A microencapsulated microalgal astaxanthin replacement additive for weaning stress in piglets and a method of preparation thereof

Abstract

The application discloses a kind of microencapsulated microalgae astaxanthin replacement additives for piglet weaning stress and a preparation method thereof, and belongs to the technical field of animal nutrition and feed additive.The microencapsulated microalgae astaxanthin replacement additive includes the following components by mass fraction: microencapsulated compound 30-45 parts, antioxidant 0.3-0.5 parts, immune enhancer 0.8-1.2 parts, prebiotic 3-3.5 parts, carrier 55-65 parts.First, high-activity, high-purity microalgae astaxanthin extract and high-activity bacteria number compound probiotic concentrate are prepared by using optimized extraction and activation process;then, by means of ion gel method and chitosan coating technology with accurate control, the two are simultaneously embedded in double-layer microcapsules, which not only improves the stability of microalgae astaxanthin, but also improves the survival rate of probiotics in gastric acid environment and is beneficial to its release in intestinal tract.

Description

Technical Field

[0001] This invention belongs to the field of animal nutrition and feed additive technology, specifically relating to a microencapsulated microalgae astaxanthin antibiotic alternative for weaning stress in piglets and its preparation method. Background Technology

[0002] Weaning of piglets, a crucial step in modern pig production, typically occurs between 21 and 28 days of age. During this time, piglets face multiple stressors, including sudden environmental changes, feed conversions, social reorganization, and immune gaps. This leads to a series of problems such as digestive system disorders (sudden drop in feed intake, damaged intestinal structure), gut microbiota imbalance (reduced beneficial bacteria, increased harmful bacteria), immunosuppression, oxidative stress, and growth retardation, resulting in increased mortality and significant economic losses. For a long time, antibiotics have been used to prevent and treat weaning-related diseases, but long-term use has led to serious problems such as bacterial resistance, drug residues, environmental pollution, and gut microbiota disruption, prompting the global implementation of "antibiotic-free" policies. Since 2020, my country has completely banned the use of growth-promoting drug feed additives, marking the industry's official entry into the "antibiotic-free" era, necessitating the development of safe and efficient alternatives. However, existing antibiotic alternatives such as probiotics, plant extracts, acidifiers, enzyme preparations, and antimicrobial peptides all have significant limitations: probiotics suffer from insufficient live bacteria counts, poor tolerance to gastric acid, weak colonization ability, and poor stability; plant extracts have unstable active ingredients, low bioavailability, and a single mechanism of action; acidifiers have poor palatability and a limited range of action; enzyme preparations are insufficient in improving immunity and oxidative stress; and antimicrobial peptides are difficult to use on a large scale due to excessively high production costs. Overall, existing products are mostly single-ingredient or simply compounded, lacking effective protection, exhibiting poor stability, and having limited mechanisms of action, failing to completely replace the comprehensive effects of antibiotics.

[0003] Against this backdrop, microalgal astaxanthin, as a natural and potent antioxidant, has shown great potential. Its unique molecular structure endows it with excellent antioxidant, immunomodulatory, anti-inflammatory, intestinal protective, and growth-promoting functions. However, its practical application faces severe challenges: extremely poor stability (sensitive to light, heat, and oxygen, and easily degraded), low bioavailability (highly lipid-soluble and poorly soluble in water), and high cost, resulting in limited dosage and difficulty in fully realizing its effects. Recent studies have found that microalgal astaxanthin and probiotics have a synergistic effect. The two complement each other in terms of target sites (cell protection and microbiome regulation) and timing of action (rapid onset and sustained action), and can enhance each other's efficacy through mechanisms such as reducing oxidative stress, improving the colonization environment, and promoting absorption. However, current technologies often simply mix the two without effective protection, leading to degradation of active ingredients and death of probiotics, thus failing to fully realize the synergistic effect. Although microencapsulation technology can provide some protection for active substances, monolayer calcium alginate gels have problems such as insufficient mechanical strength, poor acid resistance, and imprecise release control. Therefore, developing a novel encapsulation technology that can simultaneously protect microalgal astaxanthin and probiotics, improve stability and bioavailability, and fully leverage their synergistic effects has become a key breakthrough in solving weaning stress and promoting antibiotic-free aquaculture. Summary of the Invention

[0004] In view of this, the purpose of this invention is to provide a microencapsulated microalgae astaxanthin antibiotic alternative for weaning stress in piglets and its preparation method.

[0005] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a microencapsulated microalgae astaxanthin alternative to antibiotics, comprising the following components in parts by weight: 30-45 parts of microencapsulated complex, 0.3-0.5 parts of antioxidant, 0.8-1.2 parts of immune enhancer, 3-3.5 parts of prebiotic, and 55-65 parts of carrier.

[0006] Preferably, the microencapsulated complex is prepared by the following method: S1. Mix sodium alginate solution, microalgae astaxanthin extract and probiotic concentrate evenly to obtain a mixture; S2. The mixture is dripped into a calcium chloride solution for cross-linking and curing to obtain primary microcapsules; S3. The primary microcapsules are immersed in a chitosan solution for coating, and then filtered, washed and dried to obtain the microencapsulated complex.

[0007] Preferably, the mass ratio of sodium alginate solution, microalgae astaxanthin extract, and probiotic concentrate in S1 is 100:10:12.

