A selenium-enriched lactic acid bacteria drink and a preparation method thereof

Abstract

The present application relates to the technical field of fermented dairy products, and particularly relates to a selenium-rich lactic acid bacteria drink and a preparation method thereof.The present application provides a selenium-rich lactic acid bacteria drink, which comprises the following raw material components by weight: 25-65 parts of cow milk, 0.1-2 parts of walnut peptide, 0.01-0.1 parts of first fermentation inoculum, and 0.08-0.16 parts of second fermentation inoculum, wherein the second fermentation inoculum is selenium-rich swiss lactic acid bacteria.The present application uses selenium-rich swiss lactic acid bacteria as a carrier to bring selenium into the selenium-rich lactic acid drink, thereby improving the selenium content in the selenium-rich lactic acid drink, and the selenium content is greater than or equal to 8.0 mu g / 100 g, which is much higher than the selenium content of traditional dairy products; meanwhile, the taste, flavor, texture stability and shelf life of the drink are significantly improved, and the influence of the addition of selenium in the existing lactic acid bacteria drink on the fermentation performance is overcome.

Description

Technical Field

[0001] This invention relates to the field of fermented dairy product technology, and in particular to a selenium-enriched lactic acid bacteria beverage and its preparation method. Background Technology

[0002] Lactic acid bacteria beverages are fermented acidic milk-containing beverages, usually made from milk or dairy products as raw materials. The milk is sterilized, cooled, and fermented with lactic acid bacteria to obtain an emulsion, which is then mixed with other ingredients to make the beverage. The protein content of the product is not less than 0.7g / 100g or 100ml.

[0003] Selenium is an essential trace element for the human body, possessing various functions such as antioxidation, antibacterial properties, and immune enhancement. However, the human body cannot synthesize it and must obtain it from external sources. Currently available dairy products have low selenium content, far below the Chinese Dietary Reference Intake (60 μg / day). Furthermore, the selenium content in natural foods is relatively low, and most selenium-enriched foods on the market achieve a certain selenium level through additives or biochemical conversion. Bioconversion carries certain risks; for example, incomplete conversion of inorganic selenium to organic selenium can lead to inorganic selenium residues with high toxicity.

[0004] Chinese Patent CN 104255916 A discloses a home-based production method for selenium-enriched yogurt, comprising the following steps: (1) preparing a sodium selenite aqueous solution; (2) sterilizing fresh milk, and adding the sodium selenite aqueous solution to the sterilized fresh milk to obtain mixed raw milk, wherein the amount of sodium selenite aqueous solution added is 10-2500 μg of selenium per liter of fresh milk, and the fresh milk is fresh cow's milk or fresh goat's milk; (3) adding 5-15g of probiotic powder to the mixed raw milk to obtain raw milk fermentation material; (4) fermenting the raw milk fermentation material at 38-43℃ for 4-10 hours to obtain the finished product. This invention has the advantages of simple process, low cost and short preparation time, but the addition of inorganic sodium selenite has low bioavailability and high toxicity, posing a food safety risk.

[0005] Chinese patent CN 107873839 A discloses a method for preparing selenium-enriched tea yogurt. This method involves extracting selenium from tea leaves rich in organic selenium to obtain a selenium-enriched tea extract. This extract is then mixed with pretreated fresh milk and fermented to obtain selenium-enriched tea yogurt. In this invention, the amount of selenium-enriched tea extract added is 0.3% of the weight of the fresh milk. Another invention involves soaking tea leaves in hot water and then freeze-drying them to obtain an extract, which is then added to milk to make selenium-enriched yogurt. However, hot water extraction results in a relatively low selenium content that can be dissolved from the tea leaves.

[0006] In addition, based on people's requirements for health and / or taste, new types of lactic acid bacteria beverages are being created by adding different flavored nutrients to lactic acid bacteria beverages, which are becoming a development trend. Some of these lactic acid bacteria beverages contain added vitamins, minerals, fruit juices, or some amino acids, peptides, or functional factors with health benefits.

[0007] Walnut protein has been prepared into bioactive peptide products of varying fragment sizes using enzymatic hydrolysis technology, attracting widespread attention due to its diverse bioactivities. Studies have shown that walnut peptides possess multiple effects such as scavenging free radicals, relieving fatigue, and improving memory. A typical example of commercially available walnut-related dairy products involves grinding walnut kernels and adding them to milk, then inoculating with lactic acid bacteria for fermentation. Directly grinding walnut kernels results in a high oil content, which not only affects the stability of the final dairy product system but also leads to poor solubility and lack of bioactivity of the walnut protein in the walnut paste, impacting product uniformity. Furthermore, the large molecular weight of walnut protein makes it difficult for the human body to digest and absorb. Summary of the Invention

[0008] The technical problem to be solved by the present invention is to provide a selenium-enriched lactic acid bacteria beverage and its preparation method. To achieve the above objective, the present invention provides the following technical solution:

[0009] In a first aspect, the present invention provides a selenium-enriched lactic acid bacteria beverage, comprising the following raw materials in parts by weight: 25-65 parts of milk, 0.1-2 parts of walnut peptide, 0.01-0.1 parts of a first fermentation agent, and 0.08-0.16 parts of a second fermentation agent, wherein the second fermentation agent is selenium-enriched Lactobacillus helveticus.

[0010] In one embodiment of the present invention, the selenium-enriched lactic acid bacteria beverage comprises the following raw materials in parts by weight: 35-65 parts of milk, 0.1-2 parts of walnut peptide, 0.01-0.1 parts of first fermentation agent, and 0.08-0.16 parts of second fermentation agent, wherein the second fermentation agent is selenium-enriched Lactobacillus helveticus.

[0011] In one embodiment of the present invention, the selenium-enriched lactic acid bacteria beverage comprises the following raw materials in parts by weight: 35-40 parts of milk, 0.1-1 parts of walnut peptide, 0.08-0.1 parts of first fermentation agent, and 0.08-0.15 parts of second fermentation agent, wherein the second fermentation agent is selenium-enriched Lactobacillus helveticus.

[0012] In one embodiment of the present invention, the selenium-enriched Lactobacillus helveticus is prepared by fermentation culture of Lactobacillus helveticus in a selenium-containing medium; preferably, the Lactobacillus helveticus is Lactobacillus helveticus Zhegu LBH-VI, wherein Lactobacillus helveticus Zhegu LBH-VI was deposited at the China Center for Type Culture Collection (CCTCC) on February 7, 2023, with accession number CCTCC NO: M 2023095.

[0013] In one embodiment of the present invention, the organic selenium content in the selenium-enriched Lactobacillus helveticus is 100-125 μg / g.

[0014] In one embodiment of the present invention, the selenium content in the selenium-enriched lactic acid bacteria beverage is ≥8.0 μg / 100g based on its weight; preferably, the number of live bacteria in the selenium-enriched lactic acid bacteria beverage is ≥10 during its shelf life. 9 CFU / ml.

[0015] In one embodiment of the present invention, the milk is selected from one or more of whole milk, low-fat milk, or skim milk.

[0016] In one embodiment of the present invention, the molecular weight of the walnut peptide is 800-1000 Da; preferably, the walnut peptide is obtained by enzymatic hydrolysis of walnuts with protease.

[0017] In one embodiment of the present invention, the first fermentation agent is selected from one or more of Streptococcus salivarius subsp. thermophilus, Lactobacillus delbrueckii subsp. lactis, Bifidobacterium lactis, Lactococcus lactis subsp. lactis, and Lactobacillus paracasei, preferably Streptococcus salivarius subsp. thermophilus and Lactobacillus delbrueckii subsp. lactis, and more preferably, the mass ratio of Streptococcus salivarius subsp. thermophilus and Lactobacillus delbrueckii subsp. lactis is 2-3:1-1.5 based on the dry matter of the bacteria.

[0018] In one embodiment of the present invention, the thermophilic subsp. *Streptococcus salivarias* 932 is deposited at the China Center for Type Culture Collection (CCTCC) on June 5, 2023, with accession number CCTCC NO: M 2023902.

[0019] And / or, the Lactobacillus delbrueckii subsp. lactis Dangxiong LB VⅢ is deposited at the China Center for Type Culture Collection (CCTCC) on March 23, 2023, with accession number CCTCC NO: M2023396;

[0020] And / or, the lactococcus lactis subsp. Lactis 954 is a lactococcus lactis subsp. Lactis 954, which was deposited at the China Center for Type Culture Collection (CCTCC) on June 5, 2023, with accession number CCTCC NO: M2023904.

[0021] And / or, the Lactobacillus paracasei is Lactobacillus paracasei ALI Plateau LPA-Ⅰ.

[0022] In one embodiment of the present invention, the raw materials further include 5.0-8.5 parts of sweetener;

[0023] Preferably, the sweetener is selected from one or more of sucrose, erythritol, xylitol, maltitol, sucralose, or steviol glycosides, with sucrose being the most preferred.

[0024] In one embodiment of the present invention, the raw material further includes 0.3-0.7 parts of a stabilizer;

[0025] Preferably, the stabilizer is selected from one or more of sodium carboxymethyl cellulose, pectin, sodium tripolyphosphate, and sodium citrate;

[0026] More preferably, the mass ratio of sodium carboxymethyl cellulose, pectin, sodium tripolyphosphate and sodium citrate is 0.5-1.3:1.5-2.5:0.2-0.5:0.3-0.7.

[0027] In one embodiment of the present invention, the raw materials further include 0.11-0.6 parts of an acidulant;

[0028] Preferably, the acidulant is selected from citric acid, lactic acid and sodium citrate, and the mass ratio of citric acid, lactic acid and sodium citrate is preferably 5-25:5-15:1-20.

[0029] In one embodiment of the present invention, the selenium-enriched lactic acid bacteria beverage is prepared by temperature-switching fermentation of a fermentation base comprising milk and walnut peptides.

[0030] Secondly, the present invention also provides a method for preparing the above-mentioned selenium-enriched lactic acid bacteria beverage, comprising the following steps:

[0031] (1) Mix raw materials including milk and walnut peptides to prepare a fermentation base;

[0032] (2) The fermentation substrate obtained in step (1) is sequentially inoculated with the first fermentation agent and the second fermentation agent to carry out variable temperature fermentation to obtain fermented milk.

[0033] (3) The fermented milk obtained in step (2) is blended to obtain a selenium-enriched lactic acid bacteria beverage.

[0034] In one embodiment of the present invention, in the above preparation method, the formulation in step (3) includes adding stabilizers and / or acidulants to the selenium-enriched lactic acid bacteria beverage obtained in step (2) for mixing and homogenization.

[0035] In one embodiment of the present invention, the fermentation base material further includes a sweetener in the above preparation method. Preferably, the sweetener is selected from one or more of sucrose, erythritol, xylitol, maltitol, sucralose or steviol glycosides, with sucrose being the most preferred.

[0036] In one embodiment of the present invention, in the above preparation method, the step (2) of sequentially inoculating the first fermentation agent and the second fermentation agent for variable temperature fermentation includes first inoculating the first fermentation agent and fermenting at 40-45°C, and then inoculating the second fermentation agent and fermenting at 35-39°C.

[0037] Preferably, the first fermentation agent is first inoculated and fermented at 40-45℃ for 10-12 hours until the final acidity is 90-100°T, and then the second fermentation agent is inoculated and fermented at 35-39℃ for 8-10 hours until the final acidity is 160-180°T.

[0038] In one embodiment of the present invention, the above preparation method further includes a step of homogenizing the fermentation substrate before inoculation, preferably at a homogenization temperature of 50-65°C, and more preferably at a homogenization pressure of 10-50 MPa.

[0039] Preferably, the homogenization process further includes a step of sterilizing the fermentation substrate, with a preferred sterilization temperature of 90-100℃ and a more preferred sterilization time of 200-400s.

[0040] In one embodiment of the present invention, the above preparation method further includes a step of stirring and breaking the emulsion after the first fermentation and after the second fermentation to obtain fermented milk. Preferably, the stirring speed is 20-40 r / min, and more preferably, the stirring time is 10-20 min.

[0041] The beneficial effects of this invention are:

[0042] 1. This invention uses selenium-enriched Lactobacillus helveticus as a carrier to introduce selenium into the fermentation process of selenium-enriched lactic acid beverages, thereby increasing the selenium content in the beverages. The resulting lactic acid bacteria beverages contain a selenium content greater than or equal to 8.0 μg / 100g, which is much higher than the selenium content of traditional dairy products (≤0.6 μg / 100g). At the same time, the taste, flavor, textural stability, and shelf-life viable bacteria count of the selenium-enriched lactic acid beverages are significantly improved, overcoming the impact of selenium addition on fermentation performance in existing lactic acid bacteria beverages.

