A strain of brettanomyces, a microecological preparation thereof and application thereof

Abstract

The present application relates to the fields of animal nutrition and microbial fermentation technology, and particularly relates to a strain of Saccharomyces boulardii, a microecological preparation thereof and application thereof.The Saccharomyces boulardii is named as NCUTY 002, has been preserved in China Center for Type Culture Collection, and the preservation number is CCTCC NO:M 2025648.The Saccharomyces boulardii NCUTY 002 screened in the present application has excellent acid tolerance (pH≤2.5) and bile salt tolerance (0.5%), and shows high self-aggregation capacity, so that it can survive and stably colonize in the extreme environment of digestive tract of egg ducks, and can be used as a special feed additive for egg ducks.

Description

Technical Field

[0001] This invention relates to the fields of animal nutrition and microbial fermentation technology, specifically to a strain of Saccharomyces boulardii, its microecological preparations, and their applications. Background Technology

[0002] As an important part of animal husbandry, the economic benefits of duck farming are highly dependent on the production performance of the flock, especially in the later stages of egg production (over 500 days old). During this stage, caged ducks commonly experience physiological functional decline: such as weakened ovarian function leading to disrupted egg-laying cycles and a sharp drop in egg production; intestinal flora imbalance causing digestive and absorptive disorders; and problems such as decreased eggshell quality, brittleness, and insufficient synthesis of flavor compounds. These factors severely impact the economic benefits of duck farming and the market competitiveness of the eggs.

[0003] Currently, common probiotic preparations on the market still face the following technical limitations in addressing the aforementioned issues: First, insufficient strain compatibility. While probiotics (lactic acid bacteria and Bacillus) are widely used, their survival rate is low in the extreme environment of high bile salts (>0.3%) and low pH (<3.0) in the hindgut of laying ducks, making it difficult to effectively colonize and exert their functions. Although *Saccharomyces boulardii* has application potential, there is a lack of targeted screening and domestication for the gut microecological characteristics of laying ducks, preventing the full realization of its application potential. Second, high fermentation substrate costs. Existing processes largely rely on YPD media with glucose as a carbon source, failing to fully utilize agricultural byproducts such as soybean residue and wheat bran. This model not only leads to high production costs but also low resource utilization, resulting in waste and severely restricting the industrial production and large-scale application of probiotic preparations. Third, poor preparation stability. Existing freeze-dried powder preparations exhibit a high rate of viable cell attenuation during storage, significantly reducing their effectiveness. Furthermore, these formulations are unable to effectively resist the erosion of gastric acid and bile salts, making it difficult to achieve targeted release into the intestines. This results in insufficient live bacteria reaching the target site, severely impacting practical application effectiveness. Although microencapsulation technology can be used to protect live bacteria, existing technologies mostly employ a single wall material, making it difficult to simultaneously achieve high live bacteria encapsulation, long-term protection, and intestinal environment responsiveness. Therefore, developing a cost-effective, high-performance, and cost-efficient microecological formulation that can effectively improve the egg production performance of laying ducks has significant industrial importance and application value. Summary of the Invention

[0004] This invention addresses the shortcomings of existing technologies by providing a strain of *Saccharomyces boulardii*, its microecological preparation, and its applications. It has excellent acid resistance (pH≤2.5) and bile salt resistance (0.5%), and exhibits high self-polymerization ability, ensuring its high survival and stable colonization in the extreme environment of the duck's digestive tract. It can be used as a special feed additive for laying ducks.

[0005] To achieve the above objectives, the first aspect of the present invention provides a strain of *Saccharomyces boulardii*. It was deposited at the China Center for Type Culture Collection on March 31, 2025, with accession number CCTCC NO: M 2025648, and the deposit address is No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0006] The present invention first successfully screened a strain of Saccharomyces boulardii NCUTY 002, which has excellent acid resistance (pH≤2.5) and bile salt resistance (0.5%), and exhibits high self-aggregation ability, ensuring its high survival and stable colonization in the extreme environment of the digestive tract of laying ducks.

[0007] In a second aspect, the present invention provides a microecological preparation comprising the above-mentioned Saccharomyces boulardii NCUTY 002.

[0008] Furthermore, the microecological preparation contains microcapsules prepared from Saccharomyces boulardii NCUTY 002.

[0009] Furthermore, the microecological preparation also contains Aspergillus niger ATCC 16404.