[0008] Preferably, the concentration of the sodium alginate solution is 2.5~3.5%wt.

[0009] Preferably, the preparation method of the microalgae astaxanthin extract is as follows: Dry the Haematococcus pluvialis powder at 60-70℃ for 2-3 hours until the moisture content is ≤5%, and then pulverize it through a 120-mesh sieve. After immersing the pretreated algal powder in liquid nitrogen at ~196℃ for 25~35 min, it was homogenized under high pressure three times within 2~5 min, each time for 45 s, with a 2 min interval, to complete the cell wall disruption. Mix the cell wall broken algae powder with 85-95% ethanol at a solid-liquid ratio of 1g:(15-20)mL, and extract it by ultrasonication at 45-55℃ in the dark for 30-40 min. The ultrasonic power is 300-400 W and the frequency is 40-50 kHz. After filtration, the astaxanthin extract was concentrated under reduced pressure to 1 / 4 of its original volume at 40-50℃ and a vacuum degree of ~0.08 to ~0.09 MPa to obtain microalgae astaxanthin extract.

[0010] Furthermore, the microalgae astaxanthin extract contains 12-18% microalgae astaxanthin, with all-trans microalgae astaxanthin accounting for >85% and oxidation products <5%.

[0011] Preferably, the probiotic concentrate in S1 is composed of *Lactobacillus plantarum*, *Bacillus subtilis*, and *Clostridium butyricum* in a live bacteria ratio of 1:1:0.8; the live bacteria count of the probiotic concentrate is 1.5 × 10⁻⁶. 10 CFU / mL.

[0012] Furthermore, the preparation method of the probiotic concentrate is as follows: Prepare a solid culture medium, inoculate the bacterial strain for initial activation, and culture at 28-32℃ for 2-3 days to obtain an initially activated bacterial strain. Prepare a sodium alginate gel protective solution containing 0.1-0.15% wt calcium chloride and a chitosan solution containing 0.6-0.8% wt chitosan; The first-activated bacterial strain was added to a mixture of sodium alginate gel protection solution and chitosan solution at a ratio of 1 g:(90~110) mL, with a volume ratio of 1:1; it was then soaked at 28~32℃ for 50~60 min, and a pulsed electric field was applied simultaneously: field strength 25kV / cm, pulse time 2000~2500 μs, pulse interval 5~8 s, to complete the second activation. The reactivated bacterial strain was cultured in MRS liquid medium at 30-35℃ and pH 6.5-7.0 for 1-2 days until the viable count reached (4-6) × 10⁻⁶. 9 CFU / mL; Collect bacterial cells by centrifugation, resuspend in a protective solution containing 5-8% trehalose, and the viable count is 1.5 × 10⁻⁶. 10CFU / mL, total bacterial count 1.8 × 10⁻⁶ 12 CFU.

[0013] Preferably, in step S2, a nozzle with an orifice diameter of 0.3~0.5 mm is used to drip the mixture into a calcium chloride solution with a mass concentration of 2.0~2.5% at a rate of 30~40 drops / min, and the nozzle is 8~12 cm above the liquid surface. Preferably, the crosslinking and curing time in step S2 is 25 min.

[0014] Preferably, the chitosan solution in S3 has a concentration of 0.8-1.2%wt and a pH of 5.0-5.5.

[0015] Preferably, the coating time in step S3 is 12 minutes.

[0016] Preferably, the antioxidant is selected from one or more of vitamin E, tea polyphenols, and rosemary extract; The immune enhancer is selected from one or more of β-glucan, astragalus polysaccharide, and yeast cell wall; The prebiotics are selected from one or more of fructooligosaccharides, xylooligosaccharides, and mannan oligosaccharides; The carrier is selected from one or more of wheat bran powder, rice bran powder, and corn powder.

[0017] This invention also provides a method for preparing the above-mentioned microencapsulated microalgae astaxanthin alternative, comprising the following steps: Weigh the raw materials according to the stated weight proportions; After uniformly mixing the microencapsulated complex with antioxidants, immune enhancers, prebiotics and carriers, the mixture is dried to obtain the microencapsulated microalgae astaxanthin alternative to antibiotics.

[0018] It contains at least the following beneficial technical effects: This invention provides a microencapsulated microalgae astaxanthin antibiotic alternative for weaning stress in piglets and its preparation method. Through an innovative double-layer microencapsulation structure (inner layer: calcium alginate gel; outer layer: chitosan membrane) and scientifically formulated functional components, it successfully solves the key problems of poor stability, low bioavailability, and low survival rate of probiotics in the acidic gastric environment of existing technologies. The technology first utilizes optimized extraction and activation processes to prepare a highly active, high-purity microalgae astaxanthin extract and a high-viable-bacteria compound probiotic concentrate. Subsequently, through precisely controlled ionogelation and chitosan coating technology, both are simultaneously encapsulated in double-layer microcapsules. This not only significantly improves the light, heat, and oxygen stability of the microalgae astaxanthin (with a significantly higher retention rate under simulated harsh conditions than the unencapsulated and single-layer encapsulated groups), but also effectively protects the probiotics from gastric acid erosion, enabling targeted release into the intestines and a significantly improved survival rate. This design fully leverages the synergistic effect of microalgal astaxanthin and probiotics: the potent antioxidant and immunomodulatory effects of microalgal astaxanthin create a favorable intestinal environment for probiotic colonization, while short-chain fatty acids and other substances produced by probiotic metabolism promote the emulsification and absorption of fat-soluble microalgal astaxanthin, forming a virtuous cycle. Combined with exogenously added antioxidants, immune enhancers, and prebiotics, the product comprehensively improves weaning stress in piglets through antioxidant effects, immune regulation, gut microbiota balancing, and anti-inflammation. The results of the examples show that, under the experimental conditions described in this application, the application of this additive can significantly improve the growth performance of weaned piglets, reduce diarrhea rates, enhance immune and antioxidant levels, alleviate inflammatory responses, and improve gut microbiota structure. Its antibiotic alternative effect is comparable to that of the antibiotic group, and it has the advantages of reducing the risk of drug resistance and drug residues. In summary, this invention provides a feed additive solution with good stability, clear synergistic effects, and can be used for weaning stress intervention in piglets, providing a technical reference for antibiotic-free farming. Attached Figure Description