[0043] 2. This invention uses milk and walnut peptides, which have fatigue-relieving effects, as fermentation bases to produce lactic acid bacteria beverages. Animal experiments have shown that the lactic acid bacteria beverages prepared by this invention have significant fatigue-relieving effects.

[0044] Strain description

[0045] The *Lactobacillus helveticus* Zhegu LBH-VI used in this invention was deposited at the China Center for Type Culture Collection (CCTCC) on February 7, 2023, with accession number CCTCC NO: M 2023095. The deposit address is: Wuhan University, Wuhan, China, Postcode: 430072; Telephone: (027)-68754052.

[0046] The *Streptococcus salivarias* subsp. *thermophilus* 932 used in this invention was deposited on June 5, 2023, at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO: M 2023902, address: Wuhan University, Wuhan, China, postcode: 430072; telephone: (027)-68754052.

[0047] The *Lactobacillus delbrueckii* subsp. *lactis* Dangxiong LB VⅢ used in this invention was deposited on March 23, 2023, at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO: M2023396, address: Wuhan University, Wuhan, China, postcode: 430072; telephone: (027)-68754052.

[0048] The *Lactococcus lactis* subsp. *Lactis 954* used in this invention was deposited on June 5, 2023, at the China Center for Type Culture Collection (CCTCC), with accession number CCTCC NO: M2023904, address: Wuhan University, Wuhan, China, postcode: 430072; telephone: (027)-68754052.

[0049] The *Lactobacillus paracasei* AL1 Plateau LPA-1 used in this invention was deposited on October 25, 2021, at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO: M20211312, address: Wuhan University, Wuhan, China, postcode: 430072; telephone: (027)-68754052. This strain has been described in Chinese patent application CN202210217216.8 (publication number CN114480214A). Detailed Implementation

[0050] To make the objectives, technical solutions, and technical effects of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely. The embodiments described below are some embodiments of the present invention, but not all embodiments. In conjunction with the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0051] Currently, the existing technical approach for selenium-enriched lactic acid beverages involves directly adding inorganic selenium or selenium-enriched raw materials into milk for fermentation. However, inorganic selenium has low bioavailability and high toxicity, and the amount of added selenium-enriched raw materials is limited, resulting in a low selenium content in the final product. Furthermore, existing walnut dairy products are made by directly juicing walnuts or adding walnut protein to milk to create fermented dairy products. However, walnut pulp has a high fat content, and walnut protein has a large molecular weight, poor solubility, and is not easily digested and absorbed. Therefore, it is necessary to find walnut products with better solubility and easier absorption as an ingredient to ensure that the fermented dairy products produced have a good texture while maintaining the original taste and functional characteristics of lactic acid bacteria beverages.

[0052] In a first aspect, in one specific embodiment of the present invention, the present invention provides a selenium-enriched lactic acid bacteria beverage, comprising the following raw materials in parts by weight: 25-65 parts of milk, 0.1-2 parts of walnut peptide, 0.01-0.1 parts of a first fermentation agent, and 0.08-0.16 parts of a second fermentation agent, wherein the second fermentation agent is selenium-enriched Lactobacillus helveticus.

[0053] It should be noted that the selenium-enriched Lactobacillus helveticus is obtained by fermenting Lactobacillus helveticus in a selenium-containing culture medium. The dosage form of the selenium-enriched Lactobacillus helveticus can be liquid, powder, or granules. The selenium-enriched Lactobacillus helveticus can be obtained through the following steps:

[0054] The *Lactobacillus helveticus* strain preserved in glycerol tubes was activated. For the first activation, 1-5% of the culture was inoculated into MRS broth medium and cultured at 35-40°C for 12-36 hours. For the second activation, 1-3% of the culture was inoculated and cultured at 35-40°C for 6-24 hours. The activated bacterial solution was then inoculated into MRS broth medium at 5-15% of the culture and fermented at 35-40°C. After 3-5 hours of fermentation, sterile sodium selenite solution was added to a final concentration of 30 μg / mL for selenium enrichment, and fermentation continued for 19-21 hours. The fermented bacterial solution was transferred to a sterile centrifuge bottle and centrifuged at 5000×g at 4°C for 10 minutes. The supernatant was discarded, and the bacterial cells were washed twice with sterile physiological saline to obtain selenium-enriched *Lactobacillus helveticus* sludge.

[0055] The weight percentages of each component of the freeze-drying protectant are as follows: 5-15% skim milk powder, 4-8% maltodextrin, 2-8% sucrose, and the remainder is pure water. The above protectant components are thoroughly stirred and mixed, and then sterilized by keeping them in a 90℃ constant temperature water bath for 30 minutes.

[0056] Cleaned selenium-enriched Lactobacillus helveticus bacterial sludge was mixed with a freeze-drying protectant at a weight ratio of 1:1-5. The sludge was then added to the sterilized freeze-drying protectant and thoroughly mixed in a sterile freeze-drying bottle. The mixture was pre-frozen at -30°C for 12 hours, and then freeze-dried in a vacuum freeze dryer for 48 hours to obtain selenium-enriched Lactobacillus helveticus freeze-dried bacterial powder. Testing showed that the selenium content of the powder was 100-125 μg / g.

[0057] In some embodiments of the present invention, the above-mentioned selenium-enriched lactic acid bacteria beverage raw materials may comprise 25-65 parts by weight, 30-65 parts by weight, 35-65 parts by weight, 40-65 parts by weight, 45-65 parts by weight, 50-65 parts by weight, 25-60 parts by weight, 25-55 parts by weight, 25-50 parts by weight, 25-45 parts by weight, or 25-40 parts by weight of milk. In some embodiments, the above-mentioned selenium-enriched lactic acid bacteria beverage raw materials may comprise 25 parts by weight, 30 parts by weight, 35 parts by weight, 35 parts by weight, 40 parts by weight, 45 parts by weight, 50 parts by weight, 55 parts by weight, 60 parts by weight, or 65 parts by weight of milk, or milk within a numerical range defined by any two of the above specific values ​​as endpoints. It should be understood that, in specific embodiments, any of the above ranges can be combined with any other ranges, as long as the selenium-enriched lactic acid bacteria beverage with the desired performance of the present invention can be obtained.

[0058] In some embodiments of the present invention, the above-mentioned selenium-enriched lactic acid bacteria beverage raw materials may contain 0.1-2 parts by weight, 0.5-2 parts by weight, 1-2 parts by weight, 0.1-1.5 parts by weight, or 0.1-1 parts by weight of walnut peptides. In some embodiments, the above-mentioned selenium-enriched lactic acid bacteria beverage raw materials may contain 0.1 parts by weight, 0.5 parts by weight, 1 part by weight, 1.5 parts by weight, or 2 parts by weight of walnut peptides, or milk within the numerical range defined by any two of the above specific values ​​as endpoints. It should be understood that, in specific embodiments, any of the above ranges can be combined with any other ranges, as long as the selenium-enriched lactic acid bacteria beverage with the desired performance of the present invention can be obtained.

[0059] In some embodiments of the present invention, the above-mentioned selenium-enriched lactic acid bacteria beverage raw materials may include 0.01-0.1 parts by weight, 0.01-0.09 parts by weight, 0.01-0.08 parts by weight, 0.01-0.07 parts by weight, 0.01-0.06 parts by weight, 0.01-0.05 parts by weight, 0.02-0.1 parts by weight, 0.03-1 parts by weight, 0.04-0.1 parts by weight, or 0.05-1 parts by weight of a first fermentation agent. In some embodiments, the above-mentioned selenium-enriched lactic acid bacteria beverage raw materials may include 0.01 parts by weight, 0.02 parts by weight, 0.03 parts by weight, 0.04 parts by weight, 0.05 parts by weight, 0.06 parts by weight, 0.07 parts by weight, 0.08 parts by weight, 0.09 parts by weight, or 0.1 parts by weight of a first fermentation agent, or milk within the numerical range defined by any two of the above specific values ​​as endpoints. It should be understood that, in specific embodiments, any of the above ranges can be combined with any other ranges, as long as the selenium-enriched lactic acid bacteria beverage with the performance required by the present invention can be obtained.

[0060] In some embodiments of the present invention, the above-mentioned selenium-enriched lactic acid bacteria beverage raw materials may contain 0.08-0.16 parts by weight, 0.08-0.15 parts by weight, 0.08-0.14 parts by weight, 0.08-0.13 parts by weight, 0.08-0.12 parts by weight, 0.09-0.16 parts by weight, 0.10-0.16 parts by weight, 0.11-0.16 parts by weight, or 0.12-0.1 parts by weight of a second fermentation agent. In some embodiments, the above-mentioned selenium-enriched lactic acid bacteria beverage raw materials may contain 0.08 parts by weight, 0.09 parts by weight, 0.10 parts by weight, 0.11 parts by weight, 0.12 parts by weight, 0.13 parts by weight, 0.14 parts by weight, 0.15 parts by weight, or 0.16 parts by weight of a second fermentation agent, or milk within the numerical range defined by any two of the above specific values ​​as endpoints. It should be understood that, in specific embodiments, any of the above ranges can be combined with any other ranges, as long as the selenium-enriched lactic acid bacteria beverage with the performance required by the present invention can be obtained.

[0061] In some embodiments of the present invention, the Lactobacillus helveticus is Lactobacillus helveticus Zhegu LBH-VI, wherein Lactobacillus helveticus Zhegu LBH-VI was deposited at the China Center for Type Culture Collection (CCTCC) on February 7, 2023, with accession number CCTCC NO: M 2023095.

[0062] In some embodiments of the present invention, the selenium content in the selenium-enriched lactic acid bacteria beverage is ≥8.0 μg / 100g based on the weight of the beverage; preferably, the number of live bacteria in the selenium-enriched lactic acid bacteria beverage during its shelf life is ≥10. 9 CFU / ml.

[0063] It should be noted that the shelf life of the selenium-enriched lactic acid bacteria beverage refers to the period during which, under storage conditions of 2-6℃, the beverage maintains textural stability while ensuring its taste, sensory qualities, and microbial content remain in an ideal state, retaining all nutritional values ​​declared on the label. The shelf life of this selenium-enriched lactic acid bacteria beverage is 30 days.

[0064] In some embodiments of the present invention, the milk used in the present invention mainly refers to fresh milk or reconstituted milk that meets my country's standards for the purchase of raw milk. The milk includes, but is not limited to, one or more combinations of whole milk, low-fat milk or skim milk.

[0065] In some embodiments of the present invention, the molecular weight of the walnut peptide is 800-1000 Da; preferably, the walnut peptide is obtained by defatting walnuts and enzymatic hydrolysis with protease. The preparation method of the walnut peptide includes the following steps: (1) walnuts are defatted to obtain defatted walnut meal (fat content ≤1%), water is added to prepare a feed solution, the pH is adjusted to 8-10, the mixture is stirred for 10-30 min, the precipitate is separated and removed, the pH is adjusted to 4-5, the mixture is stirred for 10-30 min, and the precipitate is separated to obtain walnut protein; (2) walnut protein is prepared into a dispersion, the pH is adjusted to 8-10, 1-3% of protease is added, preferably alkaline protease and papain, the mass ratio of alkaline protease to papain is 1-2:0.5-1.5, the mixture is enzymatically hydrolyzed at 45-60℃ for 4-6 h, and after the enzymatic hydrolysis is completed, the walnut peptide is obtained by enzyme inactivation, separation and drying.

[0066] In some embodiments of the present invention, the first fermentation agent is selected from one or more of Streptococcus salivarius subsp. thermophilus, Lactobacillus delbrueckii subsp. lactis, Lactobacillus paracasei, Bifidobacterium lactis, and Lactococcus lactis subsp. lactis, preferably Streptococcus salivarius subsp. thermophilus and Lactobacillus delbrueckii subsp. lactis, more preferably the mass ratio of Streptococcus salivarius subsp. thermophilus and Lactobacillus delbrueckii subsp. lactis is 2-3:1-1.5.

[0067] In some embodiments of the present invention, the raw materials further include 5.0-8.5 parts of a sweetener; preferably, the sweetener is selected from one or more of sucrose, erythritol, xylitol, maltitol, sucralose or steviol glycosides, preferably sucrose.

