Probiotic preparations for promoting selenium-enriched eggs in laying hens and their preparation methods

Abstract

This invention discloses a probiotic preparation for promoting the production of selenium-enriched eggs in laying hens and its preparation method, relating to the field of microbiology. It includes live cells of *Ligilactobacillus salivarius* Z4 strain (accession number CGMCC No. 37648) and / or its fermentation products. The *Ligilactobacillus salivarius* Z4 strain can convert inorganic selenium into organic selenium. This probiotic preparation for promoting the production of selenium-enriched eggs in laying hens and its preparation method utilize *Ligilactobacillus salivarius* Z4, a novel avian strain with outstanding acid and bile salt tolerance, capable of successfully passing through the gastrointestinal tract and colonizing the intestines. It also exhibits strong inhibitory effects against major avian pathogens (such as *Escherichia coli*), superior to many previously reported lactic acid bacteria strains.

Description

Technical Field

[0001] This invention relates to microbial technology, specifically to probiotic preparations and their preparation methods for promoting the production of selenium-enriched eggs in laying hens. Background Technology

[0002] Selenium is an essential trace element for humans and animals, playing a crucial role in antioxidation, immune regulation, and thyroid hormone metabolism. Supplementing with bioavailable organic selenium through diet is an effective way to prevent selenium deficiency. Eggs, as a popular consumer product, are an ideal carrier for selenium fortification. Currently, the main method for producing selenium-enriched eggs is to add inorganic selenium (such as sodium selenite) or selenium-enriched yeast to the feed of laying hens. However, inorganic selenium is highly toxic, has low bioavailability, and easily causes environmental pollution; while products such as selenium-enriched yeast are effective, they are expensive, and their selenium forms are complex, making quality control difficult.

[0003] On the other hand, against the backdrop of "antibiotic-free feed," the search for green feed additives that can maintain animal gut health and improve production performance has become an urgent need in the livestock industry. Probiotics, especially lactic acid bacteria, have been extensively studied and proven to have the functions of regulating gut microbiota balance, inhibiting pathogens, and enhancing immunity. Combining the gut-regulating function of probiotics with the biotransformation function of the trace element selenium to develop a microecological preparation that can both promote the health of laying hens and efficiently produce selenium-enriched eggs has significant economic and social benefits.

[0004] In existing technologies, supplementing with inorganic selenium alone has problems such as high toxicity and low bioavailability; using conventional probiotics alone does not have the function of selenium enrichment; although products such as selenium-enriched yeast have good selenium supplementation effects, they are expensive and have no beneficial effects. Summary of the Invention

[0005] The purpose of this invention is to provide a probiotic preparation and its preparation method for promoting the production of selenium-enriched eggs in laying hens, in order to solve the following problems existing in the prior art: supplementing inorganic selenium alone has the problems of high toxicity and low bioavailability; using conventional probiotics alone does not have the function of selenium enrichment; although products such as selenium-enriched yeast have good selenium supplementation effect, they are expensive and have no beneficial effects.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] In a first aspect, the present invention provides a strain of avian-derived *Ligilactobacillus salivarius* Z4, which was deposited on February 4, 2026, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 37648.

[0008] This invention provides a probiotic preparation for promoting the production of selenium-enriched eggs in laying hens, comprising live cells of *Ligilactobacillus salivarius* Z4 strain with accession number CGMCC No. 37648 and / or its fermentation products, wherein the *Ligilactobacillus salivarius* Z4 strain is capable of converting inorganic selenium into organic selenium.

[0009] Furthermore, the 16S rRNA gene sequence of the Lactobacillus salivarius Z4 strain is shown in SEQ ID NO: 1.

[0010] The *Lactobacillus salivarius* Z4 strain described in this invention was isolated and screened from the feces of antibiotic-free healthy laying hens. Colonies of the strain on MRS solid medium are milky white, raised, round, with regular edges and a smooth surface. The bacteria are Gram-positive and appear spherical or short rod-shaped under a microscope, without spores or capsules.

[0011] This strain has the following biological characteristics:

[0012] (1) Growth and acid production characteristics: In MRS liquid medium, it enters the logarithmic growth phase after 5 hours of culture at 37°C and reaches the stationary phase after 12 hours; the pH value drops rapidly during the culture process and is maintained at around 4.0.

