A fermented milk, its preparation method and application

Abstract

This invention relates to the field of biotechnology, specifically disclosing a fermented milk, its preparation method, and its application. The method involves inoculating a fermentation substrate with *Bifidobacterium longum* BBMN68 and a starter culture, followed by temperature-dynamic frequency-modulated ultrasonic intervention during fermentation. Specifically, at the start of fermentation, a first stage of ultrasonic treatment is performed at 25±1℃, followed by warming to the fermentation temperature. When the pH of the fermentation system reaches 5.4-5.6, the temperature is lowered to 25±1℃ for a second stage of ultrasonic treatment, followed by warming to continue fermentation. When the pH of the fermentation system reaches 4.5-4.7, the temperature is lowered to 25±1℃ for a third stage of ultrasonic treatment, followed by warming to continue fermentation. The ultrasonic frequency increases and the power density decreases at each stage. This invention increases the number of *Bifidobacterium longum* BBMN68 and lactic acid bacteria, the content of beneficial metabolites, and improves the taste and texture of the product.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically, to a fermented milk, its preparation method, and its application. Background Technology

[0002] As a widely consumed carrier of probiotics, yogurt's functional attributes are receiving increasing attention. While traditional yogurt possesses basic nutritional value, it still falls short in terms of the accumulation of functional components. To improve yogurt quality, current technologies often rely on complex additives or multi-strain formulations, which not only deviates from the consumer trend of "clean labels" but also makes it difficult to achieve targeted and efficient enhancement of functional metabolites.

[0003] In recent years, ultrasound-assisted fermentation technology has attracted research attention due to its regulatory effect on microbial metabolism. Studies have shown that ultrasound treatment during the lag or logarithmic growth phases of lactic acid bacteria fermentation often has a positive impact, and the 20-100 kHz frequency range is frequently used to stimulate cell activity. However, the practical application of this technology faces significant challenges: First, the effectiveness of ultrasound is highly dependent on the characteristics of the bacterial strain, and key parameters such as frequency and power lack universal patterns, requiring tedious trial and error. More importantly, improper ultrasound treatment can easily cause physical damage to the cell membrane due to cavitation and mechanical stress, leading to irreparable pores on the surface and severely impairing cell viability and fermentation performance. This risk has led most existing studies to tend to use only a conservative single ultrasound treatment during fermentation, limiting the potential for improving the regulatory effect.

[0004] Furthermore, existing ultrasound-assisted technologies generally employ a limited range of applications, primarily using fixed frequencies or simple alternating modes. This fixed-parameter ultrasound mode cannot respond to the real-time state of the microbial community in the fermentation system, often resulting in singular effects (e.g., accelerating fermentation but easily damaging microbial activity) or instability. Some European patents mention improving the sensory characteristics and texture of yogurt through two ultrasound treatments at fixed frequencies and power, but these do not achieve a systematic enhancement of functional metabolites. All these methods fail to fundamentally solve the core problem of the compatibility between ultrasound parameters and the dynamic fermentation process—fixed ultrasound modes cannot respond to the real-time state of the microbial community in the fermentation system, thus the effects remain unsatisfactory. Moreover, for sensitive probiotics such as Bifidobacterium longum, improper ultrasound treatment can inhibit their metabolism, hindering the formation of target functional products (such as specific organic acids and fatty acids), and failing to actually improve product efficacy.

[0005] Furthermore, most related studies remain at the level of observing process phenomena, lacking systematic identification of key metabolites promoted by ultrasound, and also lacking effective verification of their claimed functions through reliable experimental models. As a result, the health efficacy claims of the products are not adequately supported and cannot truly guide the fermentation production of different strains.

[0006] Therefore, it is still necessary to conduct further research on the preparation methods of fermented milk products. Summary of the Invention

[0007] One of the objectives of this invention is to provide a method that can effectively enhance the synthesis of specific functional metabolites and the final efficacy of animal milk in the Bifidobacterium longum BBMN68 complex fermentation flora without relying on complex formulations.

[0008] This invention provides a method for preparing fermented milk, in which Bifidobacterium longum BBMN68 and a starter culture are inoculated into a fermentation substrate for fermentation, and temperature-dynamic frequency conversion ultrasound intervention is performed during the fermentation process. The temperature-dynamic frequency conversion ultrasound intervention method includes: At the start of formal fermentation, the temperature of the fermentation system is controlled at 25±1℃ before the first stage of ultrasonic treatment is performed. After ultrasonic treatment, the temperature is raised to the fermentation temperature for fermentation. The fermentation temperature is 41-43℃. When the pH value of the fermentation system reaches 5.4-5.6, the temperature of the fermentation system is lowered to 25±1℃ and then the second stage of ultrasonic treatment is carried out. After the ultrasonic treatment is completed, the temperature is raised to the fermentation temperature to continue fermentation. When the pH value of the fermentation system reaches 4.5-4.7, the temperature of the fermentation system is lowered to 25±1℃ and then the third stage of ultrasonic treatment is carried out. After the ultrasonic treatment, the temperature is raised to the fermentation temperature and then kept at a constant temperature until the end point. The conditions for the first stage of ultrasonic treatment are: The frequency is 18-26kHz, the power density is 42-50W / L, and the action time is 4-6min; The conditions for the second stage of ultrasonic treatment are as follows: Increase the frequency of the first-stage ultrasonic treatment by 8-12 kHz; decrease the power density of the first-stage ultrasonic treatment by 15-18 W / L; and reduce the treatment time by 4-6 min. The conditions for the third stage of ultrasonic treatment are as follows: Increase the frequency of the second-stage ultrasonic treatment by 8-12 kHz; decrease the power density of the second-stage ultrasonic treatment by 15-18 W / L; and reduce the treatment time by 1-3 min. All three stages of ultrasound treatment adopted an intermittent pulse mode, with a pulse period of 20-30s, an ultrasound time of 8-12s, and an interval of 12-18s; The preservation number of the Bifidobacterium longum BBMN68 is CGMCC No. 2265.

[0009] This invention reveals that existing ultrasound-assisted yogurt fermentation technologies mostly employ fixed single-frequency or alternating dual-frequency modes, which suffer from poor parameter adaptability and damage to probiotics. Prolonged application of single-frequency ultrasound easily leads to cavitation effect attenuation, making it difficult to adapt to the substrate viscosity at different fermentation stages; high-intensity ultrasound can damage sensitive probiotics such as *Bifidobacterium longum*, reducing their activity and affecting the content of metabolites and the texture of yogurt. Existing ultrasound-assisted fermentation methods cannot be effectively applied to improving the fermentation effect of *Bifidobacterium longum* BBMN68.

[0010] Specifically, studies have shown that sonication during the lag or logarithmic phases of bacterial fermentation has positive effects, but this invention found that sonication in both phases yielded limited improvement (Comparative Example 3). Other studies have indicated that periodic sonication every two hours during the latter half of the fermentation culture can stimulate cell growth and increase biomass, but this method also failed to significantly improve the fermentation effect of *Bifidobacterium longum* BBMN68 (Comparative Example 4). Furthermore, existing technologies have reported that sonication during or near the later stages of yogurt fermentation negatively impacts the integrity of the yogurt gel network structure and the smooth texture of the product. This further increases the technical challenge of achieving both optimal fermentation results for *Bifidobacterium longum* BBMN68 and ensuring the final product's texture.

