Fermented milk and preparation method and application thereof

Abstract

This invention relates to the field of biotechnology, specifically disclosing a fermented milk, its preparation method, and its application. The method includes preparing a yogurt base and a latex solution separately, then mixing and sterilizing them using an ultrasonic process. The yogurt base includes BBMN68 postbiotics or live BBMN68 bacteria; the latex solution includes yeast β-glucan. After sterilization, the mass ratio of BBMN68 postbiotics to yeast β-glucan in the fermented milk is 1:(2-6). The ultrasonic process conditions are: frequency 80-100kHz, ultrasonic power 300-400w, and time 10-15min. The fermented milk of this invention achieves ideal immune-enhancing effects with a low amount of added active ingredients, thereby significantly saving costs and indirectly reducing stability problems caused by insoluble raw materials.

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] In recent years, the market demand for functional fermented milk has shown a significant growth trend. Consumers' core demands have gradually shifted from basic nutritional supply to health benefits such as regulating gut microbiota balance and enhancing immunity. Postbiotics are complexes of non-living microorganisms and / or their components that can bring health benefits to the human body, and may or may not contain metabolites or cellular components. Postbiotics typically have good storage and transportation stability and processing tolerance, which aligns with the processing characteristics, storage conditions, and shelf life of room-temperature yogurt.

[0003] Existing studies have shown that consuming room-temperature yogurt containing inactivated BBMN68 (post-biotic) or added probiotic BBMN68 after double sterilization can restore the body's immune function, reduce damage to immune organs, alleviate inflammation, and regulate gut microbiota. However, achieving these effects requires high dosages, resulting in high costs and hindering the large-scale promotion and industrial application of inactivated BBMN68 / probiotics. Therefore, it is necessary to explore technical solutions that can synergistically enhance the effects of inactivated BBMN68 / probiotics.

[0004] Yeast beta-glucan is one of the most recognized functional ingredients in the food and health product industries. Its core function is focused on immune regulation; it can enhance the body's non-specific immunity by activating immune cells such as macrophages and neutrophils. However, yeast beta-glucan is a supercoiled, insoluble biomolecule with low bioavailability, posing the challenge of requiring relatively high dosages to achieve its effects. Clinical trials have shown that its effective dose is 250-900 mg / day. However, the current recommended daily intake of yeast beta-glucan is ≤250 mg, making it difficult to achieve ideal results in practical applications. Furthermore, yeast beta-glucan has poor solubility in yogurt; direct addition to fermented milk products can cause clumping and sedimentation, severely limiting its application.

[0005] Therefore, it is necessary to further study how to prepare functional fermented milk. Summary of the Invention

[0006] One of the objectives of this invention is to provide a room-temperature dairy product that achieves ideal immune-enhancing effects with low levels of added immune-enhancing components.

[0007] This invention provides a method for preparing fermented milk, which includes the steps of preparing yogurt base material and latex solution separately, mixing them by ultrasonic process, and then sterilizing them. The yogurt base contains BBMN68 postbiotic or BBMN68 live bacteria; the number of inactivated bacterial cells in the BBMN68 postbiotic is ≥2×10⁻⁶. 11 cell / g; the viable count of the BBMN68 live bacteria is ≥2×10⁻⁶. 11 cfu / g; The latex solution includes yeast β-glucan; After sterilization, the mass ratio of BBMN68 postbiotic to yeast β-glucan in the fermented milk is 1:(2-6).

[0008] The conditions for the ultrasonic process are: frequency of 80-100kHz, ultrasonic power of 300-400w, and time of 10-15min. The preservation number of BBMN68 live bacteria is CGMCC No. 2265.

[0009] This invention reveals that when BBMN68 postbiotic and yeast β-glucan are combined in a specific ratio in room-temperature fermented milk, along with a specific ultrasonic mixing process, a non-linear synergistic effect on enhancing immune function can be achieved. This improves their bioavailability and synergistic effect, allowing for ideal immune-enhancing efficacy even when the added amounts are far lower than the individual effective amounts. This indirectly reduces the impact of the poor solubility of yeast β-glucan on product stability (the effective amount of BBMN68 postbiotic alone is 5-20 billion cells, and the effective amount of yeast β-glucan alone is 250-900 mg / day). Changing the ratio of the two or the mixing method of the yogurt base and latex solution will affect the product efficacy.