[0068] In some embodiments of the present invention, the raw materials further include 0.3-0.7 parts of a stabilizer; the addition of a stabilizer can improve the stability and quality of the selenium-enriched lactic acid bacteria beverage. Preferably, the stabilizer is selected from one or more of sodium carboxymethyl cellulose, pectin, sodium tripolyphosphate, and sodium citrate. More preferably, the mass ratio of sodium carboxymethyl cellulose, pectin, sodium tripolyphosphate, and sodium citrate is 0.5-1.3:1.5-2.5:0.2-0.5:0.3-0.7.

[0069] In some embodiments of the present invention, the raw materials further include 0.11-0.6 parts of an acidulant; preferably, the acidulant is selected from citric acid and / or lactic acid, and more preferably the mass ratio of citric acid to lactic acid is 5-25:5-15.

[0070] In some embodiments of the present invention, the selenium-enriched lactic acid bacteria beverage is prepared by temperature-switching fermentation of a fermentation base comprising milk and walnut peptides.

[0071] In some embodiments of the present invention, the present invention provides a selenium-enriched lactic acid bacteria beverage comprising the following raw materials in weight percentages: 25-65% milk, 0.1-2% walnut peptide, 0.01-0.1% first fermentation agent, 0.08-0.16% second fermentation agent, and 33-73% water, wherein the second fermentation agent is selenium-enriched Lactobacillus helveticus.

[0072] In some embodiments of the present invention, the present invention provides a selenium-enriched lactic acid bacteria beverage comprising the following raw materials in weight percentages: 25-40% milk, 0.1-2% walnut peptide, 0.01-0.1% first fermentation agent, 0.08-0.16% second fermentation agent, and 50-69% water, wherein the second fermentation agent is selenium-enriched Lactobacillus helveticus.

[0073] In some embodiments of the present invention, the raw materials further include 5-8.5% sweetener.

[0074] In some embodiments of the present invention, the raw material further includes 0.3-0.7% of a stabilizer.

[0075] In some embodiments of the present invention, the raw material further includes 0.11-0.6% acidulant.

[0076] In some embodiments of the present invention, the above-mentioned selenium-enriched lactic acid bacteria beverage raw materials may contain 25-65%, 30-65%, 35-65%, 40-65%, 45-65%, 50-65%, 25-60%, 25-55%, 25-50%, 25-45%, or 25-40% by weight of milk. In some embodiments, the above-mentioned selenium-enriched lactic acid bacteria beverage raw materials may contain 25%, 30%, 35%, 35%, 40%, 45%, 50%, 55%, 60%, or 65% by weight of milk, or milk within a numerical range defined by any two of the above specific values ​​as endpoints. It should be understood that, in specific embodiments, any of the above ranges can be combined with any other ranges, as long as the selenium-enriched lactic acid bacteria beverage with the desired performance of the present invention can be obtained.

[0077] In some embodiments of the present invention, the above-mentioned selenium-enriched lactic acid bacteria beverage raw materials may contain 0.1-2%, 0.5-2%, 1-2%, 0.1-1.5%, or 0.1-1% by weight of walnut peptides. In some embodiments, the above-mentioned selenium-enriched lactic acid bacteria beverage raw materials may contain 0.1%, 0.5%, 1%, 1.5%, or 2% by weight of walnut peptides, or milk within the numerical range defined by any two of the above specific values ​​as endpoints. It should be understood that, in specific embodiments, any of the above ranges can be combined with any other ranges, as long as the selenium-enriched lactic acid bacteria beverage with the desired performance of the present invention can be obtained.

[0078] In some embodiments of the present invention, the above-mentioned selenium-enriched lactic acid bacteria beverage raw materials may contain 0.01-0.1%, 0.01-0.09%, 0.01-0.08%, 0.01-0.07%, 0.01-0.06%, 0.01-0.05%, 0.02-0.1%, 0.03-1%, 0.04-0.1%, or 0.05-1% by weight of a first fermentation agent. In some embodiments, the above-mentioned selenium-enriched lactic acid bacteria beverage raw materials may contain 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, or 0.1% by weight of a first fermentation agent, or milk within the numerical range defined by any two of the above specific values ​​as endpoints. It should be understood that, in specific embodiments, any of the above ranges can be combined with any other ranges, as long as the selenium-enriched lactic acid bacteria beverage with the performance required by the present invention can be obtained.

[0079] In some embodiments of the present invention, the above-mentioned selenium-enriched lactic acid bacteria beverage raw materials may contain 0.08-0.16%, 0.08-0.15%, 0.08-0.14%, 0.08-0.13%, 0.08-0.12%, 0.09-0.16%, 0.10-0.16%, 0.11-0.16%, or 0.12-0.16% by weight of a second fermentation agent. In some embodiments, the above-mentioned selenium-enriched lactic acid bacteria beverage raw materials may contain 0.08%, 0.09%, 0.10%, 0.12%, 0.13%, 0.14%, 0.15%, or 0.16% by weight of a second fermentation agent, or milk within the numerical range defined by any two of the above specific values ​​as endpoints. It should be understood that, in specific embodiments, any of the above ranges can be combined with any other ranges, as long as the selenium-enriched lactic acid bacteria beverage with the desired performance of the present invention can be obtained.

[0080] Secondly, the present invention also provides a method for preparing the above-mentioned selenium-enriched lactic acid bacteria beverage, comprising the following steps:

[0081] (1) Mix raw materials including milk and walnut peptides to prepare a fermentation base;

[0082] (2) The first fermentation agent and the second fermentation agent are sequentially added to the fermentation base material obtained in step (1) to carry out variable temperature fermentation to obtain fermented milk.

[0083] (3) The fermented milk obtained in step (2) is blended to obtain a selenium-enriched lactic acid bacteria beverage.

[0084] In some embodiments of the present invention, the preparation of step (3) above may be selected as follows, such as adding a stabilizer to the fermented milk for stabilization, adding an acidulant for acidification, adjusting the volume with water, and homogenizing.

[0085] In some embodiments of the present invention, the fermentation base also includes a sweetener.

[0086] This invention employs a two-stage inoculation and variable-temperature fermentation process, which significantly improves the flavor, taste, and textural stability of selenium-enriched lactic acid bacteria beverages and increases the number of live bacteria during shelf life.

[0087] In some embodiments of the present invention, the step (2) of sequentially introducing the first fermentation agent and the second fermentation agent for variable temperature fermentation means first inoculating the first fermentation agent and fermenting at 40-45°C, and then inoculating the second fermentation agent and fermenting at 35-39°C; preferably, first inoculating the first fermentation agent and fermenting at 40-45°C for 10-12 hours until the final acidity is 90-100°T, and then inoculating the second fermentation agent and fermenting at 35-39°C for 8-10 hours until the final acidity is 160-180°T.

[0088] It should be noted that, in this invention, the first fermentation refers to fermentation carried out at 40-45℃ after inoculation with the first fermentation agent; the second fermentation refers to fermentation carried out at 35-39℃ after inoculation with the second fermentation agent.

[0089] This invention employs a two-stage inoculation and variable-temperature fermentation process to prepare live-culture lactic acid bacteria beverages, giving them a rich taste and good texture, and increasing the number of live bacteria during the shelf life of the beverages.

[0090] In some embodiments of the present invention, the preparation method further includes a step of homogenizing the fermentation substrate before inoculation, preferably at a homogenization temperature of 50-65°C, and more preferably at a homogenization pressure of 10-50 MPa; preferably, the homogenization treatment further includes a step of sterilizing the fermentation substrate, preferably at a sterilization temperature of 90-100°C, and more preferably at a sterilization time of 200-400 s.

[0091] In some embodiments of the present invention, after each fermentation is completed, a step of stirring and demulsifying the fermentation liquid is included. Demulsification is to disperse any possible curds so that the subsequently added ingredients are evenly mixed in the liquid. Preferably, the stirring speed is 20-40 r / min, and more preferably, the stirring time is 10-20 min.

[0092] The beneficial effects of the present invention will be further illustrated below through specific embodiments.

[0093] All raw materials or reagents used in this invention are purchased from mainstream manufacturers on the market. Those without specified manufacturers or concentrations are all analytical grade raw materials or reagents that can be obtained routinely. There are no special restrictions as long as they can achieve the expected effect.

[0094] Unless otherwise specified in this embodiment, the techniques or conditions described in the literature in this field or in accordance with the product manual shall apply.

[0095] The present invention will now be described in more detail with reference to examples and comparative examples, but the scope of the present invention is not limited to these examples. It should be noted that, unless otherwise specified, all percentages, parts, and ratios used in the present invention are based on mass.

[0096] The sources of the reagents and instruments used in the following examples are shown in Table 1.

[0097] Table 1. Raw material information used in the embodiments.

[0098] raw material Model / Purity Manufacturer for sale Citric acid Purity ≥ 99.5% Wuhan Shengze Food Ingredients Co., Ltd. lactic acid Purity ≥ 95% Henan Jindan Lactic Acid Technology Co., Ltd. Sodium citrate Purity ≥ 99% Wuhan Shengze Food Ingredients Co., Ltd. Sodium carboxymethyl cellulose low viscosity Xiamen Oukai Technology Co., Ltd. alkaline protease AP-200A Angel Yeast Co., Ltd. Papain 2021070301 Angel Yeast Co., Ltd. pectin Low ester Xiamen Oukai Technology Co., Ltd. Sodium tripolyphosphate Purity ≥ 85% Wuhan Shengze Food Ingredients Co., Ltd. sucrose Level 1 Guangxi Fengtang Luocheng Sugar Co., Ltd. Xylitol Purity ≥ 98.5% Shandong Futian Technology Group Co., Ltd. Erythritol Purity ≥ 99.5% Shandong Futian Technology Group Co., Ltd. Steviosides RA96%, 400 times Wuhan Shengze Food Ingredients Co., Ltd. Maltitol Purity ≥ 98% Shandong Futian Technology Group Co., Ltd.

[0099] Example 1

[0100] 1. Preparation of walnut peptides

[0101] Dry, mold-free walnuts were selected and placed in a 0.5% NaOH solution. After soaking in a 65℃ water bath for 30 minutes, the shells were removed. The shelled walnut kernels were dried in an oven at 40℃, then pulverized and wrapped in gauze. Oil was extracted using a small hydraulic oil press at 35 MPa for 30 minutes to obtain walnut meal, which was then pulverized to 20 mesh. Supercritical fluid extraction was used to remove the oil from the walnut meal: extraction pressure 35 MPa, extraction temperature 40℃, extraction time 3 hours; separation vessel 1 pressure 10-12 MPa, temperature 40℃; separation vessel 2 pressure 5-7 MPa, temperature 40℃. This yielded de-oiled walnut meal (fat content ≤1%).

[0102] Distilled water was added to the defatted walnut meal at a mass ratio of 1:20 to obtain a liquid. The pH of the liquid was adjusted to 9.0 using 0.1% NaOH solution. The mixture was stirred at 100 r / min for 10 min. The treated liquid was then transferred to a centrifuge bottle and centrifuged at 4000 r / min for 10 min. The supernatant was collected. The pH of the liquid was then adjusted to 4.5 using 0.1% hydrochloric acid solution. The mixture was stirred at 100 r / min for 10 min. The treated liquid was then transferred to a centrifuge bottle and centrifuged at 4000 r / min for 10 min. The precipitate was collected.

[0103] Distilled water was added to the precipitate at a mass ratio of 1:10. The pH of the solution was adjusted to 9.0 using 0.1% NaOH solution. 2% protease (alkaline protease:papain = 1.5:1) was added. The temperature of the solution was raised to 50℃ and enzymatic hydrolysis was performed for 5 hours. After the enzymatic hydrolysis was completed, the solution was subjected to enzyme inactivation treatment (90℃, 10 minutes). The solution was transferred to a centrifuge bottle and centrifuged at 4000 rpm for 10 minutes. The supernatant was collected, filtered through a membrane, and concentrated by rotary evaporation at 50℃. The concentrate was pre-frozen in a -30℃ freezer for 12 hours. The pre-frozen concentrate was then freeze-dried in a vacuum freeze dryer for 48 hours to obtain walnut polypeptide with a molecular weight of 952.433 Da.

[0104] 2. Screening and identification methods and results of Lactobacillus helveticus Zhegu LBH-VI.

