Kluyveromyces marxianus complex for high production of gamma-aminobutyric acid and its use in bird's nest milk products

Abstract

This invention discloses a Kluyveromyces martensii complex rich in organic selenium and high-yielding γ-aminobutyric acid (GABA) and its application in bird's nest dairy products, belonging to the field of biotechnology. The complex was obtained through ARTP mutagenesis screening to produce a high-GABA-producing Kluyveromyces martensii strain (GABA yield ≥2000 mg / kg). After selenium enrichment culture, the organic selenium content was ≥200 mg / kg and the organic selenium ratio was >99%. When this yeast complex was applied to the preparation of bird's nest milk, the synergistic effect of organic selenium, GABA, and bird's nest significantly improved the sleep rate in mice (90%), solving the problems of single function and low bioavailability in existing products. It is suitable for the field of health foods for sleep aid and immune regulation.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a Kluyveromyces martensii complex rich in organic selenium and high in γ-aminobutyric acid (GABA) production, and its application in bird's nest dairy products, particularly suitable for aiding sleep and enhancing immunity. Background Technology

[0002] With the fast pace of modern life, sleep disorders and weakened immunity have become common health problems plaguing modern people. Sleep is closely related to the immune system; insufficient sleep directly weakens immune function, while chronic activation of the immune system affects sleep quality. Although various sleep aids and immune-boosting health foods are available on the market, most are single-ingredient or chemically synthesized, resulting in limited effectiveness, potential side effects, or low consumer acceptance.

[0003] Kluyveromyces martensii is a probiotic yeast known to accumulate organic selenium (a key component of glutathione peroxidase, enhancing antioxidant and immunomodulatory functions) and produce high levels of gamma-aminobutyric acid (GABA, an inhibitory neurotransmitter that promotes relaxation and sleep). Furthermore, it is rich in B vitamins and glutathione, suggesting potential benefits in boosting immunity and improving sleep quality. While there are existing examples of yeast being used in food, such as adding yeast to fermented dairy products to improve flavor, there are no reports of synergistic enhancement of sleep and immunity using Kluyveromyces martensii complexes.

[0004] Bird's nest, a traditional tonic, is rich in sialic acid and epidermal growth factor, which can enhance immunity and improve skin complexion. Dairy products, such as milk, are excellent sources of protein and calcium.

[0005] Currently, there are no known examples of synergistic compounding of the active ingredients of Kluyveromyces martensii with bird's nest and its application in dairy products. Therefore, developing a Kluyveromyces martensii complex that can simultaneously regulate sleep and immunity and applying it to bird's nest milk is of great significance. Moreover, developing a product preparation method that can simultaneously retain multiple active ingredients, improve bioavailability, and achieve good stability and taste has significant market value and application prospects. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art and provide an innovative Max Kluyveromycin complex, thereby achieving a functional upgrade of dairy products through the complementary advantages of Max Kluyveromycin and bird's nest.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] On the one hand, the present invention provides a Kluyveromyces martensii yeast that produces high levels of γ-aminobutyric acid (GABA). Kluyveromyces marxianus (), of which the accession number is CCTCC NO: M 20242474.

[0009] In some embodiments, the *Kluyveromyces martensii* is obtained by ARTP mutagenesis screening, wherein the starting strain for ARTP mutagenesis screening is... Kluyveromyces marxianus CJ3113, with accession number CCTCC NO: M 20211265.

[0010] In some embodiments, the *Kluyveromyces martensii* is obtained by inoculating the starting strain into YPD solid medium and then subjecting it to ARTP mutagenesis.

[0011] In some implementations, the ARTP mutagenesis parameters are: helium flow rate 12 SLM, irradiation distance 2.5 mm, and treatment time 45 s.

[0012] In some implementations, the strains obtained by ARTP mutagenesis are subjected to primary and secondary screening.

[0013] In some embodiments, the Kluyveromyces martensii produces ≥2000 mg / kg of GABA in a glutamic acid-containing medium, for example, ≥2100 mg / kg, ≥2200 mg / kg, ≥2300 mg / kg, etc., preferably greater than ≥2300 mg / kg.

[0014] On the other hand, the present invention also provides a Kluyveromyces macrocephala complex rich in organic selenium and GABA, wherein it is obtained by selenium enrichment culture of any of the above-mentioned Kluyveromyces macrocephala.

[0015] In some embodiments, the Kluyveromyces macrocephala complex contains ≥200 mg / kg of organic selenium and >99% organic selenium, and ≥2000 mg / kg of GABA.

[0016] In some implementations, the selenium enrichment culture uses sodium selenite as the selenium source.

[0017] In some implementations, the selenium enrichment culture temperature is 30°C.