[0011] Therefore, through continuous exploration, this invention has finally discovered that during the fermentation of the Bifidobacterium longum BBMN68 complex fermentation flora, by adjusting the ultrasonic intervention conditions according to the fermentation curve and the substrate state at different fermentation stages, and based on the dynamic progress of the fermentation stage, it is possible to significantly increase the number of Bifidobacterium longum BBMN68 bacteria, directionally regulate and enhance the production of anti-inflammatory and immune-related substances, and also optimize the product's texture and taste, thereby ultimately obtaining a product with better efficacy and ideal taste.

[0012] Specifically, this invention combines different ultrasonic conditions at three specific stages: the initial fermentation (located in the lag phase of the strain), the fermentation at a specific stage (located in the logarithmic growth phase of the lactic acid strain), and the fermentation nearing its end (located in the stable phase of the lactic acid strain). The final effect can be achieved through the synergistic effect of these three stages.

[0013] During the research of this invention, it was discovered that not only does the timing of ultrasonic treatment significantly affect the fermentation of the Bifidobacterium longum BBMN68 complex fermentation flora, but the specific conditions for ultrasonic treatment at each stage are also necessary to ensure the final ideal result. If the ultrasonic conditions at each stage are not properly designed and coordinated, the desired effect cannot be obtained.

[0014] In detail, the three specific ultrasonic treatments of this invention first use a low frequency and high power density method for ultrasonic treatment. Then, following the principle of gradually increasing frequency and generally decreasing power density in a stepwise manner, ultrasonic treatment is performed in subsequent specific stages. Especially in the third stage, during the strain stabilization period, a high-frequency combined with ultra-low power ultrasonic treatment method is specifically designed. This further promotes the increase of bacterial count without damaging the texture of the fermented milk; instead, it achieves gel network refinement and improved water retention. If the frequency and power density of each ultrasonic treatment are not properly matched (Comparative Examples 5 and 6), or if the variation pattern of the frequency and power density conditions of the three ultrasonic treatments is inappropriate (Comparative Examples 7 and 8), the desired effect cannot be obtained.

[0015] Furthermore, this invention specifically incorporates a cooling process during ultrasound administration to prevent the temperature rise caused by ultrasound from affecting cell activity, causing excessive protein denaturation, and resulting in a coarse gel network in the product. This approach leverages the physical promoting effect of ultrasound while avoiding thermal damage. Moreover, the timing of ultrasound administration in this invention is primarily determined by pH changes in the fermentation system. Compared to ultrasound administration based on a fixed fermentation time, this method effectively targets and positively regulates the synthesis of key functional metabolites in the fermentation system, achieving efficient and stable enrichment of beneficial substances. This avoids the problems of timing deviations, significant batch-to-batch fluctuations, and poor effect stability caused by the difficulty in precisely synchronizing the ultrasound intervention mode with the metabolic rhythms of different batches of fermentation microorganisms.

[0016] This invention, through the coordinated use of ultrasonic temperatures and conditions at each stage, promotes bacterial proliferation and the generation of anti-inflammatory and immunomodulatory metabolites while avoiding damage to the bacteria. It balances metabolic promotion with cell integrity, controlling the efficient fermentation cycle to within 7 hours, while simultaneously improving the texture of the final product and enhancing both efficacy and taste. Furthermore, the preparation method of this invention is simple and requires no complex additives, meeting the current market demand for clean-label health foods.

[0017] In this invention, Bifidobacterium longum BBMN68 ( Bifidobacterium longum The accession number is CGMCC No. 2265, the accession date is November 26, 2007, and the depositary institution is the China General Microbiological Culture Collection Center (CGMCC). The depositary location is the Institute of Microbiology, Chinese Academy of Sciences, Datun Road, Chaoyang District, Beijing, China. It is published in Chinese Patent CN101649303B.

[0018] Preferably, the conditions for the first stage of ultrasonic treatment are: frequency of 22kHz, power density of 46W / L, and treatment time of 5min; The conditions for the second stage of ultrasonic treatment are: frequency of 32kHz, power density of 30W / L, and treatment time of 5min; The conditions for the third stage of ultrasonic treatment are: frequency of 42kHz, power density of 14W / L, and treatment time of 2min. All three stages of ultrasound treatment used an intermittent pulse mode with a pulse period of 25s, including an ultrasound time of 10s and an interval of 15s.

[0019] In the method of this invention, the fermentation substrate comprises, by weight: 91-93 parts animal milk, 7.5-8 parts sweetener, 0.2-0.3 parts stabilizer, and 0-0.025 parts functional substance; the inoculum amount of both *Bifidobacterium longum* BBMN68 and the fermentation agent is 10. 6 -10 8 cfu / g; Preferably, the inoculum size of the starter culture is 1-2 × 10⁻⁶. 7 The inoculum size of *Bifidobacterium longum* BBMN68 was 2.5-3.5 × 10⁻⁶ cfu / g. 7 cfu / g; The stabilizer is one or more of the following: pectin, gellan gum, carrageenan, soybean polysaccharide, sodium carboxymethyl cellulose, xanthan gum, modified starch, and agar. The functional substances are one or more of the following: vitamin A, vitamin C, vitamin D, yeast-β-glucan, chitosan oligosaccharide, and kiwi powder; And / or, the fermenting agent includes *Lactobacillus delbrueckii* subsp. *bulgaricus* and *Streptococcus salivarius* subsp. *thermophilus*.

[0020] In this invention, a conventional commercially available starter containing Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus salivarius subsp. thermophilus can be used, with the ratio of viable bacteria between the two generally being 1:(1-1000), preferably 1:(10-100).

[0021] Preferably, when the fermentation substrate includes functional substances, the amount of functional substances used is 0.015-0.025 parts.

[0022] In this invention, animal milk refers to the milk of mammals, such as cow's milk, sheep's milk, horse's milk, deer's milk, camel's milk, donkey's milk, etc.

[0023] In this invention, the sweetener is preferably white granulated sugar.

[0024] The present invention can be carried out using methods known in the art for the preparation of fermentation substrate.

[0025] In one specific embodiment, the present invention adds each component to preheated animal milk (e.g., 45°C) and mixes it thoroughly (e.g., stirring for 15 minutes); the mixed material is then homogenized at a temperature of 60°C, with the total homogenization pressure controlled at 140-160 bar (preferably 150-160 bar) and the secondary pressure at 30-50 bar (preferably 30-35 bar); the homogenized material is then sterilized (e.g., kept at 93-96°C for 300-350 seconds, preferably at 93-95°C for 300-320 seconds), and finally cooled to obtain the fermentation substrate.

[0026] In the method of the present invention, when heating is involved, the heating rate is 5 ± 0.5 °C / min; when cooling is involved, the cooling rate is 3 ± 0.5 °C / min.

[0027] In the method of the present invention, the final pH value of the fermentation is 4.25-4.35 (preferably 4.3-4.35).

[0028] In the method of the present invention, the temperature of the fermentation substrate is 41-43℃; after the Bifidobacterium longum BBMN68 and the fermentation agent are inoculated into the fermentation substrate, the substrate is first stirred and activated for 8-12 minutes (preferably 8-10 minutes) before the formal fermentation begins, and then the temperature of the fermentation system is lowered to 25±1℃ before the first stage of ultrasonic treatment is performed.

[0029] In this invention, the fermentation substrate is preferably mixed and sterilized, then cooled to the fermentation temperature (41-43℃) before inoculation with the microbial agent. After inoculation, the agent is stirred to ensure full dispersion and activation, which facilitates subsequent fermentation. At the start of formal fermentation, the temperature of the fermentation system is higher than the temperature required for the first stage of ultrasonic treatment. Therefore, cooling (temperature control of the fermentation system) is necessary before commencing the first stage of ultrasonic treatment.