[0013] (2) Acid resistance: After being cultured in MRS medium with pH values ​​of 3.0, 4.0, 5.0 and 6.0 for 16 hours, the survival rates were 18%, 30%, 82% and 91%, respectively, indicating that it has good tolerance to the acidic environment of the gastrointestinal tract.

[0014] (3) Bile salt tolerance: After culturing in MRS medium with bile salt concentrations of 0.1%, 0.3%, and 0.5% (w / v) for 16 hours, the survival rates were 30%, 23%, and 20%, respectively, indicating that it is tolerant to the intestinal bile salt environment.

[0015] (4) Antibacterial properties: Its fermentation supernatant has a significant inhibitory effect on both Escherichia coli and Staphylococcus aureus, and the diameter of the inhibition zone is comparable to that of the antibiotic ciprofloxacin.

[0016] (5) Selenium enrichment characteristics: When grown in MRS medium containing 5-20 μg / mL sodium selenite, it can efficiently absorb inorganic selenium and convert it into organic selenium, and the organic selenium content of the bacteria can reach 150-300 μg / g.

[0017] Secondly, the present invention provides a probiotic preparation containing the above-mentioned *Lactobacillus salivarius* Z4 strain, wherein the preparation is a freeze-dried bacterial powder with a live bacteria content of not less than 1 × 10⁻⁶. 10 CFU / g.

[0018] Furthermore, the freeze-dried bacterial powder also contains a freeze-drying protectant and / or a pharmaceutically acceptable carrier; the freeze-drying protectant is a mixture of skim milk powder and trehalose, and the carrier is selected from at least one of corn starch and kaolin.

[0019] Preferably, the freeze-drying protectant is added at a ratio of 5–15 g of skim milk powder and 2–8 g of trehalose per 100 mL of protectant solution. The carrier is a pharmaceutically acceptable carrier, such as corn starch, kaolin, or microcrystalline cellulose, used to dilute and improve the physical properties of the bacterial powder.

[0020] Thirdly, the present invention provides a method for preparing a probiotic preparation, comprising the following steps:

[0021] S1. Strain activation: Inoculate Lactobacillus salivarius Z4 strain onto MRS solid medium and incubate at 35–39°C for 24–48 hours, then pick single colonies.

[0022] In a constant temperature incubator, preferably at 37°C, incubate for 24–48 hours until a single colony with a typical morphology grows.

[0023] S2. Seed culture preparation: The single colony activated in step S1 is inoculated into MRS liquid culture medium and cultured with shaking at 35-39℃ and 100-200 rpm for 12-18 hours to obtain the seed culture.

[0024] Pick a single colony activated in step S1 using an inoculation loop and inoculate it into an Erlenmeyer flask containing sterile MRS liquid medium. Incubate with shaking at 35–39°C and 150–200 rpm for 12–18 hours to obtain primary seed culture. Further propagation to a seed tank is possible as needed.

[0025] S3. Selenium-enriched fermentation culture: The seed culture from step S2 is inoculated into MRS liquid medium containing sodium selenite at a volume ratio of 1% to 5%, and cultured at 35 to 39°C and 100 to 200 rpm for 24 to 36 hours to obtain selenium-enriched fermentation broth; the final concentration of sodium selenite in the MRS liquid medium containing sodium selenite is 5 to 20 μg / mL.

[0026] S4. Microbial collection: Centrifuge the selenium-enriched fermentation broth from step S3 to collect the microbial sludge, and wash it with sterile physiological saline to remove residual sodium selenite.

[0027] Collect the bacterial cells from the fermentation broth in step S3 by centrifugation (4000–8000 × g, 10–15 minutes), discard the supernatant, and obtain bacterial sludge. Wash the bacterial sludge 2–3 times with sterile physiological saline to thoroughly remove any residual unconverted sodium selenite in the culture medium.

[0028] S5. Preparation of formulation: The bacterial mud washed in step S4 is mixed with the freeze-drying protectant solution at a mass ratio of 1:0.5-2. After pre-freezing and freeze-drying, it is pulverized and passed through a 40-60 mesh sieve to obtain selenium-enriched probiotic freeze-dried bacterial powder.