[0010] This invention has found that when BBMN68 live bacteria and starter culture are fermented together during the preparation of yogurt base, the number of live bacteria does not increase significantly (it is basically the same as the number of live bacteria when added). Moreover, the final product is sterilized fermented milk, and the BBMN68 live bacteria will be inactivated and become post-biotics at this time. Therefore, the effect of using BBMN68 live bacteria in the preparation of yogurt base is basically the same as directly adding an equal amount of BBMN68 post-biotics.

[0011] In this invention, BBMN68 live bacteria refers to Bifidobacterium (Bifidobacterium) Bifidobacterium longum BBMN68, with accession number CGMCC No. 2265, deposited on November 26, 2007, by the China General Microbiological Culture Collection Center (CGMCC). Location: Institute of Microbiology, Chinese Academy of Sciences, Chaoyang District, Beijing, China. Published in Chinese Patent CN101649303B.

[0012] BBMN68 postbiotics are inactivated BBMN68 live bacteria, which may or may not contain metabolites.

[0013] In the method for preparing fermented milk according to the present invention, the raw materials for preparing the yogurt base material, by weight, include: 90-95 parts of animal milk and / or reconstituted milk, 4-8 parts of sweetener, 0.14-0.18 parts of hydrophilic colloid, 1.2-1.5 parts of thickener, 0.005-0.015 parts of BBMN68 postbiotic or BBMN68 live bacteria, 0.03-0.05 parts of starter culture, and 0-0.1 parts of functional component; wherein the functional component is one or more of vitamin A, vitamin C, vitamin D, vitamin E, human milk oligosaccharides, and lactoferrin; Preferably, the total weight of the raw materials for preparing the yogurt base is 100 parts.

[0014] The raw materials for preparing the latex solution, by weight, include: 94-97 parts animal milk and / or reconstituted milk, 0.1-0.75 parts yeast β-glucan, and 3.5-5.5 parts stabilizer; Preferably, the total weight of the raw materials for preparing the latex solution is 100 parts.

[0015] The volume ratio of the yogurt base to the latex solution is 9:1.

[0016] In the method for preparing fermented milk of the present invention, the sweetener is one or more selected from white sugar, acesulfame potassium, erythritol, xylitol, fructose or sucralose; The hydrophilic colloid is one or more of the following: pectin, agar, gellan gum, citrus fiber, gelatin, xanthan gum, propylene glycol alginate, guar gum, and locust bean. The thickener is one or more of sodium carboxymethyl cellulose, hydroxypropyl distarch phosphate, agar, acetylated distarch phosphate, and starch. The starter culture includes Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus salivarius subsp. thermophilus; 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).

[0017] The stabilizer is one or more of pectin, gelatin, xanthan gum, gellan gum, propylene glycol alginate, and soybean polysaccharides.

[0018] 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. Reconstituted milk is liquid milk obtained by reconstituted milk powder with water.

[0019] In this invention, the molecular weight of yeast β-glucan is generally 200,000 to 4,000,000 Da.

[0020] In the method for preparing fermented milk of the present invention, the method for preparing the yogurt base material includes: When the yogurt base material includes BBMN68 post-biotic, the animal milk and / or reconstituted milk, sweetener, hydrocolloid, thickener, and BBMN68 post-biotic are mixed evenly, sterilized and cooled to 37-44℃, and then the starter is added aseptically for fermentation. When the yogurt base material includes BBMN68 live bacteria, the animal milk and / or reconstituted milk, sweetener, hydrocolloid, and thickener are mixed evenly, sterilized and cooled to 40-43°C, and then the starter and BBMN68 live bacteria are added aseptically for fermentation. The fermentation conditions are as follows: fermentation temperature of 40-43℃, and final pH value of 4.25-4.45.

[0021] Preferably, the fermentation conditions are: a fermentation temperature of 42-43℃ and a final pH value of 4.3-4.45.

[0022] In the method for preparing fermented milk of the present invention, the preparation method of the latex solution includes: thoroughly mixing the raw materials and then sterilizing them.