[0019] Figure 1 Flowchart of the preparation process for microencapsulated microalgae astaxanthin antibiotic alternative; Figure 2 This is a schematic diagram of the double-layer microencapsulation structure of the present invention; Figure 3 This is a comparison chart showing the retention rate changes of astaxanthin from microalgae under light, high temperature, and low temperature conditions for unmicroencapsulated, monolayer microencapsulated, and bilayer microencapsulated microalgae according to the present invention. Figure 4 This is a comparative diagram showing the effect of the double-layer microencapsulation of the present invention on the gastric acid tolerance of probiotics; Figure 5 This is a comparative diagram showing the effects of the product of this invention on the intestinal flora structure of piglets; Figure 6 The effects of double-layered microencapsulated microalgae astaxanthin on the growth performance and health indicators of livestock and poultry.

[0020] Figure 7 This is a graph showing the effect of the product of this invention on serum immunity and antioxidant indicators in piglets. Detailed Implementation

[0021] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0022] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0023] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0024] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This application specification and embodiments are merely exemplary.

[0025] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0026] Unless otherwise specified, "room temperature" and "normal temperature" in this invention refer to 25±2℃.

[0027] Example 1 Unless otherwise specified, all raw materials or instruments used in the following embodiments of the present invention are commercially available.

[0028] Preparation of raw materials: 1. Preparation of microalgae astaxanthin extract In this embodiment, the microalgae astaxanthin extract used can be prepared by the aforementioned method; for the convenience of experimental implementation, commercially available natural microalgae astaxanthin extract from Haematococcus pluvialis with the same indicators can also be used, with a microalgae astaxanthin content of 12~18 wt%, an all-trans microalgae astaxanthin content of ≥85%, and oxidation products of ≤5 wt%.

[0029] 2. Preparation of compound probiotic concentrate (1) Prepare solid culture medium and activate it once: Weigh out the following ingredients according to the formula: 20 g glucose, 20 g agar, 5 g yeast extract, 12 g peptone, 2 g trisodium citrate, 5 g sodium acetate, 2 g potassium dihydrogen phosphate, 0.05 g magnesium sulfate, 0.3 g potassium chloride, and 0.3 g Tween 80.

[0030] Add deionized water to a final volume of 1000 mL, then heat and stir to dissolve.

[0031] Adjust the pH to 6.8 using 1 mol / L HCl.

[0032] Dispense into 500 mL Erlenmeyer flasks, 300 mL per flask.

[0033] The bottle opening was plugged with cotton and wrapped with kraft paper, then autoclaved at 121℃ for 22 minutes.

[0034] After sterilization, remove and cool to room temperature (approximately 25°C).

[0035] Preparation of compound probiotic suspension: In a clean bench, weigh out the freeze-dried Lactobacillus plantarum powder (live count ≥ 1 × 10^ ) 11 1.0 g of Bacillus subtilis lyophilized powder (live count ≥ 1 × 10^ 10^ 1000 g) 11 1.0 g of Clostridium butyricum lyophilized powder (live count ≥ 1 × 10^ 11 Add 0.8 g of CFU / g to 100 mL of sterile saline and mix well to obtain a compound probiotic suspension; after appropriate dilution of the suspension, the bacterial cell concentration is approximately 1 × 10^6 g. 7 CFU / mL inoculation suspension.

[0036] Inoculation and culture: Pour the solid culture medium into sterile Petri dishes (90 mm in diameter), about 18 mL per dish, and allow it to solidify.

[0037] Use a sterile inoculation loop to pick up the compound probiotic suspension and perform three-zone streak inoculation on a petri dish.

[0038] Place the petri dishes in a 30℃ constant temperature incubator for incubation.

[0039] Observe every 12 hours: small colonies appear after 24 hours, colonies enlarge after 48 hours, and colonies become full (2-5 mm in diameter, with neat edges, moist surface, milky white or pale yellow) after 60 hours, with no contamination.

[0040] After 2.5 days of incubation, colonies were picked up with an inoculation loop, transferred to a 50 mL centrifuge tube, and 20 mL of sterile physiological saline was added. The mixture was then vortexed for 2 min to prepare a bacterial suspension.

[0041] The bacterial cell concentration was measured to be 2 × 10⁻⁶. 8 CFU / mL, store at 4℃ for later use.