[0010] In a third aspect, the present invention provides a method for preparing the above-mentioned microecological preparation, comprising the following steps:

[0011] (1) Activate Aspergillus niger and Saccharomyces boulardii separately to prepare activated bacterial solutions for later use;

[0012] (2) Prepare the fermentation culture medium;

[0013] (3) First, inoculate Aspergillus niger into the fermentation medium and ferment at 30℃-35℃ for 2-3 days. Then, inoculate with Saccharomyces boulardii and continue fermentation at the same temperature for 2-3 days to obtain the fermentation broth.

[0014] (4) Mix the fermentation broth with the wall material solution, stir magnetically for 15-20 minutes, and then spray dry to obtain core microcapsule powder;

[0015] (5) Place the core microcapsule powder in a fluidized bed coating machine for spray coating to obtain a microcapsule microecological preparation.

[0016] This invention constructs a synergistic fermentation system of *Aspergillus niger* and *Saccharomyces boulardii*. This system utilizes the powerful extracellular enzyme system of *Aspergillus niger* ATCC16404 (with cellulase CMCase activity reaching 8.2 U / mL and xylanase activity reaching 5.7 U / mL) to pre-degrade soybean residue and wheat bran, converting complex macromolecules into sugars and amino acids more readily utilized by *Saccharomyces boulardii*. This significantly improves raw material utilization, reduces production costs, and achieves high-value utilization of agricultural by-products, effectively solving the problems of low nutrient utilization efficiency, high costs, and waste of agricultural by-product resources in traditional culture media. Simultaneously, by preparing this fermentation broth into a novel intestinal-targeted microcapsule, a microcapsule formulation with a high viable cell count was successfully prepared. This not only achieves efficient encapsulation and protection of live bacteria, but its enteric coating design also effectively resists gastric acid erosion and enables precise release in the intestinal environment, significantly improving the bioavailability of live bacteria.

[0017] Furthermore, in step (2), the inoculation amount of Aspergillus niger is 1% of the total volume of the fermentation medium; the inoculation amount of Saccharomyces boulardii is 3.5% of the total volume of the fermentation medium; the fermentation medium is prepared by the following method: 36 parts by weight of soybean residue, 36 parts by weight of molasses, and 28 parts by weight of wheat bran are mixed, 1000 parts by weight of deionized water is added, stirred evenly, the pH is adjusted to 6.0, and sterilized at 121℃ for 20 minutes.

[0018] Furthermore, in step (4), the volume ratio of the fermentation broth to the wall material solution is 6:4; the wall material solution contains 250 g / L of resistant starch, 200 g / L of concentrated whey protein, 125 g / L of trehalose, and 5 g / L-12.5 g / L of xanthan gum or carrageenan; the inlet air temperature of the spray dryer is 150°C, the outlet air temperature is 75°C-80°C, and the feed rate is 10 mL / min-15 mL / min.

[0019] Furthermore, in step (5), the coating solution used contains the following raw material components in parts by weight: 100 parts of polyacrylic acid resin II, 15 parts of triethyl citrate, 50 parts of talc, 80 parts of polysorbate, and 834 parts of purified water; the inlet air temperature of the fluidized bed is 35℃-40℃, the spray pressure is 0.1MPa-0.2MPa, and the spray rate is 2mL / min-5mL / min.

[0020] In a fourth aspect, the present invention provides a feed additive comprising the above-mentioned microecological preparation or the microecological preparation obtained by the above-mentioned preparation method.

[0021] In a fifth aspect, the present invention provides the use of the above-described microecological preparation or the above-described feed additive in any of the following:

[0022] (1) Used in the preparation of feed additives for laying ducks;

[0023] (2) Used for duck egg farming.

[0024] The beneficial effects of this invention are:

[0025] The *Bretschneidera sinensis* strain NCUTY 002 obtained by screening in this invention has excellent acid resistance (pH≤2.5) and bile salt resistance (0.5%), which can ensure its high survival and stable colonization in the extreme environment of the digestive tract of laying ducks. At the same time, it has antioxidant capacity, self-aggregation capacity and co-aggregation capacity with pathogens, which can effectively improve the intestinal microecological environment of laying ducks and enhance the body's disease resistance.

[0026] This invention employs a co-culture system of Aspergillus niger and Saccharomyces boulardii, which form a synergistic effect through metabolic interaction and functional complementarity, significantly improving cell proliferation efficiency and the overall quality of fermentation products. It also uses inexpensive food processing by-products such as soybean residue, molasses, and wheat bran as the main fermentation raw materials, significantly reducing production costs.

[0027] The microecological preparation of the present invention uses microencapsulation technology, which further significantly improves the survival rate of Saccharomyces boulardii in the gastrointestinal tract and the storage stability of the product.