[0105] 1) Take 25g of homemade yak milk residue from the Zhegu Grassland in Tibet and mix it with 225mL of sterile physiological saline to obtain a homogeneous sample solution. Dilute the sample solution serially and take 10g of each solution. -3 10 -4 10 -5 10 -6 The diluted solution was spread on MRS medium plates and incubated at 37°C for 48 hours, after which colonies grew on the MRS medium.

[0106] 2) Initial screening of bacterial strains

[0107] Based on the standard colony characteristics of Lactobacillus helveticus, single colonies were selected for isolation, purification, and further culture. The isolation and purification were repeated at least three times to obtain purified colonies.

[0108] Culture characteristics: The optimal growth temperature is 37℃. It is a facultative anaerobe and grows in MRS medium.

[0109] 3) Acid production experiment

[0110] The single colonies obtained from the initial screening of the bacterial strain were cultured on MRS medium plates containing 0.2% CaCO3. After culturing at 37°C for 48 hours, the presence of no clear zone around the colonies was observed. Single colonies with strong acid production capacity and large clear zones were selected for further isolation and purification.

[0111] Morphological characteristics: The growth state in MRS agar medium is as follows: The growth morphology of the colonies in MRS agar medium is milky white, round, with smooth edges, raised cells, and rough surface.

[0112] 4) Gram staining

[0113] The strains obtained from the secondary screening were subjected to Gram staining. Those showing typical Gram-positive staining were identified as the target strain. Microscopic observation revealed that the cells were short rod-shaped, non-flagellated, non-spore-forming, and non-motile.

[0114] 5) Identification of strains

[0115] The isolated and purified strain was Gram-positive, H2O2 catalase-negative, acid-producing, and non-gas-producing. 16S rDNA gene sequencing was performed, and the results were compared with those in the NCBI GenBank database for homology analysis. The results showed that this strain is *Lactobacillus helveticus*. The 16S rDNA gene sequence of this strain is shown in SEQ ID NO.1:

[0116]

[0117] The strain was named Lactobacillus helveticus Zhegu LBH-VI and was deposited at the China Center for Type Culture Collection (CCTCC) on February 7, 2023, with accession number CCTCC NO:M2023095.

[0118] 3. Preparation of selenium-enriched Lactobacillus helveticus

[0119] The *Lactobacillus helveticus* strain Zhegu LBH-VI (Latin name: *Lactobacillus shelveticus* Zhegu LBH-VI) preserved in glycerol tubes was activated. For the first activation, a 3% inoculum was added to MRS broth medium and cultured at 37°C for 24 h. For the second activation, a 2% inoculum was added and cultured at 37°C for 12 h. The activated bacterial solution was then inoculated at a 10% inoculum into 2 L of MRS broth medium and fermented at 37°C. After 4 h of fermentation, sterile sodium selenite solution was added to a final concentration of 30 μg / mL for selenium enrichment, and fermentation continued for another 20 h. The fermented bacterial solution was transferred to a sterile centrifuge bottle and centrifuged at 5000 × g at 4°C for 10 min. The supernatant was discarded, and the bacterial cells were washed twice with sterile physiological saline to obtain selenium-enriched *Lactobacillus helveticus* bacterial sludge. The moisture content of the sludge was measured.

[0120] The mass percentages of the freeze-drying protectant components are as follows: 10% skim milk powder, 6% maltodextrin, and 4% sucrose. Dissolve each protectant in 55% pure water, stir thoroughly, and sterilize in a 90℃ constant temperature water bath for 30 minutes before use.

[0121] Cleaned selenium-enriched Lactobacillus helveticus bacterial sludge was mixed with a freeze-drying protectant at a ratio of 1:3. The sludge was then added to the sterilized freeze-drying protectant and thoroughly mixed in a sterile freeze-drying bottle. The mixture was pre-frozen at -30°C for 12 hours, and then freeze-dried in a vacuum freeze dryer for 48 hours to obtain selenium-enriched Lactobacillus helveticus freeze-dried bacterial powder. Testing showed that the selenium content of the powder was 100-125 μg / g.

[0122] 4. Screening, identification methods, and identification results of *Streptococcus salivarias* subsp. *thermophilus* 932.

[0123] 1) Take 25g of homemade yak milk residue from the Zhegu Grassland in Tibet and mix it with 225mL of sterile physiological saline to obtain a homogeneous sample solution. Dilute the sample solution serially and take 10g of each solution. -3 10 -4 10 -510 -6 The diluted solution was spread on MRS medium plates and incubated at 37°C for 48 hours, after which colonies grew on the MRS medium.

[0124] 2) Initial screening of bacterial strains

[0125] Based on the standard colony characteristics of Streptococcus salivarius subsp. thermophilus, single colonies were selected for isolation, purification, and further culture. The isolation and purification were repeated at least three times to obtain purified colonies.

[0126] Culture characteristics: The optimal growth temperature is 42℃. It is a facultative anaerobe and grows in MRS medium.

[0127] 3) Acid production experiment

[0128] The single colonies obtained from the initial screening of the bacterial strain were cultured on MRS medium plates containing 0.2% CaCO3. After culturing at 42℃ for 48 hours, the presence or absence of a clear zone around the colony was observed. Single colonies with strong acid production capacity and large clear zones were selected for further isolation and purification.

[0129] Morphological characteristics: The growth state in MRS agar medium is as follows: The colony morphology in MRS agar medium is slightly yellowish at the bottom, milky white on the surface, with irregular edges, raised cells, and a relatively smooth surface.

[0130] 4) Gram staining

[0131] The strains obtained from the secondary screening were subjected to Gram staining. Those showing typical Gram staining positivity were identified as the target strain. Microscopic observation revealed that the cells were rod-shaped, non-flagellated, non-spore-forming, and non-motile.

[0132] 5) Identification of strains

[0133] The isolated and purified strain was Gram-positive, H2O2 catalase-negative, acid-producing, and non-gas-producing. 16S rDNA gene sequencing was performed, and the results were compared with those in the NCBI GenBank database for homology analysis. The results showed that this strain belongs to the thermophilic subspecies of *Streptococcus salivarius*. The 16S rDNA gene sequence of this strain is shown in SEQ ID NO.2:

[0134]

[0135] The strain was named Streptococcus salivarius subsp. thermophilus 932 and was deposited at the China Center for Type Culture Collection (CCTCC) on June 5, 2023, with accession number CCTCC NO: M 2023902.

[0136] 5. Preparation of Streptococcus salivarius subsp. thermophilus 932 inoculum

[0137] The *Streptococcus salivarias* subsp. *thermophilus* 932 strain preserved in glycerol tubes was activated. For the first activation, a 3% inoculum was added to MRS broth and incubated at 37°C for 24 h. For the second activation, a 2% inoculum was added and incubated at 37°C for 12 h. After fermentation, the bacterial culture was transferred to a sterile centrifuge bottle and centrifuged at 5000×g at 4°C for 10 min. The supernatant was discarded, and the bacterial cells were washed twice with sterile physiological saline to obtain *Streptococcus salivarias* subsp. *thermophilus* 932 bacterial sludge.

[0138] The mass percentages of the freeze-drying protectant components are as follows: 10% skim milk powder, 6% maltodextrin, and 4% sucrose. Dissolve each protectant in 55% pure water, stir thoroughly, and sterilize in a 90℃ constant temperature water bath for 30 minutes before use.

[0139] The cleaned Streptococcus thermophilus subsp. 932 bacterial sludge was mixed with a freeze-drying protectant at a ratio of 1:3. The bacterial sludge was added to the sterilized freeze-drying protectant and thoroughly mixed in a sterile freeze-drying bottle. The mixture was then pre-frozen at -30°C for 12 hours. The pre-frozen bacterial milk was then freeze-dried in a vacuum freeze dryer for 48 hours to obtain freeze-dried Streptococcus thermophilus subsp. 932 bacterial powder.

[0140] 6. Screening, identification methods, and identification results of *Lactobacillus delbrueckii* subsp. *lactis* Dangxiong LB VⅢ.

[0141] 1) Enrichment of bacterial strains: Take 5g of milk sample made by herders in Damxung County, Tibet (altitude 4350m), crush it, add 45ml of sterile physiological saline and disperse it evenly. Take 2ml of the above dispersion and add it to 20ml of sterile litmus milk medium. Place it in a 37℃ incubator for static incubation until the medium becomes acidic.

[0142] Remove from heat when it has solidified and turned pink.

[0143] 2) Cultivating single colonies: Dilute the litmus milk culture medium to 10... -5 Take 100 μl of the solution and spread it onto a BL solid medium plate. Incubate at 37°C until colonies form. On BL plates, colonies are smooth, slightly raised, moist, with rough edges, colorless to slightly white, 1-3 mm in diameter, and pale yellow on the reverse side.

[0144] 3) Screening of acid-producing bacteria: The single colonies obtained from the initial screening were streaked on BL solid medium plates containing 0.2% CaCO3 and incubated at 37℃ for 36 hours. After the incubation, the presence of a clear zone around the colony was observed. Single colonies with a clear zone diameter of 3-3.5 cm were selected for further isolation and purification.

[0145] 4) Preliminary Gram staining identification: Gram staining was performed on the strain from step (3). If the bacterial cells stained purple, they were Gram-positive and retained for further research. If the staining was red, the strain was Gram-negative and was directly eliminated. The identification results showed that the obtained strain was Gram-positive, non-motile, non-spore-forming, non-flagellated, and non-capsulated. The bacterial cells were thin rods, appearing as single rods or in chains. This strain was the target strain.

[0146] 5) Identification of strains

[0147] The isolated and purified strain was Gram-positive, H2O2 catalase-negative, acid-producing, and non-gas-producing. 16S rDNA gene sequencing was performed, and the results were compared with those in the NCBI GenBank database for homology analysis. The results showed that this strain belongs to *Lactobacillus delbrueckii* subsp. *delbrueckii*. The 16S rDNA gene sequence of this strain is shown in SEQ ID NO.3:

[0148]

[0149] The strain was named Lactobacillus delbrueckii subsp. lactis Dangxiong LB VⅢ and was deposited at the China Center for Type Culture Collection (CCTCC) on March 23, 2023, with accession number CCTCC NO:M2023396.

[0150] 7. Preparation of Lactobacillus delbrueckii subsp. *Dangxiong* LB VIII bacterial inoculum

[0151] The *Lactobacillus delbrueckii* subsp. *Dangxiong* LB VIII strain preserved in glycerol tubes was activated. For the first activation, a 3% inoculum was added to MRS broth medium and cultured at 37°C for 24 h. For the second activation, a 2% inoculum was added and cultured at 37°C for 12 h. After fermentation, the bacterial culture was transferred to a sterile centrifuge bottle and centrifuged at 5000×g at 4°C for 10 min. The supernatant was discarded, and the bacterial cells were washed twice with sterile physiological saline to obtain *Lactobacillus delbrueckii* subsp. *Dangxiong* LB VIII bacterial sludge. The moisture content of the sludge was measured.

[0152] The mass percentages of the freeze-drying protectant components are as follows: 10% skim milk powder, 6% maltodextrin, and 4% sucrose. Dissolve each protectant in 55% pure water, stir thoroughly, and sterilize in a 90℃ constant temperature water bath for 30 minutes before use.

[0153] Cleaned Lactobacillus delbrueckii subsp. lactis LB VIII bacterial sludge was mixed with a freeze-drying protectant at a ratio of 1:3. The bacterial sludge was added to the sterilized freeze-drying protectant and thoroughly mixed in a sterile freeze-drying bottle. The mixture was then pre-frozen at -30°C for 12 hours. The pre-frozen bacterial milk was then freeze-dried in a vacuum freeze dryer for 48 hours to obtain Lactobacillus delbrueckii subsp. lactis LB VIII freeze-dried bacterial powder.

[0154] 8. Preparation of selenium-enriched lactic acid bacteria beverages

[0155] 1) Preparation of fermentation substrate

[0156] Raw milk was inspected according to the requirements of GB 19301. The qualified milk was then standardized to achieve a milk protein content of 2.9g / 100g. 650g of standardized milk was taken and heated to 60℃. 20g of walnut peptide and 65g of sucrose were mixed evenly and added to the standardized milk. The mixture was sheared at 3000r for 15min using a shear emulsifier. Then, homogenization was performed at 60℃ and a homogenization pressure of 25MPa (5MPa at the low pressure). After homogenization, the temperature was raised to 95℃ and held for 300s for sterilization. The milk was then transferred to a fermentation tank.