[0042] (2) Preparation of gel protective film: Weigh out 10 g of sodium alginate powder (food grade, purity ≥98%).

[0043] Add 450 mL of a 0.12% (w / v) calcium chloride solution (weigh 0.54 g of CaCl2·2H2O and dissolve it in 450 mL of deionized water) at a solid-liquid ratio of 1:45.

[0044] Place it on a thermostatic magnetic stirrer, with a water bath temperature of 45℃ and a stirring speed of 450 r / min, and stir continuously for 30 min.

[0045] Sodium alginate dissolves completely, and the solution is a uniform, transparent, viscous consistency.

[0046] Let it stand at room temperature (22℃) for 2 hours to allow bubbles to escape and sodium alginate to fully cross-link with calcium ions.

[0047] A uniform, semi-transparent gel protective film was obtained, transferred to a sterile container, and stored at 4°C for later use.

[0048] (3) Preparation of chitosan solution: Weigh out 3.5 g of chitosan powder (degree of deacetylation 90%, molecular weight 300,000).

[0049] Dissolve in 500 mL of 1% acetic acid solution (5 mL glacial acetic acid: 495 mL deionized water).

[0050] Stir magnetically at room temperature for 3.5 h at a stirring speed of 350 r / min until the chitosan is completely dissolved.

[0051] Prepare a chitosan solution with a concentration of 0.7% by mass.

[0052] Using a pH meter, add 1 mol / L NaOH solution dropwise to adjust the pH to 5.8.

[0053] Continuous stirring is required during the adjustment process to prevent localized excessively high pH levels from causing chitosan precipitation.

[0054] After adjustment, continue stirring for 12 minutes to ensure the pH is uniform.

[0055] The solution was filtered through a 0.45 μm filter membrane to obtain a clear and transparent chitosan solution, which was then transferred to a sterile bottle and stored at 4°C for later use.

[0056] (4) Secondary activation (pulsed electric field assisted): Prepare the pulse electric field processing device: Pulse generator (output voltage adjustable from 0 to 10 kV, pulse width adjustable from 100 to 5000 μs).

[0057] A pair of stainless steel parallel plate electrodes (size 10 cm × 10 cm, adjustable spacing).

[0058] Polypropylene beaker (500 mL, insulating material).

[0059] Operating steps: Take 200 mL of the gel protective film and 200 mL of the chitosan solution, and slowly mix them under sterile conditions (stir gently while pouring) to obtain 400 mL of the mixture.

[0060] Take a single activated bacterial suspension (containing approximately 4 g of bacterial cells by wet weight) and add it to 400 mL of mixed solution, so that the volume ratio of bacterial cells to solution is approximately 1 g: 100 mL.

[0061] Transfer to a 500 mL polypropylene beaker and place in a 30°C constant temperature water bath.

[0062] Place the beaker between two parallel plate electrodes and adjust the electrode spacing to 0.25 cm.

[0063] Set the pulse generator parameters: Output voltage: 6.25 kV (field strength = 6.25 kV / 0.25 cm = 25 kV / cm).

[0064] Pulse waveform: square wave.

[0065] Pulse width: 2200 μs.

[0066] Pulse interval: 6.5 s.

[0067] Soak for 55 minutes, gently stirring with a sterile glass rod every 10 minutes, while simultaneously applying pulsed electric field treatment. Monitor the solution temperature during the process to ensure it is maintained at 30°C.

[0068] After processing: Immediately transfer the mixture out of the electric field region and let it stand for 7 minutes.

[0069] Collect the bacterial cells by centrifugation at 4℃ and 5000 r / min for 9 min.

[0070] Wash twice with sterile saline, centrifuging for 8 minutes each time to remove residual gel membrane and chitosan.

[0071] The secondary activated compound probiotic cells were obtained (wet weight approximately 4.5 g).

[0072] The cells were resuspended in sterile physiological saline, and the bacterial concentration was determined to be 2.7 × 10⁻⁶. 8 CFU / mL (approximately 35% higher than single activation).

[0073] Store at 4℃ and perform expansion culture within 12 hours.

[0074] (5) Expanded culture of compound probiotics: Preparation of MRS liquid culture medium: The formula per liter is as follows: 10 g peptone, 10 g beef extract, 5 g yeast extract, 20 g glucose, 5 g sodium acetate, 2 g triammonium citrate, 2 g dipotassium hydrogen phosphate, 0.2 g magnesium sulfate, and 0.05 g manganese sulfate. Dissolve in approximately 900 mL of deionized water, stir to dissolve, and adjust the pH to 6.6 with HCl or NaOH; Add 1 mL of Tween 80 and bring the volume to 1000 mL with deionized water. Dispense into 2 L Erlenmeyer flasks, each containing 1.2 L (the volume shall not exceed 2 / 3 of the capacity). The bottle mouth was plugged with cotton, wrapped with kraft paper, and autoclaved at 121℃ for 18 minutes. Cool to room temperature before use.

[0075] Preparation of seed solution: Prepare 940 mL of MRS liquid culture medium and inoculate 60 mL of the secondary activated bacterial suspension (inoculation amount 6%). Place in a 30℃ constant temperature incubator for static incubation; Samples were taken every 4 hours to measure the OD600 value. After 18 hours of cultivation, the OD600 reached 1.0, indicating that the seed culture was complete. At this point, the bacterial solution is cloudy, has a sour taste, and has a pH of approximately 5.3.