[0030] Preferably, the stirring speed is 30-40 rpm, more preferably 30-35 rpm.

[0031] The method of the present invention further includes demulsification and sterilization steps after the fermentation is completed.

[0032] Demulsification can be performed using methods known in the art, such as stirring continuously at 20-25 rpm for 1 minute.

[0033] In the method of the present invention, the sterilization temperature is 72-75°C and the time is 50-70 seconds (preferably 60-65 seconds).

[0034] The present invention also provides a fermented milk prepared by the above method.

[0035] The present invention also provides the application of the above method in any of the following aspects: (1) Increase the content of γ-aminobutyric acid in fermented milk; (2) Increase the content of indole-3-lactic acid in fermented milk; (3) Increase the content of 2,3-butanedione in fermented milk; (4) Increase the content of 2-hydroxy-3-methylbutyric acid in fermented milk; (5) Increase the content of micranthin in fermented milk; (6) Increase the content of phloric acid in fermented milk; (7) Enhance the immune regulation function of fermented milk.

[0036] The beneficial effects of this invention are at least as follows: This invention utilizes a specific temperature-dynamic frequency-conversion ultrasound intervention method to increase the bacterial count of Bifidobacterium longum (BBMN68) and lactic acid bacteria in the prepared fermented milk, as well as the content of anti-inflammatory and antioxidant metabolites and the texture of the product, thus balancing the product's taste and immune-enhancing effects. Furthermore, the preparation method of this invention is simple, efficient, and uses clean ingredients. Attached Figure Description

[0037] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0038] Figure 1 The content of γ-aminobutyric acid in fermentation products under different treatment conditions.

[0039] Figure 2 The content of indole-3-lactic acid in fermentation products under different treatment conditions.

[0040] Figure 3 The content of 2,3-butanedione in the fermentation products under different treatment conditions.

[0041] Figure 4 The content of 2-hydroxy-3-methylbutyric acid in fermentation products under different treatment conditions.

[0042] Figure 5 The content of medaconic acid in fermentation products under different treatment conditions.

[0043] Figure 6 The content of phlorenic acid in fermentation products under different treatment conditions.

[0044] Figure 7The results show the fluorescence intensity (pixels) of zebrafish T cells after processing fermented milk samples in this example.

[0045] Figure 8 The number of residual fluorescent microspheres from phagocytes after processing the fermented milk sample in this example.

[0046] Figure 9 This is a typical image showing the number of residual fluorescent microspheres in zebrafish after processing fermented milk samples in this example. The red fluorescent particles are fluorescent microspheres.

[0047] In each diagram (if any), represent p <0.05, represent p <0.01, represent p <0.001. Detailed Implementation

[0048] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0049] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples can be obtained commercially or prepared according to conventional methods in the art. The fermentation agent used in the specific embodiments of this invention was provided by Beijing Ketuo Hengtong Biotechnology Co., Ltd., with the fermentation agent number PYS-V141B. The fermentation agent's bacterial composition consists of *Lactobacillus delbrueckii* subsp. bulgaricus and *Streptococcus salivarius* subsp. thermophilus. The pectin has an esterification degree of 23% and an amidation degree of 24%. Yeast-β-glucan raw material was purchased from Angel Yeast Co., Ltd. The compound vitamins were purchased from Shanghai Licheng Nutrition Technology Co., Ltd., product number: Lidewei B-2603159-Vit.

[0050] Example 1: This embodiment provides a method for preparing fermented milk, as detailed below: (1) Raw material selection and matrix pretreatment: Probiotics: Bifidobacterium longum BBMN68 was selected, with a live bacteria count of 8×10⁻⁶. 10 CFU / g; Fermentation agent: viable count 3.6 × 10⁻⁶ 6CFU / g; Fermentation substrate: 91.685 parts raw milk, 8 parts white sugar, 0.3 parts pectin, and 0.015 parts yeast-β-glucan.

[0051] Preheat raw milk to 45°C, add granulated sugar, pectin, and yeast-β-glucan, and mix for 15 minutes. Heat the mixture to 60°C and homogenize it, with the total homogenization pressure controlled at 150 bar and the secondary pressure at 35 bar. Then, heat the homogenized material to 95°C and sterilize it by holding it at that temperature for 300 seconds. Finally, cool it to 42°C to obtain the fermentation substrate.

[0052] (2) Co-fermentation process: The above-mentioned Bifidobacterium longum BBMN68 and starter culture were inoculated into the fermentation substrate. The inoculum size of lactic acid bacteria (starter culture) was 1.0 × 10⁻⁶. 7 cfu / mL, Bifidobacterium (BBMN68) inoculum amount was 2.8 × 10⁻⁶. 7 cfu / mL; turn on the stirrer (30 rpm), and continue stirring for 10 minutes after inoculation to ensure the inoculum is fully dispersed and activated. Stop stirring and start recording the fermentation time.

[0053] (3) Temperature-dynamic frequency conversion ultrasound intervention process: After stopping the stirring, the temperature was lowered to 25°C at 3°C / min for the first stage of ultrasonic treatment. After the ultrasonic treatment, the temperature was raised to 42°C at 5°C / min for fermentation.

[0054] The conditions for the first stage of ultrasonic treatment are: ultrasonic frequency of 22kHz, ultrasonic power density of 46W / L, treatment time of 5min, and pulse period of 25s (working time of 10 seconds and stopping time of 15 seconds).

[0055] The pH value of the fermentation system was monitored in real time using the AMS Alliance iCinac acidification monitoring system. When the pH dropped to 5.5, the temperature was lowered to 25°C at a rate of 3°C / min for the second stage of ultrasonic treatment. After ultrasonic treatment, the temperature was raised to 42°C at a rate of 5°C / min to continue fermentation.

[0056] The conditions for the second stage of ultrasonic treatment are: ultrasonic frequency of 32kHz, ultrasonic power density of 30W / L, treatment time of 5min, and pulse period of 25s (working time of 10 seconds and stopping time of 15 seconds).

[0057] When the pH continued to drop to 4.6, the temperature was lowered to 25°C at 3°C / min for the third stage of ultrasonic treatment. After the ultrasonic treatment, the temperature was raised to 42°C at 5°C / min and fermented at a constant temperature until the end.

[0058] The conditions for the third stage of ultrasonic treatment are: ultrasonic frequency of 42kHz, ultrasonic power density of 14W / L, treatment time of 2min, and pulse period of 25s (working time of 10 seconds and stopping time of 15 seconds).

[0059] (4) Demulsification: After fermentation, when the pH value of the system reaches 4.35, start stirring (25 rpm) and continue stirring for 1 minute to break up the curd.

[0060] (5) Secondary sterilization treatment: After demulsification, the milk is sterilized at 75°C for 60 seconds and then cooled to 20°C to obtain room temperature fermented milk.

[0061] Example 2: This embodiment provides a method for preparing fermented milk, as detailed below: (1) Raw material selection and matrix pretreatment: Probiotics: Bifidobacterium longum BBMN68 was selected, with a live bacteria count of 8×10⁻⁶. 10 CFU / g; Fermentation agent: viable count 3.6 × 10⁻⁶ 6 CFU / g; Fermentation substrate: 91.775 parts raw milk, 8 parts white sugar, 0.2 parts pectin, 0.015 parts yeast-β-glucan, and 0.01 parts compound vitamins (vitamin A and vitamin D in a mass ratio of 35:1).