[0157] 2) First fermentation

[0158] Cool the fermentation substrate prepared in step 1) to 42°C, add freeze-dried Streptococcus salivarius subsp. thermophilus 932 and freeze-dried Lactobacillus delbrueckii subsp. lactis Dangxiong LB VⅢ (the amount of Streptococcus salivarius subsp. thermophilus 932 and Lactobacillus delbrueckii subsp. lactis Dangxiong LB VⅢ added is 0.1g based on bacterial dry matter, and the mass ratio of Streptococcus salivarius subsp. thermophilus 932 to Lactobacillus delbrueckii subsp. lactis Dangxiong LB VⅢ is 2:1), stir at 30r / min for 5min to disperse it evenly, ferment at 42°C, and terminate at an acidity of 100°T; after fermentation, stir at 30r / min for 15min to break the emulsion.

[0159] 3) Second fermentation

[0160] After the first fermentation, the fermented milk was cooled to 37°C, and selenium-enriched Lactobacillus helveticus freeze-dried powder was added (the amount of selenium-enriched Lactobacillus helveticus added was 1g based on the dry matter of the bacteria). The mixture was stirred at 30r / min for 5 minutes to disperse it evenly, and fermented at 37°C until the acidity was terminated at 160°T. After fermentation, the mixture was stirred at 30r / min for 15 minutes to break the emulsion. The mixture was then cooled to 25°C.

[0161] 4) Secondary mixing

[0162] Dissolve 6g of stabilizer in 75℃ hot water. The stabilizer includes sodium carboxymethyl cellulose, pectin, sodium tripolyphosphate, and sodium citrate, with a mass ratio of sodium carboxymethyl cellulose: pectin: sodium tripolyphosphate: sodium citrate = 1:2:0.3:0.5. Shear emulsifier at 3000r for 15min and sterilize the liquid at 75℃ for 15s. After sterilization, cool the liquid to 25℃ and mix it with the fermented milk after the second fermentation in step 3) at 30r / min for 20min.

[0163] 5) Add acidulant

[0164] Prepare an acid solution with a solid content of 10% by mixing 1.8g citric acid, 1g lactic acid and 0.3g sodium citrate. Sterilize the acid solution at 75°C for 15s and then cool it to 25°C. Adjust the volume of the solution from step 4) with sterile distilled water (the amount of sterile distilled water added is such that the weight of the solution after adjusting the acidity is 1000g). Adjust the acidity of the solution to 65°T using the prepared sterile acid solution.

[0165] Finally, the above liquid material was homogenized at 25°C and 20MPa. Then, quantitative filling and sealing were carried out under aseptic conditions at a filling temperature of 15°C. After filling, the liquid was transferred to a cold storage and refrigerated at 6°C.

[0166] Example 2

[0167] Preparation of selenium-enriched lactic acid bacteria beverages

[0168] 1) Preparation of fermentation substrate

[0169] Raw milk was inspected according to the requirements of GB 19301. The qualified milk was standardized to achieve a milk protein content of 2.9g / 100g. 350g of standardized milk was taken and heated to 60℃. 1g of walnut peptide prepared in step 1 of Example 1 and 50g of sucrose were mixed evenly and added to the standardized milk. The mixture was sheared at 3000r for 15min using a shear emulsifier and then homogenized at 60℃ and 25MPa (with a low pressure of 5MPa). After homogenization, the temperature was raised to 95℃ and held for 300s for sterilization. The milk was then transferred to a fermentation tank.

[0170] 2) First fermentation

[0171] The fermentation substrate prepared in step 1) was cooled to 45°C, and the freeze-dried bacterial powders of *Streptococcus salivarius* subsp. *thermophilus* 932 and *Lactobacillus delbrueckii* subsp. *Dangxiong* LB VIII prepared in Example 1 were added (wherein, based on bacterial dry matter, the amount of *Streptococcus salivarius* subsp. *thermophilus* 932 and *Lactobacillus delbrueckii* subsp. *Dangxiong* LB VIII added was 1g, and the mass ratio of *Streptococcus salivarius* subsp. *thermophilus* 932 and *Lactobacillus delbrueckii* subsp. *Dangxiong* LB VIII was 3:1). The mixture was stirred at 30 r / min for 5 min to disperse it evenly, and fermented at 45°C until the acidity was terminated at 90°T. After fermentation, the mixture was stirred at 30 r / min for 15 min to break the emulsion.

[0172] 3) Second fermentation

[0173] The fermented milk after the first fermentation was cooled to 39°C, and the selenium-enriched Lactobacillus helveticus freeze-dried bacterial powder prepared in Example 1 was added (wherein, the amount of selenium-enriched Lactobacillus helveticus added was 0.8g based on the dry matter of the bacterial cells). The mixture was stirred at 30r / min for 5min to disperse it evenly, and fermented at 39°C until the acidity was terminated at 180°T. After fermentation, the mixture was stirred at 30r / min for 15min to break the emulsion. The mixture was then cooled to 25°C.

[0174] 4) Secondary mixing

[0175] Dissolve 3g of stabilizer in 75℃ hot water. The stabilizer includes sodium carboxymethyl cellulose, pectin, sodium tripolyphosphate, and sodium citrate, with a mass ratio of sodium carboxymethyl cellulose: pectin: sodium tripolyphosphate: sodium citrate = 0.5:1.5:0.2:0.3. Shear emulsifier at 3000r for 15min and sterilize the liquid at 75℃ for 15s. After sterilization, cool the liquid to 25℃ and mix it with the fermented milk after the second fermentation in step 3) at 30r / min for 20min.

[0176] 5) Add acidulant

[0177] Prepare an acid solution with a solid content of 10% by mixing 0.5g citric acid, 0.5g lactic acid, and 0.1g sodium citrate. Sterilize the acid solution at 75°C for 15s and then cool it to 25°C. Adjust the volume of the solution from step 4) with sterile distilled water (the amount of sterile distilled water added is such that the weight of the solution after adjusting the acidity is 1000g). Adjust the acidity of the solution to 65°T using the prepared sterile acid solution.

[0178] Finally, the above liquid material was homogenized at 25°C and 20MPa. Then, quantitative filling and sealing were carried out under aseptic conditions at a filling temperature of 15°C. After filling, the liquid was transferred to a cold storage and refrigerated at 6°C.

[0179] Example 3

[0180] 1. Screening, identification methods, and identification results of Lactococcus lactis subsp. lactis 954

[0181] 1) Take 25g of homemade yak milk residue from the Zhegu Grassland in Tibet and mix it with 225mL of sterile physiological saline to obtain a homogeneous sample solution. The sample solution is serially diluted, and the diluted solutions with concentrations of 10⁻³, 10⁻⁴, 10⁻⁵ and 10⁻⁶ are spread on MRS medium plates for incubation. After incubation at 30℃ for 48h, colonies grow on the MRS medium.

[0182] 2) Initial screening of bacterial strains

[0183] Based on the standard colony characteristics of Streptococcus salivarius subsp. thermophilus, single colonies were selected for isolation, purification, and further culture. The isolation and purification were repeated at least three times to obtain purified colonies.

[0184] Culture characteristics: The optimal growth temperature is 30℃. It is a facultative anaerobe and grows in MRS medium.

[0185] 3) Acid production experiment

[0186] The single colonies obtained from the initial screening of the bacterial strain were cultured on MRS medium plates containing 0.2% CaCO3. After culturing at 30°C for 48 hours, the presence of no clear zone around the colonies was observed. Single colonies with strong acid production capacity and large clear zones were selected for further isolation and purification.

[0187] Morphological characteristics: The growth state in MRS agar medium is as follows: The growth morphology of the colony is slightly yellow at the bottom, with a pale yellow and opaque surface, neat edges, low convexity of the cells, and smooth surface.

[0188] 4) Gram staining

[0189] The strains obtained from the secondary screening were subjected to Gram staining. Those showing typical Gram staining positivity were identified as the target strain. Microscopic observation revealed that the cells were rod-shaped, non-flagellated, non-spore-forming, and non-motile.

[0190] 5) Identification of strains

[0191] The isolated and purified strain was Gram-positive, H2O2 catalase-negative, acid-producing, and non-gas-producing. 16S rDNA gene sequencing was performed, and the results were compared with those in the NCBI GenBank database for homology analysis. The results showed that this strain belongs to *Lactococcus lactis* subsp. *lactococcus*. The 16S rDNA gene sequence of this strain is shown in SEQ ID NO.4:

[0192]

[0193] The strain was named Lactococcus lactis subsp. lactis strain 954 and was deposited at the China Center for Type Culture Collection (CCTCC) on June 5, 2023, with accession number CCTCC NO: M2023904.

[0194] 2. Preparation of Lactococcus lactis subsp. 954 inoculum

[0195] The *Lactococcus lactis* subsp. *lactophores* strain 954 preserved in glycerol tubes was activated. For the first activation, a 3% inoculum was added to MRS broth medium and cultured at 37°C for 24 hours. For the second activation, a 2% inoculum was added and cultured at 37°C for 12 hours. After fermentation, the bacterial culture was transferred to a sterile centrifuge bottle and centrifuged at 5000×g at 4°C for 10 minutes. The supernatant was discarded, and the bacterial cells were washed twice with sterile physiological saline to obtain *Lactococcus lactis* subsp. *lactophores* 954 bacterial sludge.

[0196] The mass percentages of the freeze-drying protectant components are as follows: 10% skim milk powder, 6% maltodextrin, and 4% sucrose. Dissolve each protectant in 55% pure water, stir thoroughly, and sterilize in a 90℃ constant temperature water bath for 30 minutes before use.

[0197] The cleaned Lactococcus lactis subsp. 954 bacterial sludge was mixed with a freeze-drying protectant at a ratio of 1:3. The bacterial sludge was added to the sterilized freeze-drying protectant and thoroughly mixed in a sterile freeze-drying bottle. The mixture was then pre-frozen in a -30°C freezer for 12 hours. The pre-frozen bacterial milk was then freeze-dried in a vacuum freeze dryer for 48 hours to obtain Lactococcus lactis subsp. 954 freeze-dried bacterial powder.

[0198] 3. Preparation of selenium-enriched lactic acid bacteria beverages

[0199] 1) Preparation of fermentation substrate

[0200] Raw milk was inspected according to the requirements of GB 19301. The qualified milk was standardized to achieve a milk protein content of 2.9g / 100g. 250g of standardized milk was heated to 60℃. 5g of walnut peptides obtained in step 1 of Example 1, 20g of maltitol, 20g of xylitol, 40g of erythritol and 5g of steviol glycosides were mixed evenly and added to the standardized milk. The mixture was sheared at 3000r for 15min using a shear emulsifier and then homogenized at 60℃ and a homogenization pressure of 25MPa (5MPa for the low pressure). After homogenization, the milk was heated to 95℃ and held for 300s for sterilization, and then transferred to a fermentation tank.

[0201] 2) First fermentation

[0202] Cool the fermentation substrate prepared in step 1) to 40°C, add the freeze-dried Lactococcus lactis subsp. lactis 954 bacterial powder (the amount of Lactococcus lactis subsp. lactis 954 added is 0.5g based on the dry matter of the bacterial cells), stir at 30r / min for 5min to disperse it evenly, ferment at 40°C, and terminate the fermentation at an acidity of 100°T; after the fermentation is completed, stir at 30r / min for 15min to break the emulsion.

[0203] 3) Second fermentation

[0204] After the first fermentation, the fermented milk was cooled to 35°C, and the selenium-enriched Lactobacillus helveticus freeze-dried powder prepared in Example 1 was added (the amount of selenium-enriched Lactobacillus helveticus added was 1.2g based on the dry matter of the bacteria). The mixture was stirred at 30r / min for 5min to disperse it evenly, and fermented at 35°C until the acidity was terminated at 160°T. After fermentation, the mixture was stirred at 30r / min for 15min to break the emulsion. The mixture was then cooled to 25°C.

[0205] 4) Secondary mixing

[0206] Dissolve 5g of stabilizer in 75℃ hot water. The stabilizer includes sodium carboxymethyl cellulose, pectin, sodium tripolyphosphate, and sodium citrate, with a mass ratio of sodium carboxymethyl cellulose: pectin: sodium tripolyphosphate: sodium citrate = 1.3:2.5:0.5:0.7. Shear emulsifier at 3000r for 15min and sterilize the liquid at 75℃ for 15s. After sterilization, cool the liquid to 25℃ and mix it with the fermented milk after the second fermentation in step 3) at 30r / min for 20min.