[0028] The microecological preparation of this invention does not contain antibiotics and has been proven by experiments to have no hemolytic activity. It has extremely high biosafety, is highly compatible with the requirements of green aquaculture development, and has good market application prospects. Attached Figure Description

[0029] Figure 1 This is a diagram illustrating the physiological and biochemical characteristics of the *Brasilaria sinensis* strain of this invention.

[0030] Figure 2 This is a laser confocal microscopy (CLSM) image of the microencapsulation core of the microecological preparation of this invention;

[0031] Figure 3 This is a scanning electron microscope (SEM) image of the microecological preparation of the present invention;

[0032] Figure 4 This is a comparison of the tolerance of the microecological preparation of this invention and free *Saccharomyces boulardii* in an in vitro simulated digestion experiment;

[0033] Figure 5 This is a slice of intestinal epithelial tissue from the duck egg production experiment of this invention, where A is the control group and B is the experimental group.

[0034] The present invention will be further described below with reference to the accompanying drawings and embodiments. Detailed Implementation

[0035] To better illustrate the technical solution of the present invention, the following will explain the solution of the present invention in conjunction with embodiments. Those skilled in the art should understand that the following embodiments are only for illustrating the present invention and should not be considered as limiting the scope of the present invention. Where specific techniques or conditions are not specified in the embodiments, they are all performed in accordance with the techniques described in literature or reference books in the field or in accordance with product instructions. Reagents or instruments used without specified manufacturers are all conventional products, and reagents used without special instructions are all prepared by conventional methods.

[0036] The above-described technical features of the present invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions.

[0037] Example 1: Screening of Saccharomyces boulardii

[0038] 1. Isolation, purification and identification of bacterial strains

[0039] Fresh rambutan bark was crushed and an appropriate amount of the crushed rambutan bark was inoculated into YPD liquid medium containing 50 μg / mL ampicillin. The medium was cultured at 35℃ under aerobic conditions until turbidity was reached. An appropriate amount of the culture was diluted and spread onto plates. Based on colony morphology, typical yeast colonies (white, 1-2 mm in size, moist and raised, with an alcoholic aroma) were selected for preliminary screening under a microscope. Through physiological and biochemical analysis and 26S rDNA sequencing, a strain of *Saccharomyces boulardii* was finally screened, and its 26S rDNA sequence (SEQ ID NO:1) is shown below:

[0040]

[0041] This strain was deposited at the China Center for Type Culture Collection (CCTCC), No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, on March 31, 2025, with accession number CCTCC NO: M 2025648. The suggested classification name is... .

[0042] The colonies of *NCUTY 002*, a strain of *Saccharomyces boulardii*, formed on YPD agarose solid medium were large, moist, and milky white, with smooth surfaces and regular edges, and exhibited rapid growth. Under a microscope, the cells were nearly spherical, relatively large, and reproduced via polygonal budding. These morphological characteristics are consistent with the typical biological characteristics of the *Saccharomyces boulardii* genus and the classification criteria for *Saccharomyces cerevisiae* subspecies in Bergey's Manual of Systematic Bacteriology.

[0043] 2. Physiological and biochemical characteristics

[0044] (1) Growth characteristics: The growth cycle of *NCUTY 002* is divided into a lag phase (0-4h), a logarithmic growth phase (4h-18h), and a stationary phase (after 18h). Figure 1 As can be seen from 'a' in the figure, the OD during the stable period 600 The corresponding viable count was maintained at 8.0 × 10⁻⁶. 7 CFU / mL, it is recommended to harvest the cells at this stage to ensure optimal activity.

[0045] (2) Temperature adaptability: Saccharomyces boulardii NCUTY 002 grows well in the temperature range of 26℃-42℃, and maintains high metabolic activity, especially at 37℃-42℃ (e.g., Figure 1 (b) Its temperature tolerance range is significantly wider than that of ordinary brewer's yeast (25℃-30℃), and it can adapt to the body temperature environment of laying ducks (41.5±0.5℃), reducing temperature competition with the intestinal symbiotic flora.

[0046] (3) Acid tolerance: Saccharomyces boulardii NCUTY 002 can proliferate stably in the pH range of 2.0-6.0, but its activity decreases significantly at pH 1.5 (e.g., Figure 1 (c) Its acid-resistance mechanism helps maintain cell membrane integrity and adapt to the acidic environment of duck gastric juice (pH 3.2±0.3), providing a theoretical basis for the development of microecological preparations suitable for poultry.

[0047] (4) Salt tolerance: The survival rate of Saccharomyces boulardii NCUTY 002 after 3 hours of treatment in an environment with 0%-0.75% bile salts can reach over 77.44% (e.g., Figure 1 (d) When the bile salt concentration rises to 1%, the bacterium exhibits certain survival pressure, but the survival rate is still 60.62%, indicating that the strain has good bile salt tolerance.