[0018] In some embodiments, the pH of the selenium enrichment culture is 6.0-6.5, for example, 6.0, 6.1, 6.2, 6.3, 6.4 or 6.5, preferably 6.2.

[0019] In some implementations, the fermentation culture time for the selenium enrichment culture is 48-72 h, for example, 48, 54, 60, 66 or 72 h.

[0020] In some implementation schemes, the selenium enrichment culture adopts a selenium source supplementation strategy, adding 50% sodium selenite at 0 h of fermentation and supplementing the remaining 50% at 24 h, to avoid the toxic inhibition of cells by high concentrations of selenium.

[0021] In some embodiments, the final concentration of sodium selenite in the selenium enrichment culture is 50-100 μg / mL, for example, 50, 60, 70, 80, 90 or 100 μg / mL.

[0022] In some implementations, glucose is added via a fed-batch method during the selenium enrichment culture process.

[0023] In some embodiments, the sugar concentration of the fermentation broth for selenium enrichment culture is 5-10 g / L, for example, 5, 6, 7, 8, 9 or 10 g / L.

[0024] In another aspect, the present invention provides a bird's nest dairy product made from the above-mentioned Kluyveromyces macrocephala complex, wherein the bird's nest dairy product comprises Kluyveromyces macrocephala complex, bird's nest, camel milk, and tremella.

[0025] In some embodiments, the bird's nest dairy product contains 1%-5% Kluyveromycin complex by weight percentage, for example, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5% or 5%, etc.

[0026] In some embodiments, the bird's nest dairy product contains 0.3%-1.0% bird's nest by weight, for example, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9% or 1.0%.

[0027] In some embodiments, the bird's nest dairy product contains 40%-60% camel milk by weight percentage, for example, 40%, 45%, 50%, 55% or 60%.

[0028] In some embodiments, the bird's nest dairy product contains 20%-40% milk by weight percentage, for example, 20%, 25%, 30%, 35% or 40%.

[0029] In some embodiments, the bird's nest dairy product contains 0.1%-1% tremella by weight, for example, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9% or 1.0%.

[0030] In some embodiments, the bird's nest dairy product further comprises one or more physiologically acceptable excipients and / or excipients, including other edible ingredients, seasonings, solvents, diluents, disintegrants, precipitation inhibitors, surfactants, flow aids, binders, lubricants, dispersants, suspending agents, isotonic agents, thickeners, emulsifiers, preservatives, stabilizers, hydrating agents, emulsification accelerators, buffers, colorants, flavorings, sweeteners, ion exchangers, flavoring agents, or antioxidants.

[0031] In some embodiments, the physiologically acceptable excipients and / or excipients comprise one or more selected from stabilizers, sweeteners, and water.

[0032] In some embodiments, the stabilizer is agar, and the amount used is 0.02%-0.1% by weight, for example, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09% or 0.1%, etc.

[0033] In some implementations, the sweetener is fructooligosaccharide.

[0034] In some embodiments, the sweetener is erythritol, used in an amount of 0.5%-2.0% by weight, for example, 0.5%, 1%, 1.5% or 2.0%.

[0035] In some embodiments, the bird's nest dairy product comprises the following ingredients (per 1000 mL of finished product): 400 mL camel milk, 300 mL cow milk, 5 g tremella, ≥3 g bird's nest, 10 g Kluyveromyces macrocephala complex, 10 g erythritol, 0.4 g agar, with the remainder being purified water.

[0036] In some implementations, the bird's nest dairy product contains ≥26 μg of organic selenium and ≥21.9 mg of GABA per 100 g of product.

[0037] Furthermore, the present invention provides a method for preparing the above-mentioned bird's nest dairy products, comprising the following steps:

[0038] (1) Pretreatment of white fungus: Soak and clean the dried white fungus, add water and boil, then filter to obtain white fungus gel;

[0039] (2) Pretreatment of bird's nest: After soaking and cleaning the dried bird's nest, add water and stew it in a double boiler to obtain bird's nest stewing liquid;

[0040] (3) Base material mixing: Mix camel milk, cow milk, tremella gelatin, sweetener and stabilizer, heat and stir to form a uniform base material;

[0041] (4) Functional addition and homogenization: Add the bird's nest stewing liquid obtained in step (2) and the above-mentioned Max Kluyveromycin complex to the base material, stir and disperse, and then perform high-pressure homogenization;

[0042] (5) Sterilization and filling: The homogenized liquid is subjected to ultra-high temperature instantaneous sterilization (UHT), then cooled and aseptically filled.

[0043] In some implementations, the amount of tremella in step (1) is 0.1%-1.0% by weight, for example, it can be 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9% or 1.0%.