[0062] Preheat raw milk to 45°C, add granulated sugar, pectin, yeast-β-glucan, and multivitamins, and mix for 15 minutes. Heat the mixture to 60°C and homogenize it, with the total homogenization pressure controlled at 160 bar and the secondary pressure at 30 bar. Then, heat the homogenized material to 93°C and sterilize it by holding it at that temperature for 320 seconds. Finally, cool it to 42°C to obtain the fermentation substrate.

[0063] (2) Co-fermentation process: The above-mentioned Bifidobacterium longum BBMN68 and starter culture were inoculated into the fermentation substrate, with an inoculum size of 2.0 × 10⁻⁶. 7 cfu / mL, Bifidobacterium inoculum amount was 3.5 × 10⁻⁶. 7 cfu / mL; turn on the stirrer (35 rpm), and continue stirring for 8 minutes after inoculation to ensure the inoculum is fully dispersed and activated. Stop stirring and start recording the fermentation time.

[0064] (3) Temperature-dynamic frequency conversion ultrasound intervention process: After stopping the stirring, the temperature was lowered to 25°C at 3°C / min for the first stage of ultrasonic treatment. After the ultrasonic treatment, the temperature was raised to 42°C at 5°C / min for fermentation.

[0065] The conditions for the first stage of ultrasonic treatment are: ultrasonic frequency of 18kHz, ultrasonic power density of 42W / L, treatment time of 4min, and pulse period of 26s (8 seconds of operation and 18 seconds of rest).

[0066] The pH value of the fermentation system was monitored in real time using the AMS Alliance iCinac acidification monitoring system. When the pH dropped to 5.5, the temperature was lowered to 25°C at a rate of 3°C / min for the second stage of ultrasonic treatment. After ultrasonic treatment, the temperature was raised to 42°C at a rate of 5°C / min to continue fermentation.

[0067] The conditions for the second stage of ultrasonic treatment are: ultrasonic frequency of 26kHz, ultrasonic power density of 27W / L, treatment time of 4min, and pulse period of 26s (8 seconds of operation and 18 seconds of rest).

[0068] When the pH continued to drop to 4.6, the temperature was lowered to 25°C at 3°C / min for the third stage of ultrasonic treatment. After the ultrasonic treatment, the temperature was raised to 42°C at 5°C / min and fermented at a constant temperature until the end.

[0069] The conditions for the third stage of ultrasonic treatment are: ultrasonic frequency of 34kHz, ultrasonic power density of 12W / L, action time of 1min, and pulse period of 26s (8 seconds of operation and 18 seconds of rest).

[0070] (4) Demulsification: After fermentation, when the pH value of the system reaches 4.30, start stirring (20 rpm) and continue stirring for 1 minute to break up the curd.

[0071] (5) Secondary sterilization treatment: After demulsification, the milk is sterilized at 72°C for 65 seconds and then cooled to 20°C to obtain room temperature fermented milk.

[0072] Example 3: This embodiment provides a method for preparing fermented milk, as detailed below: (1) Raw material selection and matrix pretreatment: Probiotics: Bifidobacterium longum BBMN68 was selected, with a live bacteria count of 8×10⁻⁶. 10 CFU / g; Fermentation agent: viable count 3.6 × 10⁻⁶ 6 CFU / g; Fermentation substrate: 92.3 parts raw milk, 7.5 parts white sugar, and 0.2 parts pectin.

[0073] Preheat raw milk to 45°C, add granulated sugar and pectin and mix for 15 minutes; heat the mixture to 60°C and homogenize it, with the total homogenization pressure controlled at 150 bar and the secondary pressure at 30 bar; then heat the homogenized material to 95°C and keep it at that temperature for 300 seconds to sterilize it, and finally cool it to 42°C to obtain the fermentation substrate.

[0074] (2) Co-fermentation process: The above-mentioned Bifidobacterium longum BBMN68 and starter culture were inoculated into the fermentation substrate, with an inoculum size of 1.0 × 10⁻⁶. 7 cfu / mL, Bifidobacterium inoculum amount was 2.8 × 10⁻⁶ 7 cfu / mL; turn on the stirrer (30 rpm), and continue stirring for 10 minutes after inoculation to ensure the inoculum is fully dispersed and activated. Stop stirring and start recording the fermentation time.

[0075] (3) Temperature-dynamic frequency conversion ultrasound intervention process: After stopping the stirring, the temperature was lowered to 25°C at 3°C / min for the first stage of ultrasonic treatment. After the ultrasonic treatment, the temperature was raised to 42°C at 5°C / min for fermentation.

[0076] The conditions for the first stage of ultrasonic treatment are: ultrasonic frequency of 26kHz, ultrasonic power density of 50W / L, treatment time of 6min, and pulse period of 24s (working time of 12 seconds and stopping time of 12 seconds).

[0077] The pH value of the fermentation system was monitored in real time using the AMS Alliance iCinac acidification monitoring system. When the pH dropped to 5.5, the temperature was lowered to 25°C at a rate of 3°C / min for the second stage of ultrasonic treatment. After ultrasonic treatment, the temperature was raised to 42°C at a rate of 5°C / min to continue fermentation.

[0078] The conditions for the second stage of ultrasonic treatment are: ultrasonic frequency of 38kHz, ultrasonic power density of 32W / L, treatment time of 6min, and pulse period of 24s (working time of 12 seconds and stopping time of 12 seconds).

[0079] When the pH continued to drop to 4.6, the temperature was lowered to 25°C at 3°C / min for the third stage of ultrasonic treatment. After the ultrasonic treatment, the temperature was raised to 42°C at 5°C / min and fermented at a constant temperature until the end.

[0080] The conditions for the third stage of ultrasonic treatment are: ultrasonic frequency of 50kHz, ultrasonic power density of 14W / L, action time of 3min, and pulse period of 24s (working time of 12 seconds and stopping time of 12 seconds).

[0081] (4) Demulsification: After fermentation, when the pH value of the system reaches 4.35, start stirring (20 rpm) and continue stirring for 1 minute to break up the curd.

[0082] (5) Secondary sterilization treatment: After demulsification, the milk is sterilized at 72°C for 65 seconds and then cooled to 20°C to obtain room temperature fermented milk.

[0083] Comparative Example 1 (No Ultrasound Group): This comparative example provides a method for preparing fermented milk, which is basically the same as the method in Example 1. The only difference is that after step (2), constant temperature fermentation is carried out directly at 42°C without temperature-dynamic frequency conversion ultrasonic intervention.

[0084] Comparative Example 2 (only the second stage of ultrasound treatment was performed according to the teachings of the existing technology): This comparative example provides a method for preparing fermented milk, which is basically the same as the method in Example 1, except that in step (3), ultrasonic treatment is applied only once when the pH value of the fermentation broth drops to 5.5. Ultrasonic parameters: frequency 20kHz, power density 65W / L, treatment time 2min, continuous ultrasonic treatment.

[0085] Comparative Example 3 (only the first and second stages of ultrasound treatment were performed): This comparative example provides a method for preparing fermented milk, which is basically the same as the method in Example 1. The only difference is that in step (3), only the first and second stages of ultrasonic treatment are performed, and after the second stage of ultrasonic treatment, the fermentation is carried out directly at a constant temperature until the end point.

[0086] Comparative Example 4 (periodic sonication was performed every two hours during the latter half of the culture): This comparative example provides a method for preparing fermented milk, which is basically the same as the method in Example 1. The only difference is that in step (3), ultrasonic treatment (ultrasonic frequency 20kHz, ultrasonic power 3W / L, ultrasonic time 5min) is performed every two hours only when the pH drops to 4.9 until the fermentation ends.