[0076] Large-scale expansion: Take 45 mL of seed culture (4% inoculum) and inoculate it into 1 L of fresh MRS medium; Place it in a constant temperature shaker at 32℃ and rotate at 190 r / min.

[0077] Samples are taken and tested every 6 hours. Measure the OD600 value; Measure the pH value and adjust the pH to 6.7 using a sterile 1 mol / L NaOH solution; Plate count method for detecting viable bacteria; After 36 hours of incubation, the OD600 reached 2.5, and the viable cell count reached 5 × 10⁻⁶. 9 CFU / mL, expansion culture complete; The bacterial solution is cloudy and milky white, with a distinct fermented odor.

[0078] (6) Collect concentrated probiotics: Transfer the cultured bacterial solution to a centrifuge bottle and centrifuge at 9000 r / min for 12 min in a refrigerated centrifuge at 4℃. Pour off the supernatant and collect the precipitated bacterial cells (approximately 15 g wet weight). Add 250 mL of sterile saline, gently pipette to resuspend, and centrifuge at 9000 r / min for 12 min. Wash three times, and discard the supernatant after the last centrifugation. Weigh 8.4 g of trehalose (≥99% purity), dissolve it in 120 mL of sterile physiological saline to prepare a 7% trehalose protective solution, and sterilize it at 121℃ for 20 min. Resuspend the bacterial cells in trehalose protection solution and mix thoroughly. The viable bacterial count was 1.5 × 10⁻⁶. 10 CFU / mL (plate count method); Aliquot into sterile cryovials, store at 4°C, and use within 24 hours.

[0079] Example 1 S1. Preparation of microencapsulated compounds (1) Preparation of sodium alginate solution: Weigh out 30 g of sodium alginate powder (pharmaceutical grade, purity ≥98%, molecular weight 200,000). Dissolve in 1000 mL of deionized water; Place the mixture in a thermostatic magnetic stirrer at a water bath temperature of 55℃ and a stirring speed of 550 r / min for 35 min. Sodium alginate was completely dissolved to prepare a 3.0% wt sodium alginate solution; Turn off the heating and continue stirring while cooling to 27°C for later use.

[0080] (2) Preparation of the mixture: Transfer 1000g of sodium alginate solution cooled to 27°C to a beaker; Under light-proof and sterile conditions (in a clean bench, with the UV lamp off and only the fan on), slowly add 100g of microalgae astaxanthin extract. Stir with a magnetic stirrer for 18 minutes at a speed of 350 r / min to ensure uniform dispersion of microalgal astaxanthin and a uniform orange-red solution. Then add 120 g (about 120 mL) of probiotic concentrate. Continue stirring for 12 minutes, then reduce the stirring speed to 250 r / min (to avoid shearing damage to the bacterial cells). Ensure the probiotics are evenly distributed to obtain a mixed solution that is orange-red and slightly turbid. Maintain the temperature at 27°C and proceed to the next step immediately.

[0081] (3) Preparation of crosslinking curing solution: Weigh out 275 g of calcium chloride (food grade); Dissolve in 12 L of deionized water to prepare a calcium chloride solution with a concentration of 2.3% by mass. The volume of the solution is approximately 10 times the volume of the mixture; Store at room temperature (25℃) for later use.

[0082] (4) Microencapsulated drip addition: Preparation of equipment: Peristaltic pump (flow rate adjustable 50 mL / min); Stainless steel microencapsulated nozzle (inner diameter 0.4 mm); Connect the silicone tubing (3 mm inner diameter); Stand (height adjustable); A 2 L beaker contains a calcium chloride solution; Small magnetic stir bar.

[0083] Operating steps: Connect the beaker containing the mixture to the inlet of the peristaltic pump, and connect the outlet of the peristaltic pump to the nozzle through a silicone tube; Fix the nozzle at a height of 10 cm above the surface of the calcium chloride solution using a stand (measure precisely with a ruler). Place a magnetic stir bar in the calcium chloride solution and turn on the magnetic stirrer at a speed of 125 r / min (gentle stirring). Start the peristaltic pump and set the flow rate to 4.2 mL / min; The mixture drips slowly from the nozzle, forming droplets at a rate of approximately 35 drops / min. The moment the droplet enters the calcium chloride solution, the sodium alginate on the surface reacts with Ca²⁺. + Rapid cross-linking forms a gel shell, keeping the droplets spherical; After the microcapsules sank to the bottom of the solution, they continued to solidify for 25 minutes.

[0084] Quality control: Every 15 minutes, a few microcapsules were removed and observed under a microscope (40x magnification). Qualified microcapsules: spherical, smooth surface, no tails, uniform size, diameter 2.0~2.3 mm, uniform orange-red color; If the microcapsule morphology is found to be unsatisfactory, adjust the dropping speed or stirring speed in a timely manner. After all the drops were added (approximately 4.8 h), the drops were stopped, and the microcapsules were allowed to solidify in the calcium chloride solution for another 25 min.