[0048] Example 2: Co-fermentation process of Aspergillus niger and Saccharomyces boulardii

[0049] 1. Activation of bacterial strains: Aspergillus niger ATCC 16404 and Saccharomyces boulardii NCUTY 002 were inoculated into YPD medium (10g yeast extract, 20g peptone, 20g glucose, water added to 1L) and cultured at 30℃ with shaking for 24h to obtain activated bacterial solutions.

[0050] 2. Fermentation medium: Weigh 36g soybean residue, 36g molasses, and 28g wheat bran, add 1L of deionized water, adjust the pH to 6.0, and autoclave at 121℃ for 20min.

[0051] 3. Batch fermentation: The activated Aspergillus niger culture was inoculated into the fermentation medium at a 1% (v / v) inoculation rate. After fermentation at 30℃ for 48 hours in shake flasks, a Saccharomyces boulardii NCUTY 002 culture was inoculated at a 3.5% (v / v) inoculation rate, and fermentation continued at 30℃. Yeast concentration was measured every 12 hours during this period. The results showed that after inoculation with Saccharomyces boulardii and continued fermentation for 48 hours, the yeast concentration reached 4.15 × 10⁻⁶. 8 The CFU / mL yield is 4.19 times that of Saccharomyces boulardii in conventional YPD medium.

[0052] Example 3: Preparation process of intestinal-targeted probiotic microcapsules

[0053] This preparation process consists of two main stages: the preparation of the core microcapsules and the external enteric coating.

[0054] 1. Preparation of kernel microcapsules

[0055] (1) Formulation and proportions (based on 1L suspension)

[0056] Probiotic fermentation broth: approximately 600 mL (live bacteria count ≥ 1.0 × 10⁻⁶) 10 CFU / mL);

[0057] Resistant starch: 100g (approximately 10% w / v);

[0058] Whey protein concentrate: 80g (approximately 8% w / v);

[0059] Trehalose: 50g (approximately 5% w / v);

[0060] Xanthan gum or carrageenan: 2g-5g (approximately 0.2-0.5% w / v);

[0061] Sterile water: Add to 1L.

[0062] (2) Preparation of wall material solution

[0063] Add approximately 300 mL of sterile water to a clean beaker and stir continuously (400 rpm). Add the resistant starch and whey protein concentrate sequentially, avoiding clumping. Heat the solution to 60°C and maintain this temperature for 30 minutes to promote starch gelatinization and protein dissolution. Slowly add trehalose and xanthan gum (or carrageenan), continuing to stir until completely dissolved; the solution viscosity will increase significantly. Cool the solution to approximately 4°C for later use.

[0064] (3) Preparation of probiotic suspension

[0065] Add the cooled wall material solution to the probiotic fermentation broth and stir with a magnetic stirrer at low speed (approximately 100 rpm) for 15 minutes to ensure the probiotics are evenly dispersed in the wall material solution. Low-pressure homogenization (approximately 50 bar) can be performed using a homogenizer to improve the stability of the suspension.

[0066] (4) Preparation of kernel microcapsules

[0067] Transfer the mixed suspension to the spray dryer feeding system and set the parameters as follows:

[0068] Inlet air temperature: 150℃;

[0069] Air outlet temperature: 75℃-80℃;

[0070] Feed pump speed: 10mL / min-15mL / min (adjustable according to equipment model and ambient humidity to ensure stable outlet air temperature);

[0071] Atomization pressure: 0.2MPa-0.3MPa;

[0072] After spray drying, collect the core microcapsule powder, seal it immediately, and store it at 4°C.

[0073] (5) Analysis of embedding effect

[0074] The prepared core microcapsule powder was analyzed by fluorescence imaging using laser confocal microscopy to visually understand its encapsulation. Two dyes, DAPI (blue fluorescence) and FITC (green fluorescence), were used to stain the samples. The results... Figure 2 As shown in a, b, and c.

[0075] Figure 2Image a shows a superimposed fluorescence image of the composite microcapsules. This image is a superposition of the blue fluorescent signal (DAPI staining) labeling probiotic cells and the green fluorescent signal (FITC labeling) labeling the microcapsule wall material. Image b shows the blue fluorescent signal of the bacterial cells inside the microcapsules. DAPI is a blue fluorescent dye that specifically binds to DNA and can penetrate the cell membrane to label the probiotic cells inside the microcapsules. Image c shows the green fluorescent signal of the microcapsule wall material. FITC is a green fluorescent dye that covalently binds to the proteins and polysaccharides that make up the microcapsule wall material. This image shows the outline and structure of the microcapsules.