[0023] In the method for preparing fermented milk according to the present invention, the method for preparing the yogurt base material, including the uniform mixing and sterilization of the raw materials, comprises: mixing the raw materials at a temperature of 45-50℃ and then homogenizing and sterilizing them; the conditions for homogenization are: a temperature of 55-65℃, a total homogenization pressure of 150-240 bar, and a secondary pressure of 30-50 bar; the conditions for sterilization are: a temperature of 121-137℃ and a time of 4-6 seconds.

[0024] Preferably, the homogenization conditions are: temperature of 64-65℃, total homogenization pressure of 190-210 bar, and secondary pressure of 40-50 bar; In the method for preparing fermented milk of the present invention, the method for fully mixing the raw materials during the preparation of the latex solution includes: first mixing the raw materials at a temperature of 80-85℃, and then homogenizing or ultrasonically treating them. The homogenization conditions are: temperature 55-65℃, total homogenization pressure 150-240 bar, and secondary pressure 30-50 bar. Preferably, the homogenization conditions are: temperature of 52-57℃, total homogenization pressure of 190-210 bar, and secondary pressure of 40-50 bar; The conditions for ultrasonic treatment are as follows: temperature 50-60℃, simultaneous ultrasonic treatment at dual frequencies of 20-40kHz and 50-70kHz with a frequency difference of 30-40kHz, ultrasonic power 600-800w, and time 35-45min.

[0025] This invention has found that, during the preparation of latex solutions, products with similar average particle size, viscosity, and stability index can be obtained through both homogenization and ultrasonic treatment. However, ultrasonic treatment is preferred as it can further improve the long-term stability of the product. Specifically, under the ultrasonic treatment conditions specified in this invention, a shelf life of 8 months at room temperature can be achieved. If the frequency difference in the dual-frequency ultrasonic treatment is changed, for example, using dual-frequency ultrasonic frequencies of 40kHz and 60kHz with a frequency difference of 20kHz, or using dual-frequency ultrasonic frequencies of 20kHz and 70kHz with a frequency difference of 50kHz, although the resulting product still possesses the effect of enhancing immunity, its long-term stability will decrease (to 5 months and 6 months, respectively).

[0026] In this invention, room temperature refers to 20-30℃.

[0027] The sterilization conditions for preparing the latex solution are a temperature of 121-137℃ and a time of 4-6 seconds.

[0028] In the method for preparing fermented milk of the present invention, the sterilization conditions after mixing the yogurt base material and the latex solution are: temperature of 75±2℃ and time of 20-35 seconds.

[0029] In large-scale industrial production, it is preferable to cool the ultrasonically treated mixture to 18-22°C before sterilization to avoid continued fermentation that would result in a sour taste.

[0030] The present invention also provides a fermented milk prepared by the above method.

[0031] The fermented milk obtained by this invention has a low amount of added active ingredients, which can significantly enhance immunity, has good stability, a refreshing and sweet taste, and can be stored at room temperature for a long time.

[0032] The rheological viscosity of the fermented milk of this invention is generally 260-320 mPa·s.

[0033] The present invention also provides the application of the above-mentioned fermented milk in the preparation of products that enhance immunity.

[0034] The beneficial effects of this invention are at least as follows: This invention, through a specific mixing process and material combination, yields a room-temperature fermented milk that achieves ideal immune-enhancing effects despite low levels of added active ingredients. This significantly reduces costs and indirectly minimizes stability issues caused by insoluble raw materials. Furthermore, the fermented milk of this invention has an ideal taste and can be stored at room temperature for an extended period (at least 4 months). Detailed Implementation

[0035] 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.

[0036] 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 are commercially available or prepared according to conventional methods in the art.

[0037] The BBMN68 postbiotic and BBMN68 probiotics (live bacteria) used in the specific embodiments of this invention were provided by Sanhe Fucheng Biotechnology Co., Ltd., and the BBMN68 postbiotic specification was 2×10. 11 cell / g (number of inactivated bacterial cells); BBMN68 probiotics are available in 2×10⁶ g samples. 11 CFU / g. The starter culture was provided by Beijing Ketuo Hengtong Biotechnology Co., Ltd., with the starter culture number PYS-V141B. The starter culture consisted of *Lactobacillus delbrueckii* subsp. bulgaricus and *Streptococcus salivarius* subsp. thermophilus. Hydroxypropyl distarch phosphate (REZISTA® 682 NG), acetylated distarch phosphate (Kol Guard® 383 NGStarch), and starch were provided by Tailai Trading (Shanghai) Co., Ltd. The yeast β-glucan raw material was purchased from Angel Yeast Co., Ltd., and its active ingredient, yeast β-glucan, contained 80%. Therefore, the mass of the active ingredient yeast β-glucan in the latex solution in each example and comparative example should be converted by multiplying the formula addition amount by 80%.