[0085] (5) Washing the primary microcapsules: Use a stainless steel sieve (1.5 mm aperture) to remove the microcapsules and drain off most of the calcium chloride solution; Approximately 1100 g (wet weight) of primary microcapsules were obtained. Transfer the microcapsules to a 3 L beaker and add 3.3 L of deionized water (3 times the volume of the microcapsules). Stir gently for 3 minutes to dissolve any remaining calcium chloride on the surface; Filter with a sieve and discard the washing water; Wash 4 times. The conductivity of the wash water was measured after the final wash and was close to that of deionized water (<50 μS / cm), indicating that calcium chloride had been removed. Drain the microcapsules and place them on filter paper to absorb the surface moisture.

[0086] (6) Chitosan coating: Preparation of chitosan coating solution: Weigh out 11 g of chitosan powder (degree of deacetylation 90%, molecular weight 300,000). Dissolve in 1100 mL of 1% acetic acid solution; Magnetic stirring was performed at room temperature for 3.5 h at a stirring speed of 350 r / min. Prepare a chitosan solution with a concentration of 1.0% by mass. Using a pH meter, add 1 mol / L NaOH solution dropwise to adjust the pH to 5.3; Stir continuously during the adjustment process to prevent sedimentation; After adjustment, continue stirring for 12 minutes to ensure the pH is uniform; The solution was filtered through a 0.45 μm filter membrane to obtain a clear coating solution.

[0087] Coating process: Add approximately 1000 g (wet weight) of the washed and drained primary microcapsules to the chitosan coating solution (solid-liquid ratio 1 g: 17.5 mL). Place in a thermostatic magnetic stirrer at 27°C and a stirring speed of 90 r / min (stir gently). Soak the coating for 12 minutes; During the coating process, the surface of the microcapsules gradually changed from transparent to slightly milky white, and the texture changed from soft to slightly tough. After coating is complete, use a sieve to remove the microcapsules and drain the coating solution (the coating solution can be recycled and reused twice). Transfer the microcapsules to a beaker, add deionized water (3.5 times the volume of the microcapsules), and stir gently for 2.5 min. Wash away unbound chitosan from the surface; Filter through a sieve, drain, and wash three times. After the final wash, drain the microcapsules and place them on filter paper to absorb surface moisture. Approximately 950 g of the bilayer microencapsulated complex was obtained (wet weight, water content approximately 65%).

[0088] Quality inspection: The microcapsules are orange-red spherical particles with a smooth, slightly milky-white luster. Approximately 2.2~2.5 mm in diameter (slightly larger than primary microcapsules); It has a springy and resilient feel and is not easily broken when squeezed; When the cross-section is observed under a microscope (by cutting open the microcapsule), the orange-red core inside and the translucent chitosan membrane on the outside are clearly visible, with distinct layers. S2. Preparation of microencapsulated microalgae astaxanthin antibiotic alternatives Weigh out 332.5 g (38%) of microencapsulated complex (dry weight), 3.5 g (0.4%) of vitamin E, 8.8 g (1.0%) of β-glucan, 30.6 g (3.5%) of fructooligosaccharides, and 499.6 g (57.1%) of wheat bran.

[0089] First, add the wheat bran powder to the V-type mixer (5 L capacity); Add vitamin E, beta-glucan, and fructooligosaccharides, and mix for 5 minutes; Finally, add the microencapsulated complex, set the rotation speed to 27 r / min, and mix for 18 min. Every 6 minutes, the machine was stopped to take samples to check the mixing uniformity; no obvious stratification was observed.

[0090] Add the mixture to the spray dryer hopper; to ensure atomization and pumping performance, add an appropriate amount of deionized water to adjust the slurry before spray drying.

[0091] Set process parameters: Feed rate: 4.8 mL / min; Inlet temperature: 160℃; Outlet temperature: 70℃; Atomization pressure: 0.18 MPa; Carrier gas flow rate: 260 L / h; Drying time is approximately 40 minutes; After spray drying, approximately 980 g of powdered product was obtained (approximately 920 g of water was lost during drying). Immediately circulate cold air (room temperature 20℃) to cool to room temperature for 4 minutes; Collect the product and store it in a sealed container away from light.

[0092] Determine moisture content: The oven drying method was used (drying at 105℃ to constant weight). The moisture content is 6.5%, which meets the requirements (5~8%).

[0093] Grind and sieve: Put the dried product (with a small amount of lumps) into a universal grinder (2 L capacity); Set the rotation speed to 22000 r / min and grind for 4 min; At the same time, cold air is introduced to cool down the temperature (a fan blows in room temperature air); The temperature inside the pulverizer is controlled at 25~28℃ (monitored with an infrared thermometer); After crushing, immediately pour out the material and cool it to room temperature; Passed through a 70-mesh sieve (approximately 210 μm aperture); Approximately 180 g of material (coarse particles) remained on the sieve; it was returned to the grinder for further grinding for 2 minutes. Collect approximately 800 g of the sieved material.

[0094] Second screening check: Approximately 10 g of material remains on the sieve (a very small amount of coarse particles, which should be discarded or crushed again). The final product was 790 g (the process introduced solvent and water to form wet microcapsules, and normal loss and sieve residue were generated during spray drying and sieving).

[0095] Detection of particle size distribution: The particle size was determined using a laser particle size analyzer. D50 = 45 μm (median particle size); 85% of the particles are in the range of 20~95 μm; It has uniform particle size and good flowability.