[0076] The results show that: Figure 2 As shown in Figure a, the blue fluorescent signal (representing probiotics) and the green fluorescent outline (representing the microcapsule wall material) exhibit a high degree of spatial overlap, clearly confirming that the probiotic cells are completely embedded inside the microcapsule wall material, demonstrating a good embedding effect.

[0077] 2. Preparation of external enteric coating

[0078] (1) Preparation of coating solution

[0079] The formulation of the coating solution is shown in Table 1.

[0080] Table 1. Coating solution formulation (taking the preparation of 1000g of coating solution as an example)

[0081]

[0082] (2) Operating steps

[0083] a. Preparation of coating solution:

[0084] Prepare phase A: In a clean container, add about 400g of purified water, turn on the stirrer (using a high-speed shear or homogenizer), add Tween 80, and stir until completely dissolved. While stirring continuously, slowly add triethyl citrate (TEC), and shear at high speed for 10-15 minutes until a uniform white emulsion is formed. Reduce the stirring speed to medium, slowly sift in talc powder, and continue stirring for 15-20 minutes to ensure that the talc powder is completely wetted and evenly dispersed to form a lumpy suspension.

[0085] Preparing Phase B: In another larger main preparation tank, add 333.3 g of polyacrylic acid resin (II) aqueous dispersion;

[0086] Pour phase A into phase B and mix. Start the stirrer in the main preparation tank and maintain a slow and steady stirring state. Rinse the container of phase A with the remaining approximately 200g of purified water and add the rinsing solution into the main preparation tank to ensure complete transfer of materials. Continue to stir slowly for 30-60 minutes to ensure that the entire system is fully mixed and homogeneous. Before use, filter the prepared coating solution through a sieve of approximately 180mm.

[0087] b. Fluidized bed coating: The core microcapsule powder prepared in the first stage is loaded into a fluidized bed coating machine.

[0088] The fluidized bed parameters are set as follows:

[0089] Inlet air temperature: 35℃-40℃;

[0090] Fluidization velocity: Adjusted according to the fluidization state of the microcapsule powder to ensure that the powder is uniformly suspended in the cavity;

[0091] Spray pressure: 0.1MPa-0.2MPa;

[0092] Spray rate: 2 mL / min-5 mL / min (slow spraying is required to prevent microcapsule adhesion and clumping);

[0093] Start spraying the coating solution. When the coating weight gain reaches 5%-100% of the total weight of the microcapsules, stop spraying. Continue fluidization for 10-20 minutes to completely evaporate any remaining moisture.

[0094] Collection and Storage: The final intestinal-targeting probiotic microcapsules were collected, sealed, and stored at 4°C. The probiotic preparation was observed using field emission environmental scanning electron microscopy, and the results are as follows: Figure 3 As shown, after fluidized bed coating, the probiotic core microcapsule powder forms spherical microcapsules with smooth surfaces and uniform particle size. The particles are regular and round ellipsoids without obvious depressions or damage, with clear edges and good dispersibility, exhibiting a dense and continuous coating layer structure.

[0095] Example 4: In vitro simulated digestion experiment of probiotic preparations

[0096] 1. Preparation of simulated gastric juice

[0097] Prepare artificial gastric juice, the components of which include: sodium chloride (NaCl) 125 mmol / L, sodium bicarbonate (NaHCO3) 45 mmol / L, potassium chloride (KCl) 7 mmol / L, and pepsin 3 g / L. After dissolving the above components, adjust the pH of the solution to 2.0, filter it through a 0.22 μm filter membrane and sterilize it for later use.

[0098] 2. Preparation of simulated intestinal fluid

[0099] The artificial intestinal fluid is prepared, and its components include:

[0100] Buffer system: Add 1% pancreatic enzyme and 0.3% bile salts.

[0101] The above components were placed in phosphate-buffered saline (PBS), the pH of the solution was adjusted to 8.0, and the solution was sterilized by filtration through a 0.22 μm filter membrane for later use.

[0102] 3. Sequential digestion assay

[0103] Phase 1: Simulating gastric digestion

[0104] The microecological preparation samples and control bacterial suspensions were centrifuged and resuspended in 1:10 artificial gastric juice. They were placed in a water bath (37℃, 100 rpm) for continuous digestion for 2 hours. 200 μL samples were taken every 0.5 hours for each of the following concentrations: 0, SGF (simulated gastric juice)-0.5, SGF-1, SGF-1.5, and SGF-2. The viable count of *Saccharomyces boulardii* was determined using the serial dilution plate count method. After gastric digestion, the pH of the samples and control bacterial suspensions was adjusted to 7.0 ± 0.2 with NaOH solution to terminate the digestion. The samples were centrifuged (4℃, 8000 rpm, 10 min), and the supernatant was discarded.

