An antioxidant fermented milk and a method for preparing the same

Abstract

The present application belongs to the technical field of fermented milk products and their preparation, and in particular relates to an antioxidant fermented milk and a preparation method thereof. According to mass percentage, the raw materials for preparing the fermented milk comprise: 3-8% of a sweetening agent, 0.0005-0.002% of L-type ergothioneine, 0.1-0.5% of a thickening agent, 0.001-0.005% of a fermenting agent, and the rest is original selenium-rich milk or original selenium-rich concentrated milk treated by ultrafiltration membrane concentration. The present application uses the membrane concentration technology to prepare high-protein and high-selenium concentrated intermediate milk from the original selenium-rich milk, which is used as a fermentation substrate. At the same time, L-type ergothioneine is added, which consumes residual oxygen during fermentation and produces a synergistic effect with bifidobacterium, thereby jointly constructing a high-antioxidant system. The obtained fermented milk has high protein, is rich in selenium and has high antioxidant properties.

Description

Technical Field

[0001] This invention belongs to the field of fermented dairy products and their preparation technology, and more specifically, relates to an antioxidant fermented milk and its preparation method. Background Technology

[0002] With the upgrading of consumers' health awareness, fermented milk with antioxidant and anti-aging functions has gradually become a hot topic in industry research and development. Currently, functional yogurts on the market mainly have the following technical approaches and shortcomings: ① Exogenous selenium-added type: This involves directly adding sodium selenite or selenium-enriched yeast to ordinary milk (with extremely low selenium content). This method not only easily results in a metallic taste, but also leaves exogenous selenium in a free state in the milk system, leading to poor stability. ② Direct utilization of naturally selenium-enriched milk type: Although there are naturally selenium-enriched milk sources obtained through biotransformation (selenium content ≥9.0μg / 100mL), there are two pain points in industrial applications: First, their protein content is usually only around 3.2%, resulting in a thinner yogurt texture from direct fermentation, lacking the rich and creamy taste of high-end yogurt; second, due to insufficient protein substrate, the hydrolysis of organic selenoproteins by bifidobacteria during fermentation is insufficient, leading to limited activity of the released endogenous antioxidant peptides (selenium-peptides), failing to maximize the antioxidant potential of the milk source. ③ Limitations of single antioxidants: Vitamin C (VC) is easily degraded, plant polyphenols (such as tea polyphenols) can damage the milk protein network and cause whey to separate, and traditional antioxidants mostly act on the extracellular space and are difficult to remove intracellular reactive oxygen species (ROS).

[0003] Addressing the limitations of traditional antioxidants added to dairy products, L-ergothioneine (EGT), a naturally occurring chiral thiol, can enter cells via specific transport proteins to scavenge reactive oxygen species. Furthermore, it is proposed for inclusion in the list of new food ingredients (recommended daily intake ≤20mg), providing a compliant and efficient pathway for developing novel dual-antioxidant fermented milk products.

[0004] Therefore, there is an urgent need in this field to develop a high-end fermented milk product that can take advantage of the natural selenium-rich nature of native milk sources, enhance the density of protein and selenium through processing, and achieve dual "intracellular + extracellular" antioxidant effects in conjunction with ergothioneine (EGT), while also being texturally stable. Summary of the Invention

[0005] The purpose of this invention is to provide an antioxidant fermented milk and its preparation method, so as to realize the preparation of fermented dairy products with a high antioxidant system.

[0006] Therefore, the present invention provides the following technical solution.

[0007] The first aspect of the present invention provides an antioxidant fermented milk, wherein the raw materials for preparing the fermented milk, by weight percentage, comprise: 3-8% sweetener, 0.0005-0.002% L-ergothioneine, 0.1-0.5% thickener, 0.001-0.005% starter culture, and the balance being native selenium-enriched milk or native selenium-enriched concentrated milk after ultrafiltration membrane concentration treatment.

[0008] In a preferred embodiment of the present invention, the sweetener is a mixture of erythritol and white sugar in a weight ratio of (1-3):1.

[0009] In a preferred embodiment of the present invention, the thickener is composed of agar and pectin in a weight ratio of 1:(1-3).

[0010] In a preferred embodiment of the present invention, the fermentation agent comprises Lactobacillus bulgaricus, Streptococcus thermophilus, and Bifidobacterium, and the ratio of the number of viable Lactobacillus bulgaricus, Streptococcus thermophilus, and Bifidobacterium is (1-2):(1-2):(0.5-1).

[0011] In a preferred embodiment of the present invention, the Bifidobacterium is Bifidobacterium lactis subsp. animalis.