[0207] 5) Add acidulant

[0208] Prepare an acid solution with a solid content of 10% by mixing 2.5g citric acid, 1.5g lactic acid, and 2g sodium citrate. Sterilize the acid solution at 75°C for 15s and then cool it to 25°C. Adjust the volume of the solution from step 4) with sterile distilled water (the amount of sterile distilled water added is such that the weight of the solution after adjusting the acidity is 1000g). Adjust the acidity of the solution to 65°T using the prepared sterile acid solution.

[0209] Finally, the above liquid material was homogenized at 25°C and 20MPa. Then, quantitative filling and sealing were carried out under aseptic conditions at a filling temperature of 15°C. After filling, the liquid was transferred to a cold storage and refrigerated at 6°C.

[0210] Example 4

[0211] 1. Lyophilized Lactobacillus paracasei AL1 Plateau LPA-1 bacterial powder

[0212] The *Lactobacillus paracasei* AL1 Plateau LPA-1 strain preserved in glycerol tubes was activated. For the first activation, a 3% inoculum was added to MRS broth medium and cultured at 37°C for 24 h. For the second activation, a 2% inoculum was added and cultured at 37°C for 12 h. After fermentation, the bacterial culture was transferred to a sterile centrifuge bottle and centrifuged at 5000×g at 4°C for 10 min. The supernatant was discarded, and the bacterial cells were washed twice with sterile physiological saline to obtain *Lactobacillus paracasei* AL1 Plateau LPA-1 bacterial sludge.

[0213] The mass percentages of the freeze-drying protectant components are as follows: 10% skim milk powder, 6% maltodextrin, and 4% sucrose. Dissolve each protectant in 55% pure water, stir thoroughly, and sterilize in a 90℃ constant temperature water bath for 30 minutes before use.

[0214] The cleaned Lactobacillus paracasei AL1 Plateau LPA-1 bacterial sludge was mixed with a freeze-drying protectant at a ratio of 1:3. The bacterial sludge was added to the sterilized freeze-drying protectant and thoroughly mixed in a sterile freeze-drying bottle. The mixture was then pre-frozen at -30°C for 12 hours. The pre-frozen bacterial milk was then freeze-dried in a vacuum freeze dryer for 48 hours to obtain Lactobacillus paracasei AL1 Plateau LPA-1 freeze-dried bacterial powder.

[0215] 2. Preparation of selenium-enriched lactic acid bacteria beverages

[0216] 1) Preparation of fermentation substrate

[0217] Raw milk was inspected according to the requirements of GB 19301. The qualified milk was then standardized to achieve a milk protein content of 2.9g / 100g. 400g of standardized milk was taken and heated to 60℃. 10g of walnut peptides obtained in step 1 of Example 1, 30g of sucrose, 20g of xylitol, and 20g of sucrose were mixed evenly and added to the standardized milk. The mixture was sheared at 3000r for 15min using a shear emulsifier and then homogenized at 60℃ and a homogenization pressure of 25MPa (5MPa for the low pressure). After homogenization, the milk was heated to 95℃ and held for 300s for sterilization, and then transferred to a fermentation tank.

[0218] 2) First fermentation

[0219] Cool the fermentation substrate prepared in step 1) to 42°C, add lyophilized Lactobacillus paracasei AL1 Plateau LPA-1 bacterial powder (the amount of Lactobacillus paracasei AL1 Plateau LPA-1 added is 0.8g based on the dry matter of the bacterial cells), stir at 30r / min for 5min to disperse it evenly, ferment at 40°C, and terminate at an acidity of 100°T; after fermentation, stir at 30r / min for 15min to break the emulsion.

[0220] 3) Second fermentation

[0221] The fermented milk after the first fermentation was cooled to 37°C, and the selenium-enriched Lactobacillus helveticus freeze-dried bacterial powder prepared in Example 1 was added (wherein, the amount of selenium-enriched Lactobacillus helveticus added was 1.5g based on the dry matter of the bacterial cells). The mixture was stirred at 30r / min for 5min to disperse it evenly, and fermented at 37°C until the acidity was terminated at 160°T. After fermentation, the mixture was stirred at 30r / min for 15min to break the emulsion. The mixture was then cooled to 25°C.

[0222] 4) Secondary mixing

[0223] Dissolve 7g of stabilizer in 75℃ hot water. The stabilizer includes sodium carboxymethyl cellulose, pectin, sodium tripolyphosphate, and sodium citrate, with a mass ratio of sodium carboxymethyl cellulose: pectin: sodium tripolyphosphate: sodium citrate = 1:2:0.3:0.5. Shear emulsifier at 3000r for 15min and sterilize the liquid at 75℃ for 15s. After sterilization, cool the liquid to 25℃ and mix it with the fermented milk after the second fermentation in step 3) at 30r / min for 20min.

[0224] 5) Add acidulant

[0225] Prepare an acid solution with a solid content of 10% by mixing 2g citric acid, 1g lactic acid and 1g sodium citrate. Sterilize the acid solution at 75°C for 15s and then cool it to 25°C. Adjust the volume of the solution from step 4) with sterile distilled water (add sterile distilled water to make the weight of the solution after adjusting the acidity 1000g). Adjust the acidity of the solution to 65°T using the prepared sterile acid solution.

[0226] Finally, the above liquid material was homogenized at 25°C and 20MPa. Then, quantitative filling and sealing were carried out under aseptic conditions at a filling temperature of 15°C. After filling, the liquid was transferred to a cold storage and refrigerated at 6°C.

[0227] Comparative Example 1

[0228] The difference between Comparative Example 1 and Example 1 is that no walnut peptide was added; otherwise, they are the same as in Example 1.

[0229] Comparative Example 2

[0230] The difference between Comparative Example 2 and Example 1 is that selenium-enriched Lactobacillus helveticus was not added during the second fermentation; otherwise, they were the same as in Example 1. The specific preparation method is as follows:

[0231] 1) Preparation of fermentation substrate

[0232] Same as Example 1

[0233] 2) First fermentation

[0234] The fermentation substrate prepared in step 1) was cooled to 42°C, and the freeze-dried bacterial powders of *Streptococcus salivarius* subsp. *thermophilus* 932 and *Lactobacillus delbrueckii* subsp. *Dangxiong* LB VIII prepared in Example 1 were added (wherein, based on bacterial dry matter, the amount of *Streptococcus salivarius* subsp. *thermophilus* 932 and *Lactobacillus delbrueckii* subsp. *Dangxiong* LB VIII added was 0.1g, and the mass ratio of *Streptococcus salivarius* subsp. *thermophilus* 932 and *Lactobacillus delbrueckii* subsp. *Dangxiong* LB VIII was 2:1). The mixture was stirred at 30 r / min for 5 min to disperse it evenly, and fermented at 42°C until the acidity was terminated at 100°T. After fermentation, the mixture was stirred at 30 r / min for 15 min to break the emulsion.

[0235] 2) Second fermentation

[0236] After the first fermentation, the fermented milk was cooled to 37°C and stirred at 30 r / min for 5 min to disperse it evenly. Fermentation was continued at 37°C until the acidity was 160°T. After fermentation, the milk was broken by stirring at 30 r / min for 15 min. The temperature was then lowered to 25°C.

[0237] The other steps are the same as in Example 1.

[0238] Comparative Example 3

[0239] Preparation of selenium-enriched lactic acid bacteria beverages

[0240] 3) Preparation of fermentation substrate

[0241] Same as Example 1

[0242] 4) First fermentation

[0243] The fermentation substrate prepared in step 1) was cooled to 42°C, and the freeze-dried Streptococcus salivarius subsp. thermophilus 932 and Lactobacillus delbrueckii subsp. lactis Dangxiong LB VIII freeze-dried bacterial powders prepared in Example 1 were added (wherein, based on bacterial dry matter, the amount of Streptococcus salivarius subsp. thermophilus 932 and Lactobacillus delbrueckii subsp. lactis Dangxiong LB VIII added was 0.1g, and the mass ratio of Streptococcus salivarius subsp. thermophilus 932 and Lactobacillus delbrueckii subsp. lactis Dangxiong LB VIII was 2:1) and the freeze-dried selenium-enriched Lactobacillus helveticus prepared in Example 1 (wherein, based on bacterial dry matter, the amount of selenium-enriched Lactobacillus helveticus added was 1g). The mixture was stirred at 30 r / min for 5 min to disperse it evenly, and fermented at 42°C until the acidity was terminated at 100°T. After fermentation, the mixture was stirred at 30 r / min for 15 min to break the emulsion.

[0244] 2) Second fermentation

[0245] After the first fermentation, the fermented milk was cooled to 37°C and stirred at 30 r / min for 5 min to disperse it evenly. Fermentation was continued at 37°C until the acidity was 160°T. After fermentation, the milk was broken by stirring at 30 r / min for 15 min. The temperature was then lowered to 25°C.

[0246] The other steps are the same as in Example 1.

[0247] Comparative Example 4

[0248] The difference from Example 1 is that the temperature of the second fermentation in the preparation of the selenium-enriched lactic acid bacteria beverage is 42°C, while the rest is the same as in Example 1.

[0249] Comparative Example 5

[0250] The difference from Example 1 is that the temperature of the first fermentation in the preparation of the selenium-enriched lactic acid bacteria beverage is 37°C, while the rest is the same as in Example 1.

[0251] Comparative Example 6

[0252] The difference from Example 1 is that the amount of walnut peptide used in the preparation of the selenium-enriched lactic acid bacteria beverage is 25g, while the rest is the same as in Example 1.

[0253] The lactic acid bacteria beverages of Examples 1-4 and Comparative Examples 1-6 of the present invention were tested using the following methods:

[0254] 1. Determination of selenium content

[0255] The selenium content (μg / 100g) in the lactic acid bacteria beverages prepared in Examples 1-4 and Comparative Examples 1-6 was determined using the following method, and the results are shown in Table 2.

[0256] Method for determining selenium content: The selenium content in selenium-enriched lactic acid bacteria beverages was determined according to GB 5009.93-2017 (Method I) hydride atomic fluorescence spectrometry.

[0257] Table 2 shows the selenium content in the lactic acid bacteria beverages prepared in Examples 1-4 and Comparative Examples 1-6.

[0258]

[0259]

[0260] 2. Sensory evaluation

[0261] Ten dairy professionals who had received sensory training were randomly selected as sensory evaluators, and the scoring criteria are shown in Table 3 below.

[0262] Table 3 Sensory Evaluation Criteria for Lactic Acid Bacteria Beverages

[0263]

[0264] The average score from the sensory evaluators was taken, and the scores of each indicator were added together to obtain the total score. The higher the score, the higher the product satisfaction. The results are shown in Table 4.

[0265] Table 4 Sensory test results of lactic acid bacteria beverages

[0266]

[0267]

[0268] 3. Determination of viable bacteria count during shelf life

[0269] The lactic acid bacteria beverages prepared in Examples 1-4 and Comparative Examples 1-6 were stored at a low temperature of 2-6 degrees Celsius for 30 days. Samples were taken on days 1, 10, 20 and 30 to detect the viable bacteria count (CTU / g) in the lactic acid bacteria beverages. The specific methods are as follows, and the results are shown in Table 5.

[0270] Method for detecting viable bacteria: The total number of lactic acid bacteria colonies was determined using the dilution plate method.