[0105] Phase Two: Simulating Intestinal Digestion

[0106] The bacterial pellet after the first stage of treatment was resuspended in a 1:10 artificial intestinal fluid and placed in a 37°C water bath for further digestion for 2 hours. 200 μL samples were taken every 0.5 hours, with samples taken once each for SIF (simulated intestinal fluid)-0, SIF-0.5, SIF-1, SIF-1.5, and SIF-2, and the viable count of *Saccharomyces boulardii* was determined using the serial dilution plate count method.

[0107] 4. Viable bacteria count determination

[0108] The viable bacterial count (CFU / g) in samples at different time points was determined using the serial dilution plate count method. Results are as follows: Figure 4 As shown.

[0109] Experimental results showed that after simulated gastrointestinal digestion, the number of viable free yeast cells decreased by 3.76 log CFU / g, while the number of viable cells in the microencapsulated product decreased by only 1.13 log CFU / g, indicating that microencapsulation significantly improved the strain's tolerance to the gastrointestinal environment.

[0110] Example 5: Egg-laying duck rearing experiment

[0111] 1. Experimental animals: 300 healthy caged ducks of similar age, weight, and egg production performance were selected.

[0112] 2. Experimental design: The laying ducks were randomly divided into two treatment groups, with three replicates in each group and 50 laying ducks in each replicate.

[0113] Control group (A): fed a basal diet without any added preparations;

[0114] Experimental group (B): fed a basal diet with 0.1% of the microecological preparation of this invention added.

[0115] 3. Test cycle

[0116] Pre-trial period: 7 days, to allow the laying ducks to adapt to the new feeding environment and diet;

[0117] The trial period is 90 days, during which the system records changes in various indicators.

[0118] 4. Measurement Indicators and Methods

[0119] (1) Production performance indicators (daily record)

[0120] Daily egg production rate: Collect eggs from each duplicate cage twice a day, morning and evening, record the total number of eggs produced, and calculate the egg production rate (%).

[0121] Daily feed intake: Feed in the morning and collect the feed the next morning. Weigh the remaining feed and record the daily feed intake (g / bird) for each replicate cage.

[0122] Feed conversion ratio: The total weight of eggs laid and the amount of feed consumed in each repeat cage are counted weekly to calculate the feed conversion ratio (g / g).

[0123] Egg weight: 50 eggs were randomly selected from each duplicate cage each week, and the average egg weight (g) was calculated.

[0124] Live weight: Weigh the live weight of each replicate cage monthly and calculate the average weight (kg).

[0125] (2) Egg quality indicators (sampled and tested every 15 days)

[0126] Egg weight: Weighed using an electronic balance (g);

[0127] Egg shape index: Measure the transverse and longitudinal diameters of the egg using vernier calipers, and calculate the egg shape index = (transverse diameter / longitudinal diameter) × 100;

[0128] Haugh Unit: Measured using an egg quality analyzer to measure albumen height (mm) and egg weight, and automatically calculated in Haugh units;

[0129] Yolk height: The height of the highest point of the yolk (mm) was measured using a micrometer.

[0130] Eggshell thickness: The thickness of the eggshell at three points on the equator was measured using a micrometer, and the average value (mm) was taken.

[0131] Eggshell strength: The force (N) required to break an eggshell is measured using an eggshell strength tester.

[0132] Shell weight percentage: Weigh the eggshell using an electronic balance and calculate the shell weight percentage (%) = (eggshell weight / egg weight) × 100.

[0133] (3) Immune and antioxidant indicators (samples taken at the end of the experiment)

[0134] Blood sample collection: After the trial period, 10 laying ducks were randomly selected from each replicate, and blood samples were collected using the cardiac blood collection method.

[0135] Serum separation: After the blood sample is left to stand at 4℃ for 4 hours, it is centrifuged at 3000 rpm for 15 minutes to separate the serum and store it in a -80℃ refrigerator for later use.

[0136] Antioxidant indicators: The content or activity of MDA, SOD, CAT, GSH-PX and T-AOC in serum were determined using commercial kits;

[0137] Immunological indicators: The levels of interferon-gamma, interleukin-2 (IL-2), IgA, IgG, and IgM in serum were measured using enzyme-linked immunosorbent assay (ELISA).

[0138] (4) Intestinal health indicators (samples taken at the end of the trial)

[0139] Intestinal contents collection: After sacrificing the ducks and collecting blood samples, immediately and aseptically remove the cecum, squeeze out the contents, and store them in a -80℃ refrigerator for later use;

[0140] Microbial community analysis: 16S rRNA gene high-throughput sequencing technology was used to analyze the microbial community in the cecal contents and assess microbial diversity (α diversity) and composition (β diversity).