[0087] Comparative Example 5 (Changing the ultrasound conditions in the second stage): This comparative example provides a method for preparing fermented milk, which is basically the same as the method in Example 1. The only difference is that in step (3), the ultrasonic conditions of the second stage are changed to: ultrasonic frequency of 40kHz, ultrasonic power density of 22W / L, action time of 5min, and pulse period of 25s (working for 10 seconds and stopping for 15 seconds).

[0088] Comparative Example 6 (Changing the ultrasound conditions in the third stage): This comparative example provides a method for preparing fermented milk, which is basically the same as the method in Example 1. The only difference is that in step (3), the ultrasonic conditions of the third stage are changed to: ultrasonic frequency of 42kHz, ultrasonic power density of 10W / L, action time of 4min, and pulse period of 25s (working for 10 seconds and stopping for 15 seconds).

[0089] Comparative Example 7 (using the same ultrasound conditions in all three stages): This comparative example provides a method for preparing fermented milk, which is basically the same as the method in Example 1, except that in step (3), the ultrasonic conditions at each stage are set as follows: The ultrasonic frequency is 42kHz, the ultrasonic power density is 14W / L, the action time is 2min, and the pulse period is 25s (working for 10 seconds and stopping for 15 seconds).

[0090] Comparative Example 8 (Changing the pattern of ultrasound conditions in three stages): This comparative example provides a method for preparing fermented milk, which is basically the same as the method in Example 1, except that in step (3), the ultrasonic conditions at each stage are changed as follows: Phase 1: Ultrasonic frequency 42kHz, ultrasonic power density 14W / L, action time 2min, pulse period 25s (working time 10 seconds, vibration stop time 15 seconds).

[0091] Second stage: The ultrasonic frequency is 32kHz, the ultrasonic power density is 30W / L, the action time is 5min, and the pulse period is 25s (working for 10 seconds and stopping for 15 seconds).

[0092] The third stage: the ultrasonic frequency is 42kHz, the ultrasonic power density is 14W / L, the action time is 2min, and the pulse period is 25s (working for 10 seconds and stopping for 15 seconds).

[0093] Experimental Example 1 This experiment tested the basic fermentation performance indicators of the bacterial count and the final product during the preparation process of each embodiment and comparative example. The evaluation indicators included: viable count (lactic acid bacteria count, bifidobacteria count), textural properties (viscosity), stability (particle size, clarification index), and sensory score. The results are shown in Table 1.

[0094] For viscosity testing methods, please refer to: Hu Jiajie, Wu Xiuying, Mu Zhishen, Guo Yan, Wang Bin, Xue Jianbin, Yin Yongzhi. Effects of thickeners on sensory and rheological properties of stirred yogurt [J]. China Food Additives, 2017, (10): 135-140. Test temperature: 25℃.

[0095] For particle size testing methods, please refer to: Hao Yanping, Liu Zhenmin, Zhang Huanchang, et al. Effect of Lactobacillus paracasei on the storage stability of fermented milk [J]. Food Industry Technology, 2026, 47(01):381-391.

[0096] For the clarification index test method, see: Feng Wei, Qin Yu, Huang Yin, et al. Analysis of the stability of coconut jelly acidic milk beverage based on LUMiSizer [J]. Food and Fermentation Industries, 2021, 47(23).

[0097] For the sensory evaluation method, please refer to: Hu Jiajie, Wu Xiuying, Mu Zhishen, Guo Yan, Wang Bin, Xue Jianbin, Yin Yongzhi. Effects of thickeners on sensory and rheological properties of stirred yogurt [J]. China Food Additives, 2017, (10): 135-140. The product was evaluated based on its appearance, flavor, smoothness, and consistency.

[0098] The determination of the number of lactic acid bacteria and bifidobacteria shall be in accordance with GB4789.35-2023 "National Food Safety Standard for Microbiological Examination of Food - Lactic Acid Bacteria Examination".

[0099] Table 1: Results of various indicators in fermented milk products obtained from the examples and comparative examples

[0100] The experimental results show that the fermented milk prepared in the embodiments of the present invention can simultaneously achieve efficient proliferation of probiotics and obtain better texture, stability and sensory evaluation.

[0101] This invention, through a combination of "staged pH-triggered ultrasound + precise temperature and ultrasound condition control", significantly increases the number of lactic acid bacteria and probiotic BBMN68 live bacteria in a shorter fermentation time. The fermented milk gel is stable with no whey separation, has excellent sensory appeal, and balances product efficiency and quality. For the first time, it achieves a dual synergistic gain of probiotic proliferation and gel structure optimization in fermented milk fermentation.

[0102] Experiment Example 2 This experiment employed non-targeted metabolomics technology to detect metabolites in the fermented milk prepared in each example (S1-S3) and the comparative examples 1, 3, and 5-8 (D1, D3, D5-D8) that performed reasonably well in Experimental Example 1. The details are as follows: Metabolomics testing: The prepared room-temperature fermented milk samples were quickly placed in an ultra-low temperature freezer at -80℃ for subsequent metabolomics detection and analysis.

[0103] ① Metabolite extraction: Take 100 μL of the sample after thawing in an ice-water bath, add 400 μL of extraction solution (methanol:acetonitrile = 1:1 (v / v), containing isotope internal standard), vortex mix for 30 s; sonicate in an ice-water bath for 10 min, and let stand at -40℃ for 1 h; centrifuge the sample at 4℃, 12000 rpm (centrifugal force 13800 (×g), radius 8.6 cm) for 15 min; take 100 μL of supernatant into a sample vial; take an equal amount of supernatant from all samples and mix them to form a QC sample for instrumental analysis.

[0104] ② Analytical Separation: For polar metabolites, a Vanquish (Thermo Fisher Scientific) ultra-high performance liquid chromatograph was used. The target compounds were separated by chromatographic separation using a Waters ACQUITY UPLC BEH Amide (2.1 mm × 50 mm, 1.7 μm) column. Gradient elution mode was employed. The mobile phase consisted of two phases, A and B. Phase A was aqueous containing 25 mmol / L ammonium acetate and 25 mmol / L ammonia, while phase B was acetonitrile. Sample tray temperature: 4℃; injection volume: 2 μL; flow rate: constant at 0.5 mL / min; column temperature: 30℃. The gradient program is as follows: 0-0.25 min: mobile phase maintains 5% A / 95% B; 0.25-3.50 min: phase A linearly increases from 5% to 35% (phase B linearly decreases from 95% to 65%); 3.50-4.00 min: phase A linearly increases from 35% to 60% (phase B linearly decreases from 65% to 40%); 4.00-4.50 min: mobile phase maintains 60% A / 40% B; 4.50-4.55 min: phase A linearly decreases from 60% to 5% (phase B linearly increases from 40% to 95%); 4.55-6.00 min: mobile phase maintains 5% A / 95% B for system equilibration.

[0105] ③ Data processing after logging off the machine: The raw data was converted into mzXML format using ProteoWizard software (V3.0.24054), and then metabolite identification was performed using R package 7 with BiotreeDB (V3.0) as the database. Finally, visualization analysis was performed.

[0106] Specifically, relative quantification and normalization are performed based on the ion peak area of ​​the metabolites. In the examples, the differentially expressed metabolites are mainly upregulated, and the main categories include amino acids and their derivatives, lipids, carbohydrates, organic acids and phenylpropanoids.

[0107] Specific results show that, after processing with the present invention, the amino acid differential metabolites upregulated in the examples mainly include leucine, methionine, phenylalanine, pantothenic acid, threonine, L-homoserine, creatine anhydride, serine, γ-aminobutyric acid (GABA), and indole-3-lactic acid (ILA).