[0044] In some implementations, the temperature for cooking the white fungus in step (1) is 90℃-100℃, for example, it can be 90℃, 91℃, 92℃, 93℃, 94℃, 95℃, 96℃, 97℃, 98℃, 99℃ or 100℃, preferably 95℃.

[0045] In some implementations, the cooking time of the white fungus in step (1) is 1-2 hours, for example, 1, 1.5 or 2 hours, preferably 1.5 hours.

[0046] In some implementations, the amount of bird's nest in step (2) is 0.3-1.0% by weight, for example, it can be 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9% or 1.0%.

[0047] In some implementations, the temperature for double-boiling the bird's nest in step (2) is 90℃-100℃, for example, it can be 90℃, 91℃, 92℃, 93℃, 94℃, 95℃, 96℃, 97℃, 98℃, 99℃ or 100℃, preferably 95℃.

[0048] In some implementations, the double-boiling time for bird's nest in step (2) is 20-60 minutes, for example, 20, 30, 40, 50 or 60 minutes, preferably 30 minutes.

[0049] In some implementations, the stabilizer in step (3) is agar, and the amount used is 0.02%-0.1% by weight, for example, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09% or 0.1%, etc.

[0050] In some implementations, the sweetener in step (3) is fructooligosaccharide.

[0051] In some implementations, the sweetener in step (3) is erythritol, and the amount used is 0.5%-2% by weight, for example, 0.5%, 1%, 1.5% or 2.0%.

[0052] In some implementations, the camel milk in step (3) is used in an amount of 40%-60% by weight, for example, 40%, 45%, 50%, 55% or 60%.

[0053] In some implementations, the amount of milk used in step (3) is 20%-40% by weight, for example, 20%, 25%, 30%, 35% or 40%.

[0054] In some embodiments, the stirring speed in step (3) is 200-600 rpm, for example, 200, 300, 400, 500 or 600 rpm, preferably 300 rpm.

[0055] In some embodiments, the stirring temperature in step (3) is 60°C-70°C, for example, it can be 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C or 70°C, preferably 65°C.

[0056] In some implementations, the stirring time in step (3) is 20-60 minutes, for example, 20, 30, 40, 50 or 60 minutes, preferably 30 minutes.

[0057] In some implementations, the amount of the Kluyveromyces macrocephala complex in step (4) is 1%-5% by weight, for example, it can be 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5% or 5%.

[0058] In some implementations, the stirring speed in step (4) is 400-600 rpm, for example, 400, 450, 500, 550 or 600 rpm, preferably 500 rpm.

[0059] In some implementations, the stirring time in step (4) is 10-30 minutes, for example, 10, 15, 20, 25 or 30 minutes, preferably 15 minutes.

[0060] In some implementations, the first-stage pressure of high-pressure homogenization in step (4) is 15-25 MPa, for example, it can be 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 MPa, preferably 20 MPa.

[0061] In some implementations, the second-stage pressure of high-pressure homogenization in step (4) is 3-10 MPa, for example, it can be 3, 4, 5, 6, 7, 8, 9 or 10 MPa, preferably 5 MPa.

[0062] In some implementations, the temperature of the high-pressure homogenized liquid in step (4) is maintained at 60-65°C, for example, 60°C, 61°C, 62°C, 63°C, 64°C or 64°C.

[0063] In some implementations, the particle size of the liquid after high-pressure homogenization in step (4) should be ≤2 μm.

[0064] In some implementations, the ultra-high temperature instantaneous sterilization step (5) is as follows: the homogenized liquid is rapidly heated to 137°C, held for 4 seconds, and then immediately cooled to below 25°C.

[0065] In another aspect, the present invention provides the use of the above-mentioned Max Kluyveromyces complex, the above-mentioned bird's nest dairy product, or the bird's nest dairy product prepared according to the above-mentioned preparation method in the preparation of food or health products for sleep aid or immune enhancement.

[0066] In some implementations, the Max Kluyveromycin complex or the bird's nest dairy product can improve sleep rates in mice.

[0067] In some implementations, the Max Kluyveromycin complex or the bird's nest dairy product can increase the average sleep time of mice.

[0068] In some embodiments, the Kluyveromycin complex or the bird's nest dairy product can enhance immunity by increasing NK cell activity, IL-2 levels, and IgG levels.

[0069] Advantages of this invention

[0070] The yeast complex provided by this invention can be applied to the preparation of bird's nest milk. Through the synergistic effect of organic selenium, GABA and bird's nest, it can significantly improve the sleep rate (90%) of mice and enhance the immunity of mice. This solves the problems of single function and low bioavailability of existing products and is suitable for the field of health food for sleep aid and immune regulation.