[0012] In a preferred embodiment of the present invention, the native selenium-enriched milk source is milk produced by dairy cows fed with hydroxymethionine selenium, with a protein content of 3.2g / 100g and a natural selenium content of ≥9.0μg / 100mL.

[0013] In a preferred embodiment of the present invention, the ultrafiltration conditions are: temperature 10-50°C, concentration factor of 1.5 times, and membrane molecular weight cutoff of 10kDa-30kDa.

[0014] In a preferred embodiment of the present invention, the ultrafiltration membrane material used is polyethersulfone or polyvinylidene fluoride.

[0015] In a preferred embodiment of the present invention, the antioxidant fermented milk has a protein content ≥3.0g / 100g, a selenium content ≥8.7μg / 100mL, an L-ergothioneine content of 0.5-2.0mg / 100g, and does not contain any exogenously added inorganic or organic selenium fortifiers.

[0016] A second aspect of the present invention provides a method for preparing antioxidant fermented milk, comprising the following steps: S1: Pretreatment: Standardize the raw selenium-enriched milk or raw selenium-enriched concentrated milk after ultrafiltration membrane concentration, add sweetener and thickener to mix and dissolve to obtain the liquid; S2: Homogenization: Heat the liquid to 60-65℃, perform the first stage of homogenization at 15-20MPa, and then perform the second stage of homogenization at 5-10MPa to obtain a homogenized liquid. S3: Sterilization and cooling: Sterilize the homogenized liquid at 90-95℃ for 5-10 minutes, and cool it to 42-44℃ to obtain the sterilized liquid; S4: Inoculation and addition of ergothioneine: Under aseptic conditions, inoculate the fermentation agent into the obtained sterilized liquid and simultaneously add an aqueous solution of ergothioneine, stir evenly to obtain a pre-fermentation liquid; S5: Constant temperature fermentation: Ferment the obtained pre-fermented liquid at 42-43℃ until the acidity reaches 70-75°T to obtain fermented milk; S6: Demulsification and Step-by-Step Cooling: The obtained fermented milk is slowly stirred to demulsify, first cooled to 18-20℃, then filled and placed in a cold storage at 2-6℃ for post-maturation for 12-24 hours to obtain the finished antioxidant fermented milk product.

[0017] In a preferred embodiment of the present invention, the ergothioneine aqueous solution has a mass concentration of 5-10% and is prepared by sterilization treatment through a 0.22μm microporous membrane.

[0018] In a preferred embodiment of the present invention, the antioxidant fermented milk exhibits a DPPH scavenging rate retention rate ≥90% after 21 days of storage, and a Bifidobacterium viable count ≥10. 8 CFU / mL.

[0019] This invention utilizes membrane concentration technology to prepare high-protein, high-selenium concentrated intermediate milk from native selenium-enriched milk, which is then used as a fermentation substrate. Simultaneously, L-ergothioneine is added, which consumes residual oxygen during fermentation and produces a synergistic effect with Bifidobacteria, jointly constructing a high-antioxidant system. The resulting fermented milk is high in protein, rich in selenium, and possesses high antioxidant properties. Specifically, the beneficial effects of this invention include: (1) Explosive activity resulting from substrate enrichment (high-protein substrate effect): This invention uses membrane concentration technology to increase the intermediate milk protein content to 4.8%, providing Bifidobacteria with a protein substrate far exceeding that of ordinary yogurt. At high substrate concentrations, the protease activity secreted by Bifidobacteria is enhanced, enabling deep hydrolysis of the enriched organic selenoproteins and releasing more bioactive "selenium-peptide complexes." This is the core reason why the DPPH clearance rate of this invention remains as high as 91.00% at the end of its shelf life, and its antioxidant activity far exceeds that of ordinary selenium-enriched yogurt.

[0020] (2) Dual Antioxidant Mechanism of "Intracellular + Extracellular": This invention achieves three-dimensional antioxidant protection: Extracellular / matrix defense: dominated by the selenium-peptide complex produced by fermentation, it scavenge environmental free radicals. Intracellular defense: the synergistically added L-ergothioneine enters the cell through the OCTN1 transporter protein to scavenge intracellular ROS. The combination of the two constructs a comprehensive defense system.

[0021] (3) Extreme safety (0 added selenium fortifiers): This invention relies entirely on high-quality milk source (≥9.0μg / 100mL) and does not add any exogenous selenium preparations, which not only ensures the natural organic form of selenium (easy to absorb and non-toxic) but also meets the market demand for high-end clean labels.