[0029] The washed bacterial sludge is mixed with a pre-prepared and sterilized freeze-drying protectant solution at a mass ratio of 1:0.5–2 (preferably 1:1) to obtain a bacterial suspension. The bacterial suspension is dispensed into freeze-drying trays (with a liquid layer thickness of approximately 1.0–1.5 cm) and pre-frozen at -45°C to -35°C for 3–5 hours. Then, it is transferred to a freeze dryer and freeze-dried under a vacuum of 10–30 Pa. The freeze-drying program is as follows: maintain at -40°C for 2 hours, then raise the temperature to 0°C at a rate of 0.5–1°C / min and maintain for 18–20 hours, then raise the temperature to 20–30°C and maintain for 4–6 hours. After freeze-drying, the freeze-dried cake is pulverized in an environment with a relative humidity below 20% and passed through a 40–60 mesh sieve to obtain selenium-enriched probiotic freeze-dried bacterial powder.

[0030] The bacterial powder prepared by this method has a high survival rate of live bacteria and is rich in highly bioactive organic selenium.

[0031] Furthermore, the freeze-drying protectant solution in step S5 is an aqueous solution containing 5%–15% w / v skim milk powder and 2%–8% w / v trehalose.

[0032] Furthermore, the freeze-drying conditions described in step S5 are as follows: after pre-freezing at -45°C to -35°C for 3 to 5 hours, under a vacuum of 10 to 30 Pa, the shelf temperature is gradually increased from -40°C to 20 to 30°C, and the total freeze-drying time is 20 to 30 hours.

[0033] Furthermore, the selenium-enriched probiotic freeze-dried bacterial powder obtained in step S5 has an organic selenium content of 150–300 μg / g.

[0034] Fourthly, the present invention provides the application of the probiotic preparation in the preparation of a feed additive for laying hens to increase the selenium content in eggs.

[0035] Furthermore, the probiotic preparation is used in the preparation of veterinary microecological preparations for improving the intestinal health of laying hens and enhancing egg production performance.

[0036] Compared with the prior art, the probiotic preparation for promoting the production of selenium-enriched eggs in laying hens and its preparation method provided by the present invention have the following beneficial effects:

[0037] The Lactobacillus salivarius Z4 provided by this invention is a novel avian strain with outstanding acid and bile salt tolerance, which can pass smoothly through the gastrointestinal tract and colonize the intestine. It also has a strong inhibitory effect on major avian pathogens (such as Escherichia coli), which is superior to many reported lactic acid bacteria strains.

[0038] Strain Z4 not only efficiently converts inorganic selenium into organic selenium (with a selenium content as high as 150–300 μg / g in bacterial cells), but also possesses probiotic functions such as regulating intestinal flora and promoting nutrient absorption. This overcomes the limitations of existing technologies that simply supplement selenium or probiotics, achieving multiple benefits with a single dose.

[0039] Animal studies have confirmed that laying hens fed the formulation of this invention produce eggs with significantly increased selenium content, meeting the standards for selenium-enriched eggs, and the selenium in the yolks is mainly present in the form of highly active selenomethionine. Simultaneously, the laying rate, intestinal morphology, and immune function of the hens are all improved.

[0040] The preparation method provided by this invention has a clear process, well-defined parameters, stable fermentation and freeze-drying processes, and a high live bacteria yield, making it suitable for large-scale industrial production.

[0041] The strain was isolated from the intestines of healthy laying hens and belongs to the native gut microbiota of animals. Attached Figure Description

[0042] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in this invention. For those skilled in the art, other drawings can be obtained based on these drawings.

[0043] Figure 1 Photographs of colony morphology of Lactobacillus salivarius Z4 strain on MRS solid medium;

[0044] Figure 2 Gram-stained microscopic image of Lactobacillus salivarius Z4 strain (100× oil immersion).

[0045] Figure 3 Growth curve of Lactobacillus salivarius strain Z4;

[0046] Figure 4 Acid production curve of Lactobacillus salivarius Z4 strain;

[0047] Figure 5 Bar chart showing the survival rate of Lactobacillus salivarius Z4 strain under different pH conditions;

[0048] Figure 6 Bar chart showing the survival rate of Lactobacillus salivarius Z4 strain at different bile salt concentrations;

[0049] Figure 7 Photograph of the inhibition zone of the fermentation supernatant of Lactobacillus salivarius Z4 strain against Escherichia coli. Detailed Implementation

[0050] To enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings.

[0051] Experimental methods not described in detail in the following examples were performed under conventional experimental conditions in the art or under conditions recommended by the manufacturer.