[0141] Intestinal tissue morphology: Tissue from the mid-duodenal segment was taken and fixed with 4% paraformaldehyde; tissue sections were prepared and stained with hematoxylin-eosin (HE); villus height and crypt depth were measured under a microscope, and the villus / crypt ratio (V / C) was calculated.

[0142] 5. Results and Analysis

[0143] (1) Production performance and egg quality indicators

[0144] The test results are shown in Table 2.

[0145] Table 2 Production performance and egg quality indicators

[0146]

[0147] Note: a and b are markers of significant difference; different letters in the same row indicate significant differences at the p-value level.

[0148] The test results show that the experimental group significantly outperformed the control group in several key production performance indicators, including egg production rate, egg weight, Haugh units, and eggshell strength (P<0.05). In particular, the eggshell strength was significantly improved, indicating that the microecological preparation prepared in this invention effectively enhances the absorption and utilization of calcium and phosphorus in laying ducks, reducing the egg breakage rate. The feed conversion ratio in the experimental group was significantly lower, meaning that the microecological preparation prepared in this invention can effectively improve feed conversion efficiency, bringing direct economic benefits to farmers. There were no significant differences in live weight and daily feed intake between the experimental and control groups, indicating that the microecological preparation prepared in this invention achieves improved production performance without affecting the growth and appetite of laying ducks.

[0149] (2) Antioxidant and immune indicators

[0150] The test results are shown in Tables 3 and 4.

[0151] Table 3 Antioxidant Indicators

[0152]

[0153] Note: a and b are markers of significant difference; different letters in the same row indicate significant differences at the p-value level.

[0154] Table 4 Immune Indicators

[0155]

[0156] Note: a and b are markers of significant difference; different letters in the same row indicate significant differences at the p-value level.

[0157] From the perspective of antioxidant indicators, the serum MDA content in the experimental group was significantly reduced, while the SOD and T-AOC activities were significantly increased (P<0.05). This indicates that the microecological preparation prepared in this invention can effectively scavenge free radicals, enhance the stress resistance and antioxidant capacity of laying ducks, help delay aging, and maintain high production. From the perspective of immune indicators, the experimental group was significantly better than the control group in all immune indicators (P<0.05), and most of them showed significant improvement, especially the IgG content, which increased significantly (P<0.01). This indicates that the microecological preparation prepared in this invention can systematically enhance the immune function of laying ducks.

[0158] (3) Intestinal health indicators

[0159] The test results are shown in Tables 5-7 and Figure 5 As shown.

[0160] Table 5. Differences in relative abundance of species at the phylum level

[0161]

[0162] Firmicutes ( Firmicutes ) and Bacteroidetes ( Bacteroidetes Microbes are the most abundant microbial group in the animal gut, and their community diversity index is usually positively correlated with host productivity. This association may be related to the synergistic effect of richer metabolic pathways, and also stems from their competitive exclusion effect on pathogens, thereby inhibiting the colonization of harmful bacteria in the gut.

[0163] As shown in Table 5, compared with the control group (74.92%), the relative abundance of Bacteroidetes in the probiotic preparation group was significantly reduced (69.30%, P<0.05), a decrease of 5.62%. Bacteroidetes are the main producers of carbohydrate-active enzymes in the gut, and their decrease in abundance may reflect a slight adjustment in the intestinal fermentation pattern. Meanwhile, Firmicutes significantly increased to 25.21% in the experimental group, an increase of 2.96% compared with the control group (22.25%), indicating that probiotic intervention has a regulatory effect on the intestinal flora structure and helps maintain microecological balance. Furthermore, the Firmicutes / Bacteroidetes ratio (F / B value) in the probiotic preparation group increased from 0.30 in the control group to 0.36. This ratio is positively correlated with energy acquisition efficiency, and its increase may be related to improved feed utilization efficiency, further demonstrating the potential role of the probiotic preparation prepared in this invention in improving host nutritional metabolism.

[0164] Table 6. Differences in relative abundance of species at the genus level

[0165]

[0166] As can be seen from the data in Table 6, at the genus level, Bacteroides ( Bacteroides Prevostii was the most dominant bacterial genus in both groups, but its relative abundance in the probiotic preparation group was 39.89%, significantly higher than the control group (33.30%) by 6.59%, showing a clear probiotic-promoting effect. Meanwhile, Prevostii spp. (…) Prevotella )and Prevotellaceae The abundance of the _Ga6A1_group in the microecological preparation group was 4.27% and 3.13%, respectively, which were significantly lower than those in the control group (7.65% and 7.44%) by 3.38% and 4.31%, respectively. This change may be related to the microecological preparation promoting the production of antimicrobial metabolites such as short-chain fatty acids, thereby indirectly inhibiting the growth of potential pathogenic bacteria such as Prevotella.

