A compound microbial agent for yak butter fermentation and its application

Abstract

This invention relates to the field of microbial directed fermentation processing technology for yak milk fat products, and discloses a compound microbial agent for yak butter fermentation and its application. The compound microbial agent includes *Lactococcus gasseri* (…). Lactococcus garvieae CGMCC No. 32756, Enterococcus faecium ( Enterococcus faecium CGMCC NO.36378 and Kluyveromyces martensii ( Kluyveromyces Marxian (CGMCC NO.39019) All three strains exist in live form. This invention also discloses a method for preparing the composite microbial agent, a method for fermenting yak ghee using the composite microbial agent, and the resulting fermented yak ghee. The three strains possess environmental tolerance and single-strain growth characteristics suitable for optimizing the synergistic fermentation process of yak ghee; the interaction of the three strains can enhance the growth performance of the strains. Inoculating the composite microbial agent into yak milk fat substrate for fermentation can improve the flavor of yak ghee, reduce acid value, peroxide value, and biogenic amine accumulation, and improve the controllability of the yak ghee fermentation process and the stability of product quality.

Description

Technical Field

[0001] This invention belongs to the field of microbial directional fermentation processing technology for yak milk fat products. Specifically, it relates to a compound microbial agent composed of three preserved strains: Lactococcus garvieae (Lac), Enterococcus faecium (Ent), and Kluyveromyces marxianus (Klu). The invention also relates to the application of this compound microbial agent in the fermentation of yak milk fat as a base material to prepare yak ghee, improve the flavor of yak ghee, control oil rancidity, reduce the accumulation of biogenic amines, and optimize the co-fermentation process of yak ghee. Background Technology

[0002] Yak butter is typically made from high-fat milk fat obtained through fat separation from yak milk, through fermentation, washing, and kneading. Traditional yak butter fermentation relies heavily on the raw milk and natural microorganisms in the processing environment, or on empirical processing to create regional flavors. While this method can produce certain characteristics such as milk fat aroma, sour aroma, and cheese aroma, the sources of the microorganisms involved in fermentation are complex, and the composition and metabolic activity of the microbial community are easily affected by season, raw material batch, processing environment, fermentation temperature, and operating conditions. This makes it difficult to standardize and control the fermentation process, resulting in batch-to-batch variations in the flavor and quality of the final product.

[0003] To improve the controllability of dairy product fermentation, current practices typically involve lactic acid bacteria, yeast, or commercial compound starter cultures. These cultures allow the dairy base to form volatile flavor compounds such as acids, alcohols, esters, and ketones through microbial metabolism, thus improving the product's flavor to some extent. However, conventional commercial bacterial strains or compound starter cultures are usually designed for general dairy products or ordinary dairy fat fermentation, and are not specifically screened and optimized for yak milk fat matrix, ghee post-processing, and the target flavor of yak ghee. This can lead to problems in yak ghee fermentation such as a heavy flavor, a strong cheese odor, a noticeable greasiness, or insufficient control over rancidity.

[0004] In addition to the flavor and physicochemical quality of the finished product, the strains used for co-fermentation of yak ghee should also possess basic properties suitable for the fermentation environment. The fermentation of yak milk fat substrate may involve changes in temperature, acidity, salt concentration, and pH. If a single strain lacks sufficient environmental tolerance, it can easily lead to slow growth in the early stages of fermentation, imbalance in the microbial community ratio in the middle and later stages, or instability in the fermentation process. Therefore, simply confirming that a particular strain originates from ghee or can produce specific flavor substances is insufficient to support stable industrial fermentation; it is also necessary to clarify the tolerance of the tested single strain to environmental conditions such as temperature, lactic acid, salt, and pH, as well as the characteristics of its single-strain growth curve.

[0005] Meanwhile, the technical effectiveness of compound microbial agents depends not only on the performance of individual strains but also on the interactions during co-cultivation of multiple strains. If there is a lack of synergistic effect between strains, competitive inhibition may occur after compounding, leading to a decrease in total bacterial count, a shortened stationary period, or insufficient formation of target metabolites. Therefore, the compound microbial agent for yak ghee still needs to address whether the strains can form growth-promoting interactions during co-cultivation.

[0006] Therefore, there is still a need for a compound microbial agent with a well-defined composition, suitable for high-fat yak milk fat base, good environmental tolerance and single-strain growth characteristics, synergistic proliferation effect of strains in the compound microbial agent, and reusability, so that it can simultaneously achieve basic strain compatibility, synergistic fermentation process, target flavor formation, acid value and peroxide value control, and reduction of biogenic amine accumulation in yak butter fermentation. Summary of the Invention

[0007] To address the aforementioned technical problems, this invention provides a compound microbial agent for yak butter fermentation and its application. This invention not only solves the problems of unstable flavor, insufficient rancidity control, and accumulation of biogenic amines in finished yak butter, but also addresses the issues of unclear environmental tolerance of single strains used in yak butter fermentation and insufficient or uncertain synergistic effects of multiple strains in combination.

[0008] To achieve the above objectives, in a first aspect, the present invention provides a compound microbial agent for fermenting and preparing yak ghee, the compound microbial agent comprising the following three preserved bacterial strains: *Lactococcus garvieae* strain, with preservation number CGMCC No. 32756; *Enterococcus faecium* strain, with preservation number CGMCC No. 36378; and *Kluyveromyces marxianus* strain, with preservation number CGMCC No. 39019; wherein all three preserved bacterial strains exist in live form.

[0009] In a second aspect, the present invention provides a method for preparing the composite microbial agent described in the first aspect, the method comprising activating and culturing Lactococcus gasseri strain, Enterococcus faecalis strain and Kluyveromyces martensii strain respectively, and mixing them in a predetermined ratio to obtain the composite microbial agent.

[0010] Thirdly, the present invention provides a method for preparing yak ghee by fermentation, the method comprising: sterilizing or sterilizing yak milk fat base material; inoculating the sterilized or sterilized yak milk fat base material with the compound microbial agent described in the first aspect; fermenting to obtain fermented yak milk fat; and post-processing the fermented yak milk fat to obtain fermented yak ghee.

[0011] Fourthly, the present invention provides the application of the compound microbial agent described in the first aspect in improving the flavor of yak ghee, reducing the acid value of yak ghee, reducing the peroxide value of yak ghee, reducing the accumulation of biogenic amines in yak ghee, and / or improving the storage stability of yak ghee.

[0012] Fifthly, the present invention provides a fermented yak ghee, which is prepared by the method described in the third aspect.

[0013] The technical solution of the present invention can achieve the following beneficial effects:

[0014] I. Core characteristics and advantages of the strain

[0015] The compound microbial agent has a clearly defined composition: it is a compound of three strains with preservation numbers, which solves the problems of unclear composition of natural fermentation microorganisms and large batch differences, and realizes the standardization and controllability of the fermentation system.

[0016] Excellent environmental tolerance: The tested strains can grow stably at 25-35℃ and maintain a high viable count. The optimal pH is 5-7. They exhibit good stability under lactic acid and salt stress, solving the problem of unstable single-strain growth caused by fluctuations in temperature, acidity, alkali and salt during fermentation.