[0038] This invention provides a fermented milk and its preparation method, wherein the liquid used to make it comprises two parts: yogurt base and latex solution.

[0039] By weight, the raw materials of the yogurt base include: 90-95 parts raw milk and / or reconstituted milk, 4-8 parts sweetener, 0.14-0.18 parts hydrocolloid, 1.2-1.5 parts thickener, 0.005-0.015 parts BBMN68 postbiotic / probiotic, 0.03-0.05 parts starter culture, and 0-0.1 parts functional component; the functional component is one or more of vitamin A, vitamin C, vitamin D, vitamin E, human milk oligosaccharides, and lactoferrin.

[0040] The sweetener is one or more of white sugar, acesulfame potassium, erythritol, xylitol, fructose or sucralose, with white sugar being preferred, as it helps to obtain yogurt with a full-bodied, sweet, refreshing and delicious taste.

[0041] The hydrophilic colloid is one or more of the following: pectin, agar, gellan gum, citrus fiber, gelatin, xanthan gum, propylene glycol alginate, guar gum, and locust bean. The thickener is one or more of sodium carboxymethyl cellulose, hydroxypropyl distarch phosphate, agar, acetylated distarch phosphate, and starch.

[0042] The raw materials of the latex solution, by weight, include: 94-97 parts raw milk and / or reconstituted milk, 0.1-0.75 parts yeast β-glucan, and 3.5-5.5 parts stabilizer.

[0043] The stabilizer is one or more of pectin, gelatin, xanthan gum, gellan gum, propylene glycol alginate, and soybean polysaccharides.

[0044] The method for preparing the fermented milk described above in this invention includes: preparing yogurt base material, preparing latex solution, mixing the yogurt base material and latex solution, sterilizing, and filling. Specifically, as follows: 1. Yogurt base preparation: When BBMN68 post-biotic is used in the raw materials, the preparation method is as follows: Heat raw milk / reconstituted milk to 45-50℃ for melting. Turn on the high-efficiency online mixer and start the shear pump. While it is running, add sweetener, hydrocolloid, thickener, and BBMN68 post-biotic, and stir for 10-15 minutes. Homogenize: heat to 55-65℃, with a total homogenization pressure of 150-240 bar and a secondary pressure of 30-50 bar to obtain a mixed product. Sterilize: sterilize at 121-137℃ for 4-6 seconds. Cool to fermentation temperature of 40-43℃, inoculate with starter culture, stir for 10-15 minutes, turn off the stirring and start the fermentation timer. Start measuring the pH value after 4.0 hours of fermentation, and the final pH value is 4.25-4.45.

[0045] When BBMN68 probiotics are used in the raw materials, the preparation method is basically the same as above. The only difference is that BBMN68 postbiotics are not added during the mixing stage, but BBMN68 probiotics are added at the same time as the fermentation agent.

[0046] 2. Preparation of latex solution: Heat raw milk / reconstituted milk to 80-85℃ for mixing. Turn on the high-efficiency online mixer and start the shear pump. While it is running, add stabilizer and yeast β-glucan, and stir for 10-15 minutes. After homogenization or ultrasonic treatment, sterilize and cool to 15-18℃.

[0047] Homogenization method: Heat to 55-65℃, the total homogenization pressure is 150-240 bar, and the secondary pressure is 30-50 bar.

[0048] Ultrasonic treatment method: Heat to 50-60℃, perform simultaneous ultrasonic treatment at dual frequencies of 20-40kHz and 50-70kHz, with a frequency difference preferably of 30-40kHz, ultrasonic power of 600-800w, and time of 35-45min to better improve the stability of the final product.

[0049] Sterilization method: Sterilization temperature: 121-137℃, time: 4-6s.