[0071] Preservation Information

[0072] The strains used in this invention include Kluyveromyces martensii. Kluyveromyces marxianus CJ3113, accession number CCTCC NO: M 20211265, was deposited at the China Center for Type Culture Collection (CCTCC) on October 13, 2021.

[0073] The strains used in this invention include Kluyveromyces martensii. Kluyveromyces marxianus CJA0021, accession number CCTCC NO: M 20242474, was deposited at the China Center for Type Culture Collection (CCTCC) on November 7, 2024. Detailed Implementation

[0074] For the purpose of clarity and concise description, features are described herein as part of some identical or separate embodiments; however, it will be understood that the scope of the invention may include some embodiments having combinations of all or some of the described features. The technical solutions of the present invention will now be described clearly and completely. Obviously, based on the specific embodiments of the present invention, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of the present invention.

[0075] I. Definition

[0076] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly used in the field to which this invention pertains. For the purposes of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural forms, and vice versa.

[0077] As used herein, the terms “contains,” “has,” “includes,” and “contains” should be interpreted as open-ended terms (i.e., meaning “including but not limited to”), referring to the inclusion of explicitly specified features but not excluding other elements. “And / or” describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A alone, A and B simultaneously, or B alone. The character “ / ” generally indicates an “or” relationship between the preceding and following related objects, while the character “+” generally indicates an “and” relationship.

[0078] As used herein, the terms “optional,” “any,” “arbitrary,” or “any item” mean that the event or situation described below may, but does not have to, occur, including the circumstances in which the event or situation occurs or does not occur. As used herein, “a,” “an item,” and “a kind” are used to refer to one or more grammatical objects. “Multiple,” “various,” or “multiple items” as used herein refer to two or more grammatical objects, such as two, three, four, five, etc. The terms “one or more / a kind or more / an item or more” and “at least one / a kind / item” are used interchangeably.

[0079] As used herein, references to “some embodiments,” “some implementations,” or “some implementation schemes” describe a subset of all possible embodiments, but it is understood that “some embodiments” may be the same subset or different subsets of all possible embodiments and may be combined with each other without conflict.

[0080] As used herein, the term “about” generally refers to a variation of 0.5% to 10% above or below a specified value, such as a variation of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below a specified value.

[0081] As used in this article, the term "feedback" refers to the addition of specific nutrients or other components to a fermentation system or production process in a continuous or intermittent manner, according to a certain method or rate, in order to maintain the conditions required for cell growth and metabolism. This method is often used in large-scale cell culture.

[0082] As used herein, the term "physiologically acceptable excipient" includes, but is not limited to, solvents, diluents, disintegrants, precipitation inhibitors, surfactants, flow aids, binders, lubricants, dispersants, suspending agents, isotonic agents, thickeners, emulsifiers, preservatives, stabilizers, hydrating agents, emulsification accelerators, buffers, colorants, flavorings, sweeteners, ion exchangers, flavoring agents, or antioxidants. These components work together to provide a more comprehensive effect. In some embodiments, the physiologically acceptable excipient and / or excipient comprises one or more selected from sweeteners, stabilizers, and water; in some preferred embodiments, the stabilizer is agar. In some preferred embodiments, the sweetener is fructooligosaccharides. In some preferred embodiments, the sweetener is erythritol.

[0083] II. Examples

[0084] The present invention will be described in detail below through specific embodiments. It should be understood that the following embodiments are for explanation and illustration only and do not limit the scope of the present invention in any way.

[0085] Where specific methods, techniques, or conditions are not specified in the examples, they shall be performed in accordance with the methods, techniques, or conditions described in the literature in this field or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0086] Example 1: Screening of high-GABA-producing Kluyveromyces marxoiris strains

[0087] 1.1 Materials and Reagents

[0088] Starting strain: Kluyveromyces marxianus CJ3113 (CCTCC NO: M 20211265, deposited at the China Center for Type Culture Collection on October 13, 2021)

[0089] Culture medium:

[0090] YPD medium (solid medium): yeast extract 10 g / L, peptone 20 g / L, glucose 20 g / L, agar 20 g / L.

[0091] Screening medium: glucose 30 g / L, sodium glutamate 10 g / L, yeast extract 5 g / L, KH2PO4 1 g / L, MgSO4·7H2O 0.5 g / L, pH 6.5.

[0092] Instrument: ARTP mutagenesis instrument: Beijing Siqingyuan Biotechnology ARTP-M100 model.