[0022] (4) Perfect Synergy Between EGT and Microbial Strains: If membrane filtration is chosen, the concentration is increased by 1.5 times, resulting in a selenium content exceeding 12.0 μg / 100mL (more than 130% of the national standard requirement for "rich in selenium"). Although the membrane concentration system is nutrient-rich, its high viscosity may affect microbial growth. The EGT added in this invention rapidly consumes residual oxygen in the system during inoculation, reducing the redox potential and creating an excellent anaerobic microenvironment for Bifidobacteria. Data shows that this significantly increases the viable count of Bifidobacteria at the fermentation endpoint and maintains extremely high activity in a high-viscosity system.

[0023] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it in accordance with the contents of the specification, the preferred embodiments of the present invention are described in detail below. Detailed Implementation

[0024] To make the technical means, creative features, achieved objectives, and effects of this invention readily understandable, the technical solutions in the embodiments of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are merely some embodiments of this invention, and 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.

[0025] The following embodiments involve and refer to the following: 1. Experimental Materials Naturally enriched selenium milk: protein content 3.2g / 100g, selenium content 9.5μg / 100mL; Plain milk protein: protein content 3.2g / 100g, selenium content 2.50μg / 100mL, organic selenium (food-grade hydroxymethionine selenium content is 40%).

[0026] Regular raw milk: protein content 3.2%, selenium content 2.5μg / 100mL.

[0027] L-ergothioneine: purity ≥98%, purchased from Jiangsu Jinsan Biotechnology Co., Ltd.

[0028] Compound fermentation agent: total viable count is 1.5 × 10⁻⁶ 12 CFU / g; of which, Lactobacillus bulgaricus + Streptococcus thermophilus: the basic starter culture of the YoFlex series, viable count 1.2 × 10⁻⁶. 12 CFU / g, derived from Danisco; Bifidobacterium animalis subsp. lactis: BB-12, viable count 3.0 × 10⁻⁶ 11 CFU / g, sourced from Chr. Hansen.

[0029] 2. Detection Method DPPH free radical scavenging rate determination method: Take 2 mL of sample supernatant and add 2 mL of 0.1 mmol / L DPPH-anhydrous ethanol solution, mix well and react in the dark for 30 min, and measure the absorbance at 517 nm (A1); use 2 mL of anhydrous ethanol instead of DPPH solution as a sample background blank tube to measure the absorbance (A2); use 2 mL of anhydrous ethanol instead of sample supernatant as a control tube to measure the absorbance (A0).

[0030] The clearance rate is calculated using the formula: [1 - (A1 - A2) / A0] × 100% Protein content: Kjeldahl method as per GB 5009.5.

[0031] Selenium content: ICP-MS method according to GB5009.93.

[0032] The following is a detailed description of specific embodiments.

[0033] Example 1: Antioxidant Fermented Milk I and its Preparation This embodiment provides an antioxidant fermented milk I, the formula of which (based on a total of 1000 kg) is: 30 kg of white sugar, 20 kg of erythritol, 1.2 kg of pectin, 0.8 kg of agar, 0.01 kg of L-ergothioneine, and 0.02 kg of starter culture (of which the ratio of live bacteria of Lactobacillus bulgaricus: Streptococcus thermophilus: Bifidobacterium animalis subsp. lactis is 1:1:0.5), and the original selenium-enriched milk is added to make up to 1000 kg.

[0034] The preparation method of this antioxidant fermented milk I includes the following steps: (1) Pretreatment: Standardize the original selenium-enriched milk, add white sugar, erythritol, pectin and agar and stir to dissolve at 40°C to obtain the liquid; (2) Two-stage homogenization: The liquid is heated to 65°C, and the first stage of homogenization is carried out at 20MPa, followed by the second stage of homogenization at 5MPa to obtain the homogenized liquid. (3) Sterilization and cooling: The homogenized liquid was sterilized at 95°C for 5 minutes, and then cooled to 43°C to obtain the sterilized liquid; (4) Inoculation and addition of ergothioneine: Prepare an aqueous solution of L-ergothioneine with a mass concentration of 10%, and sterilize it by passing it through a 0.22μm microporous membrane. Add it to the sterilized solution along with the fermentation agent and stir evenly to obtain a pre-fermentation solution. (5) Constant temperature fermentation: The obtained pre-fermented liquid was fermented at 43℃ for 4.2h until the acidity was 75°T to obtain fermented milk; (6) Post-maturation: The obtained fermented milk is slowly stirred to break the emulsion, cooled to 18°C ​​and filled, and then placed in a 4°C cold storage for post-maturation for 24 hours to obtain the antioxidant fermented milk product.