[0108] GABA content: The detection results of GABA in the fermentation products of each example and comparative example are shown in the figure. Figure 1 One-way ANOVA showed highly significant differences in GABA content among the groups. p =1.32×10 -21 The results indicate that different treatment methods have a significant regulatory effect on GABA synthesis. In the example groups (S1-S3), Example 2 (S2) had the highest GABA content, followed by Examples 1 (S1) and 3 (S3). Compared with Comparative Example 1 (D1), the GABA content of Examples 2, 1, and 3 was approximately 7 times, 5.5 times, and 5.1 times that of D1, respectively. These results demonstrate that the ultrasonic treatment scheme of the present invention can significantly upregulate GABA accumulation. GABA is a naturally occurring bioactive compound produced by microbial fermentation, possessing physiological activities such as sedation, anti-anxiety, sleep improvement, blood pressure regulation, and anti-inflammation. Previous studies have shown that GABA can reduce the release of pro-inflammatory factors such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) by binding to GABA receptors on immune cells (such as T cells and macrophages), while simultaneously promoting the production of anti-inflammatory factors such as interleukin-10 (IL-10). This mechanism has been verified in in vitro and in vivo studies of GABA-enriched fermented foods.

[0109] ILA content: Specific test results of ILA content in fermentation products under different treatment conditions in each example and comparative example are shown in [the original text]. Figure 2 One-way ANOVA showed highly significant differences in ILA levels among the groups. p =1.76×10 -21The results indicate that different treatment methods have a significant regulatory effect on the synthesis of this metabolite. In the example groups (S1-S3), the ILA content in Example 1 (S1) was the highest, followed by Example 2 (S2) and Example 3 (S3). Compared with Comparative Example 1 (D1), the ILA expression level in Example 1 was approximately 11 times higher, and the ILA expression levels in Examples 2 and 3 were approximately 7.7 times higher, indicating that the ultrasound treatment scheme of the present invention can significantly upregulate the accumulation of this metabolite. In the comparative examples, the ILA content in Comparative Example 1 (D1) was extremely low, and the ILA content in the other comparative examples was also significantly lower than that in the example groups. ILA is a characteristic product of tryptophan metabolism by microorganisms such as lactic acid bacteria. As an endogenous ligand of the aryl hydrocarbon receptor (AhR), it can upregulate the expression of tight junction proteins by activating the AhR signaling pathway, thereby enhancing the intestinal epithelial barrier function. At the same time, ILA can inhibit the nuclear factor κB (NF-κB)-mediated inflammatory response and reduce the production of pro-inflammatory cytokines such as TNF-α and IL-6, thereby significantly alleviating the inflammatory response of intestinal epithelial cells. Furthermore, ILA can regulate the intestinal flora structure, increase the relative abundance of beneficial bacteria such as Bifidobacterium and Faecalibacterium, and promote the production of short-chain fatty acids (especially butyric acid), forming a virtuous cycle of the intestinal microecology. ILA has been widely detected in fermented dairy products, and its content is closely related to the metabolic characteristics of the fermentation strains, providing a theoretical basis for the product application of the present invention.

[0110] Metabolomics analysis also showed that the specific ultrasound intervention strategy used in this invention can significantly regulate the generation of various differential metabolites in the fermentation system, especially enriching a series of short-chain and medium-chain fatty acids and their derivatives, including glyceric acid, 4-methylvaleric acid, hexanoic acid, caprylic acid, myristic acid, 2,3-butanedione, 2-hydroxy-3-methylbutyric acid and mesocarboxylic acid. The above metabolites are important flavor and functional components in fermented foods.

[0111] 2,3-Butanedione content: The detection results of 2,3-butanedione in the fermentation products under different treatment conditions in each example and comparative example are shown in [the table below]. Figure 3 After treatment with the ultrasonic method of this invention, the content of 2,3-butanedione in the example groups was significantly higher than that in the comparative example. Specifically, Example 3 had the highest content, followed by Examples 1 and 2, while Comparative Example 1 had the lowest. Compared to Comparative Example 1, the 2,3-butanedione content in Examples 3, 1, and 2 was approximately 9 times, 8.6 times, and 8 times that of Comparative Example 1, respectively, indicating that ultrasonic treatment can significantly promote the accumulation of 2,3-butanedione. 2,3-Butanedione, also known as diacetyl, is a crucial flavor metabolite in yogurt, mainly synthesized by lactic acid bacteria and other microbial communities through the citric acid metabolic pathway. It is a key compound that imparts the typical creamy aroma and flavor to dairy products. In addition to its sensory contribution, emerging research indicates that diacetyl also possesses potential physiological activities such as antioxidant and antifungal activity.

[0112] 2-Hydroxy-3-methylbutyric acid content: The detection results of 2-hydroxy-3-methylbutyric acid in the fermentation products under different treatment conditions in each example and comparative example are shown in the figure. Figure 4 Its expression level showed highly significant differences among the groups (ANOVA). p =5.29×10 -17 Previous studies have confirmed that this metabolite can promote intestinal epithelial cell proliferation by upregulating the MYC signaling pathway, thereby playing an important role in the repair of intestinal mucosal damage and the maintenance of intestinal barrier function. Furthermore, given its structurally branched hydroxy acid nature, combined with the existing general understanding of the anti-inflammatory function of short-chain fatty acids, this metabolite may also participate in immune regulation through related signaling pathways (such as HDAC inhibition or GPRs activation), possessing potential anti-inflammatory activity. Increased expression levels will further enhance the intestinal health function of fermented products. The ultrasound intervention mode used in this invention (Examples 1-3, i.e., S1, S2, S3) significantly improved the synthesis efficiency of 2-hydroxy-3-methylbutyric acid. The overall expression level of this metabolite in all ultrasound intervention groups was significantly higher than that in the control group without ultrasound intervention (Comparative Example 1, D1), with the highest expression level in Example 1, followed by Examples 2 and 3. Specifically, the expression level of 2-hydroxy-3-methylbutyric acid in Example 1 was approximately 8.5 times that of Comparative Example 1, and the expression levels in Examples 2 and 3 were also more than 6.8 times that of Comparative Example 1. Although the expression levels of the other comparative samples increased, none of them reached the level of any embodiment of the present invention.

[0113] Mesoconic acid content: The detection results of mesoconic acid in the fermentation products under different treatment conditions in each example and comparative example are shown in [the table below]. Figure 5 One-way ANOVA showed highly significant differences in canonical expression levels among the groups (p = 1.24 × 10⁻⁶). -18 Compared with Comparative Example 1 (D1) without ultrasound intervention, the expression level of medconic acid was significantly increased in all ultrasound-treated example groups (Examples 1-3). Example 2 showed the highest expression level, followed by Examples 1 and 3. Specifically, the expression level of medconic acid in Example 2 was approximately 5.4 times that of Comparative Example 1, while the expression levels in Examples 1 and 3 were more than 4 times that of Comparative Example 1. Although the expression levels in the other comparative examples were increased, none reached the level of any of the embodiments of this invention. These results indicate that ultrasound treatment of this invention can significantly promote the accumulation of medconic acid. Medconic acid is a functional metabolite with significant anti-inflammatory and anti-aging potential. Previous studies have shown that medconic acid can regulate gut microbiota, alleviate oxidative stress, and maintain the integrity of the intestinal barrier. Mechanistically, medconic acid can synergistically delay aging by regulating inflammatory responses, inhibiting the NLRP3 inflammasome-mediated pyroptosis pathway, and activating the Nrf2 antioxidant signaling pathway. These findings provide a theoretical basis for the functional applications corresponding to the upregulation of medconic acid in this invention.

