-4The preliminary effective ranges for each parameter were determined: fermentation temperature 36-44℃, fermentation time 8-16 hours, and inoculum size of the compound lactic acid bacteria agent 1-5% (v / v), with the viable count ratio of the five lactic acid bacteria in the compound lactic acid bacteria agent being Lactobacillus bulgaricus: Streptococcus thermophilus: Lactobacillus helveticus: Lactobacillus plantarum: Lactobacillus casei = 1:1:(0.1-0.9):(0.1-0.9):(0.1-0.9). Based on this, using the aforementioned ratio (1:1:0.5:0.5:0.5), within the effective range, three key factors—fermentation temperature, fermentation time, and inoculum size—were selected, and a Box-Behnken design was used for response surface optimization (Table 1), with the viable count at the fermentation endpoint as the response value. Analysis of variance of the obtained model showed that the model was highly significant (P<0.0001), and its three-dimensional response surface plot is shown in Table 1. Figure 5-7 As shown in Table 2, the optimal process parameters predicted by the model analysis are: fermentation temperature 40.6℃, fermentation time 12.035 hours, and inoculum size 2.8%. Based on the model prediction results and considering production convenience, the optimized process parameters are determined to be: fermentation temperature 40-41℃, preferably 40.5℃; fermentation time 11-13 hours, preferably 12 hours; and inoculum size 2.5-3.5%, preferably 3%.

[0052] E. Methodology 5: Product Quality Testing and Evaluation To systematically evaluate the overall quality of the products obtained from this optimized process, especially its global impact on flavor and functional substances, non-targeted metabolomics technology was used to analyze the fermentation products. The study was divided into three experimental groups: UM (Raw Mare's Milk) consisted of fresh mare's milk without any fermentation treatment; FM (Five-strain Mixed Starter Culture) used a composite lactic acid bacteria inoculum (Lactobacillus bulgaricus, Streptococcus thermophilus, Lactobacillus helveticus, Lactobacillus plantarum, and Lactobacillus casei) screened in this study, and fermented strictly according to the optimal process parameters obtained through response surface methodology; FS (Traditional Two-starter Culture) used only the two core traditional strains used in making regular yogurt (Lactobacillus bulgaricus + Streptococcus thermophilus).

[0053] The technical solution of the present invention will be further described in detail below with reference to the embodiments.

[0054] Example 1 (Optimal Process) Process parameters: Five types of lactic acid bacteria, including Lactobacillus bulgaricus (… Lactobacillus delbrueckii subsp. bulgaricus (Accession number CICC 6045, Linyi Hongkun Biotechnology Co., Ltd.), Streptococcus thermophilus ( Streptococcus thermophilusAccession number BNCC362702, Suzhou Beina Chuanglian Biotechnology Co., Ltd.), Lactobacillus helveticus ( Lactobacillus helveticus (Catalogue number CICC 6032, Linyi Hongkun Biotechnology Co., Ltd.), Lactobacillus plantarum ( Lactiplantibacillus plantarum (Accession number ATCC 8014, Linyi Hongkun Biotechnology Co., Ltd.) and Lactobacillus casei ( Lacticaseibacillus casei The bacteria were provided by Inner Mongolia Agricultural University. Reference: Analysis of Changes in Fatty Acids in the Lower Membrane of *Lactobacillus casei* Zhang in Simulated Artificial Gastric Juice (Merlan). A compound lactic acid bacteria agent was prepared by mixing bacteria in a live bacteria ratio of 1:1:0.5:0.5:0.5. This agent was inoculated into pasteurized mare's milk at an inoculation rate of 3% (v / v). After fermentation at a constant temperature of 40.5℃ for 12 hours, the mixture was cooled to 4℃ to obtain the fermented mare's milk product.