[0096] Surface sterilization: Spread 790g of the product evenly in a stainless steel tray (40 cm × 30 cm); The material thickness is approximately 0.8 cm; The ultraviolet lamp (254 nm, 30 W) was suspended 30 cm above the tray; Irradiate for 18 minutes (control the irradiation dose to reduce the number of viable bacteria by ≤5%, and ensure that the packaging hygiene indicators are qualified); Turn the material over with a sterile scraper every 6 minutes to ensure that all parts are irradiated evenly.

[0097] Samples were taken for microbial testing: Total bacterial count: ≤3.2×10³ CFU / g (standard <10) 4 CFU / g, qualified); Mold: <10² CFU / g (qualified); Escherichia coli: Not detected (qualified).

[0098] Repackaging: The process is carried out in a clean packaging room (a 100,000-level cleanroom). Use a semi-automatic packaging machine; Aluminum foil composite bag specifications: inner layer PE 60 μm, middle layer aluminum foil 25 μm, outer layer PET 40 μm, bag size 18cm×25cm.

[0099] Packaging steps: Open the packaging bag; Inflate with high-purity nitrogen (purity ≥ 99.9%) for 2 seconds to displace the air inside the bag; Quantitative filling of product 50 g (accuracy ±1 g); Refill with nitrogen for 2 seconds (to reduce the oxygen content inside the bag to <2%). Heat sealing: temperature 165℃, time 2.5 s, sealing width 9 mm; Leak test: Immerse the sealed bag in water and press it gently. If no bubbles come out, the seal is good. Labeling: Indicate the product name, ingredients, live bacteria count, production date (January 20, 2024), shelf life (12 months), usage method (mix into feed, add 0.3-0.5%), storage conditions (4-8℃, protected from light), manufacturer, etc.

[0100] Packaging complete: For the small-scale trial, the product was divided into 15 bags (50 g per bag, totaling 750 g), with the remaining approximately 40 g used for sample retention / testing and process loss; the small-scale trial consisted of one box (15 bags / box). Storage: The products were transferred to cold storage; Storage conditions: Temperature 6℃, relative humidity 55%, protected from light; Under these conditions, the shelf life is 12 months.

[0101] Quality Inspection: Take 3 bags of samples for comprehensive quality testing: (1) Microalgae astaxanthin content: 1.82% (target 1.5~2.5%, qualified), all-trans microalgae astaxanthin ratio: 86.8% (target ≥85%, qualified).

[0102] (2) Viable bacteria count (plate count method): Lactobacillus plantarum: 1.48 × 10⁻⁶ 8 CFU / g (target 1.2~1.8×10⁻⁶) 8 ,qualified).

[0103] Bacillus subtilis: 1.25 × 10⁻⁶ 8 CFU / g (target 1.0~1.5×10⁻⁶) 8 ,qualified).

[0104] Clostridium butyricum: 2.72 × 10⁻⁶ 7 CFU / g (target 2.5~3.0×10) 7 ,qualified).

[0105] (3) Moisture content: 6.5% (target 5~8%, qualified).

[0106] (4) Microbiological indicators: The number of miscellaneous bacteria is ≤3.2×10⁻⁶. 3 CFU / g (standard <10) 4 ,qualified).

[0107] Mold: <10 2 CFU / g (qualified).

[0108] Escherichia coli: Not detected (qualified).

[0109] (5) Particle size distribution: D50=45 μm, 85% are in the range of 20~95 μm (qualified).

[0110] (6) Appearance: Orange-red fine powder, with good fluidity and no lumps.

[0111] Conclusion: The product quality meets the design requirements and can be used for subsequent animal experiments and application trials.

[0112] Test Example 1: Stability Comparison Test Unmicroencapsulated astaxanthin, monolayer microencapsulated astaxanthin (microcapsules prepared in step 5 of Example 1), and bilayer microencapsulated astaxanthin (bilayer microencapsulated complex prepared in Example 1) were subjected to stability tests under the same conditions. Parallel experiments were conducted for each group of samples, and the average values ​​of the results were compared and analyzed.

[0113] (1) Photostability test: The sample was placed in a light incubator (illuminance 4500±500 lx, 25℃) and samples were taken at 0, 5, 10, 15, 20, 25 and 30 days. The astaxanthin content was determined by HPLC and the retention rate was calculated based on the initial content.

[0114] (2) Thermal stability test: The sample was placed under constant temperature of 60℃ and samples were taken at 0, 5, 10, 15, 20, 25 and 30 min. The astaxanthin content was determined by HPLC and the retention rate was calculated.

[0115] (3) Cold storage stability test: After sealing the sample, place it in a refrigerator at 4-8℃ and take samples at 0, 2, 4, 6, 8, 10 and 12 months to determine the astaxanthin content (HPLC method) and viable bacteria count (plate count method) respectively, and calculate the retention rate / survival rate.