[0093] 2. Strain Screening Procedure

[0094] 2.1 Activation of the starting strain

[0095] The frozen starting strain was inoculated onto YPD solid medium and cultured at 30°C for 48 h. A single colony was picked and inoculated into 50 mL of YPD liquid medium and cultured at 30°C with shaking at 200 rpm for 18 h. The bacterial concentration was then adjusted to OD0.05. 600 =1.0 (for backup)

[0096] 2.2 ARTP mutagenesis treatment

[0097] Take 100 μL of bacterial suspension and spread it on a sterile metal slide. Place it in the sample chamber of the ARTP mutagenesis instrument and set the parameters as follows: helium flow rate 12 SLM, irradiation distance 2.5 mm, and treatment time 45 s (lethality rate approximately 75%).

[0098] After mutagenesis, the bacterial culture was serially diluted with 0.85% physiological saline, spread on YPD plates, and incubated at 30°C for 72 h. Single colonies were then picked for later use.

[0099] 2.3 Screening of high-yielding strains

[0100] Initial screening: The mutant colonies were inoculated on screening medium plates containing 1.0% glutamic acid and incubated at 30℃ for 96 h. The amount of GABA produced was detected by thin layer chromatography (TLC) (developing solvent: n-butanol-glacial acetic acid-water = 4:1:1, colorimetric reagent: ninhydrin ethanol solution). 50 colonies with darker spots than the starting strain were selected.

[0101] Secondary screening: The primary screening strains were inoculated into 50 mL of screening medium and cultured at 30℃ and 200 rpm for 72 h with shaking. GABA content was detected by HPLC (column: C18 column, mobile phase: 0.05 mol / L potassium dihydrogen phosphate buffer, detection wavelength: 210 nm). A high-yielding strain, CJA0021, was obtained, with a yield of 2380 mg / kg (compared to 830 mg / kg for the starting strain). The high-GABA-producing strain CJA0021 was classified as *Kluyveromyces martensii*. Kluyveromyces marxianus The accession number is CJA0021, and its accession number is CCTCCNO: M 20242474. It was deposited on November 7, 2024 at the China Center for Type Culture Collection (CCTCC, Wuhan University, Wuhan, China).

[0102] Example 2: Preparation of Marks Kluyveromyces protein rich in organic selenium and high in GABA

[0103] 1. Materials and Instruments

[0104] Strains: Kluyveromyces marxianus CJA0021 (obtained through screening in Example 1, CCTCC NO: M20242474)

[0105] Culture medium:

[0106] Seed culture medium: glucose 20 g / L, yeast extract 10 g / L, peptone 15 g / L, KH2PO4 2 g / L, MgSO4·7H2O 0.5 g / L, pH 6.0 (sterilized at 121℃ for 20 min).

[0107] Selenium-enriched fermentation medium: glucose 40 g / L, sodium glutamate 20 g / L, yeast extract 8 g / L, (NH4)2SO4 5 g / L, KH2PO4 3 g / L, MgSO4·7H2O 1 g / L, sodium selenite (selenium source) 80 μg / mL, pH 6.2 (sterilized at 115℃ for 30 min).

[0108] Instruments: 5 L fermenter (BIOTECH-5BG), ultrasonic crusher (JY92-IIN), ultrafiltration system (3 kDa filtration membrane), spray dryer (SD-1000).

[0109] 2. Selenium-enriched yeast fermentation process

[0110] 2.1 Seed Culture

[0111] CJA0021 strain, frozen at -80℃, was streaked onto YPD solid plates and incubated at 30℃ for 48 h.

[0112] A single colony was picked and inoculated into 100 mL of seed culture medium, and cultured at 30°C with shaking at 200 rpm for 16 h until OD reached the target value. 600 =2.0 (live bacteria count ≈ 5 × 10⁻⁶) 8 (CFU / mL).

[0113] 2.2 Selenium-enriched fermentation culture

[0114] Fermentation tank parameters: inoculum size 5% (v / v), initial pH 6.2, temperature 30℃, stirring speed 300 rpm, aeration rate 1.0 vvm.

[0115] Selenium source supplementation strategy: Add 50% sodium selenite (40 μg / mL) at 0 h of fermentation, and add the remaining 50% (40 μg / mL) at 24 h to avoid the toxic inhibition of cells by high concentrations of selenium.

[0116] Fermentation process control: The sugar concentration of the fermentation broth was maintained at 5-10 g / L by adding 200 g / L glucose. After fermentation for 72 h, the broth was removed from the tank (residual sugar <1 g / L).

[0117] 3. Cell collection and drying

[0118] The fermentation broth was filtered through a plate and frame filter (0.22 μm filter membrane) to collect yeast cells, which were then washed three times with deionized water to obtain wet cells (moisture content approximately 85%). After sterilization, the cells were spray-dried at an inlet air temperature of 180℃, an outlet air temperature of 80℃, and a feed rate of 15 mL / min to obtain a pale yellow yeast protein powder with a moisture content of <5%.