[0055] result: (1) Excellent and stable fermentation performance: As shown in the repeated experiments, the endpoint viable count stabilized at 2.05–2.12 × 10⁻⁶. 11 High levels of CFU / mL (average approximately 2.08 × 10⁻⁶) 11 The product pH was 4.3 and the acidity was 85°T (CFU / mL). This indicates that the optimized process of this invention has high stability and reproducibility. GC-MS and non-targeted metabolomics analysis showed that principal component analysis and orthogonal partial least squares discriminant analysis consistently indicated a significant separation of the volatile compound profiles between the UM group and the fermented mare's milk groups (FM and FS groups). Figure 8 a and Figure 8 b). Among them, the constructed PLS-DA model exhibits good predictive ability (Q). 2 > 0.8)( Figure 8 c). The product has an elegant aroma (vanillin OAV > 1, indole OAV < 1) and is enriched with various functional metabolites (such as pregnanelonone, tauroursodeoxycholic acid, and the characteristic short peptide Tyr-Ile-Val, etc.). Figure 9 , Figure 10 ) (2) Significant improvement in flavor quality (compared to the traditional FS group): Unpleasant flavors are drastically suppressed: the flavor activity value (OAV) of indole, a key defective flavor compound, is only 0.49 in this product, far lower than the 24.18 of the product fermented under the same conditions by traditional starter cultures (Lactobacillus bulgaricus + Streptococcus thermophilus) (FS group). This indicates that the present invention completely solves the unpleasant "fecal odor" problem of traditional mare's milk fermentation products. Figure 11 a).

[0056] b. Specific generation of elegant aroma: Vanillin, which has an elegant vanilla sweet aroma, was successfully detected in this product, with an OAV>1, while this substance was almost undetectable in FS group products.

[0057] c. Significantly enhanced positive flavor profile: This product has a higher enrichment of terpenoids such as borneol, which have a fresh woody aroma and a cooling sensation, and a stronger ability to accumulate key flavor substances such as 2-undecanoic acid, n-decanoic acid, and 1-undecanoic acid, which contribute to fruity, fatty, and floral aromas, forming a unique flavor profile with richer layers and more elegant quality.

[0058] (3) Targeted enrichment of functional components (compared with the traditional FS group): a. Enrichment of high-value functional factors: Non-targeted metabolomics analysis showed that the content of high-value metabolites such as pregnanelone (neuroactive precursor) and tauroursodeoxycholic acid (potential hepatobiliary protective effect) in this product was significantly higher than that in the FS group product.

[0059] b. Abundant release of bioactive peptides: This product contains several characteristic bioactive short peptides that are almost undetectable in the FS group, such as Tyr-Ile-Val (FC=208.99 compared to the FS group) and Ile-Ser-Thr.

[0060] c. Enhanced antioxidant system: Metabolic pathway analysis shows that this product can more efficiently drive tyrosine metabolism towards the synthesis of antioxidant phenolic compounds such as rhodioloside. Figure 11 b).

[0061] Conclusion: Example 1 confirms that the specific ratio (1:1:0.5:0.5:0.5) of the compound lactic acid bacteria agent and the optimized process described in this invention not only achieves stable and efficient fermentation, but more importantly, it synergistically produces superior effects that traditional two-strain fermentation cannot achieve: namely, while maximally suppressing undesirable flavors, it specifically creates elegant aromas and directionally enriches a variety of high-value functional metabolites. This demonstrates the unique synergistic metabolic advantages of the strain combination of this invention.

[0062] Examples 2 and 3 (Example points within the optimized process range) Objective: To illustrate that the process of the present invention has a reasonable operational tolerance near the optimal value, two sets of parameter combinations located at the design point of the response surface and with a high viable count are provided as examples.

[0063] Example 2 Process parameters: inoculum size 2.5%, fermentation temperature 40℃, fermentation time 11 hours. Other conditions are the same as in Example 1.

[0064] Results are based on: Item 9 in the response surface design table (Table 1). Under these conditions, the viable count reached 1.88 × 10⁻⁶. 11The CFU / mL level remains high.

[0065] Example 3 Process parameters: inoculum size 3.5%, fermentation temperature 40℃, fermentation time 13 hours. Other conditions are the same as in Example 1.

[0066] Results are based on: this combination corresponds to sequence number 12 in the response surface design table (Table 1). Under these conditions, the viable count is 1.76 × 10⁻⁶. 11 CFU / mL.

[0067] Overall results show that, according to Examples 2 and 3, by finely adjusting the parameters near the optimal process point (40.5℃, 12h, 3%), the fermentation system can still maintain a high viable cell count (>1.75×10⁻⁶). 11 The product's basic indicators (pH, acidity) are within acceptable limits (CFU / mL). This verifies the reliability of the response surface methodology prediction and demonstrates that the process parameters of this invention have good operational flexibility.

[0068] Comparative Examples 1 and 2 (comparative examples outside the optimal strain combination and the ratio range) Objective: To illustrate the specificity and superiority of the strain combination and ratio of the present invention, the strain combination and the strain ratio were changed as comparative examples.

[0069] Comparative Example 1 The same batch of mare's milk was fermented using a commercially available yogurt starter (FS) containing Lactobacillus bulgaricus and Streptococcus thermophilus, with the same inoculum (3%), temperature (40.5°C), and time (12 hours).