[0050] 3. Mixing, sterilizing, and aseptically filling yogurt base and latex solution: Yogurt base and latex solution are mixed in a 9:1 (V / V) ratio, ultrasonically treated, cooled, sterilized, and then filled to obtain fermented milk. The filling specification is 100g / bottle.

[0051] Ultrasonic treatment method: frequency 80-100kHz, ultrasonic power 300-400w, time 10-15min.

[0052] The cooling temperature after ultrasonic treatment is 18-22℃.

[0053] Sterilization method: Sterilization temperature is 75±2℃, time is 20-35 seconds; aseptic filling.

[0054] Examples 1-3 This embodiment prepares fermented milk according to the above method. The specific raw material formulas for each embodiment are shown in Table 1, and the specific preparation process conditions are shown in Table 2.

[0055] Table 1. Formula (Unit: g for each raw material)

[0056] Table 2

[0057] Example 4 This embodiment provides a method for preparing fermented milk. The raw material formula and method are basically the same as those in Example 2, except that instead of adding BBMN68 postbiotic during the yogurt base preparation stage, 0.01111g of BBMN68 probiotics (live bacteria) are added at the same time as the starter culture. After the yogurt base fermentation is completed, the number of live BBMN68 bacteria is basically unchanged from before fermentation. After the product is sterilized, the content of BBMN68 postbiotic is basically the same as in Example 2.

[0058] Example 5 This embodiment provides a method for preparing fermented milk. The raw material formula and method are basically the same as those in Example 2. The only difference is that when preparing the latex solution, homogenization is performed after the materials are dissolved. The homogenization method is as follows: the latex solution is heated to 55°C, the total homogenization pressure is 200 bar, and the secondary pressure is 45 bar.

[0059] Example 6 This embodiment provides a method for preparing fermented milk. The raw material formula and method are basically the same as those in Example 2, except that 0.07g of vitamin A and 0.002g of vitamin D3 are added in addition during the yogurt base material preparation stage.

[0060] Example 7 This embodiment provides a method for preparing fermented milk. The raw material formula and method are basically the same as those in Example 2. The only difference is that 0.1g of human milk oligosaccharide (2'-fucosylated lactose) is added in the yogurt base material preparation stage.

[0061] Comparative Example 1 This comparative example provides a method for preparing fermented milk. The raw material formula and method are basically the same as those in Example 2. The only difference is that, while keeping the total mass of BBMN68 post-biotic and yeast β-glucan in the final product unchanged, the mass ratio of the two added is adjusted to 1:1.

[0062] Comparative Example 2 This comparative example provides a method for preparing fermented milk. The raw material formula and method are basically the same as those in Example 2. The only difference is that, while keeping the total mass of BBMN68 post-biotic and yeast β-glucan in the final product unchanged, the mass ratio of the two added is adjusted to 1:7.

[0063] Comparative Example 3 This comparative example provides a method for preparing fermented milk. The raw material formula and method are basically the same as those in Example 2. The only difference is that yeast β-glucan is not added, but the amount of BBMN68 postbiotic added is increased so that it is the same as the total mass of BBMN68 postbiotic and yeast β-glucan in the final product in Example 2.

[0064] Comparative Example 4 This comparative example provides a method for preparing fermented milk. The raw material formula and method are basically the same as those in Example 2. The only difference is that BBMN68 post-biotic is not added, but the amount of yeast β-glucan added is increased so that it is the same as the total mass of BBMN68 post-biotic and yeast β-glucan in the final product in Example 2.

[0065] Comparative Example 5 This comparative example provides a method for preparing fermented milk. The raw material formula and method are basically the same as those in Example 2, except that when preparing the latex solution, 0.03g of vitamin C is used to replace the yeast β-glucan in the latex solution.

[0066] In this comparative example, the amount of vitamin C added was 50% of the effective amount when used alone, while in Example 2, the amount of yeast β-glucan added was only 9.6% of the effective amount when used alone.

[0067] Comparative Example 6 This comparative example provides a method for preparing fermented milk. The raw material formula and method are basically the same as those in Example 2. The only difference is that after mixing the yogurt base and the latex solution, ultrasonic treatment is not used, but homogenization is used instead. The homogenization conditions are: a total homogenization pressure of 100 bar and a secondary pressure of 25 bar.