[0114] This invention significantly modulates the expression of various phenylpropanoid metabolites, including 3-dehydroquinic acid, 2,6-dihydroxybenzoic acid, 2,3-dihydroxybenzoic acid, benzoic acid, and phloroglucinic acid (deaminotyrosine), through specific ultrasonic intervention in the fermentation system. The upregulation effect on phloroglucinic acid was particularly pronounced. The results of one-way ANOVA are shown below. Figure 6 The results showed that the differences in root bark acid content among the experimental groups were statistically highly significant. p The result (<0.01) confirms the clear technical effectiveness of the ultrasound intervention in regulating this metabolite. Compared with Comparative Example 1, all embodiments of the present invention achieved a significant increase in phloric acid expression: Example 1 had the highest phloric acid content, approximately 5.8 times that of Comparative Example 1; the phloric acid content in Examples 2 and 3 was more than 4 times that of Comparative Example 1, indicating that the ultrasound intervention scheme of the present invention can stably and efficiently promote the accumulation of this functional metabolite. Phloric acid, as a key product of intestinal microorganisms or fermentation systems metabolizing dietary flavonoids, has had its physiological functions confirmed by multiple studies: this metabolite can exert immunomodulatory and anti-inflammatory effects by enhancing macrophage phagocytosis and bactericidal activity and inhibiting the release of pro-inflammatory factor IL-6; at the same time, it can enhance the body's antiviral defense capabilities by activating the type I interferon signaling pathway, and improve cellular stress tolerance by scavenging free radicals, regulating the insulin / IGF-1 signaling pathway and autophagy. The significant upregulation of phloric acid content in the fermented milk of the present invention directly confirms the core advantages of the ultrasound intervention scheme of the present invention in enhancing the anti-inflammatory, antioxidant, and immunomodulatory functional properties of fermented dairy products. In contrast, the overall levels of phloretic acid in each of the parallel samples were significantly lower than those in the example.

[0115] Metabolomics analysis showed that the embodiments of this invention significantly enriched dipeptides and organic acid metabolites. The enriched dipeptides included glycylproline, prolyl-proline (Pro-Pro), and phenylalanyl-proline, etc., these small molecule peptides are bioactive peptides released from milk proteins by lactic acid bacteria proteases. The enriched organic acids included pyruvic acid, fumaric acid, and decanoic acid. Pyruvic acid is a key intermediate product of glycolysis and an important precursor of diacetyl (a core component of butter aroma). Decanoic acid (C10:0), as a medium-chain fatty acid, can enhance the creaminess and improve the flavor and texture of fermented milk by increasing its content. Furthermore, medium-chain fatty acids have the potential to regulate the gut microbiota, and decanoic acid may act as a functional component, synergistically contributing to gut health benefits with other metabolites.

[0116] In summary, the innovative "dynamic variable frequency ultrasound-assisted fermentation process" of this invention, combined with the specific "Bifidobacterium BBMN68" complex fermentation microbial community, not only increases the yield of conventional metabolites, but also can directionally induce and enrich a series of specific metabolites with clear anti-inflammatory, antioxidant, intestinal barrier enhancement, and immune regulation functions, such as γ-aminobutyric acid, indole-3-lactic acid, 2,3-butanedione, 2-hydroxy-3-methylbutyric acid, medronic acid, and phloroglucinogenic acid.

[0117] Experiment Example 3: Zebrafish Experiment to Verify T Cell and Phagocyte Function This experiment verified the immunomodulatory activity of the fermented milk prepared in Examples 1, 2 and 3 by constructing an immunodeficiency model. The detection indicators included T cell fluorescence intensity and the number of residual fluorescent microspheres of phagocytes.

[0118] Zebrafish functional verification experimental method: ① Laboratory animals Zebrafish were raised in aquarium water at 28℃ (water quality: 200mg of instant sea salt added per 1L of reverse osmosis water, conductivity 450-550μS / cm; pH 6.5-8.5; hardness 50-100mg / L CaCO3), laboratory animal use license number: SYXK (Zhejiang) 2022-0004, husbandry and management met the requirements of international AAALAC certification (certification number: 001458), IACUC ethics review number: IACUC-2025-202510160015-01.

[0119] ② Instruments, consumables and reagents Dissecting microscope (SZX7, OLYMPUS, Japan); CCD camera (VertA1, Shanghai Tusen Vision Technology Co., Ltd., China); motorized focusing continuous zoom fluorescence microscope (AZ100, Nikon, Japan); precision electronic balance (CP214, OHAUS, USA); microinjection apparatus (IM-300, Narishige, Japan); needle puller (PC-10, Narishige, Japan); 6-well plate (Zhejiang Beilanbo Biotechnology Co., Ltd., China).

[0120] Methylcellulose (batch number C2004046, Shanghai Aladdin Biochemical Technology Co., Ltd., China); 0.9% sodium chloride injection (batch number S21091609, Hunan Kelun Pharmaceutical Co., Ltd., China); vinorelbine tartrate injection (batch number 600211003, Jiangsu Hansoh Pharmaceutical Co., Ltd., China); fluorescent microspheres (batch number MKCB2036V, Sigma, USA).

[0121] ③ Measurement of T cell fluorescence intensity Three-day-old (3dpf) transgenic T-cell red fluorescent zebrafish were randomly selected and placed in 6-well plates, with 30 zebrafish treated in each well (experimental group). Samples were administered via water-soluble injection. A normal control group, a positive control group, and a model control group were also included. The volume of each well was 3 mL, and the sample concentrations for each group are shown in Table 2. Except for the normal control group, all other experimental groups received an intravenous injection of 2 ng / zebrafish vinorelbine tartrate injection (diluted to 0.2 mg / mL with 0.9% sodium chloride injection, then injected in 10 nL) to establish a zebrafish immunodeficiency model. After treatment at 28℃ for 48 h, 10 zebrafish from each experimental group were randomly selected, fixed with methylcellulose, and photographed under a fluorescence microscope. Data were collected using NIS-Elements D 3.20 advanced image processing software, and the fluorescence intensity of zebrafish T cells was analyzed. The statistical analysis results of this index were used to evaluate the immune-enhancing efficacy of the samples. Statistical results are expressed as mean ± SE. Statistical analysis was performed using SPSS software. p A value <0.05 indicates that the difference is statistically significant.

[0122] Table 2

[0123] ④ Determination of the phagocytic function of zebrafish macrophages (residual number of fluorescent microspheres) in fermented milk samples Wild-type AB zebrafish, 3 days post-fertilization (3dpf), were randomly selected and placed in 6-well plates, with 30 zebrafish treated in each well (experimental group). Samples were administered via water-soluble injection. A normal control group, a positive control group, and a model control group were also included. The volume of each well was 3 mL, and the sample concentrations for each group are shown in Table 2. Except for the normal control group, all other experimental groups received intravenous injections of 2 ng / zebrafish vinorelbine tartrate injection (diluted to 0.2 mg / mL with 0.9% sodium chloride injection, then injected in 10 nL) and 1% fluorescent microspheres to establish a zebrafish immunodeficiency model. After treatment at 28℃ for 96 h, 10 zebrafish from each experimental group were randomly selected, fixed with methylcellulose, and photographed under a fluorescence microscope. Data were collected using NIS-Elements D 3.20 advanced image processing software, and the residual number of fluorescent microspheres in the zebrafish was analyzed. The statistical analysis results of this index were used to evaluate the immune-enhancing efficacy of the samples. Statistical results are expressed as mean ± SE. Statistical analysis was performed using SPSS software. p A value <0.05 indicates that the difference is statistically significant.