[0271] Table 5 Viable bacteria count during shelf life

[0272] Storage days 1 day 10 days 20 days 30 days Example 1 <![CDATA[5.9×10 10 ]]> <![CDATA[5.8×10 10 ]]> <![CDATA[5.6×10 10 ]]> <![CDATA[5.4×10 10 ]]> Example 2 <![CDATA[7.7×10 9 ]]> <![CDATA[7.5×10 9 ]]> <![CDATA[7.3×10 9 ]]> <![CDATA[7.1×10 9 ]]> Example 3 <![CDATA[5.8×10 10 ]]> <![CDATA[5.8×10 10 ]]> <![CDATA[5.7×10 10 ]]> <![CDATA[5.6×10 10 ]]> Example 4 <![CDATA[9.3×10 10 ]]> <![CDATA[9.2×10 10 ]]> <![CDATA[9.2×10 10 ]]> <![CDATA[9.0×10 10 ]]> Comparative Example 1 <![CDATA[2.4×10 9 ]]> <![CDATA[2.3×10 9 ]]> <![CDATA[2.0×10 9 ]]> <![CDATA[1.9×10 9 ]]> Comparative Example 2 <![CDATA[7.4×10 7 ]]> <![CDATA[7.1×10 7 ]]> <![CDATA[6.9×10 7 ]]> <![CDATA[6.7×10 7 ]]> Comparative Example 3 <![CDATA[3.7×10 8 ]]> <![CDATA[3.6×10 8 ]]> <![CDATA[3.4×10 8 ]]> <![CDATA[3.2×10 8 ]]> Comparative Example 4 <![CDATA[4.4×10 8 ]]> <![CDATA[4.2×10 8 ]]> <![CDATA[4.1×10 8 ]]> <![CDATA[3.8×10 8 ]]> Comparative Example 5 <![CDATA[3.5×10 8 ]]> <![CDATA[3.3×10 8 ]]> <![CDATA[3.1×10 8 ]]> <![CDATA[2.9×10 8 ]]> Comparative Example 6 <![CDATA[5.3×10 10 ]]> <![CDATA[5.2×10 10 ]]> <![CDATA[5.0×10 10 ]]> <![CDATA[4.8×10 10 ]]>

[0273] 4. Sedimentation rate of lactic acid bacteria beverages

[0274] The sedimentation rate (%) of the lactic acid bacteria beverages prepared according to the following test examples 1-4 and comparative examples 1-6 is determined by the following method:

[0275] Take the lactic acid bacteria beverage out of the 2-6℃ refrigerator, mix it evenly, weigh 10g of the sample into a 10mL centrifuge tube, put the sample into a centrifuge, centrifuge at 3000rpm for 10min, weigh the precipitate, and calculate the precipitation rate of the lactic acid bacteria beverage according to the following formula.

[0276] Sedimentation rate of lactic acid bacteria beverages = m1 / m × 100%

[0277] m1 — the mass of the precipitate, in grams (g);

[0278] m — the mass of the sample taken, in grams (g);

[0279] Repeat three times and take the average.

[0280] The results are shown in Table 6.

[0281] Table 6. Sedimentation rates of lactic acid bacteria beverages in Examples 1-4 and Comparative Examples 1-6

[0282] product Sedimentation rate (%) of lactic acid bacteria beverages Example 1 0.51％ Example 2 0.70％ Example 3 0.59％ Example 4 0.62％ Comparative Example 1 1.89％ Comparative Example 2 2.23％ Comparative Example 3 4.30％ Comparative Example 4 3.20％ Comparative Example 5 3.71％ Comparative Example 6 4.96％

[0283] As shown in Table 2, the selenium content in the lactic acid bacteria beverages prepared in Examples 1-4 of this invention is 8.5-9.7 μg / 100g, all exceeding 8 μg / 100g. Comparative Example 2, without the addition of selenium-enriched Lactobacillus helveticus, had a selenium content of only 0.5 μg / 100g in its lactic acid bacteria beverage. Furthermore, as shown in Table 4, the lactic acid bacteria beverages prepared in Examples 1-4 of this invention have good fineness, without any grainy or powdery texture, a balanced sweet and sour taste, and a smooth mouthfeel. Comparative Example 1, without the addition of walnut peptides, had poor smoothness, with a slightly grainy or powdery texture and a thinner mouthfeel. Comparative Example 2, without the addition of selenium-enriched Lactobacillus helveticus, had poor smoothness and taste in addition to its low selenium content. Comparative Example 3, with both the first and second fermentation agents added to the fermentation base for fermentation, produced a lactic acid bacteria beverage with poor smoothness, a grainy or powdery texture, and a thinner mouthfeel. Comparative Examples 4 and 5 both used constant temperature fermentation, and regardless of whether it was high-temperature or low-temperature fermentation, the smoothness and taste of the lactic acid bacteria beverages produced were not as good as those in Example 1. Comparative Example 6, with its higher addition of walnut peptides, also resulted in a decrease in the smoothness and taste of the lactic acid beverage.

[0284] As shown in Table 5, the number of live bacteria in the lactic acid bacteria beverages prepared in Examples 1-4 of this invention is ≥10 during the shelf life. 9 (CTU / g) indicates that the lactic acid beverage prepared using the formula and process of this invention has a relatively high lactic acid bacteria content.

[0285] As shown in Table 6, the sedimentation rate of the lactic acid bacteria beverages prepared in Examples 1-4 of this invention is less than 1%. Comparative Example 1, without the addition of walnut peptide, has a sedimentation rate of 1.89%; Comparative Example 2, without the addition of selenium-enriched Lactobacillus helveticus, has a sedimentation rate of 2.23%; Comparative Example 3, with both the first and second fermentation agents added to the fermentation substrate for fermentation, has a sedimentation rate of 4.3%; Comparative Examples 4 and 5 both use constant temperature fermentation, and regardless of whether it is high-temperature or low-temperature fermentation, the sedimentation rates of the lactic acid bacteria beverages prepared are 3.2% and 3.71%, respectively; Comparative Example 6 has a relatively large amount of walnut peptide added, and the sedimentation rate of the lactic acid bacteria beverage prepared is 4.96%.

[0286] Experimental Example 1

[0287] Animal experiments on resistance to exercise fatigue

[0288] This experiment evaluated the anti-exercise fatigue function of the lactic acid bacteria beverages of Example 1 and Comparative Example 1. Forty-eight 5-week-old male SD rats (SPF grade) were purchased from the Experimental Animal Center of Three Gorges University. They were acclimatized for 7 days before the experiment, with 16 rats per cage. Experimental groups: blank control group (gavage with physiological saline), experimental group 1 (gavage with the lactic acid bacteria beverage of Example 1), and experimental group 2 (gavage with the lactic acid bacteria beverage of Comparative Example 1).

[0289] All rats underwent daily treadmill training for four weeks of progressive overload exercise. The first week involved running at 20 m / min for 10 minutes; the second week, at 25 m / min for 20 minutes; the third week, at 30 m / min for 20 minutes; and the fourth week, at 35 m / min for 30 minutes. During the running period, all rats were administered gastric gavage. After four weeks of training, the rats were forced to run at 40 m / min until exhaustion. Running time was recorded to determine running endurance. Exhaustion was defined as a rat remaining on the electric plate for more than 10 seconds. The results are shown in Table 7.

[0290] Table 7. Exhaustion running time of rats

[0291] Blank control group Experimental group 1 Experimental group 2 Time to exhaustion during running (min) 43.1±0.1 55.0±0.7 45.2±0.6

[0292] Compared with the blank control group, the running time of both experimental groups increased by 27.9% and 4.8% respectively, indicating that the lactic acid bacteria beverage with added walnut peptides can prolong the exhaustion running time of rats, enhance exercise endurance, and has an anti-fatigue effect.

[0293] After the fourth week of endurance testing, the rats were immediately euthanized, and the corresponding tissues were taken to test the corresponding biochemical indicators.

[0294] (1) Blood was collected from the heart of rats after anesthesia, left at room temperature for 2 hours, and centrifuged for 20 minutes to separate the serum. Liver and leg skeletal muscle were collected and washed with physiological saline. All samples were stored at -80℃.

[0295] (2) Serum: Detection of lactate and urea nitrogen levels;

[0296] (3) Muscle: Take skeletal muscle from the leg, homogenize it, take 0.1g and add 5mL of 100mmol / L potassium phosphate buffer, then centrifuge the homogenate at 4℃ for 15min, take the supernatant and detect the lactic acid and glycogen content;

[0297] (4) Liver: Take 0.1g of frozen liver homogenate and soak it in phosphate buffer (pH 6.8). Then centrifuge the homogenate at 4°C for 10 min, take the supernatant, and detect the glycogen content.

[0298] The test results are shown in Table 8.

[0299] Table 8 Results of fatigue-related biochemical indicators

[0300]

[0301] The reserves of liver and muscle glycogen are decisive factors in fatigue; generally, the more energy the body stores, the later fatigue occurs. During prolonged exercise, the body consumes a large amount of ATP, leading to insufficient energy supply from aerobic oxidation. This causes anaerobic glycolysis, resulting in lactic acid buildup and a decrease in body fluid pH. Lactic acid content is an important biochemical indicator accurately reflecting the degree of fatigue. Urea, the end product of protein metabolism, has a content range that serves as an important indicator for evaluating exercise intensity and is a measure of protein metabolism in the body. Extremely strenuous exercise disrupts the nitrogen balance, leading to increased nitrogen levels. Therefore, there is a significant negative correlation between urea nitrogen levels and exercise endurance.

[0302] Table 8 shows that the levels of lactic acid in the serum and skeletal muscle of rats administered the lactic acid bacteria beverage of Example 1 by gavage were significantly lower than those in the control group. In the control group, muscle glycogen was almost completely depleted, leading to the breakdown of liver glycogen for energy. In contrast, experimental group 1 consumed less liver glycogen, primarily relying on muscle glycogen, indicating that experimental group 1 had an adequate supply of muscle glycogen. Liver glycogen, as a reserve energy source, effectively improved the rats' exercise endurance. Comparing the various indicators of experimental group 1 and experimental group 2, the data for experimental group 1 were significantly better than those for experimental group 2, while the data for experimental group 2 were not significantly different from those of the control group. This suggests that the lactic acid bacteria beverage of experimental group 1 possesses strong anti-fatigue activity.

[0303] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made to it in form and detail without departing from the scope defined by the claims of the present invention.

[0304] sequence list

[0305]

[0306]

[0307]

[0308]

[0309]

Claims

1. A selenium-enriched lactic acid bacteria beverage, characterized in that, The product comprises the following raw materials in parts by weight: 25-65 parts milk, 0.1-2 parts walnut peptide, 0.01-0.1 parts first fermentation agent, and 0.08-0.16 parts second fermentation agent. The first fermentation agent is selected from one or more of the following: *Streptococcus salivarius* subsp. *thermophilus*, *Lactobacillus delbrueckii* subsp. *lactobacter*, *Bifidobacterium lactis*, *Lactococcus lactis* subsp. *lactophilus*, and *Lactobacillus paracasei*. The second fermentation agent is selenium-enriched *Lactobacillus helveticus*, obtained by fermentation culture of *Lactobacillus helveticus* in a selenium-containing medium. *Lactobacillus helveticus* is *Zhegu LBH-VI*. Lactobacillus helveticus Zhegu LBH-VI), accession number CCTCC NO: M2023095; The preparation method of the selenium-enriched lactic acid bacteria beverage includes the following steps: (1) Mix raw materials including milk and walnut peptides to prepare a fermentation base; (2) The fermentation substrate obtained in step (1) is sequentially inoculated with the first fermentation agent and the second fermentation agent to carry out temperature-switching fermentation to obtain fermented milk; the sequential inoculation of the first fermentation agent and the second fermentation agent for temperature-switching fermentation includes first inoculating the first fermentation agent and fermenting at 40-45℃, and then inoculating the second fermentation agent and fermenting at 35-39℃. (3) The fermented milk obtained in step (2) is blended to obtain a selenium-enriched lactic acid bacteria beverage.

2. The selenium-enriched lactic acid bacteria beverage according to claim 1, characterized in that, The selenium-enriched lactic acid bacteria beverage comprises the following raw materials in parts by weight: 35-65 parts milk, 0.1-2 parts walnut peptide, 0.01-0.1 parts first fermentation agent, and 0.08-0.16 parts second fermentation agent, wherein the second fermentation agent is selenium-enriched Lactobacillus helveticus.

3. The selenium-enriched lactic acid bacteria beverage according to claim 1, characterized in that, The selenium-enriched lactic acid bacteria beverage comprises the following raw materials in parts by weight: 35-40 parts milk, 0.1-1 parts walnut peptide, 0.08-0.1 parts first fermentation agent, and 0.08-0.15 parts second fermentation agent, wherein the second fermentation agent is selenium-enriched Lactobacillus helveticus.

4. The selenium-enriched lactic acid bacteria beverage according to claim 1, characterized in that, The organic selenium content in the selenium-enriched Lactobacillus helveticus is 100-125 μg / g.

5. The selenium-enriched lactic acid bacteria beverage according to claim 2, characterized in that, The organic selenium content in the selenium-enriched Lactobacillus helveticus is 100-125 μg / g.

6. The selenium-enriched lactic acid bacteria beverage according to claim 3, characterized in that, The organic selenium content in the selenium-enriched Lactobacillus helveticus is 100-125 μg / g.

7. The selenium-enriched lactic acid bacteria beverage according to claim 1, characterized in that, Based on the weight of the selenium-enriched lactic acid bacteria beverage, the selenium content in the selenium-enriched lactic acid bacteria beverage is ≥8.0μg / 100g.