[0035] Example 2: Antioxidant Fermented Milk II and its Preparation This embodiment provides an antioxidant fermented milk II, the formula of which (based on a total of 1000 kg) is as follows: 30 kg of white sugar, 20 kg of erythritol, 1.2 kg of pectin, 0.8 kg of agar, 0.01 kg of L-ergothioneine, and 0.02 kg of starter culture (of which the ratio of live bacteria Lactobacillus bulgaricus: Streptococcus thermophilus: Bifidobacterium animalis subsp. lactis is 1:1:0.5), and the original selenium-enriched concentrated milk is added to make up to 1000 kg.

[0036] The preparation method of this antioxidant fermented milk II includes the following steps: Pretreatment of raw selenium-enriched milk: Raw selenium-enriched milk was selected and concentrated by ultrafiltration (temperature 40℃, membrane molecular weight cutoff of 10kDa polyethersulfone ultrafiltration membrane). The volumetric concentration ratio (VCR) was set to 1.5 times to obtain raw selenium-enriched concentrated milk (indicators: protein 3.2%×1.5=4.8%; selenium = 9.5×1.5=14.25μg / 100mL).

[0037] Pretreatment: Take the original selenium-enriched concentrated milk, add white sugar, erythritol, pectin and agar, and stir to dissolve at 40℃ to obtain the liquid.

[0038] Two-stage homogenization: The liquid is heated to 65°C, and the first stage of homogenization is carried out at 20MPa, followed by the second stage of homogenization at 5MPa to obtain a homogenized liquid. (4) Sterilization and cooling: The homogenized liquid was sterilized at 95°C for 5 minutes, and then cooled to 43°C to obtain the sterilized liquid; (5) Inoculation and addition of ergothioneine: Prepare an aqueous solution of L-ergothioneine with a mass concentration of 10%, sterilize it by passing it through a 0.22μm microporous membrane, add it together with the fermentation agent into the sterilized solution and stir evenly to obtain a pre-fermentation solution; (6) Constant temperature fermentation: The obtained pre-fermented liquid was fermented at 43℃ for 4.2h until the acidity was 75°T to obtain fermented milk; (7) Post-maturation: The obtained fermented milk is slowly stirred to break the emulsion, cooled to 18°C ​​and filled, and then placed in a 4°C cold storage for post-maturation for 24 hours to obtain the antioxidant fermented milk product.

[0039] Example 3: Antioxidant Fermented Milk III and its Preparation This embodiment provides an antioxidant fermented milk III, the formula of which (based on a total of 1000 kg) is: 7.5 kg of white sugar, 22.5 kg of erythritol, 0.75 kg of pectin, 0.25 kg of agar, 0.005 kg of L-ergothioneine, 0.01 kg of starter culture, and supplemented with native selenium-enriched concentrated milk to bring the total to 1000 kg.

[0040] The preparation method of this antioxidant fermented milk III includes the following steps: Pretreatment of raw selenium-enriched milk: Raw selenium-enriched milk is selected and concentrated by ultrafiltration (temperature 10℃, membrane molecular weight cutoff of 10kDa polyethersulfone ultrafiltration membrane), and the volume concentration ratio (VCR) is set to 1.5 times to obtain raw selenium-enriched concentrated milk.

[0041] (2) Pretreatment: Take the original selenium-enriched concentrated milk, add white sugar, erythritol, pectin and agar, stir and dissolve at 40°C to obtain the liquid; (3) Two-stage homogenization: The liquid is heated to 60°C, and the first stage of homogenization is carried out at 15MPa, followed by the second stage of homogenization at 5MPa to obtain the homogenized liquid. (4) Sterilization and cooling: The homogenized liquid was sterilized at 90°C for 10 min, and then cooled to 42°C to obtain the sterilized liquid; (5) Inoculation and addition of ergothioneine: Prepare an aqueous solution of L-ergothioneine with a mass concentration of 5%, and sterilize it by passing it through a 0.22μm microporous membrane. Add it to the sterilized solution along with the fermentation agent and stir evenly to obtain a pre-fermentation solution. (6) Constant temperature fermentation: The obtained pre-fermented liquid is fermented at 42℃ until the acidity is 70°T to obtain fermented milk; (7) Post-maturation: The obtained fermented milk is slowly stirred to break the emulsion, cooled to 18°C ​​and filled, and then placed in a 2°C cold storage for post-maturation for 24 hours to obtain the antioxidant fermented milk product.

[0042] Example 4: Antioxidant Fermented Milk IV and its Preparation This embodiment provides an antioxidant fermented milk IV, the formula of which (based on a total of 1000 kg) is: 20 kg of white sugar, 60 kg of erythritol, 3.75 kg of pectin, 1.25 kg of agar, 0.02 kg of L-ergothioneine, 0.05 kg of starter culture, and supplemented with native selenium-enriched concentrated milk to bring the total to 1000 kg.