[0017] Clear and controllable growth characteristics: The strain exhibits a typical growth cycle (0-10h lag phase, 10-30h logarithmic growth phase, approximately 30h stationary phase, and 40h death phase), solving the problem of unclear single-strain growth characteristics affecting activation culture, compounding time, and optimization of synergistic fermentation.

[0018] Significant synergistic fermentation effect: The three strains exhibit positive interaction when cultured together, with a viable cell count of approximately 4.5 × 10^5 CFU / mL after 48 hours, which is higher than that of dual-strain combinations and commercial triple-strain combinations. This solves the problems of competitive inhibition and insufficient synergy that may exist in compound strains, and ensures stable fermentation cell count.

[0019] II. Product Advantages and Safety Comparison

[0020] Superior flavor and quality: Compared with commercial three-strain fermentation products, the fermentation products of the three strains provided by this invention have significantly higher contents of superior flavor substances such as hexanoic acid and ethanol, lower contents of undesirable flavor substances such as 2-heptanone and butyric acid, and no detectable or lower than the detection level of n-hexadecanoic acid, thus solving the problem of heavy flavor and strong oiliness of commercial strain fermentation products.

[0021] Better safety and stability: The three-strain fermentation provided by this invention can inhibit the hydrolysis and oxidative rancidity of oils, reduce the accumulation of biogenic amines, and its acid value is lower than that of commercial strains throughout the process. The peroxide value continues to decrease, and the nitrile amine content (0.43 mg / kg) is much lower than that of commercial three-strain fermentation (1.68 mg / kg), thus improving the safety, quality and storage stability of the product.

[0022] The above results indicate that the compound microbial agent of the present invention is beneficial to reducing the accumulation of specific biogenic amines and to controlling the hydrolytic rancidity and oxidative rancidity of oils.

[0023] Biological Preservation

[0024] The Lactococcus garvieae strain provided by this invention was deposited on November 25, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 32756. The deposit address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences (CGMCC).

[0025] The Enterococcus faecium strain provided by this invention was deposited on October 28, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 36378. The deposit address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences (CGMCC).

[0026] The Kluyveromyces marxianus strain provided by this invention was deposited on October 28, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 39019. The deposit address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences (CGMCC). Attached Figure Description

[0027] Figure 1 The figure shows the temperature tolerance results of the three tested strains and their corresponding commercial strains under conditions of 15-45℃, which is used to illustrate that 25-35℃ can be regarded as the suitable growth temperature range for the strains of this invention, and to provide a basis for the selection of fermentation temperature for yak milk fat base.

[0028] Figure 2 The graph shows the tolerance results of the three tested strains and their corresponding commercial strains at different lactic acid concentrations, illustrating the stability of the tested strains in an acidified fermentation environment.

[0029] Figure 3 The graph shows the tolerance results of the three tested strains and their corresponding commercial strains at different salt concentrations, which is used to illustrate the growth stability of the tested strains under salt stress or osmotic pressure changes.

[0030] Figure 4 The figure shows the tolerance results of the three tested strains and their corresponding commercial strains under different pH conditions, illustrating that pH 5-7 is the suitable growth range for the tested strains.

[0031] Figure 5 The single-cell growth curves of the three tested strains and their corresponding commercial strains are shown to illustrate that the tested strains have good growth characteristics and can be used for subsequent optimization of the co-fermentation process.

[0032] Figure 6 The diagram shows the interaction culture results of commercial strain dual-strain combinations and commercial triple-strain combinations, used to illustrate the growth trend of the commercial triple-strain combination in co-culture.

[0033] Figure 7 The diagram shows the interaction culture results of the three strains of the present invention in the combination of two strains and the combination of three strains, which are used to illustrate that the three strains of the present invention have a better growth-promoting synergistic effect than the combination of two strains.

[0034] Figure 8 The graph shows the moisture change results of the three-strain compound inoculant of this invention and the commercial three-strain combination fermentation system, which is used to illustrate the differences in moisture change between the two fermentation systems.

[0035] Figure 9 The graph shows the pH change results of the three-strain compound inoculant of this invention and the commercial three-strain combination fermentation system, which is used to illustrate the differences in acidification and later changes between the two fermentation systems.

[0036] Figure 10 The graph shows the acid value change results of the three-strain compound microbial agent of the present invention and the commercial three-strain combination fermentation system, which is used to illustrate the control trend of the compound microbial agent of the present invention on the hydrolysis and rancidity of oils.

[0037] Figure 11 The graph shows the change in peroxide value between the three-strain compound inoculant of the present invention and a commercial three-strain combination fermentation system, which is used to illustrate the control trend of the compound inoculant of the present invention on the lipid oxidation process. Detailed Implementation

[0038] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0039] "Yak milk fat base material" refers to a milk matrix with yak milk fat as the main component, which can be used to prepare fermented yak butter. In this invention, the fat content of the yak milk fat base material can be 85%-95%. The specific method of milk fat separation is not a necessary limitation of this invention, and yak milk fat base material with the said fat content range can be obtained by conventional methods in the art.

[0040] "Compound microbial agent" refers to a microbial agent composed of two or more live bacteria used for fermentation. The compound microbial agent described in this invention specifically refers to a three-strain compound microbial agent composed of three preserved strains: Lactococcus gasseri, Enterococcus faecalis, and Kluyveromyces martensii.

[0041] "Live count ratio" refers to the relative proportion of each strain in the compound microbial agent based on the number of live bacteria. For the three-strain compound microbial agent of this invention, the live count ratio is used to characterize the inoculation ratio among Lactococcus gasseri, Enterococcus faecalis, and Kluyveromyces martensii strains.

[0042] "Environmental tolerance" refers to the ability of a strain to maintain growth or survival under different temperature, lactic acid concentration, salt concentration, or pH conditions; this ability can be used to evaluate whether the strain is suitable for further optimization of the co-fermentation process of yak butter.

[0043] "Strain interaction" refers to the relationship between two or three strains of bacteria that influence each other's growth performance under co-culture conditions. When the bacterial concentration or stationary phase of the co-culture system is better than that of the single or dual bacterial system, it can be considered that there is a tendency for growth promotion or synergistic interaction.

[0044] In a first aspect, the present invention provides a compound microbial agent for fermenting and preparing yak ghee, the compound microbial agent comprising the following three preserved strains: *Lactococcus garvieae* strain Lac, with preservation number CGMCC No. 32756; *Enterococcus faecium* strain Ent, with preservation number CGMCC NO. 36378; and *Kluyveromyces marxianus* strain Klu, with preservation number CGMCC NO. 39019; wherein all three preserved strains exist in live form.

[0045] In some embodiments, the present invention evaluated the temperature tolerance of the three provided strains within a range of 15-45°C. The results showed that the viable cell counts of all tested strains exhibited a consistent pattern with temperature changes: slow growth at 15-20°C, rapid proliferation at 20-25°C, a stable phase with a high viable cell count maintained at 25-35°C, and a slight decrease after 40°C. These results indicate that 20-40°C, particularly 25-35°C (e.g., 25°C, 28°C, 30°C, 32°C, or 35°C), can serve as a suitable growth temperature range for the tested strains and provide a basis for the fermentation temperature range of yak ghee. Compared to the corresponding commercial strains, the commercial *Lactococcus lactis* strain showed slightly higher growth activity, while the experimental strains of *Kluyveromyces martensii* and *Entococcus faecalis* showed superior activity compared to their commercial counterparts. In particular, *Kluyveromyces martensii* and *Entococcus faecium* showed a more favorable fermentation adaptation basis in terms of temperature tolerance and growth activity compared to the commercial strains.