[0119] 4. Finished Product Composition Testing and Results

[0120] 4.1 Analysis of Organic Selenium Content and Speciation

[0121] The determination of total selenium content in the sample was performed directly according to the method specified in GB 5009.93.

[0122] 4.2 Determination of Inorganic and Organic Selenium Content:

[0123] The determination of inorganic selenium and the calculation of organic selenium content shall be carried out strictly in accordance with the methods specified in Appendix A.2 of GB 1903.21-2016.

[0124] Preparation of inorganic selenium sample: Weigh approximately 1 g of yeast powder (accurate to 0.0001 g) and place it in a 50 mL volumetric flask. Add approximately 25 mL of water, sonicate for 10 min to dissolve the sample completely, and then add water to the mark and mix well. Centrifuge the solution at 5000 r / min for 10 min, accurately pipette 10.0 mL of the supernatant into a 15 mL centrifuge tube, add 2.0 mL of hydrochloric acid and 1.0 mL of potassium ferricyanide solution sequentially, mix well, and use this as the test solution. Simultaneously prepare a blank solution.

[0125] Analytical procedure: According to the provisions of GB 5009.93 on hydride atomic fluorescence spectrometry, the concentration of inorganic selenium in the above test solution was determined, and the organic selenium content was obtained by subtracting the inorganic selenium content from the total selenium content, thereby calculating the percentage content of organic selenium.

[0126] Results: The finished product contained 260 mg / kg of organic selenium and <0.5 mg / kg of inorganic selenium, therefore the proportion of organic selenium was 99.8%.

[0127] 4.3 Determination of GABA content

[0128] Detection method: OPA derivatization-HPLC method was used. Chromatographic conditions: C18 column (250 mm × 4.6 mm), mobile phase methanol-0.05 mol / L potassium dihydrogen phosphate (35:65), detection wavelength 210 nm.

[0129] Results: GABA content was 2190 mg / kg, RSD = 2.3% (n = 3).

[0130] The results above show that this embodiment achieved efficient synergistic enrichment of organic selenium (260 mg / kg) and GABA (2190 mg / kg) through stepwise selenium source supplementation and optimized crushing process, providing highly active functional raw materials for the subsequent development of bird's nest dairy products.

[0131] Example 3: Preparation of Kluwer Camel Milk and Bird's Nest Milk

[0132] 1. The formula for Kluwer camel milk bird's nest milk is shown in the table below:

[0133] Table 1. Kluwer Camel Milk and Bird's Nest Milk Formula (per 1000 mL of finished product)

[0134]

[0135] 2. Preparation process

[0136] 2.1 Pretreatment of Tremella

[0137] Soak 5g of dried white fungus in purified water until softened, rinse, trim off the root, and tear into small pieces. Add 200mL of purified water and simmer in a 95℃ water bath for 1.5 hours, stirring occasionally, until the white fungus is fully softened and the broth is slightly thickened. Filter through a 200-mesh sieve to collect the white fungus gelatinous liquid, and cool to 50℃ for later use.

[0138] 2.2 Bird's Nest Pretreatment

[0139] Weigh out at least 600 mg of dried bird's nest and soak it in purified water for 4 hours until fully expanded. Remove any fine feathers with tweezers. Drain the bird's nest and add 100 times its weight in purified water (approximately 60 mL). Place the mixture in a double boiler and steam in a 95°C water bath for 30 minutes. After steaming, cool the bird's nest mixture along with the broth to obtain the bird's nest stewing liquid, which is then ready for use.

[0140] 2.3 Base Material Mixing and Standardization

[0141] In a clean mixing container, add 400 mL of pure fresh camel milk, 300 mL of pure milk, and all of the tremella fuciformis gelatin solution prepared in step 2.1. Start stirring (300 rpm) and slowly add erythritol (10 g) and agar (0.4 g). Heat the mixture to 65°C and maintain stirring at this temperature for 30 minutes to ensure that the agar is completely swollen and the system is homogeneous.

[0142] 2.4 Functional ingredient addition and homogenization

[0143] Add the bird's nest stewing liquid (containing all bird's nest solids and broth) prepared in step 2.2 and 0.5g of Max Kluwer yeast complex to the above-mentioned mixed base at 65℃. Increase the stirring speed to 500 rpm and stir continuously for 15 minutes to ensure that the yeast is completely dispersed.

[0144] Subsequently, the mixture was subjected to high-pressure homogenization: the first stage pressure was 20 MPa, the second stage pressure was 5 MPa, and the temperature of the mixture was maintained at 60-65℃. The particle size of the homogenized mixture should be ≤2 μm.

[0145] 2.5 Sterilization and Filling

[0146] The process employs ultra-high temperature instantaneous sterilization (UHT): the homogenized liquid is rapidly heated to 137°C, held for 4 seconds, and then immediately cooled to below 25°C.