[0043] The preparation method of this antioxidant fermented milk IV includes the following steps: (1) Pretreatment of raw selenium-enriched milk: Raw selenium-enriched milk was selected and concentrated by ultrafiltration (temperature 50℃, membrane molecular weight cutoff 30kDa polyethersulfone ultrafiltration membrane), and the volume concentration ratio (VCR) was set to 1.5 times to obtain raw selenium-enriched concentrated milk.

[0044] (2) Pretreatment: Take the original selenium-enriched concentrated milk, add white sugar, erythritol, pectin and agar, stir and dissolve at 40°C to obtain the liquid; (3) Two-stage homogenization: The liquid is heated to 65°C, and the first stage of homogenization is carried out at 20MPa, followed by the second stage of homogenization at 10MPa to obtain the homogenized liquid. (4) Sterilization and cooling: The homogenized liquid was sterilized at 95°C for 5 minutes, and then cooled to 44°C to obtain the sterilized liquid; (5) Inoculation and addition of ergothioneine: Prepare an aqueous solution of L-ergothioneine with a mass concentration of 10%, sterilize it by passing it through a 0.22μm microporous membrane, add it together with the fermentation agent into the sterilized solution and stir evenly to obtain a pre-fermentation solution; (6) Constant temperature fermentation: The obtained pre-fermented liquid is fermented at 43°C until the acidity is 75°T to obtain fermented milk; (7) Post-maturation: The obtained fermented milk is slowly stirred to break the emulsion, cooled to 20°C and filled, and then placed in a 6°C cold storage for post-maturation for 12 hours to obtain the antioxidant fermented milk product.

[0045] Comparative Example 1: Fermented milk without ergothionein and its preparation This comparative example provides a fermented milk without ergothioneine, with the following formula (based on a total volume of 1000 kg): 30 kg white sugar, 20 kg erythritol, 1.2 kg pectin, 0.8 kg agar, and 0.02 kg starter culture (of which the ratio of live bacteria Lactobacillus bulgaricus: Streptococcus thermophilus: Bifidobacterium animalis subsp. lactis is 1:1:0.5), supplemented with native selenium-enriched milk to bring the total to 1000 kg.

[0046] The method for preparing this fermented milk without ergothioneine includes the following steps: (1) Pretreatment: Standardize the selenium-enriched raw milk, add white sugar, erythritol, pectin and agar and stir to dissolve at 40°C to obtain the liquid; (2) Two-stage homogenization: The liquid is heated to 65°C, and the first stage of homogenization is carried out at 20MPa, followed by the second stage of homogenization at 5MPa to obtain the homogenized liquid. (3) Sterilization and cooling: The homogenized liquid was sterilized at 95°C for 5 minutes, and then cooled to 43°C to obtain the sterilized liquid; (4) Inoculation and addition: Add the fermenting agent to the sterilized liquid and stir evenly to obtain the pre-fermentation liquid; (5) Constant temperature fermentation: The obtained pre-fermented liquid was fermented at 43℃ for 4.2h until the acidity was 75°T to obtain fermented milk; (6) Post-maturation: The obtained fermented milk is slowly stirred to break the emulsion, cooled to 18°C ​​and filled, and then placed in a 4°C cold storage for post-maturation for 24 hours to obtain the finished fermented milk product.

[0047] Comparative Example 2: Fermented milk containing ordinary milk source and its preparation This comparative example provides a fermented milk containing ordinary milk source, the formula of which (based on a total of 1000 kg) is: 30 kg of white sugar, 20 kg of erythritol, 1.2 kg of pectin, 0.8 kg of agar, 0.01 kg of L-ergothioneine, and 0.02 kg of starter culture (of which the ratio of live bacteria of Lactobacillus bulgaricus: Streptococcus thermophilus: Bifidobacterium animalis subsp. lactis is 1:1:0.5), and ordinary milk source is supplemented to 1000 kg.