[0046] In some embodiments, the acid stability of the three provided bacterial strains was evaluated using a lactic acid concentration ranging from 0% to 4%. The results showed that as the lactic acid concentration increased from 0% to 4%, the overall viable cell count of each strain decreased. Among them, the *Kluyveromyces martensii* and *Lactococcus gasseri* strains exhibited smaller fluctuations at different lactic acid concentrations, demonstrating better acid stability. Compared with commercial strains, the decrease in viable cell count and proportion of commercial *Lactococcus gasseri* and *Enterococcus faecium* was more pronounced with increasing lactic acid concentration. The *Lactococcus gasseri* and *Kluyveromyces martensii* strains of this invention showed smaller fluctuations under 0%-4% lactic acid conditions, indicating that they are more conducive to maintaining stable cell count and colony ratio in an acidified fermentation environment.

[0047] In some embodiments, the salt tolerance stability of the three strains provided in this invention was evaluated using different salt concentrations (0%-8%). The results showed that different salt concentrations had a certain inhibitory effect on the growth of the tested strains, but the strains of this invention exhibited more stable growth under salt stress, which can provide a reference for subsequent optimization of the yak butter fermentation process. Combined with the control of commercial strains, it was found that the growth fluctuations of the experimental strains under salt stress were smaller, indicating that the three strains of this invention are more suitable for fermentation of yak milk fat substrates with varying salt content or osmotic pressure; this characteristic helps to reduce the imbalance of the compound microbial agent ratio caused by changes in salt concentration during fermentation.

[0048] In some embodiments, the pH tolerance of the three provided bacterial strains was evaluated within a pH range of 3-10. The results showed that the optimal growth pH for the tested strains was 5-7, and the pH stability of the *Kluyveromyces martensii* strain was superior to that of the *Lactococcus gasseri* and *Enterococcus faecium* strains. Compared with commercial strains, the experimental strains generally showed similar performance in pH tolerance; among the three experimental strains, the *Kluyveromyces martensii* strain exhibited better pH stability. Therefore, the compound bacterial agent of this invention can maintain relatively stable growth within a suitable pH range of 5-7, and provides a basis for the synergistic effect of the three strains during fermentation acidification.

[0049] The three-strain interaction provided by this invention significantly enhances the growth performance of the strains. The bacterial concentration in the three-strain co-culture system is higher than that of each two-strain combination, indicating a synergistic growth-promoting trend when the three strains coexist. For example, in specific interaction results, the bacterial concentration of the three-strain interaction system of this invention reaches approximately 4.5 × 10^5 CFU / mL at 48 h, which is higher than that of two-strain combinations such as *Lactococcus lactis* + *Enterococcus faecium*, *Lactococcus lactis* + *Kluyveromyces oryzae*, and *Kluyveromyces oryzae* + *Enterococcus faecium*; the bacterial concentration of commercial three-strain combinations is approximately 4.3 × 10^5 CFU / mL at 48 h. Therefore, the synergistic effect produced by the interaction of the three strains of this invention is superior to that of commercial strain combinations, and can provide a stable bacterial quantity basis for subsequent co-fermentation of yak ghee.

[0050] The three-strain compound provided by this invention effectively solves the problems of unclear composition of natural fermentation microbiota, insufficient adaptability of commercial strains to yak milk fat substrate, and uncertain interaction effects of compound strains. Each of the three strains possesses the basic single-strain growth potential for subsequent synergistic fermentation and exhibits stability suitable for optimizing yak butter fermentation processes under environmental tolerance conditions such as temperature, lactic acid, salt, and pH. The bacterial concentration increase during co-culturing of the three strains is higher than that of a two-strain combination, and the synergistic effect of the three strains is superior to that of commercial three-strain combinations, thus providing a strain foundation for the controllable fermentation of yak milk fat substrate.

[0051] In the compound microbial agent provided by this invention, given that the three strains of this invention all exhibit strong tolerance compared to commercial strains, the ratio of the three strains can be selected within a wide range. However, to further improve the synergistic effect of each strain in the compound microbial agent, the ratio of *Lactococcus gasseri* strain, *Enterococcus faecium* strain, and *Kluyveromyces martensii* strain, based on the number of viable cells, is (0.5-2):(0.5-2):(0.5-2). Preferably, the ratio of the three strains is (0.8-1.2):(0.8-1.2):(0.8-1.2), for example, it can be 0.8:1:1, 1:0.8:1, 1:1:0.8, 1:1:1, 1.2:1:1, 1:1.2:1, and 1:1:1.2. More preferably, the ratio is 1:1:1. Using the above-mentioned preferred ratio can make the synergistic effect of the three strains more significant, and at the same time make the initial bacterial count of the three strains more balanced in the early stage of inoculation, thereby further reducing the fluctuation of fermentation start-up.

[0052] In some embodiments, the compound microbial agent may be a liquid microbial agent, a concentrated microbial suspension, or a freeze-dried microbial powder. The freeze-dried microbial powder may contain commonly used protectants or carriers for food fermentation microbial agents, such as skim milk powder, lactose, sucrose, trehalose, maltodextrin, gelatin, sodium caseinate, monosodium glutamate, sodium ascorbate, glycerol, sterile water, physiological saline, or combinations thereof.

[0053] In some embodiments, the compound microbial agent is a liquid microbial agent. In some embodiments, the total viable count is 1×10^6 CFU / mL to 1×10^8 CFU / mL; preferably 5×10^6 CFU / mL to 5×10^7 CFU / mL, for example, it can be 5×10^6, 8×10^6, 1×10^7, 2×10^7 and 5×10^7 CFU / mL; more preferably about 1×10^7 CFU / mL. Using this preferred viable count allows for matching with the inoculum amount of yak milk fat substrate, forming stable fermentation initiation conditions.

[0054] The compound microbial agent of the present invention, on the one hand, makes the composition of the fermentation system clear and avoids batch differences caused by the complex sources of miscellaneous bacteria and the fluctuation of dominant bacterial groups in natural fermentation; on the other hand, the three strains have good environmental tolerance and growth characteristics, and exhibit growth-promoting interactions when co-cultured, thus providing a foundation for stable and synergistic fermentation of yak milk fat base.

[0055] In a second aspect, the present invention provides a method for preparing the composite microbial agent described in the first aspect, wherein the method includes activating and culturing the *Lactococcus gasseri* strain, *Enterococcus faecium* strain, and *Kluyveromyces martensii* strain respectively, and mixing them in a predetermined ratio to obtain the composite microbial agent.