[0052] As attached Figure 1 To be continued Figure 7 As shown:

[0053] Example 1: Preparation of Lactobacillus salivarius Z4 selenium-enriched probiotic preparation

[0054] S1. Strain Activation: The cryovial of *Lactobacillus salivarius* strain Z4 (CGMCC No. 37648) preserved at -80℃ was taken out and streaked onto an MRS solid medium plate. The plate was then incubated at 37℃ for 36 hours to obtain a single colony. S2. Seed Culture Preparation: A well-grown single colony was picked and inoculated into a 250 mL Erlenmeyer flask containing 100 mL of MRS liquid medium. The flask was incubated at 37℃ and 180 rpm on a shaker for 16 hours to obtain the primary seed culture. S3. Selenium-Enriched Fermentation Culture: 3 L of MRS liquid medium (containing sodium selenite, final concentration 10 μg / mL) was added to a 5 L fully automated fermenter and sterilized at 121℃ for 20 minutes. After cooling to 37℃, the primary seed culture was inoculated at a 2% (v / v) inoculation rate. Fermentation parameters were set as follows: temperature 37℃, stirring speed 150 rpm, pH maintained at 6.2 by automatic ammonia addition, and fermentation for 28 hours. S4. Cell Collection and Washing: Centrifuge the fermentation broth at 4℃ and 6000 ×g for 15 minutes, discard the supernatant, and collect the bacterial sludge. Resuspend the cells twice with an equal volume of sterile physiological saline, and centrifuge again to collect the bacterial sludge. S5. Formulation Preparation: Prepare a lyophilization protectant solution (containing 10% w / v skim milk powder and 5% w / v trehalose), sterilize at 85℃ for 30 minutes, and then cool to room temperature. Mix the bacterial sludge and protectant solution at a mass ratio of 1:1 to prepare a bacterial suspension. Dispense the bacterial suspension into lyophilization trays (with a liquid layer thickness of approximately 1.2 cm) and pre-freeze in a -40℃ quick-freezing chamber for 4 hours. Then transfer it to a lyophilizer, setting the first drying stage: starting at -40℃, increasing the temperature to -5℃ at a rate of 0.8℃ / min, and maintaining for 20 hours; the second drying stage: continuing to increase the temperature to 25℃ and maintaining for 6 hours, with the vacuum degree maintained at 15 Pa throughout. After freeze-drying, the bacteria were pulverized in an environment with a relative humidity of less than 20% and passed through a 60-mesh sieve to obtain selenium-enriched probiotic freeze-dried powder A. Testing showed that the live bacteria content of this powder was 3.2 × 10⁻⁶. 10 The CFU / g content and the organic selenium content are 210 μg / g.

[0055] Example 2: The application effect of the formulation prepared in Example 1 in laying hen production (selenium deposition effect)

[0056] Two hundred and forty healthy 35-week-old Hy-Line Brown laying hens with similar egg production rates and body weights were randomly divided into three groups, with eight replicates per group and ten hens per replicate. The control group (Group C) was fed a basal diet (without additional selenium source, measured selenium content 0.08 mg / kg); the experimental group (Group T1) received 200 g / ton of the selenium-enriched probiotic freeze-dried bacterial powder A prepared in Example 1 in addition to its basal diet; the experimental group (Group T2) received an equal amount of a mixture of sterile carrier and sodium selenite in its basal diet, ensuring an additional inorganic selenium level comparable to Group T1 (approximately 0.04 mg / kg). The pre-trial period was 7 days, and the formal trial period was 35 days. Feed and water were freely available during the trial. At the end of the trial, three eggs were randomly selected from each replicate to determine the whole egg selenium content, average egg weight, egg production rate, feed conversion ratio, serum IgG content, and jejunal villus height / crypt depth ratio. The experimental data were analyzed by one-way ANOVA using SPSS software. The results are expressed as mean ± standard deviation, as shown in Table 1 below.

[0057] Table 1. Effects of different treatments on laying hen production performance, selenium content in eggs, and gut health indicators.

[0058]

[0059] Note: Different lowercase letters in the superscript of data in the same column indicate significant differences (P < 0.05).

[0060] As shown in Table 1, compared with the control group, the selenium content of eggs in group T1, fed with the selenium-enriched probiotic preparation of this invention, increased by 129.2% (P < 0.05), and the egg production rate also significantly improved. In contrast, group T2, fed with the same amount of inorganic selenium, showed an increase in egg selenium content, but the increase was far less than that in group T1, and the egg production rate did not improve. This demonstrates that the Z4 strain in the preparation of this invention not only efficiently converts and provides highly bioavailable organic selenium, but its own probiotic effects also promote the production performance of laying hens.