[0167] In summary, the addition of the microecological preparation prepared in this invention not only regulates the abundance of key bacterial genera, but also promotes the overall improvement of intestinal microbial diversity and richness, indicating that it has a positive regulatory effect on the intestinal microecological structure.

[0168] Table 7. Morphological Indicators of the Small Intestine

[0169]

[0170] Note: a, b, and c are markers of significant differences. Different letters in the same row indicate significant differences at the p-value level.

[0171] Analysis of the morphological indicators of the mid-duodenal tissue revealed that the villus height increased by 15.30%, the crypt depth decreased by 22.54%, and the VH / CD ratio increased by 51.47% in the experimental group. This indicates that the experimental treatment effectively improved the intestinal morphology and structure of laying ducks, expanded the nutrient absorption surface area, stabilized the intestinal mucosal renewal rate, and thus improved the overall intestinal health and feed conversion efficiency.

[0172] In summary, this invention, through an innovative three-pronged approach of "precise strain selection, fermentation process optimization, and delivery system optimization," achieves low-cost, high-density production, high-activity, stable preservation, and intestinal-targeted delivery of Saccharomyces boulardii NCUTY 002. This provides a safe, economical, and efficient new green solution for healthy duck farming, possessing significant industrial application value and broad market prospects.

[0173] Finally, it should be emphasized that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A microecological preparation, characterized in that, The microecological preparation contains Saccharomyces boulardii (Saccharomyces boulardii) Saccharomyces cerevisiae subsp. boulardii NCUTY 002 and Aspergillus niger ATCC 1640; The *Brainia bradyceps* strain NCUTY 002 is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO:M 2025648.

2. The method for preparing the probiotic preparation as described in claim 1, characterized in that, Includes the following steps: (1) Activate Aspergillus niger and Saccharomyces boulardii separately to prepare activated bacterial solutions for later use; (2) Prepare the fermentation culture medium; (3) First, inoculate Aspergillus niger into the fermentation medium and ferment at 30℃-35℃ for 2-3 days. Then, inoculate with Saccharomyces boulardii and continue fermentation at the same temperature for 2-3 days to obtain the fermentation broth. (4) Mix the fermentation broth with the wall material solution, stir magnetically for 15-20 minutes, and then spray dry to obtain core microcapsule powder; (5) Place the core microcapsule powder in a fluidized bed coating machine for spray coating to obtain the microcapsule microecological preparation.

3. The method for preparing the microecological preparation according to claim 2, characterized in that, In step (3), the inoculation amount of Aspergillus niger is 1% of the total volume of the fermentation medium; the inoculation amount of Saccharomyces boulardii is 3.5% of the total volume of the fermentation medium; the fermentation medium is prepared by the following method: 36 parts by weight of soybean residue, 36 parts by weight of molasses and 28 parts by weight of wheat bran are mixed, 1000 parts by weight of deionized water is added, stirred evenly, the pH is adjusted to 6.0, and sterilized at 121℃ for 20 minutes.

4. The method for preparing the microecological preparation according to claim 2, characterized in that, In step (4), the volume ratio of the fermentation broth to the wall material solution is 6:4; the wall material solution contains 250 g / L of resistant starch, 200 g / L of concentrated whey protein, 125 g / L of trehalose and 5 g / L-12.5 g / L of xanthan gum or carrageenan; the inlet air temperature of the spray dryer is 150°C, the outlet air temperature is 75°C-80°C, and the feed rate is 10 mL / min-15 mL / min.

5. The method for preparing the microecological preparation according to claim 2, characterized in that, In step (5), the coating solution used contains the following raw material components in parts by weight: 100 parts of polyacrylic acid resin II, 15 parts of triethyl citrate, 50 parts of talc, 1 part of polysorbate 80 and 834 parts of purified water; the inlet air temperature of the fluidized bed is 35℃-40℃, the spray pressure is 0.1MPa-0.2MPa, and the spray rate is 2mL / min-5mL / min.

6. A feed additive, characterized in that, It comprises the microecological preparation according to claim 1 or the microecological preparation prepared by any one of claims 2-5.

7. The use of a microecological preparation as described in claim 1 or a feed additive as described in claim 6 in any of the following: (1) Used in the preparation of feed additives for laying ducks; (2) Used for duck egg farming.

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