[0056] In some embodiments, the three strains can be activated and cultured separately in suitable culture media until the late logarithmic growth phase or the early stationary phase, and then the bacterial concentration is adjusted according to the number of viable cells before being compounded. Single-cell growth curves show that the three strains exhibit a typical growth process: a 0-10 h lag phase, a 10-30 h logarithmic growth phase, a stationary phase around 30 h, and gradual decline after 40 h. Therefore, setting the compounding time at a stage with high bacterial activity helps avoid initial inoculum deviations and fluctuations in the bacterial community ratio after compounding due to differences in bacterial age.

[0057] In some embodiments, the *Lactococcus gasseri* strain may be revived, activated, or amplified using MRS medium. The MRS medium, per 1 L, may include 10 g peptone, 8 g beef extract, 4 g yeast extract, 20 g glucose, 1 mL Tween-80, 2 g diammonium hydrogen citrate, 5 g sodium acetate, 2 g K₂HPO₄, 0.2 g magnesium sulfate, and 0.04 g manganese sulfate.

[0058] In some embodiments, the *Enterococcus faecalis* can be resuscitated, activated, or amplified using MRS medium. The MRS medium, per 1 L, may include 10 g peptone, 8 g beef extract, 4 g yeast extract, 20 g glucose, 1 mL Tween-80, 2 g diammonium citrate, 5 g sodium acetate, 2 g K₂HPO₄, 0.2 g magnesium sulfate, and 0.04 g manganese sulfate. After culture, the bacterial cells can be collected by centrifugation and resuspended after washing with sterile physiological saline, sterile water, or sterile buffer to reduce the impact of the medium components on the subsequent yak milk fat fermentation system.

[0059] In some embodiments, the *Kluyveromyces martensii* strain can be revived, activated, or amplified using a suitable yeast culture medium. The suitable yeast culture medium can be YPD medium, YM medium, or other yeast culture media with yeast extract, peptone, and sugar sources as the main nutrients. For example, YM medium, per 1 L, may include 5.0 g casein peptone, 3.0 g malt extract, 10.0 g glucose, and 3.0 g yeast extract.

[0060] In some embodiments, a synthetic microbial community liquid culture medium can be used to evaluate the interaction relationships of the three strains in a simulated yak milk fat fermentation environment, or for the cultivation of three-strain interactions. The synthetic microbial community liquid culture medium, per 1 L, may include 10 g glucose, 10 g tryptone, 5 g yeast extract, 10 g sterile yak milk fat, 2 g dipotassium hydrogen phosphate, 0.5 g magnesium sulfate, 0.1 g calcium chloride, and 1 mL Tween 80. This culture medium, containing sterile yak milk fat, more closely resembles the yak milk fat-based fermentation system, which is beneficial for evaluating the co-cultivation adaptability and interaction trends of the three strains in a milk fat-containing environment.

[0061] In some embodiments, when the three strains are activated and cultured separately, the culture temperature can be 20-40°C, preferably 25-35°C (for example, 25°C, 28°C, 30°C, 32°C and 35°C), and more preferably 28-30°C.

[0062] In some embodiments, the activation and culturing time of the three strains can be determined based on the single-strain growth curve. Preferably, they are cultured to the logarithmic growth phase of 10-30 hours, or to the early stage of the stationary phase of about 30 hours, before being used for compounding. The above time points are only used to illustrate that compounding is carried out at a stage with high cell activity, and do not indicate that the same fermentation effect has been obtained at each time point; in actual production, the actual harvest time of the three strains can be determined by combining OD600, plate count, or flow cytometry results.

[0063] In some implementations, the culture conditions can be adjusted according to the environmental tolerance of the three strains. pH tolerance results showed that the optimal growth pH for the tested strains was 5-7. Lactic acid tolerance results showed that as the lactic acid concentration increased from 0% to 4%, the overall viable cell count of each strain decreased, but the *Kluyveromyces martensii* and *Lactococcus gasseri* strains showed smaller changes at different lactic acid concentrations, exhibiting better acid stability. Salt concentration experiments showed that different salt concentrations had a certain inhibitory effect on the growth of the tested strains, but the experimental strains showed more stable growth under salt stress. These results indicate that the three strains possess the environmental adaptability necessary for optimizing the co-fermentation process of yak ghee.

[0064] In some embodiments, the ratio of the three strains can be (0.5-2):(0.5-2):(0.5-2), preferably (0.8-1.2):(0.8-1.2):(0.8-1.2), and more preferably 1:1:1. All ratios are based on the number of viable bacteria. Using a mixture with equal or near-equal numbers of viable bacteria helps reduce the impact of differences in initial inoculum amounts of different strains on the interaction and fermentation results, and also facilitates further optimization of the ratio based on the microbial succession and flavor compound formation during fermentation.

[0065] Preferably, the three strains are adjusted to the same or substantially the same viable cell count level before being mixed in a predetermined ratio. More preferably, the *Lactococcus gasseri* strain, *Enterococcus faecium* strain, and *Kluyveromyces martensii* strain are each adjusted to approximately 1 × 10⁻⁶. 7 After CFU / mL, the microbial agents are mixed at a live bacteria ratio of 1:1:1 to obtain the compound microbial agent. Using this method, the three strains of bacteria in the compound microbial agent enter the yak milk fat base fermentation system in a measurable and repeatable manner, thus providing stable starting conditions for subsequent interactions between the three bacteria, flavor metabolism, and quality control.

[0066] In some embodiments, the three strains can be mixed and used directly as a liquid compound microbial agent, or they can be concentrated by centrifugation, resuspended, treated with a preservative, or freeze-dried to produce a concentrated microbial suspension or freeze-dried microbial powder. When using freeze-dried microbial powder, it is preferable to add a freeze-drying preservative commonly used in food fermentation agents before freeze-drying, and to reconstitute it to the same or substantially the same viable cell count level as the liquid compound microbial agent before inoculation.

[0067] Thirdly, the present invention provides a method for preparing yak ghee through fermentation, wherein the method includes the following steps:

[0068] Sterilize or sterilize the yak milk fat base material;

[0069] The compound microbial agent described in the first aspect is inoculated into yak milk fat base material that has been sterilized or sterilized;

[0070] Fermentation is carried out to obtain fermented yak milk fat;

[0071] The fermented yak milk fat is post-processed to obtain fermented yak ghee.

[0072] In some embodiments, the yak milk fat base material is a milk fat phase, a high-fat cream phase, or a milk fat raw material obtained from yak milk through milk fat separation, or through further processing. Specifically, fresh yak milk can be mixed evenly and preheated, then separated from the skim milk phase using centrifugation or other conventional milk fat separation methods in the art, and the resulting milk fat phase or high-fat cream phase can be collected as the yak milk fat base material. The above-described milk fat separation method is only used to illustrate one way of obtaining yak milk fat base material and should not be construed as the only preparation method that must be used in this invention. In some preferred embodiments, the fat content of the yak milk fat base material is 85%-95%, for example, it can be 85%, 87%, 90%, 92%, or 95%.

[0073] In some embodiments, the yak milk fat base material is sterilized or disinfected to reduce the interference of contaminating bacteria in the raw material on the fermentation process of the three preserved bacterial strains. The sterilization or disinfection treatment can be high-temperature sterilization, pasteurization, instantaneous high-temperature treatment, or other sterilization methods suitable for milk fat base materials. Preferably, high-temperature sterilization is used to obtain sterile or low-bacterial-load yak milk fat base material.