[0048] The preparation method of this fermented milk containing ordinary milk source includes the following steps: (1) Pretreatment: Standardize ordinary raw milk, add white sugar, erythritol, pectin and agar and stir to dissolve at 40°C to obtain the liquid; (2) Two-stage homogenization: The liquid is heated to 65°C, and the first stage of homogenization is carried out at 20MPa, followed by the second stage of homogenization at 5MPa to obtain the homogenized liquid. (3) Sterilization and cooling: The homogenized liquid was sterilized at 95°C for 5 minutes, and then cooled to 43°C to obtain the sterilized liquid; (4) Inoculation and addition of ergothioneine: Prepare an aqueous solution of L-ergothioneine with a mass concentration of 10%, and sterilize it by passing it through a 0.22μm microporous membrane. Add it to the sterilized solution along with the fermentation agent and stir evenly to obtain a pre-fermentation solution. (5) Constant temperature fermentation: The obtained pre-fermented liquid was fermented at 43℃ for 4.2h until the acidity was 75°T to obtain fermented milk; (6) Post-maturation: The obtained fermented milk is slowly stirred to break the emulsion, cooled to 18°C ​​and filled, and then placed in a 4°C cold storage for post-maturation for 24 hours to obtain the finished fermented milk product.

[0049] Comparative Example 3: Fermented Milk and its Preparation This comparative example provides a fermented milk with the following formula (based on a total volume of 1000 kg): 30 kg white sugar, 20 kg erythritol, 1.2 kg pectin, 0.8 kg agar, 165 g organic selenium (hydroxymethionine selenium), 0.01 kg L-ergothioneine, and 0.02 kg starter culture (of which the ratio of live bacteria Lactobacillus bulgaricus: Streptococcus thermophilus: Bifidobacterium animalis subsp. lactis is 1:1:0.5), with ordinary raw milk added to bring the total to 1000 kg.

[0050] The preparation method of this fermented milk includes the following steps: (1) Pretreatment: Standardize ordinary raw milk, add white sugar, erythritol, pectin, agar and organic selenium and dissolve at 40°C to obtain a liquid; (2) Two-stage homogenization: The liquid is heated to 65°C, and the first stage of homogenization is carried out at 20MPa, followed by the second stage of homogenization at 5MPa to obtain the homogenized liquid. (3) Sterilization and cooling: The homogenized liquid was sterilized at 95°C for 5 minutes, and then cooled to 43°C to obtain the sterilized liquid; (4) Inoculation and addition of ergothioneine: Prepare an aqueous solution of L-ergothioneine with a mass concentration of 10%, and sterilize it by passing it through a 0.22μm microporous membrane. Add it to the sterilized solution along with the fermentation agent and stir evenly to obtain a pre-fermentation solution. (5) Constant temperature fermentation: The obtained pre-fermented liquid was fermented at 43℃ for 4.2h until the acidity was 75°T to obtain fermented milk; (6) Post-maturation: The obtained fermented milk is slowly stirred to break the emulsion, cooled to 18°C ​​and filled, and then placed in a 4°C cold storage for post-maturation for 24 hours to obtain the antioxidant fermented milk product.

[0051] Experimental Example 1: Performance Testing of Fermented Milk The fermented milk prepared in Examples 1-4 and Comparative Examples 1-3 were tested for nutritional components, antioxidant properties, and other indicators. The results are shown in Table 1.

[0052] Table 1 Results of Fermented Milk Performance Tests ; As shown in Table 1, regarding antioxidant performance, the DPPH scavenging rate of the fermented milk in Example 1 remained at 83.00% (retention rate >90%) on day 21, while that of the fermented milk in Comparative Example 1 (ergothioneine-free) decreased to 65.00%. This indicates that ergothioneine provides strong and sustained antioxidant capacity in the selenium-enriched milk system. Regarding probiotic survival, at the end of the 21-day shelf life, the number of viable Bifidobacteria in the fermented milk of Example 1 was 1.0 × 10⁻⁶. 8 CFU / mL, Comparison Example 1 (5.0 × 10⁻⁶) 7The DPPH scavenging rate of Example 1 was twice that of Comparative Example 3 (CFU / mL). This indicates that ergothioneine mitigated the damage to probiotics caused by oxidative stress to some extent, while maintaining a lower acidity of 102°T on day 21, which helped delay post-acidification. Regarding milk source selection, the DPPH scavenging rate of the fermented milk in Example 1 (83.00%) was significantly higher than that of Comparative Example 2 (52.00%), and the number of viable Bifidobacteria was also significantly higher. This suggests that ordinary milk sources, due to their low natural selenium background (only 2.4 μg / 100mL in the finished product), lack sufficient selenoprotein synergy, thus limiting the function of ergothioneine. When using native selenium-enriched milk to provide sufficient natural selenoprotein, the antioxidant and antibacterial effects of ergothioneine are more fully realized. Further comparing Example 1 and Comparative Example 3, with the same total selenium content (both 9.0 μg / 100mL), the DPPH scavenging rate of Example 1 on day 21 (83.00%) was higher than that of Comparative Example 3 (68.00%). The reason for this is speculated to be that the selenium in naturally occurring selenium-enriched milk is bound to the three-dimensional structure of milk protein in the form of natural selenoamino acids via peptide bonds, which can be released through targeted hydrolysis during deep fermentation by Bifidobacteria; while the added chemically synthesized hydroxymethionine selenium exists in a free or freely adsorbed state in the milk system, and cannot form an equally efficient synergistic antioxidant effect. Furthermore, the number of viable Bifidobacteria in the fermented milk of Example 1 on day 21 was 1.0 × 10⁻⁶. 8 The CFU / mL ratio was significantly better than that of Comparative Example 3 (4.5 × 10⁻⁶ CFU / mL). 7 (CFU / mL), further confirming the irreplaceable role of natural selenium sources in maintaining system stability and probiotic activity.