[0124] The experimental results are as follows: T cell function, results are shown in Figure 7 : Model Construction and Validation: The mean fluorescence intensity of T cells in the model control group (9751 pixels) was significantly lower than that in the normal control group (37189 pixels), and the difference was statistically significant, indicating that the immunosuppression model was successfully constructed and suitable for subsequent drug efficacy evaluation. Positive Control Efficacy: The mean fluorescence intensity of T cells in the positive control drug Bailin capsules (15.0 μg / mL) group was 28150 pixels, significantly higher than that in the model control group, confirming that this positive control drug can effectively enhance T cell activity. Effects of the Embodiments of this Invention: Example 1: The mean fluorescence intensity of T cells was 18931 pixels, significantly higher than that in the model control group (9751 pixels). Example 2: The mean fluorescence intensity of T cells was 20162 pixels, significantly higher than that in the model control group. Example 3: The mean fluorescence intensity of T cells was 13465 pixels, also significantly higher than that in the model control group, indicating that the embodiments of this invention can significantly enhance T cell activity and strengthen cellular immune response.

[0125] Phagocyte function, results are shown in Figure 8 and Figure 9 : Model Construction and Validation: The mean number of residual fluorescent microspheres in the model control group (31.2) was significantly higher than that in the normal control group (8.7), with a statistically significant difference, indicating impaired phagocytic function. This demonstrates the successful construction of the immunodeficiency model, which can be used for subsequent drug efficacy evaluation. Positive Control Efficacy: The mean number of residual fluorescent microspheres in the positive control drug Bailin capsules (15.0 μg / mL) group was 12.9, significantly lower than that in the model control group, confirming that the drug can effectively improve the phagocytic function of phagocytes. Effects of the Invention Examples: Example 1: The mean number of residual fluorescent microspheres was 17.2, significantly lower than that in the model control group. Example 2: The mean number of residual fluorescent microspheres was 15.9, significantly lower than that in the model control group. Example 3: The mean number of residual fluorescent microspheres was 21.5, also significantly lower than that in the model control group, indicating that the embodiments of the present invention have a prominent enhancing effect on the phagocytic function of phagocytes and can effectively improve the body's innate immune defense capabilities.

[0126] In summary, the embodiments of the present invention can significantly enhance T cell activity and strengthen cellular immune responses; they can also significantly improve the phagocytic function of phagocytes and enhance the body's innate immune defense level. Embodiment 1 is the optimal implementation scheme, exhibiting more significant advantages in both enhancing T cell activity and improving phagocytic function.

[0127] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing fermented milk, characterized in that, Bifidobacterium longum BBMN68 was inoculated into the fermentation substrate along with the starter culture for fermentation, and temperature-dynamic frequency conversion ultrasound intervention was performed during the fermentation process. The temperature-dynamic frequency conversion ultrasound intervention method includes: At the start of formal fermentation, the temperature of the fermentation system is controlled at 25±1℃ before the first stage of ultrasonic treatment is performed. After ultrasonic treatment, the temperature is raised to the fermentation temperature for fermentation. The fermentation temperature is 41-43℃. When the pH value of the fermentation system reaches 5.4-5.6, the temperature of the fermentation system is lowered to 25±1℃ and then the second stage of ultrasonic treatment is carried out. After the ultrasonic treatment is completed, the temperature is raised to the fermentation temperature to continue fermentation. When the pH value of the fermentation system reaches 4.5-4.7, the temperature of the fermentation system is lowered to 25±1℃ and then the third stage of ultrasonic treatment is carried out. After the ultrasonic treatment, the temperature is raised to the fermentation temperature and then kept at a constant temperature until the end point. The conditions for the first stage of ultrasonic treatment are: The frequency is 18-26kHz, the power density is 42-50W / L, and the action time is 4-6min; The conditions for the second stage of ultrasonic treatment are as follows: Increase the frequency of the first-stage ultrasonic treatment by 8-12 kHz; decrease the power density of the first-stage ultrasonic treatment by 15-18 W / L; and reduce the treatment time by 4-6 min. The conditions for the third stage of ultrasonic treatment are as follows: Increase the frequency of the second-stage ultrasonic treatment by 8-12 kHz; decrease the power density of the second-stage ultrasonic treatment by 15-18 W / L; and reduce the treatment time by 1-3 min. All three stages of ultrasound treatment adopted an intermittent pulse mode, with a pulse period of 20-30s, an ultrasound time of 8-12s, and an interval of 12-18s; The preservation number of the Bifidobacterium longum BBMN68 is CGMCC No. 2265; The fermentation substrate comprises, by weight, 91-93 parts animal milk, 7.5-8 parts sweetener, 0.2-0.3 parts stabilizer, and 0-0.025 parts functional substance; The starter culture includes Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus salivarius subsp. thermophilus.

2. The method according to claim 1, characterized in that, The inoculum amounts of both *Bifidobacterium longum* BBMN68 and the fermentation agent were 10. 6 -10 8 cfu / g; and / or, the stabilizer is one or more of pectin, gellan gum, carrageenan, soybean polysaccharide, sodium carboxymethyl cellulose, xanthan gum, modified starch, and agar; The functional substances are one or more of the following: vitamin A, vitamin C, vitamin D, yeast-β-glucan, chitosan oligosaccharide, and kiwi powder.

3. The method according to claim 1, characterized in that, The conditions for the first stage of ultrasonic treatment are: frequency of 22kHz, power density of 46W / L, and treatment time of 5min; The conditions for the second stage of ultrasonic treatment are: frequency of 32kHz, power density of 30W / L, and treatment time of 5min; The conditions for the third stage of ultrasonic treatment are: frequency of 42kHz, power density of 14W / L, and treatment time of 2min. All three stages of ultrasound treatment used an intermittent pulse mode with a pulse period of 25s, including an ultrasound time of 10s and an interval of 15s.

4. The method according to claim 1, characterized in that, When the method involves heating, the heating rate is 5±0.5℃ / min; when it involves cooling, the cooling rate is 3±0.5℃ / min.

5. The method according to claim 1, characterized in that, The final pH value of the fermentation was 4.25-4.

35.

6. The method according to claim 1, characterized in that, The temperature of the fermentation substrate is 41-43℃. After the Bifidobacterium longum BBMN68 and the starter culture are inoculated into the fermentation substrate, the substrate is first stirred and activated for 8-12 minutes before the formal fermentation begins. Then, the temperature of the fermentation system is lowered to 25±1℃ before the first stage of ultrasonic treatment is performed.

7. The method according to any one of claims 1-6, characterized in that, The fermentation process also includes demulsification and sterilization steps after it is completed.

8. The method according to claim 7, characterized in that, The sterilization temperature is 72-75℃, and the time is 50-70 seconds.

9. A fermented milk, characterized in that, Prepared by the method described in any one of claims 1-8.

10. The application of the method according to any one of claims 1-8 in any of the following aspects: (1) Increase the content of γ-aminobutyric acid in fermented milk; (2) Increase the content of indole-3-lactic acid in fermented milk; (3) Increase the content of 2,3-butanedione in fermented milk; (4) Increase the content of 2-hydroxy-3-methylbutyric acid in fermented milk; (5) Increase the content of micranthin in fermented milk; (6) Increase the content of phloric acid in fermented milk; (7) Enhance the immune regulation function of fermented milk.

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