[0061] Comparative Example 1: Comparison of the selenium enrichment capacity and selenium-enrichment effect of different lactic acid bacteria strains on laying hens

[0062] To verify the superiority of the *Lactobacillus salivarius* Z4 strain of the present invention, commercially available *Lactobacillus acidophilus* (LA) and *Lactobacillus plantarum* (LP) were selected as comparative strains. Selenium-enriched freeze-dried bacterial powders B and C were prepared according to the process of Example 1 (only the strains were replaced with LA and LP), respectively, using a sodium selenite concentration of 10 μg / mL in both preparations. The organic selenium content of bacterial powders B and C was determined to be 85 μg / g and 132 μg / g, respectively. 160 35-week-old Hy-Line Brown laying hens with similar egg production rates were randomly divided into 4 groups, with 5 replicates per group and 8 hens per replicate. Each group received the same basal diet, with 200 g / ton of bacterial powder A (Z4 group), bacterial powder B (LA group), bacterial powder C (LP group), and an equal amount of carrier (control group). Feeding and management were the same as in Example 2, and the experimental period was 35 days. The results are shown in Table 2 below.

[0063] Table 2. Effects of different lactic acid bacteria strains on selenium content in eggs and laying hen performance.

[0064]

[0065] Note: Different lowercase letters in the superscript of the same data indicate significant differences (P < 0.05).

[0066] The results showed that the selenium-enriching capacity of Lactobacillus salivarius Z4 and its effect on promoting selenium deposition in laying hens were significantly superior to those of common Lactobacillus acidophilus and Lactobacillus plantarum. Furthermore, the Z4 group also demonstrated significant advantages in improving egg production rate, feed conversion efficiency, and regulating intestinal flora (enhancing beneficial bacteria and inhibiting harmful bacteria). This fully demonstrates the uniqueness and irreplaceable nature of the Z4 strain of this invention in its dual functions of selenium enrichment and probiotics.

[0067] Comparative Example 2: Comparison of the effects of the formulation of the present invention with sodium selenite and selenium-enriched yeast on laying hens

[0068] The selenium-enriched probiotic freeze-dried bacterial powder A prepared in Example 1 of this invention was compared with commercial selenium-enriched yeast (organic selenium content 2000 μg / g) and sodium selenite (analytical grade). Two hundred and forty 35-week-old Hy-Line Brown laying hens were randomly divided into four groups, with six replicates per group and ten hens per replicate. The control group (CK) was fed a basal diet; experimental group 1 (T1) received 200 g / ton of the bacterial powder A of this invention added to the basal diet; experimental group 2 (T2) received commercial selenium-enriched yeast added to the basal diet, ensuring the additional selenium level was the same as in group T1 (approximately 0.04 mg / kg); experimental group 3 (T3) received sodium selenite added to the basal diet, also ensuring the additional selenium level was 0.04 mg / kg. The trial period was 35 days. At the end of the experiment, the selenium content of eggs, the proportion of selenomethionine in egg yolks to total selenium, serum glutathione peroxidase (GSH-Px) activity, the number of Escherichia coli in jejunal contents, and fecal selenium excretion were measured. The results are shown in Table 3.

[0069] Table 3. Comparison of the effects of the formulation of this invention with common selenium sources in laying hens.

[0070]

[0071] Note: Different lowercase letters in the superscript of the same data indicate significant differences (P < 0.05).

[0072] Table 3 shows that, at the same selenium supplementation level, the formulation of this invention (Group T1) and selenium-enriched yeast (Group T2) were comparable in increasing selenium content in eggs, the proportion of highly active selenium form (selenomethionine) in egg yolks, and serum antioxidant capacity, and were significantly superior to inorganic selenium sources (Group T3). However, only the formulation of this invention showed significant effects in improving gut health (inhibiting Escherichia coli) and increasing egg production. Furthermore, compared to the inorganic selenium group, the fecal selenium excretion of the formulation of this invention and the selenium-enriched yeast group was significantly reduced, indicating that organic selenium has higher bioavailability. These results highlight the unique advantages of the formulation of this invention, which combines the functions of highly efficient selenium supplementation and gut health enhancement, and its overall benefits are superior to single-function selenium-enriched products or ordinary probiotic products.