[0068] Comparative Example 7 This comparative example provides a method for preparing fermented milk. The raw material formula and method are basically the same as those in Example 2. The only difference is that the ultrasonic conditions after mixing the yogurt base and the latex solution are changed to: ultrasonic frequency of 70kHz, ultrasonic power of 300w, and time of 10min.

[0069] Comparative Example 8 This comparative example provides a method for preparing fermented milk. The raw material formula and method are basically the same as those in Example 2. The only difference is that the ultrasonic conditions after mixing the yogurt base and the latex solution are changed to: ultrasonic frequency of 110 kHz, ultrasonic power of 300 W, and time of 10 min.

[0070] Experimental Example This experimental example demonstrates the experiments conducted on the fermented milk prepared in each of the embodiments and comparative examples, as detailed below: 1. Product particle size: The detection method refers to Wu Weidu, Zhu Hui, Ou Kai, et al. Study on the determination of particle size of stirred yogurt by laser particle size analyzer [J]. Grain and Feed Industry, 2018(8). 2. Rheological viscosity: The detection method is based on Liu Hongxia, Guo Yanrong, Wu Xiuying, et al. Screening and characterization of combined starter cultures for fermented milk [J]. China Dairy Industry, 2025(53). 3. Stability index: The detection method is based on 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).

[0071] After placing each group of products at room temperature (25℃), observe their stability.

[0072] The results are shown in Table 3.

[0073] Table 3

[0074] After tasting, the products in Examples 1-7 have a full flavor, a refreshing and sweet taste, and a delicate and smooth texture.

[0075] 4. Zebrafish experiment to detect immune efficacy: ① Testing materials Positive control: Bailin® Bailin capsules (hereinafter referred to as Bailin capsules), batch number 2307029D, Hangzhou Sino-American East China Pharmaceutical Co., Ltd., solvent is standard dilution water.

[0076] 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.

[0077] 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).

[0078] 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), for diluting vinorelbine tartrate injection; vinorelbine tartrate injection (batch number 600211003, Jiangsu Hansoh Pharmaceutical Co., Ltd., China); fluorescent microspheres (batch number MKCB2036V, Sigma, USA).

[0079] ②Detection methods and results T cells: 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). Water-soluble samples (concentrations shown in Table 4) were administered, along with a positive control of 15.0 μg / mL of Bailin capsules. A normal control group and a model control group were also included, with a volume of 3 mL per well. Except for the normal control group, all other experimental groups received intravenous injection of 2 ng / zebrafish vinorelbine tartrate injection to establish a zebrafish immunodeficiency model. After treatment at 28℃ for 48 h, 10 zebrafish from each experimental group were randomly selected and photographed under a fluorescence microscope (zebrafish were fixed with methylcellulose). 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, with p < 0.05 indicating statistical significance. Compared with the model control group, p<0.05, p<0.01, p<0.001, results are shown in Table 4. Dosage conversion: zebrafish (ug / mL) × 6 / 1000 = human (g).

[0080] Table 4

[0081] Devour: 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). Water-soluble samples (concentrations shown in Table 5) were administered, along with a positive control of 15.0 μg / mL of Bailin capsules. A normal control group and a model control group were also included, with a volume of 3 mL per well. Except for the normal control group, all experimental groups received intravenous injections of 2 ng / zebrafish of vinorelbine tartrate injection 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 and photographed under a fluorescence microscope (zebrafish were fixed with methylcellulose). 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 < 0.05 indicated statistical significance. Compared with the model control group, p<0.01, p < 0.001. The results are shown in Table 5.

[0082] Table 5

[0083] 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 of preparing a fermented milk, characterized in that, This includes the steps of separately preparing yogurt base and latex solution, mixing them using an ultrasonic process, and then sterilizing them. The yogurt base comprises Bifidobacterium longum BBMN68 probiotic or Bifidobacterium longum BBMN68 live bacteria; the inactivated bacterial cell count of the BBMN68 probiotic is ≥ 2 x 10 11 cell / g; the viable bacterial count of the BBMN68 live bacteria is ≥ 2 x 10 11 cfu / g; The latex solution includes yeast β-glucan; After sterilization, the mass ratio of BBMN68 postbiotic to yeast β-glucan in the fermented milk is 1:(2-6). The conditions for the ultrasonic process are: frequency of 80-100kHz, ultrasonic power of 300-400w, and time of 10-15min. The preservation number of BBMN68 live bacteria is CGMCC No. 2265.