[0074] In some embodiments, the compound microbial agent is the compound microbial agent containing Lactococcus gasseri strain, Enterococcus faecalis strain and Kluyveromyces martensii strain as described in the first aspect of the present invention.

[0075] In some embodiments, the inoculation amount of the compound microbial agent is 1×10^6 to 1×10^8 CFU / 100 g yak milk fat base, based on the total number of viable bacteria in the compound microbial agent; preferably 0.8×10^7 to 1.2×10^7 CFU / 100 g yak milk fat base; more preferably about 1.0×10^7 CFU / 100 g yak milk fat base.

[0076] In some embodiments, the fermentation temperature is 25-35°C; preferably 26-32°C; more preferably 27-30°C (e.g., 27°C, 28°C, 29°C, and 30°C); and even more preferably 28°C.

[0077] In some embodiments, the fermentation time is 24-72 h; preferably 48-72 h (e.g., 24 h, 48 h and 72 h); more preferably 72 h.

[0078] In some embodiments, the fermentation can be carried out in a closed, semi-closed, or open but controlled environment. Preferably, to reduce interference from other microorganisms and improve batch stability, the present invention uses sterilized or sterilized yak milk fat as a base material and inoculates it with a compound microbial agent for fermentation under aseptic or low microbial load conditions.

[0079] In some embodiments, the post-processing includes one or more of the following: cold water washing, ice water washing, beating, kneading, and shaping. Preferably, after fermentation, the fermented yak milk fat is washed with ice water to remove some aqueous impurities and free fermentation liquid components, and then kneaded and shaped to obtain fermented yak ghee.

[0080] When using the above method, the three preserved strains can be introduced into the yak milk fat base fermentation system at a controllable inoculation amount, avoiding the problem of unstable starting microbiota in natural fermentation; the high-fat milk fat phase obtained by milk fat separation can provide a suitable matrix for the lipid metabolism, flavor precursor conversion and volatile flavor substance formation of the three strains; after fermentation and post-treatment, fermented yak ghee with improved flavor, better control of acid value and peroxide value, and lower accumulation of biogenic amines can be obtained.

[0081] Fourthly, the present invention provides the application of the compound microbial agent described in the first aspect in improving the flavor of yak ghee, reducing the acid value of yak ghee, reducing the peroxide value of yak ghee, reducing the accumulation of biogenic amines in yak ghee, and / or improving the storage stability of yak ghee.

[0082] In some embodiments, the application includes inoculating a compound microbial agent consisting of *Lactococcus gasseri* strains, *Enterococcus faecium* strains, and *Kluyveromyces martensii* strains into yak milk fat substrate for fermentation. The three strains collectively participate in lipid, sugar, and amino acid metabolism within the yak milk fat substrate, resulting in fermented yak ghee with a more harmonious milky, mellow, and cheese-like aroma, while reducing its heavy, greasy feel.

[0083] In some embodiments, improving the flavor of yak ghee includes increasing the relative content of volatile components such as hexanoic acid and ethanol, which are associated with a refreshing, mellow, and cheese-like aroma, or decreasing the proportion of components such as 2-heptanone, butyric acid, and hexadecanoic acid, which are associated with a heavy, greasy feel. According to the results of the embodiments, compared with commercial three-strain combinations, the fermentation samples of the compound microbial agent of the present invention have higher relative contents of hexanoic acid and ethanol, exhibiting a more refreshing flavor and a purer cheese aroma.

[0084] In some embodiments, the reduction of acid value and peroxide value of yak ghee refers to the fact that, under the same or substantially the same conditions of yak milk fat base material, inoculum amount, fermentation temperature and fermentation time, compared with commercial three-strain combination or non-inventory microbial agent treatment, the compound microbial agent of the present invention can slow down the process of fat hydrolysis rancidity and lipid oxidation, thereby improving the quality stability of fermented yak ghee.

[0085] In some embodiments, the reduction in biogenic amine accumulation in yak ghee means that the biogenic amine content in yak ghee fermented with the compound microbial agent of the present invention is lower than that in commercial three-strain fermentation samples or samples not treated with the present invention. Therefore, the compound microbial agent of the present invention can not only improve flavor but also reduce the risk of accumulation of adverse metabolites during fermentation.

[0086] In some embodiments, improving the storage stability of yak ghee means that the growth of acid value, peroxide value, biogenic amines, and / or microbial contamination indicators in fermented yak ghee is inhibited during storage, resulting in more stable product flavor and quality. The aforementioned improvements in flavor, acid value, peroxide value, biogenic amines, and storage stability can be achieved individually or in combination.

[0087] Fifthly, the present invention provides a fermented yak ghee, which is prepared by the method described in the third aspect.

[0088] Example

[0089] The technical solution of the present invention will be further described below with reference to embodiments and accompanying drawings. The advantages and features of the present invention will become clearer with the description. However, it should be understood that the embodiments are merely exemplary and do not constitute a limitation on the scope of the present invention. It should be noted that, unless otherwise specified, the experimental methods used in the following embodiments are conventional methods in the art. Unless otherwise defined, all scientific and technical terms used in this invention have the same meaning as commonly understood by those skilled in the art.

[0090] Commercial Enterococcus faecalis: purchased from Chr. Hansen; LACTIFERM

[0091] Commercial lactococci: purchased from Chr. Hansen; Pro-K TM

[0092] Commercial Kluyveromycin: Purchased from Chr. Hansen; CONCERTO TM

[0093] The detection of biogenic amines can be performed in accordance with GB 5009.208-2016, "National Food Safety Standard - Determination of Biogenic Amines in Food". Specifically, the dansyl chloride derivatization method is used: Weigh 10 g of ghee sample, add 20 mL of 0.1 M HCl and mix, vortex at room temperature for 2 min, centrifuge at 4000 rpm for 20 min, and then make up to 50 mL; take 1 mL of sample or standard solution for derivatization, filter through a 0.22 μm organic phase membrane, and then perform liquid chromatography detection. The chromatographic column is C18 (250 mm × 4.6 mm, 5 μm), the mobile phase is acetonitrile and ultrapure water, the flow rate is 1.5 mL / min, the UV detection wavelength is 254 nm, the injection volume is 10 μL, and the column temperature is 30℃.

[0094] Example 1 illustrates the verification of the environmental tolerance of the three strains provided by the present invention.

[0095] The *Lactococcus gasseri*, *Enterococcus faecium*, and *Kluyveromyces martensii* strains of this invention were activated and cultured separately. *Lactococcus gasseri* was cultured on MRS medium at 37°C for 24 h; *Enterococcus faecium* was cultured on MRS medium at 37°C for 24 h; and *Kluyveromyces martensii* was cultured on YM medium at 28°C for 24 h. The activated bacterial solutions were collected by centrifugation, washed with sterile physiological saline, resuspended, and the bacterial concentration was adjusted to approximately 1 × 10^7 CFU / mL for later use. Corresponding commercial strains were used as comparative controls. Each strain was inoculated into its corresponding test medium at an inoculum of 1% (v / v). The test conditions included temperature, lactic acid concentration, salt concentration, and pH. In the temperature tolerance experiment, the inoculated test system was cultured at 15℃, 20℃, 25℃, 30℃, 35℃, and 40℃ for 48 h. In the lactic acid tolerance experiment, lactic acid was added to the test medium to achieve concentrations of 0%, 1%, 2%, 3%, and 4%, and the culture was carried out at 28℃ for 48 h. In the salt concentration tolerance experiment, sodium chloride was added to the test medium to achieve concentrations of 0%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, and 8%, and the culture was carried out at 28℃ for 48 h. In the pH tolerance experiment, the pH of the test medium was adjusted to 3, 4, 5, 6, 7, 8, 9, and 10, and the culture was carried out at 28℃ for 48 h. Except for the different test factors, all other culture conditions were kept consistent. After the culture was completed, the viable cell concentration was determined by plate counting, and the environmental tolerance results were plotted.