[0053] Normally, membrane concentration systems, due to the sudden increase in viscosity and osmotic pressure, exert a stress-inhibiting effect on the growth and metabolic activity of Bifidobacteria, leading to a decrease in the viable count at the fermentation endpoint. However, in Example 2 of this invention, after the milk source was concentrated 1.5 times, the viable count of Bifidobacteria on day 21 not only did not decrease, but actually increased compared to the unconcentrated Example 1 (1.0 × 10⁻⁶). 8 The CFU / mL level defied the trend and increased to 1.5 × 10⁻⁶. 8 The CFU / mL concentration and DPPH scavenging rate approached a high level of 91%. This indicates that ergothioneine not only counteracted the stress of the high-viscosity system on the microorganisms but also formed a denser synergistic antioxidant network with the high concentration of natural selenoproteins. Further comparison of the data from Examples 2, 3, and 4 shows that the concentration factor and process parameters are not simply linearly positively correlated with system performance. Although Example 3 had the highest protein and selenium content, its DPPH scavenging rate on day 21 (87.00%) was lower than that of Example 2 (91.00%), and the viable cell count also decreased compared to Example 2. Although Example 4 had a viable cell count on day 21 (1.2 × 10⁻⁶), the DPPH scavenging rate was lower. 8The CFU / mL concentration remained at a high level, and the DPPH scavenging rate (94.00%) was slightly higher than that of Example 2, but the selenium content of the finished product (13.0 μg / 100mL) was lower than that of Example 2. This indicates that in a concentrated system, excessively high substrate concentrations or specific process parameters (such as high-temperature / high-pressure homogenization) may have a certain diminishing marginal effect on the metabolism of probiotics or the synergistic effect of the antioxidant system. Example 2 used a 1.5x concentration process, achieving an optimal system balance between nutrient density, antioxidant performance, and probiotic survival rate. This counter-cyclical synergistic effect and system balance at high substrate concentrations constitute the unexpected technical effect of this invention. The DPPH scavenging rate on day 21 of Example 1 (83.00%) was significantly different from that of Comparative Example 3 on day 21 (68.00%). This indicates that the synergistic antioxidant effect achieved by the present invention is closely related to the combined use of selenoproteins with natural structures in native selenium-enriched milk and ergothioneine. In contrast, exogenously added chemical selenium, due to its different form and mechanism of action, has failed to exhibit the same level of synergistic antioxidant effect in the system of the present invention.

[0054] Experimental Example 2: Sensory Evaluation of Different Fermented Milks According to ISO 13299:2016 "Sensory analysis—Methodology—Quantitative descriptive analysis", a 1-5 scoring system (1 = very weak / very poor, 5 = very strong / very good) was used to evaluate the fermented milk samples of Examples 1-4 and Comparative Examples 1-3. Ten trained food sensory evaluators were invited to score the color, aroma, taste, mouthfeel (texture), and texture of the samples. The scores for each indicator were averaged by the ten evaluators. The results are shown in Table 2.

[0055] Note: Samples should be refrigerated at 4℃ for 24 hours and then evaluated at 18-20℃ to avoid the influence of temperature on sensory perception.

[0056] Table 2 Sensory ratings and descriptions of fermented milk ; As shown in Table 2, Example 2 performed better in color (4.8 points), aroma (4.9 points), taste (4.7 points), mouthfeel (4.8 points), and texture (4.9 points), with a higher overall score than other examples and comparative examples. In comparison, although Examples 3 and 4 each have their own style (e.g., Example 3 has a fresh milky aroma and refreshing taste, while Example 4 has a thick texture), they are slightly inferior to Example 2 in terms of overall flavor harmony. Example 1, due to its lack of concentration, has a slightly thinner mouthfeel and whey separation in its texture, resulting in a sensory performance inferior to Example 2.