[0147] In a Class A clean environment, the sterilized and cooled liquid is aseptically filled into sterilized packages of a predetermined capacity (e.g., 200 mL) and then sealed.

[0148] 2.6 Product Features

[0149] Compared with commercially available ordinary bird's nest beverages, this product has the following characteristics:

[0150] Double milk base: Combining camel milk and cow milk, with a harmonious flavor and complementary nutrition.

[0151] Natural compatibility: Tremella and bird's nest are both traditional nourishing ingredients, with synergistic effects and a smooth taste.

[0152] Clear functionality: The product is rich in organic selenium, GABA from Kluyveromyces marxianus, as well as various functional ingredients such as sialic acid and tremella polysaccharide. These ingredients may play a synergistic role in promoting sleep, relieving fatigue, and regulating immunity.

[0153] Clean label tendency: Using erythritol to provide sweetness and agar as a single stabilizer, without adding flavors, the product ingredients are more concise.

[0154] Example 4: Experimental study on the sleep-promoting effect of Kluyveromyces camel milk bird's nest milk on mice

[0155] 1. Experimental materials and methods

[0156] 1.1 Experimental animals

[0157] Species and quantity: 100 SPF-grade ICR mice, half male and half female, 6 - 8 weeks old, weighing 18 - 22 g (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., license number SCXK(Beijing)2025 - 0001).

[0158] Rearing environment: SPF-grade animal room, temperature (22 ± 2) °C, humidity (50 ± 10)%, 12 h light-dark cycle (8:00 - 20:00 light), free access to food and water, adaptively reared for 7 days.

[0159] 1.2 Test substances and grouping

[0160] The experimental grouping is shown in Table 2. The administration method is once daily by gavage for 30 consecutive days, and the sleep experiment is carried out 45 minutes after the last gavage.

[0161] Table 2. Grouping and dose setting for the sleep-promoting experiment of mice

[0162]

[0163] 1.3 Hypnotic experiment with subthreshold dose of pentobarbital sodium

[0164] Reagent preparation: Pentobarbital sodium (Sigma) is prepared into a 3 mg / mL solution with physiological saline and prepared immediately before use.

[0165] Experimental procedure: Mice in each group were intraperitoneally injected with sodium pentobarbital 30 mg / kg (calculated based on body weight, dose that prevents 80%-90% of mice from falling asleep), and the results were recorded within 30 minutes.

[0166] Assessing sleep: The righting reflex disappears for ≥60 seconds;

[0167] Sleep rate = (Number of animals falling asleep / Number of animals per group) × 100%;

[0168] Average sleep time: the time from the disappearance of the righting reflex to its recovery.

[0169] 1.4 Statistical Analysis

[0170] SPSS 26.0 software was used. Quantitative data were expressed as mean ± standard deviation (x±s). One-way ANOVA was used for comparisons between groups, and categorical data were expressed using the chi-square test (χ²). 2 The test was performed, and p < 0.05 was considered statistically significant.

[0171] 2. Experimental Results

[0172] 2.1 Effect on sleep rate in mice

[0173] Table 3. Statistical table of sleep rate in mice of different groups

[0174]

[0175] The results of this experiment are shown in Table 3. It can be seen that the sleep rate of the Kluwer camel milk and bird's nest milk group reached 90%, which was significantly higher than that of the ordinary camel milk and bird's nest milk group containing only one ingredient (60%) and the Kluwer yeast group (50%). This indicates that organic selenium and GABA synergistically enhance the sleep-aiding effect (p<0.01).

[0176] 2.2 Effects on sleep time in mice

[0177] Table 4. Statistics on sleep time of mice in different groups

[0178]

[0179] The results of this experiment are shown in Table 4. It can be seen that the average sleep time of the experimental group (78.3 min) was 84.2% longer than that of the ordinary camel milk and bird's nest milk group (42.5 min), which verifies that the synergistic effect of "yeast complex-bird's nest" is better than that of the single ingredient.

[0180] All of the above results demonstrate that the yeast complex-bird's nest is rich in nutrients and has a good effect on promoting sleep.

[0181] Example 5: Experimental study on the immune-enhancing effects of Kluwer camel milk and bird's nest milk on mice.

[0182] 1.1 Experimental animals

[0183] Species and quantity: 100 SPF-grade ICR mice, 50 males and 50 females, 6 - 8 weeks old, weighing 18 - 22 g (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., license number SCXK(Beijing)2025 - 0001).

[0184] Feeding environment: SPF-grade animal house, temperature (22 ± 2) °C, humidity (50 ± 10)%, 12 h light-dark cycle (light from 8:00 - 20:00), free access to food and water, adaptively fed for 7 days.