[0096] Temperature tolerance results as follows Figure 1 As shown, by Figure 1 It can be seen that the viable cell count of all strains exhibits a consistent pattern with temperature changes: slow growth at 15–20 ℃, rapid proliferation at 20–25 ℃, a stable phase at 25–35 ℃ with a high viable cell count, and a slight decrease after 40 ℃. Therefore, 25–35 ℃ is the optimal growth temperature range for the tested strains. Compared with commercial strains of the same species, the commercial strain of *Lactococcus lactis* showed slightly higher growth activity, while *Kluyveromyces oryzae* and *Enterococcus faecium* showed better activity in the experimental strains. There are differences in growth ability among different strains; *Lactococcus lactis* showed the strongest overall growth activity, followed by *Kluyveromyces oryzae*, while *Enterococcus faecium* was relatively weaker. The results of this study can provide a basis for optimizing the strain ratio and selecting temperature conditions in subsequent ghee fermentation processes.

[0097] Lactic acid tolerance results as follows Figure 2 As shown, by Figure 2It can be seen that as the lactic acid concentration increased from 0% to 4%, the viable cell count of all strains generally showed a decreasing trend, indicating that lactic acid has a certain inhibitory effect on the growth of the strains. Among them, the viable cell counts of Kluyveromyces and experimental Lactococcus showed relatively small changes at different concentrations, exhibiting good acid resistance stability; the viable cell counts of commercial Lactococcus and commercial Enterococcus faecalis decreased more significantly with increasing lactic acid concentration. The above results indicate that the tested strains have different tolerances to lactic acid, providing a basis for the screening, formulation, and optimization of fermentation conditions in the subsequent butter fermentation process.

[0098] Salt concentration tolerance results as follows Figure 3 As shown, by Figure 3 It can be seen that different salt concentrations have a certain inhibitory effect on the growth of the tested strains, and the salt tolerance characteristics of the experimental strains and commercial strains differ. The results indicate that the experimental strains exhibit more stable growth under salt stress, which can provide a reference for subsequent optimization of the ghee fermentation process.

[0099] pH tolerance results are as follows Figure 4 As shown, by Figure 4 The results of pH tolerance indicate that the optimal growth pH for the tested strains is 5-7. Kluyveromyces has better pH stability than Lactococcus and Enterococcus faecalis. The experimental strains and commercial strains showed similar performance, providing a basis for optimizing the ghee fermentation process.

[0100] Example 2 This example illustrates the verification of the growth curves of three single strains provided by the present invention.

[0101] The *Lactococcus gasseri*, *Enterococcus faecium*, and *Kluyveromyces martensii* strains of this invention were activated and cultured separately. *Lactococcus gasseri* was cultured on MRS medium at 37°C for 24 h; *Enterococcus faecium* was cultured on MRS medium at 37°C for 24 h; and *Kluyveromyces martensii* was cultured on YM medium at 28°C for 24 h. The activated bacterial solutions were collected by centrifugation, washed with sterile physiological saline, resuspended, and the bacterial concentration was adjusted to approximately 1 × 10^7 CFU / mL for later use. Corresponding commercial strains were used as comparative controls. Each of the above strains was inoculated into its corresponding test medium at an inoculation rate of 1% (v / v). The test medium for *Lactococcus gasseri* and *Enterococcus faecium* was MRS medium, and the test medium for *Kluyveromyces martensii* was YM medium. A corresponding commercial strain was used as a control, and cultures were conducted under the same culture medium, inoculum size, and culture conditions. During the culture process, samples were taken at 0 h, 10 h, 20 h, 30 h, 40 h, and 48 h, and the viable cell concentration at each time point was determined using the plate count method. Single-strain growth curves were plotted based on the results. Figure 5 As shown.

[0102] Depend on Figure 5 It can be seen that the tested strains generally exhibit typical growth cycle characteristics: all strains show typical growth cycle characteristics: 0-10 h is the lag phase, with slow increase in bacterial concentration; 10-30 h is the logarithmic growth phase, with a rapid increase in bacterial concentration; around 30 h, they enter the stationary phase, with the bacterial concentration reaching its peak and remaining stable; after 40 h, they gradually enter the decline phase, with a slight decrease in bacterial concentration. Different strains show differences in growth performance: commercial *Lactococcus lactis* has the highest peak bacterial concentration, *Kluyveromyces kluyveromyces* has the fastest growth rate in the logarithmic phase, and *Lactococcus lactis* shows a more pronounced decline trend in the decline phase. The growth trends of the experimental strains are basically consistent with the corresponding commercial strains, indicating that the tested strains possess good growth characteristics and can be used for subsequent optimization of the ghee co-fermentation process.

[0103] Example 3 illustrates the preparation, interaction verification, and fermentation effect of the three-strain compound inoculant on yak butter.

[0104] Lactococcus gasseri strain Lac, Enterococcus faecium strain Ent, and Kluyveromyces martensii strain Klu were activated and cultured separately. Lactococcus gasseri and Enterococcus faecium strains were activated and cultured on MRS medium at 37°C for 24 h; Kluyveromyces martensii strain was activated and cultured on YM medium at 28°C for 24 h. After activation, the bacterial suspensions of the three strains were collected by centrifugation, washed with sterile physiological saline, and resuspended. The viable cell concentration of each bacterial suspension was determined by plate counting. Based on the results, the concentrations of the Lactococcus gasseri, Enterococcus faecium, and Kluyveromyces martensii strains were adjusted to the same or substantially the same viable cell count level. Subsequently, the three bacterial suspensions were mixed at a viable cell count ratio of 1:1:1 to prepare a three-strain compound bacterial agent. The total viable cell concentration of the three-strain compound bacterial agent was approximately 1 × 10^7 CFU / mL. To evaluate the interaction effects among the three bacterial strains, co-culture experiments were conducted using two-strain and three-strain combinations. The two-strain combinations included Lac+Ent, Lac+Klu, and Ent+Klu, each mixed at a viable count ratio of 1:1. The three-strain combinations were mixed at a viable count ratio of Lac:Ent:Klu = 1:1:1. Two-strain and three-strain combinations of corresponding commercial strains were used as comparative systems. Each combination was inoculated into synthetic microbial community liquid culture medium at a 1% (v / v) inoculum and cultured at 28°C for 48 h. Samples were taken every two hours during the culture period, and the change in viable count concentration of each combination at different culture times was determined using the plate count method. Interaction growth curves of the strains were plotted based on the results. The interaction results of the commercial strain combinations are shown below. Figure 6 As shown, the interaction results of the three bacterial strains in this invention are as follows: Figure 7 As shown.