[0070] result: (1) Comparison of key flavor compounds: Vanillin, an elegant vanilla-like aroma compound, was not detected in the FS group products. More importantly, the flavor activity value (OAV) of indole, an unpleasant flavor compound, was as high as 24.18, giving the product a distinctly unpleasant odor; while the OAV of indole in the FM group products of this invention was successfully controlled at an extremely low level of 0.49.

[0071] (2) Comparison of Functional and Non-volatile Metabolites (Core Advantages): Non-targeted metabolomics analysis shows that in the products of this technology: a) the content of various bioactive short peptides (such as Tyr-Ile-Val, Ile-Ser-Thr) is extremely low or undetectable. b) the accumulation of high-value functional factors pregnanelone and tauroursodeoxycholic acid is negligible. c) in terms of lipid remodeling and the conversion of amino acid metabolism into beneficial products, its metabolic profile is significantly less rich and beneficial than that of the FM group of this invention.

[0072] Comparative Example 2 To illustrate the importance of the specific bacterial strain ratio described in this invention, two control groups deviating from the optimized ratio (1:1:0.5:0.5:0.5) of this invention were set up. Details are as follows: Control 2A: The ratio of viable bacteria to live bacteria was 1:1:0.1:0.1:0.1 (i.e., the ratio of Lactobacillus helveticus, Lactobacillus plantarum, and Lactobacillus casei was severely weakened).

[0073] Control 2B: The ratio of viable bacteria was 1:1:0.1:0.9:0.9 (that is, although the proportion of Lactobacillus plantarum and Lactobacillus casei was increased, the proportion of Lactobacillus helveticus was severely weakened, and the balance of the bacterial community was disrupted).

[0074] result: (1) Viable bacterial count and acid production capacity ( Figure 3 The viable bacterial counts in the final products of both control groups were significantly lower than those in Example 1 (approximately 2.08 × 10⁻⁶). 11 (CFU / mL). The viable count in control 2A was only about 1.0 × 10⁻⁶. 10 CFU / mL, control 2B is approximately 1.3 × 10⁻⁶. 10 CFU / mL. Correspondingly, their acid-producing capacity is insufficient, with pH values ​​of 4.8 and 4.53, respectively. Among them, the pH value of control 2A can no longer meet the common quality requirements of fermented dairy products (usually requiring pH ≤ 4.6).

[0075] (2) Product texture: The product of control 2B has a serious problem of reduced viscosity, thin texture, and significantly deteriorated sensory quality.

[0076] Table 1. Response surface methodology and results

[0077] Table 2 Regression Model and Analysis of Variance

[0078] Note:" "Indicates a significant impact on the outcome (" P <0.05); "Indicates a highly significant impact on the outcome" P <0.01).

[0079] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A microbial strain combination for fermenting mare's milk, characterized in that, It is composed of five bacteria: Lactobacillus delbrueckii subsp. bulgaricus, Streptococcus thermophilus, Lactobacillus helveticus, Lactobacillus plantarum, and Lactobacillus casei.

2. The strain combination according to claim 1, characterized in that, The usage ratio of the five bacteria is 1:1:(0.1-0.9):(0.1-0.9):(0.1-0.9).

3. The strain combination according to claim 2, characterized in that, The ratio of the five bacteria used is 1:1:0.5:0.5:0.

5.

4. The application of any of the strain combinations described in claims 1-3 in the preparation of a compound lactic acid bacteria agent for mare's milk fermentation.

5. A compound lactic acid bacteria agent for fermenting mare's milk, characterized in that, It includes live bacteria of any combination of strains described in claims 1-3.

6. The compound lactic acid bacteria agent according to claim 5, characterized in that, The number of live bacteria is not less than 1×10⁻⁶. 10 CFU / g.

7. The application of the compound lactic acid bacteria agent according to any one of claims 5-6 in the preparation of fermented mare's milk.

8. A fermented mare's milk, characterized in that, It is made from mare's milk through fermentation with any of the compound lactic acid bacteria agents described in claims 5-6.

9. A method for preparing fermented mare's milk as described in claim 8, characterized in that, The fermentation temperature is 36-44℃, the fermentation time is 8-16 hours, and the inoculation amount of compound lactic acid bacteria is 1-5%.

10. The preparation method according to claim 9, characterized in that, The fermentation temperature is 40-41℃, the fermentation time is 11-13 hours, and the inoculation amount of the compound lactic acid bacteria agent is 2.5-3.5%.

Images