8. The selenium-enriched lactic acid bacteria beverage according to claim 2, characterized in that, Based on the weight of the selenium-enriched lactic acid bacteria beverage, the selenium content in the selenium-enriched lactic acid bacteria beverage is ≥8.0μg / 100g.

9. The selenium-enriched lactic acid bacteria beverage according to claim 3, characterized in that, Based on the weight of the selenium-enriched lactic acid bacteria beverage, the selenium content in the selenium-enriched lactic acid bacteria beverage is ≥8.0μg / 100g.

10. The selenium-enriched lactic acid bacteria beverage according to claim 4, characterized in that, Based on the weight of the selenium-enriched lactic acid bacteria beverage, the selenium content in the selenium-enriched lactic acid bacteria beverage is ≥8.0μg / 100g.

11. The selenium-enriched lactic acid bacteria beverage according to claim 5, characterized in that, Based on the weight of the selenium-enriched lactic acid bacteria beverage, the selenium content in the selenium-enriched lactic acid bacteria beverage is ≥8.0μg / 100g.

12. The selenium-enriched lactic acid bacteria beverage according to claim 6, characterized in that, Based on the weight of the selenium-enriched lactic acid bacteria beverage, the selenium content in the selenium-enriched lactic acid bacteria beverage is ≥8.0μg / 100g.

13. The selenium-enriched lactic acid bacteria beverage according to claim 1, characterized in that, The selenium-enriched lactic acid bacteria beverage has a live bacteria count ≥10 during its shelf life. 9 CFU / ml.

14. The selenium-enriched lactic acid bacteria beverage according to claim 7, characterized in that, The selenium-enriched lactic acid bacteria beverage has a live bacteria count ≥10 during its shelf life. 9 CFU / ml.

15. The selenium-enriched lactic acid bacteria beverage according to claim 1, characterized in that, The milk is selected from one or more of whole milk, low-fat milk, or skim milk.

16. The selenium-enriched lactic acid bacteria beverage according to claim 4, characterized in that, The milk is selected from one or more of whole milk, low-fat milk, or skim milk.

17. The selenium-enriched lactic acid bacteria beverage according to claim 7, characterized in that, The milk is selected from one or more of whole milk, low-fat milk, or skim milk.

18. The selenium-enriched lactic acid bacteria beverage according to claim 13, characterized in that, The milk is selected from one or more of whole milk, low-fat milk, or skim milk.

19. The selenium-enriched lactic acid bacteria beverage according to claim 1, characterized in that, The molecular weight of the walnut peptide is 800-1000 Da.

20. The selenium-enriched lactic acid bacteria beverage according to claim 4, characterized in that, The molecular weight of the walnut peptide is 800-1000 Da.

21. The selenium-enriched lactic acid bacteria beverage according to claim 7, characterized in that, The molecular weight of the walnut peptide is 800-1000 Da.

22. The selenium-enriched lactic acid bacteria beverage according to claim 13, characterized in that, The molecular weight of the walnut peptide is 800-1000 Da.

23. The selenium-enriched lactic acid bacteria beverage according to claim 15, characterized in that, The molecular weight of the walnut peptide is 800-1000 Da.

24. The selenium-enriched lactic acid bacteria beverage according to claim 1, characterized in that, The walnut peptides are obtained by enzymatic hydrolysis of walnuts using proteases.

25. The selenium-enriched lactic acid bacteria beverage according to claim 19, characterized in that, The walnut peptides are obtained by enzymatic hydrolysis of walnuts using proteases.

26. The selenium-enriched lactic acid bacteria beverage according to claim 1, characterized in that, The first fermentation agent is *Streptococcus salivarius* subsp. *thermophilus* and *Lactobacillus delbrueckii* subsp. *lactobacter*.

27. The selenium-enriched lactic acid bacteria beverage according to claim 26, characterized in that, Based on bacterial dry matter, the mass ratio of *Streptococcus salivarius* subsp. *thermophilus* to *Lactobacillus delbrueckii* subsp. *lactobacter* is 2-3:1-1.

5.

28. The selenium-enriched lactic acid bacteria beverage according to any one of claims 1-27, characterized in that, The thermophilic subspecies of Streptococcus salivarius is Streptococcus salivarius subspecies 932 (…). Straptococcus salivarias subsp. thermophilus 932), deposited on June 5, 2023 at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO: M 2023902; And / or, the Lactobacillus delbrueckii subsp. lactis is Lactobacillus delbrueckii LB VⅢ ( Lactobacillus delbrueckii subsp. lactis Dangxiong LB VⅢ), was deposited at the China Center for Type Culture Collection (CCTCC) on March 23, 2023, with accession number CCTCC NO: M2023396; And / or, the Lactococcus lactis subsp. lactis is Lactococcus lactis subsp. lactis 954 (… Lactococcus lactis subsp. Lactis 954), deposited on June 5, 2023 at the China Center for Type Culture Collection (CCTCC), accession number CCTCCNO: M2023904; And / or, the Lactobacillus paracasei is Lactobacillus paracasei AL1 Plateau LPA-1 ( lactobacillus paracasei AL1 Plateau LPA-1).

29. The selenium-enriched lactic acid bacteria beverage according to any one of claims 1-27, characterized in that, The ingredients also include 5.0-8.5 parts of sweetener.

30. The selenium-enriched lactic acid bacteria beverage according to claim 28, characterized in that, The ingredients also include 5.0-8.5 parts of sweetener.

31. The selenium-enriched lactic acid bacteria beverage according to claim 29, characterized in that, The sweetener is selected from one or more of sucrose, erythritol, xylitol, maltitol, sucralose, or steviol glycosides.

32. The selenium-enriched lactic acid bacteria beverage according to claim 29, characterized in that, The sweetener is sucrose.

33. The selenium-enriched lactic acid bacteria beverage according to any one of claims 1-27, characterized in that, The raw materials also include 0.3-0.7 parts of stabilizer.

34. The selenium-enriched lactic acid bacteria beverage according to claim 28, characterized in that, The raw materials also include 0.3-0.7 parts of stabilizer.

35. The selenium-enriched lactic acid bacteria beverage according to claim 29, characterized in that, The raw materials also include 0.3-0.7 parts of stabilizer.

36. The selenium-enriched lactic acid bacteria beverage according to claim 33, characterized in that, The stabilizer is selected from one or more of sodium carboxymethyl cellulose, pectin, sodium tripolyphosphate, and sodium citrate.

37. The selenium-enriched lactic acid bacteria beverage according to claim 36, characterized in that, The mass ratio of sodium carboxymethyl cellulose, pectin, sodium tripolyphosphate, and sodium citrate is 0.5-1.3:1.5-2.5:0.2-0.5:0.3-0.

7.

38. The selenium-enriched lactic acid bacteria beverage according to any one of claims 1-27, characterized in that, The raw materials also include 0.11-0.6 parts of acidulant.

39. The selenium-enriched lactic acid bacteria beverage according to claim 28, characterized in that, The raw materials also include 0.11-0.6 parts of acidulant.

40. The selenium-enriched lactic acid bacteria beverage according to claim 29, characterized in that, The raw materials also include 0.11-0.6 parts of acidulant.

41. The selenium-enriched lactic acid bacteria beverage according to claim 33, characterized in that, The raw materials also include 0.11-0.6 parts of acidulant.

42. The selenium-enriched lactic acid bacteria beverage according to claim 36, characterized in that, The raw materials also include 0.11-0.6 parts of acidulant.

43. The selenium-enriched lactic acid bacteria beverage according to claim 38, characterized in that, The acidulant is selected from citric acid, lactic acid, and sodium citrate.

44. The selenium-enriched lactic acid bacteria beverage according to claim 43, characterized in that, The mass ratio of citric acid, lactic acid and sodium citrate is 5-25:5-15:1-20.

45. The selenium-enriched lactic acid bacteria beverage according to any one of claims 1-27, characterized in that, The selenium-enriched lactic acid bacteria beverage is made by fermenting a fermentation base containing milk and walnut peptides at variable temperatures.

46. ​​The method for preparing the selenium-enriched lactic acid bacteria beverage according to any one of claims 1-45, characterized in that, The preparation method includes the following steps: (1) Mix raw materials including milk and walnut peptides to prepare a fermentation base; (2) The fermentation substrate obtained in step (1) is sequentially inoculated with the first fermentation agent and the second fermentation agent to carry out temperature-switching fermentation to obtain fermented milk; the sequential inoculation of the first fermentation agent and the second fermentation agent for temperature-switching fermentation includes first inoculating the first fermentation agent and fermenting at 40-45℃, and then inoculating the second fermentation agent and fermenting at 35-39℃. (3) The fermented milk obtained in step (2) is blended to obtain a selenium-enriched lactic acid bacteria beverage.

47. The method for preparing selenium-enriched lactic acid bacteria beverage according to claim 46, characterized in that, The preparation in step (3) includes adding stabilizers and / or acidulants to the selenium-enriched lactic acid bacteria beverage prepared in step (2) and mixing and homogenizing it.

48. The method for preparing selenium-enriched lactic acid bacteria beverage according to claim 46, characterized in that, The fermentation base also includes sweeteners.

49. The method for preparing selenium-enriched lactic acid bacteria beverage according to claim 47, characterized in that, The fermentation base also includes sweeteners.

50. The method for preparing selenium-enriched lactic acid bacteria beverage according to claim 48, characterized in that, The sweetener is selected from one or more of sucrose, erythritol, xylitol, maltitol, sucralose, or steviol glycosides.

51. The method for preparing selenium-enriched lactic acid bacteria beverage according to claim 48, characterized in that, The sweetener is sucrose.

52. The method for preparing a selenium-enriched lactic acid bacteria beverage according to any one of claims 46-51, characterized in that, First, inoculate with the first fermentation agent and ferment at 40-45℃ for 10-12 hours until the final acidity is 90-100ºT. Then, inoculate with the second fermentation agent and ferment at 35-39℃ for 8-10 hours until the final acidity is 160-180ºT.

53. The method for preparing a selenium-enriched lactic acid bacteria beverage according to any one of claims 46-51, characterized in that, The preparation method further includes a step of homogenizing the fermentation substrate before inoculation.

54. The method for preparing selenium-enriched lactic acid bacteria beverage according to claim 52, characterized in that, The preparation method further includes a step of homogenizing the fermentation substrate before inoculation.

55. The method for preparing selenium-enriched lactic acid bacteria beverage according to claim 53, characterized in that, The homogenization temperature is 50-65℃.

56. The method for preparing selenium-enriched lactic acid bacteria beverage according to claim 53, characterized in that, The homogenization pressure is 10-50 MPa.

57. The method for preparing a selenium-enriched lactic acid bacteria beverage according to claim 55, characterized in that, The homogenization pressure is 10-50 MPa.

58. The method for preparing selenium-enriched lactic acid bacteria beverage according to claim 53, characterized in that, The homogenization process also includes a step of sterilizing the fermentation substrate.

59. The method for preparing a selenium-enriched lactic acid bacteria beverage according to claim 58, characterized in that, The sterilization temperature is 90-100℃.

60. The method for preparing selenium-enriched lactic acid bacteria beverage according to claim 58, characterized in that, The sterilization time is 200-400 seconds.

61. The method for preparing selenium-enriched lactic acid bacteria beverage according to claim 59, characterized in that, The sterilization time is 200-400 seconds.

62. The method for preparing a selenium-enriched lactic acid bacteria beverage according to any one of claims 46-51, characterized in that, The preparation method also includes the step of stirring and breaking the emulsion after both the first and second fermentations.

63. The method for preparing selenium-enriched lactic acid bacteria beverage according to claim 52, characterized in that, The preparation method also includes the step of stirring and breaking the emulsion after both the first and second fermentations.

64. The method for preparing selenium-enriched lactic acid bacteria beverage according to claim 53, characterized in that, The preparation method also includes the step of stirring and breaking the emulsion after both the first and second fermentations.

65. The method for preparing selenium-enriched lactic acid bacteria beverage according to claim 62, characterized in that, The stirring speed is 20-40 r / min.

66. The method for preparing selenium-enriched lactic acid bacteria beverage according to claim 62, characterized in that, Stirring time is 10-20 minutes.

67. The method for preparing a selenium-enriched lactic acid bacteria beverage according to claim 65, characterized in that, Stirring time is 10-20 minutes.

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