[0105] The co-culture was performed using a synthetic microbial community liquid medium, which, per 1 L, contained 10 g glucose, 10 g tryptone, 5 g yeast extract, 10 g sterile yak milk fat, 2 g dipotassium hydrogen phosphate, 0.5 g magnesium sulfate, 0.1 g calcium chloride, and 1 mL Tween 80. During co-culture, the initial cell concentration of each participating strain was adjusted to approximately 1 × 10^7 CFU / mL. After mixing according to the set ratio, the mixture was incubated at 28 °C for 48 h, and changes in OD600 or bacterial concentration were measured.

[0106] Depend on Figure 6 It can be seen that the commercial three-strain combination reached approximately 4.3 × 10^5 CFU / mL at 48 h, which is higher than that of the commercial two-strain combination, indicating that there can also be some interactive growth between commercial strains. Figure 7It can be seen that the three-strain interaction system of this invention reaches approximately 4.5 × 10^5 CFU / mL at 48 h, which is higher than the Lac+Ent, Lac+Klu, and Klu+Ent dual-strain combinations, and higher than the commercial three-strain combination of approximately 4.3 × 10^5 CFU / mL. This result indicates that the coexistence of the three strains in this invention is not a simple parallel arrangement, but rather forms a synergistic growth-promoting effect superior to dual-strain combinations and commercial three-strain combinations, providing a more stable bacterial count and interaction basis for subsequent yak milk fat base fermentation.

[0107] Furthermore, the 1:1:1 three-strain compound microbial agent was used to ferment yak milk fat base. Fresh yak milk from the same batch was preheated and the fat was separated to obtain either a milk fat phase or a high-fat cream phase. The fat content of the resulting yak milk fat base was controlled at 90g / 100g. After sterilization, the yak milk fat base was inoculated at approximately 1×10^7 CFU / 100g of yak milk fat base, based on the total viable count of the compound microbial agent. Fermentation was carried out at 28℃ for 72 h, with samples taken at 24 h, 48 h, and 72 h. After fermentation, the mixture was washed with ice water, kneaded, and shaped according to the ghee preparation process to obtain fermented yak ghee. The physicochemical results are as follows: Figures 8-11 As shown.

[0108] For moisture determination, refer to GB 5009.3-2016 Direct Drying Method: Solid Sample: Take a clean aluminum or glass flat weighing bottle, place it in a drying oven at 101℃~105℃ with the cap tilted against the side of the bottle, heat for 1.0 h, remove and cover, place in a desiccator to cool for 0.5 h, weigh, and repeat drying until the difference between the two batches does not exceed 2 mg, which is constant weight. Quickly grind the well-mixed sample to particles smaller than 2 mm. Samples that are difficult to grind should be chopped as much as possible. Weigh 2 g~10 g of sample (accurate to 0.0001 g), place it in this weighing bottle, the sample thickness should not exceed 5 mm, if it is a loose sample, the thickness should not exceed 10 mm, cover, weigh accurately, place in a drying oven at 101℃~105℃ with the cap tilted against the side of the bottle, dry for 2 h~4 h, cover and remove, place in a desiccator to cool for 0.5 h, and weigh. Then place it in a drying oven at 101℃~105℃ for about 1 hour, remove it, cool it in a desiccator for 0.5 hours, and weigh it again. Repeat the above operation until the difference between the two weighings does not exceed 2mg, which is considered constant weight.

[0109] pH determination should be performed according to GB 5009.237-2016 using a digital pH meter: Take 10.00 g of yak milk fat base material from the fermentation process, mix thoroughly, and add 90 mL of freshly boiled and cooled distilled water or tertiary water. Homogenize in a homogenizer for 1–2 min to ensure thorough dispersion. After homogenization, let stand for 30 min, and filter or centrifuge if necessary. Use the filtrate or supernatant as the test solution. Before measurement, calibrate the digital pH meter using standard buffer solutions at pH 4.00 and pH 6.86 or pH 9.18. After calibration, insert the composite electrode into the test solution and record the pH value after the reading stabilizes. Each sample should be measured in parallel at least twice, and the average value should be used as the result, retained to the nearest 0.01 or 0.05 pH unit.

[0110] The determination of peroxide value follows the titration method of GB 5009.227-2016: Weigh 2-3 g (accurate to 0.001 g) of the prepared sample and place it in a 250 mL iodine flask. Add 30 mL of chloroform-glacial acetic acid solution and gently shake until the sample is completely dissolved. Accurately add 1.00 mL of saturated potassium iodide solution, tighten the cap, and gently shake for 0.5 min. Place in the dark for 3 min. Remove the flask, add 100 mL of water, shake well, and immediately titrate the precipitated iodine with sodium thiosulfate standard titration solution (use 0.002 mol / L standard titration solution when the estimated peroxide value is 0.15 g / 100 g or less; use 0.01 mol / L standard titration solution when the estimated peroxide value is greater than 0.15 g / 100 g). Titrate until the solution turns pale yellow, add 1 mL of starch indicator, continue titrating, and shake vigorously until the blue color disappears, which is the endpoint.

[0111] The acid value determination refers to the cold solvent indicator titration method in GB 5009.229-2016: Add 50mL~100mL of cold solvent to the weighed oil sample, gently shake the sample until it is completely dissolved, add 3 drops~4 drops of phenolphthalein indicator, and immediately titrate with the potassium hydroxide or sodium hydroxide standard titration solution of the concentration specified in Table 1 until the solution turns slightly red and does not fade within 15s, which is the endpoint.

[0112] Depend on Figure 8 It can be seen that as the fermentation time is extended, the moisture content of the three-strain group decreases significantly from 28.00% on day 1 to 4.79% on day 3; the moisture content of the commercial three-strain group remains at a low level and the change is small. The results indicate that the water metabolism rate of the three-strain fermentation system is significantly higher than that of the commercial three-strain group.

[0113] Depend on Figure 9It can be seen that the three-strain system has stronger overall metabolic activity, more significant pH changes, stronger acidification, and obvious ability to recover in the later stage; while the commercial system maintains a higher and more stable pH level, with gentler metabolic changes and more conservative characteristics.

[0114] Depend on Figure 10 It can be seen that the acid value of the three-strain group was lower than that of the commercial strain group throughout the process, and the upward trend was slower, indicating that the self-developed strain is gentler on the decomposition of oils, can reduce rancidity, and is more suitable for the flavor fermentation of ghee.

[0115] Depend on Figure 11 It can be seen that the peroxide value of the three-strain group showed a continuous downward trend during fermentation, effectively controlling the oxidation process of the oil. In contrast, although the initial value of the commercial strain was lower, its decrease was smaller. This indicates that the three strains have a more significant effect on scavenging peroxides and are more conducive to improving the shelf-life stability of the product.