[0057] In the comparative groups, the differences in components had a significant impact on sensory quality: Comparative Example 1 (without EGT) scored low, possibly due to the lack of antioxidant protection leading to flavor degradation, a weaker aroma, and a bitter taste; Comparative Example 2 (regular milk + EGT) had added EGT, but due to the limitations of regular milk base, the aroma was relatively bland, which may be related to its lack of sufficient natural selenoproteins as aroma co-factors; Comparative Example 3 (regular milk + organic selenium + EGT) exhibited a distinct metallic off-flavor and had the lowest sensory score, which is likely related to the form in which exogenous organic selenium was introduced.

[0058] The sensory evaluation results above indicate that the "concentrated milk + EGT" synergistic solution adopted in Example 2 not only improves the product texture by increasing the density of protein and selenium, but may also optimize the aroma and taste to a certain extent through the antioxidant effect of EGT and the synergy of high protein substrates, effectively reducing the risk of off-flavors, and showing comprehensive advantages in sensory quality.

[0059] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the methods and techniques disclosed above without departing from the scope of the present invention to create equivalent embodiments. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. An antioxidant fermented milk, characterized in that, The raw materials for preparing the fermented milk, by weight percentage, include: 3-8% sweetener, 0.0005-0.002% L-ergothioneine, 0.1-0.5% thickener, 0.001-0.005% starter culture, and the balance being native selenium-enriched milk or native selenium-enriched concentrated milk after ultrafiltration membrane concentration treatment.

2. The antioxidant fermented milk according to claim 1, characterized in that, The sweetener is a mixture of erythritol and white sugar in a weight ratio of (1-3):

1.

3. The antioxidant fermented milk according to claim 1, characterized in that, The thickener is composed of agar and pectin in a weight ratio of 1:(1-3).

4. The antioxidant fermented milk according to claim 1, characterized in that, The starter culture contains Lactobacillus bulgaricus, Streptococcus thermophilus, and Bifidobacterium, and the ratio of the number of viable Lactobacillus bulgaricus, Streptococcus thermophilus, and Bifidobacterium is (1-2):(1-2):(0.5-1).

5. The antioxidant fermented milk according to claim 4, characterized in that, The Bifidobacterium is Bifidobacterium lactis subspecies.

6. The antioxidant fermented milk according to claim 1, characterized in that, The original selenium-enriched milk source is milk produced by dairy cows fed with hydroxymethionine selenium, with a protein content of 3.2g / 100g and a natural selenium content of ≥9.0μg / 100mL.

7. The antioxidant fermented milk according to claim 1, characterized in that, The ultrafiltration conditions are: temperature 10-50℃, concentration factor 1.5 times, and membrane molecular weight cutoff of 10kDa-30kDa; the ultrafiltration membrane material used is polyethersulfone or polyvinylidene fluoride.

8. The antioxidant fermented milk according to claim 1, characterized in that, The antioxidant fermented milk has a protein content ≥3.0g / 100g, a selenium content ≥8.7μg / 100mL, and an L-ergothioneine content of 0.5-2.0mg / 100g, and does not contain any exogenously added inorganic or organic selenium fortifiers.

9. The method for preparing antioxidant fermented milk according to any one of claims 1-8, characterized in that, Includes the following steps: S1： Pretreatment: After standardizing the original selenium-enriched milk or the original selenium-enriched concentrated milk after ultrafiltration membrane concentration, add sweetener and thickener to mix and dissolve to obtain the liquid. S2: Homogenization: Heat the liquid to 60-65℃, perform the first stage of homogenization at 15-20MPa, and then perform the second stage of homogenization at 5-10MPa to obtain a homogenized liquid. S3: Sterilization and cooling: Sterilize the homogenized liquid at 90-95℃ for 5-10 minutes, and cool it to 42-44℃ to obtain the sterilized liquid; S4: Inoculation and addition of ergothioneine: Under aseptic conditions, inoculate the fermentation agent into the obtained sterilized liquid and simultaneously add an aqueous solution of ergothioneine, stir evenly to obtain a pre-fermentation liquid; S5: Constant temperature fermentation: Ferment the obtained pre-fermented liquid at 42-43℃ until the acidity reaches 70-75°T to obtain fermented milk; S6: Demulsification and Step-by-Step Cooling: The obtained fermented milk is slowly stirred to demulsify, first cooled to 18-20℃, then filled and placed in a cold storage at 2-6℃ for post-maturation for 12-24 hours to obtain the finished antioxidant fermented milk product.

10. The preparation method according to claim 9, characterized in that, The ergothioneine aqueous solution has a mass concentration of 5-10% and is prepared by sterilization treatment through a 0.22μm microporous membrane.