2. The method for preparing fermented milk according to claim 1, characterized in that, By weight, the raw materials for preparing the yogurt base include: 90-95 parts animal milk and / or reconstituted milk, 4-8 parts sweetener, 0.14-0.18 parts hydrophilic colloid, 1.2-1.5 parts thickener, 0.005-0.015 parts BBMN68 postbiotic or BBMN68 live bacteria, 0.03-0.05 parts starter culture, and 0-0.1 parts functional component; the functional component is one or more of vitamin A, vitamin C, vitamin D, vitamin E, human milk oligosaccharides, and lactoferrin. The raw materials for preparing the latex solution, by weight, include: 94-97 parts animal milk and / or reconstituted milk, 0.1-0.75 parts yeast β-glucan, and 3.5-5.5 parts stabilizer; The volume ratio of the yogurt base to the latex solution is 9:

1.

3. The method for preparing fermented milk according to claim 2, characterized in that, The sweetener is one or more of the following: white sugar, acesulfame potassium, erythritol, xylitol, fructose, or sucralose. The hydrophilic colloid is one or more of the following: pectin, agar, gellan gum, citrus fiber, gelatin, xanthan gum, propylene glycol alginate, guar gum, and locust bean. The thickener is one or more of sodium carboxymethyl cellulose, hydroxypropyl distarch phosphate, agar, acetylated distarch phosphate, and starch. The starter culture includes Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus salivarius subsp. thermophilus; The stabilizer is one or more of pectin, gelatin, xanthan gum, gellan gum, propylene glycol alginate, and soybean polysaccharides.

4. The method for preparing fermented milk according to any one of claims 1-3, characterized in that, The method for preparing the yogurt base includes: When the yogurt base material includes BBMN68 post-biotic, the animal milk and / or reconstituted milk, sweetener, hydrocolloid, thickener, and BBMN68 post-biotic are mixed evenly, sterilized and cooled to 37-44℃, and then the starter is added aseptically for fermentation. When the yogurt base material includes BBMN68 live bacteria, the animal milk and / or reconstituted milk, sweetener, hydrocolloid, and thickener are mixed evenly, sterilized and cooled to 40-43°C, and then the starter and BBMN68 live bacteria are added aseptically for fermentation. The fermentation conditions are as follows: fermentation temperature is 40-43℃, and the final pH value of fermentation is 4.25-4.

45.

5. The method for preparing fermented milk according to any one of claims 1-3, characterized in that, The method for preparing the latex solution includes: thoroughly mixing the raw materials and then sterilizing them.

6. The method for preparing fermented milk according to claim 4, characterized in that, The method for preparing the yogurt base material, including uniform mixing and sterilization of the raw materials, includes: mixing the raw materials at a temperature of 45-50℃ and then homogenizing and sterilizing them. The homogenization conditions are: a temperature of 55-65℃, a total homogenization pressure of 150-240 bar, and a secondary pressure of 30-50 bar. The sterilization conditions are: a temperature of 121-137℃ and a time of 4-6 seconds.

7. The method for preparing fermented milk according to claim 5, characterized in that, The method for fully mixing the raw materials during the preparation of the latex solution includes: first mixing the raw materials at a temperature of 80-85℃, and then homogenizing or ultrasonically treating them. The homogenization conditions are: temperature 55-65℃, total homogenization pressure 150-240 bar, and secondary pressure 30-50 bar. The conditions for ultrasonic treatment are as follows: temperature is 50-60℃, ultrasonic treatment is performed simultaneously at dual frequencies of 20-40kHz and 50-70kHz with a frequency difference of 30-40kHz, ultrasonic power is 600-800w, and time is 35-45min. The sterilization conditions for preparing the latex solution are a temperature of 121-137℃ and a time of 4-6 seconds.

8. The method for preparing fermented milk according to any one of claims 1-3, characterized in that, The sterilization conditions after mixing the yogurt base and the latex solution are: temperature 75±2℃, time 20-35 seconds.

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

10. The use of the fermented milk according to claim 9 in the preparation of products that enhance immunity.

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