[0073] The foregoing has only described certain exemplary embodiments of the present invention by way of illustration. Undoubtedly, those skilled in the art can modify the described embodiments in various ways without departing from the spirit and scope of the present invention. Therefore, the foregoing drawings and descriptions are illustrative in nature and should not be construed as limiting the scope of protection of the claims of the present invention.

Claims

1. A probiotic preparation for promoting the production of selenium-enriched eggs in laying hens, characterized in that, The present invention comprises live cells of *Ligilactobacillus salivarius* Z4 strain with accession number CGMCC No. 37648 and / or its fermentation products, wherein the *Ligilactobacillus salivarius* Z4 strain is capable of converting inorganic selenium into organic selenium.

2. The probiotic preparation for promoting the production of selenium-enriched eggs in laying hens according to claim 1, characterized in that, The 16S rRNA gene sequence of the Lactobacillus salivarius Z4 strain is shown in SEQ ID NO:

1.

3. The probiotic preparation for promoting the production of selenium-enriched eggs in laying hens according to claim 1 or 2, characterized in that, The preparation is a freeze-dried bacterial powder with a live bacteria content of not less than 1×10⁻⁶. 10 CFU / g.

4. The probiotic preparation for promoting the production of selenium-enriched eggs in laying hens and its preparation method according to claim 3, characterized in that, The freeze-dried bacterial powder further comprises a freeze-drying protectant and / or a pharmaceutically acceptable carrier; the freeze-drying protectant is a mixture of skim milk powder and trehalose, and the carrier is selected from at least one of corn starch and kaolin.

5. A method for preparing a probiotic preparation as described in any one of claims 1 to 4, characterized in that, Includes the following steps: S1. Strain activation: Inoculate Lactobacillus salivarius Z4 strain onto MRS solid medium and incubate at 35–39°C for 24–48 hours, then pick single colonies. S2. Seed culture preparation: The single colony activated in step S1 is inoculated into MRS liquid culture medium and cultured with shaking at 35-39℃ and 100-200 rpm for 12-18 hours to obtain the seed culture. S3. Selenium-enriched fermentation culture: The seed culture from step S2 is inoculated into MRS liquid medium containing sodium selenite at a volume ratio of 1% to 5%, and cultured at 35 to 39°C and 100 to 200 rpm for 24 to 36 hours to obtain selenium-enriched fermentation broth; the final concentration of sodium selenite in the MRS liquid medium containing sodium selenite is 5 to 20 μg / mL. S4. Microbial collection: Centrifuge the selenium-enriched fermentation broth from step S3 to collect the microbial sludge, and wash it with sterile physiological saline to remove residual sodium selenite. S5. Preparation of formulation: The bacterial mud washed in step S4 is mixed with the freeze-drying protectant solution at a mass ratio of 1:0.5-2. After pre-freezing and freeze-drying, it is pulverized and passed through a 40-60 mesh sieve to obtain selenium-enriched probiotic freeze-dried bacterial powder.

6. The method for preparing the probiotic preparation for promoting the production of selenium-enriched eggs in laying hens according to claim 5, characterized in that, The freeze-drying protectant solution mentioned in step S5 is an aqueous solution containing 5%–15% w / v skim milk powder and 2%–8% w / v trehalose.

7. The method for preparing the probiotic preparation for promoting the production of selenium-enriched eggs in laying hens according to claim 5, characterized in that, The freeze-drying conditions described in step S5 are as follows: after pre-freezing at -45°C to -35°C for 3 to 5 hours, the shelf temperature is gradually increased from -40°C to 20 to 30°C under a vacuum of 10 to 30 Pa, and the total freeze-drying time is 20 to 30 hours.

8. The method for preparing the probiotic preparation for promoting the production of selenium-enriched eggs in laying hens according to claim 5, characterized in that, The selenium-enriched probiotic freeze-dried bacterial powder obtained in step S5 contains 150–300 μg / g of organic selenium in the bacterial cells.

9. The use of a probiotic preparation as described in any one of claims 1 to 4 in the preparation of a feed additive for laying hens to increase the selenium content in eggs.

10. The use of a probiotic preparation as described in any one of claims 1 to 4 in the preparation of a veterinary microecological preparation for improving the intestinal health of laying hens and enhancing egg production performance.

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