[0116] Biogenic amine detection: The results of biogenic amine detection are shown in Table 1 below. Neither group of samples tested positive for 2-phenylethylamine, putrescine, octylamine hydrochloride, 4-hydroxyphenylethylamine, or tryptamine. The nitrileamine content in the TT-tribiotic sample was 0.43 mg / kg, lower than the 1.68 mg / kg in the TB-commercial tribiotic sample. This result indicates that the ghee samples obtained from fermentation with the compound microbial agent of this invention have a lower risk of specific biogenic amine accumulation.

[0117] Table 1

[0118]

[0119] Flavor analysis by GC-MS: The volatile components of TT-tribacterium and TB-commercial tribacterium samples were qualitatively and relatively quantitatively analyzed by gas chromatography-mass spectrometry (GC-MS). A total of 11 major categories of volatile components were detected, including amides and salts, alkanes, cyclosiloxanes, esters, alcohols, ketones, aromatics, acids, aldehydes, alkenes and other miscellaneous substances. The composition and relative content of each substance in the two bacterial agents were significantly different, as shown in Table 2 below.

[0120] Table 2

[0121]

[0122]

[0123]

[0124] Acids were the components with the highest relative abundance in both groups of samples, serving as the main flavor compounds. They primarily originated from fat degradation and microbial metabolism, exhibiting low odor thresholds and making a significant contribution to the overall flavor. The TT-triple-strain sample contained significantly higher levels of hexanoic acid (22.78%) than the TB-commercial-triple-strain sample (3.19%), imparting a pure and refreshing cheese aroma to the sample. The TB-commercial-triple-strain sample contained higher levels of butyric acid (6.92%) and n-hexadecanoic acid (32.94%), with long-chain fatty acids contributing a distinct fatty, waxy, and robust fermented flavor.

[0125] Alcohols mainly originate from carbohydrate degradation and microbial fermentation, exhibiting a high threshold but bright aroma. The TT-tri-strain contains an extremely high proportion of ethanol (12.71%), significantly higher than the TB-commercial-tri-strain (1.04%), contributing distinct floral, sweet, and ripe fruit aromas to the system, resulting in a fresher and more pleasant overall aroma.

[0126] Cyclosiloxanes and dimethylsilanediol were characteristic components of both groups of samples. The proportions of hexadecyl cyclohexasiloxane (16.15%) and dimethylsilanediol (12.11%) were significantly higher in TT-tribum, giving the system a clean, mellow silicone-like sweet aroma and a refreshing base, which can effectively neutralize the acidic irritation.

[0127] Alkenes, aldehydes, esters, and alkanes complement and modify the overall flavor. TT-triumurum has a higher total olefin content, contributing herbal, fresh, and fatty aromas.

[0128] Overall flavor differences

[0129] Gas chromatography-mass spectrometry (GC-MS) was used to detect and analyze the volatile flavor compounds in ghee fermented with TT-tri-strain and TB-commercial-tri-strain. The results showed that there were differences in the composition and relative content of volatile flavor compounds between TT-tri-strain and TB-commercial-tri-strain. Compared with TB-commercial-tri-strain, the TT-tri-strain fermented samples had higher relative contents of volatile compounds such as ethanol and hexanoic acid, indicating that TT-tri-strain can form a characteristic volatile flavor compound profile that is distinct from the commercial tri-strain combination.

[0130] Examples 4-6

[0131] Following the activation culture and bacterial suspension adjustment method of Example 3, *Lactococcus gasseri* strain, *Enterococcus faecium* strain, and *Kluyveromyces martensii* strain were mixed at viable cell ratios of 0.8:1:1.2, 1.2:0.8:1, and 1:1.2:0.8, respectively, to prepare a three-strain compound inoculum. Except for the different ratios of the three strains, the interaction culture, yak milk fat substrate treatment, inoculum size based on total viable cell count, fermentation temperature, fermentation time, and post-treatment steps were all the same as in Example 3.

[0132] The above three near-equal proportion compounding schemes all retain the synergistic fermentation basis formed by the coexistence of the three strains, which can be used for fermentation of yak milk fat base material, and obtain fermented yak ghee with improved flavor, controlled acid value and peroxide value, and reduced tendency of biogenic amine accumulation.

[0133] The above results demonstrate that, while maintaining the coexistence of the three strains, a slight adjustment to the initial viable cell ratio of Lac, Ent, and Klu can still achieve the fermentation effect required to satisfy the objectives of this invention. Therefore, 0.8:1:1.2, 1.2:0.8:1, and 1:1.2:0.8 can all be considered specific implementations of the near-equiproportional compounding scheme of the three strains in this invention.

[0134] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. A compound microbial agent for fermenting and preparing yak ghee, characterized in that, The compound microbial agent includes the following three preserved bacterial strains: Lactococcus garvieae strain, with accession number CGMCC No. 32756; The strain of Enterococcus faecium, with accession number CGMCC NO.36378; The strain Kluyveromyces marxianus, with accession number CGMCC NO.39019; All three preserved bacterial strains existed in live form.

2. The compound microbial agent according to claim 1, characterized in that, The ratio of *Lactococcus gasseri* strain, *Enterococcus faecium* strain, and *Kluyveromyces martensii* strain, based on the number of viable cells, is (0.5-2):(0.5-2):(0.5-2); preferably (0.8-1.2):(0.8-1.2):(0.8-1.2).

3. The compound microbial agent according to claim 1 or 2, characterized in that, The compound microbial agent is a liquid microbial agent, a concentrated microbial suspension, a freeze-dried microbial powder, or a combination thereof.

4. The compound microbial agent according to any one of claims 1-3, characterized in that, The compound microbial agent is a liquid microbial agent, and the total viable count in the compound microbial agent is 1×10⁻⁶. 6 CFU / mL to 1×10 8 CFU / mL.

5. A method for preparing the compound microbial agent according to any one of claims 1-4, characterized in that, The method includes activating and culturing the Lactococcus gasseri strain, Enterococcus faecalis strain, and Kluyveromyces martensii strain separately, and mixing them in a predetermined ratio to obtain the compound bacterial agent.

6. A method for preparing yak ghee through fermentation, characterized in that, The method includes the following steps: Sterilize or sterilize the yak milk fat base material; Inoculate the compound microbial agent according to any one of claims 1-4 into yak milk fat base that has been sterilized or sterilized; Fermentation is carried out to obtain fermented yak milk fat; The fermented yak milk fat is post-processed to obtain fermented yak ghee.

7. The method according to claim 6, characterized in that, The fat content of the yak milk fat base is 85%-95%.

8. The method according to claim 6 or 7, characterized in that, Based on the number of viable bacteria, the inoculation amount of the compound microbial agent is 1×10^6-1×10^8 CFU of compound microbial agent per 100 g of yak milk fat base; and / or The fermentation temperature is 25-35℃; and / or The fermentation time is 24-72 h; and / or The post-processing includes one or more of the following: cold water washing, ice water washing, beating, kneading, and shaping.

9. The application of the compound microbial agent according to any one of claims 1-4 in improving the flavor of yak ghee, reducing the acid value of yak ghee, reducing the peroxide value of yak ghee, reducing the accumulation of biogenic amines in yak ghee, and / or improving the storage stability of yak ghee.

10. A fermented yak ghee, characterized in that, The fermented yak ghee is prepared by the method described in any one of claims 6-8.

Images