[0185] 1.2 Test substances and grouping

[0186] The experimental grouping is shown in Table 5. The administration method is once daily by gavage for 30 consecutive days.

[0187] Table 5. Grouping and dose setting for the experiment on improving mouse immunity

[0188]

[0189] 1.3 Detection indexes and methods

[0190] 1.3.1 Detection of NK cell activity:

[0191] The lactate dehydrogenase (LDH) release method was used and operated according to the kit instructions (purchased from Sigma-Aldrich).

[0192] 1.3.2 Detection of IL-2 and IgG levels:

[0193] The ELISA method was used. The detection kit was purchased from Nanjing Jiancheng Bioengineering Institute and operated strictly according to the instructions.

[0194] The experimental results

[0195] The effects on mouse immune indexes are shown in Table 6.

[0196] Table 6. Detection results of immune indexes of mice in each group (x ± s, n = 20)

[0197]

[0198] Note: Compared with the blank control group, *p < 0.05, **p < 0.01; compared with control group 2, #p < 0.05, ##p < 0.01

[0199] 3. Conclusion

[0200] The experimental results showed that the NK cell activity, IL-2 level, and IgG level in the experimental group were significantly higher than those in the blank control group, the ordinary camel milk and bird's nest milk group, and the Kluyveromyces group (p<0.01). Specifically, the NK cell activity in the experimental group increased by 150%, IL-2 level by 151%, and IgG level by 138% compared to the blank control group, and all were significantly higher than those in the single-component groups, indicating that the organic selenium, GABA, and sialic acid in Kluyveromyces camel milk and bird's nest milk have a synergistic effect in enhancing immunity.

[0201] The specific embodiments of the present invention have been described in detail above, but they are merely examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions to the present invention are also within the scope of the present invention. Therefore, all equivalent changes and modifications made without departing from the spirit and scope of the present invention are covered within the scope of the present invention.

Claims

1. A bird's nest dairy product made from a Kluyveromyces macrocephala complex, characterized in that, By weight percentage, the bird's nest dairy product comprises the following components: 1%-5% Kluyveromyces martensii complex, 0.3%-1.0% bird's nest, 40%-60% camel milk, 20%-40% cow's milk, and 0.1%-1.0% tremella fuciformis. The Kluyveromyces martensii complex is derived from Kluyveromyces martensii, a high-yield producer of γ-aminobutyric acid (GABA). Kluyveromyces marxianus The *Kluyveromyces martensii* was obtained through selenium enrichment culture, and its preservation number is CCTCC NO: M 20242474.

2. The bird's nest dairy product according to claim 1, characterized in that, The Kluyveromyces macrocephala complex contains ≥200 mg / kg of organic selenium and >99% organic selenium, and ≥2000 mg / kg of GABA.

3. The bird's nest dairy product according to claim 1, characterized in that, The selenium enrichment culture uses sodium selenite as the selenium source and is fermented at 30℃ and pH 6.0-6.5 for 48-72 h. The final concentration of sodium selenite is 50-100 μg / mL, and the sugar concentration of the fermentation broth is 5-10 g / L.

4. The bird's nest dairy product according to any one of claims 1-3, characterized in that, The bird's nest dairy products also contain one or more physiologically acceptable excipients and / or excipients, including other edible ingredients, seasonings, solvents, diluents, disintegrants, surfactants, flow aids, binders, lubricants, dispersants, suspending agents, isotonic agents, thickeners, emulsifiers, stabilizers, hydrating agents, emulsification accelerators, colorants, flavorings, sweeteners, or ion exchangers.

5. The method for preparing bird's nest dairy products according to any one of claims 1-4, characterized in that, Includes the following steps: (1) Pretreatment of white fungus: Soak and clean the dried white fungus, add water and boil, then filter to obtain white fungus gel; (2) Pretreatment of bird's nest: After soaking and cleaning the dried bird's nest, add water and stew it in a double boiler to obtain bird's nest stewing liquid; (3) Base material mixing: Mix camel milk, cow milk, tremella gelatin, sweetener and stabilizer, heat and stir to form a uniform base material; (4) Functional addition and homogenization: Add the bird's nest stewing liquid obtained in step (2) and the Max Kluyveromycin complex to the base material, stir and disperse, and then perform high-pressure homogenization; (5) Sterilization and filling: The homogenized liquid is subjected to ultra-high temperature instantaneous sterilization (UHT), then cooled and aseptically filled.

6. The use of the bird's nest dairy product according to any one of claims 1-4 or the bird's nest dairy product prepared by the preparation method according to claim 5 in the preparation of health food for improving sleep or enhancing immunity.

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