[0116] The results of this test case indicate that: Depend on Figure 3 As shown in Figure A, under the same light storage conditions, the astaxanthin retention rate of each treatment group gradually decreased as the storage time increased from 0 days to 30 days, indicating that light exposure causes a certain degree of degradation of astaxanthin. Compared with unmicroencapsulated astaxanthin, monolayer microencapsulated astaxanthin showed a higher retention rate throughout the storage process, while bilayer microencapsulated astaxanthin consistently exhibited the highest retention rate. This indicates that microencapsulation treatment can effectively mitigate the loss of astaxanthin under light conditions, with the bilayer microencapsulation structure providing a more significant photoprotective effect on astaxanthin. Figure 3 As shown in Figure B, under heat treatment conditions, the astaxanthin retention rate of each group gradually decreased as the treatment time increased from 0 min to 30 min, indicating that high-temperature treatment accelerates astaxanthin degradation. The non-microencapsulated group showed a larger decrease, indicating that free astaxanthin is more sensitive to heat. The retention rate of the monolayer microencapsulated group was significantly higher than that of the non-microencapsulated group, while the double-layer microencapsulated group maintained a high level at all time points, indicating that the double-layer microencapsulation wall material can form a more effective isolation and protective barrier, thereby improving the thermal stability of astaxanthin. Figure 3 As shown in Figure C, after 12 months of storage at 4–8℃, the astaxanthin content in the samples remained at a high level, with a retention rate of 85%. This indicates that the microencapsulation system can effectively reduce the oxidation, decomposition, and migration loss of astaxanthin during long-term low-temperature storage, demonstrating good storage stability. Figure 3 As shown in Figure D, under the same low-temperature storage conditions, the number of viable bacteria in the sample decreased with the extension of storage time, but the viable bacteria retention rate still reached 72% after 12 months. This indicates that the system can not only protect astaxanthin, but also maintain the viable bacteria survival rate to a certain extent, and has good overall stability.

[0117] In summary, the results of this test case show that microencapsulation significantly improves the stability of astaxanthin under light, heat treatment, and long-term low-temperature storage conditions compared to non-microencapsulation. Furthermore, the double-layer microencapsulation treatment demonstrates superior protection compared to single-layer microencapsulation, effectively delaying astaxanthin degradation and maintaining high astaxanthin and viable cell retention rates during long-term cold storage. This indicates that the double-layer microencapsulation system possesses excellent stability protection and practical application value.

[0118] Experiment Example 2: Probiotic Tolerance Test to Artificial Gastric Fluid Unmicroencapsulated astaxanthin, monolayer microencapsulated astaxanthin (microcapsules prepared in step 5 of Example 1), and bilayer microencapsulated astaxanthin (bilayer microencapsulated complex prepared in Example 1) were subjected to tolerance tests under simulated gastric fluid conditions. Simulated gastric fluid preparation: 16.4 mL of dilute hydrochloric acid was added to approximately 800 mL of water and 10 g of pepsin. After shaking, water was added to bring the volume to 1000 mL, with a pH of approximately 1.2. The mixture was incubated at a constant temperature of 37°C. Samples were taken at 0 min, 120 min, and 240 min, and the viable bacterial count was determined using the plate count method. The survival rate was calculated based on the initial viable bacterial count. Parallel experiments were conducted for each group of samples, and the results were averaged.

[0119] The results of this test case show that: Figure 4 As shown, in the initial stage of artificial gastric fluid treatment, the survival rates of the free probiotic group, the monolayer microencapsulated probiotic group, and the bilayer microencapsulated probiotic group showed little difference. With prolonged artificial gastric fluid treatment, the survival rates of all groups decreased, but the free probiotic group showed the largest decrease, followed by the monolayer microencapsulated probiotic group, while the bilayer microencapsulated probiotic group maintained a consistently high survival rate. These results indicate that microencapsulation treatment can improve the tolerance of probiotics to the acidic gastric environment, with the bilayer microencapsulation showing a more significant protective effect.

[0120] Experimental Example 3: Effects on the Intestinal Microbiota Structure of Piglets The experiment included a control group and an experimental group. The experimental group received a basal diet supplemented with the double-layered microencapsulated product of this invention. A non-microencapsulated group and a single-layered microencapsulated group were also included for comparison. Fresh rectal fecal samples were collected after 21 days of feeding, and the quantities of *Lactobacillus plantarum*, *Bacillus subtilis*, and *Clostridium butyricum* in the intestines were determined using quantitative real-time PCR.

[0121] The results of this test case show that: Figure 5 As shown, compared with the control group, the number of Lactobacillus plantarum, Bacillus subtilis and Clostridium butyricum in the experimental group all increased, indicating that the product of the present invention helps to promote the proliferation of beneficial bacteria and improve the intestinal flora structure.

[0122] Application Example 1: Pilot-scale application in a large-scale pig farm In an application example, 6 kg of the microencapsulated microalgae astaxanthin antibiotic alternative prepared in Example 1 was weighed and added to 2000 kg of piglet diet (addition amount 0.3%). A 0.5% addition group was also included; the effect was not significantly different from the 0.3% group, and both were superior to the control. The mixture was thoroughly mixed using a biaxial paddle mixer for 25 minutes to obtain the feed containing the antibiotic alternative. The prepared feed was used to feed 28-day-old weaned piglets.

[0123] Feed formulation and mixing methods (1) Basic diet formula (per 2000 kg): 1200 kg corn, 500 kg soybean meal, 80 kg fish meal, 100 kg whey powder, 40 kg soybean oil, 20 kg dicalcium phosphate, 15 kg limestone powder, 6 kg salt, and 39 kg premix.

[0124] (2) Adding antibiotic alternatives: Mix 6 kg of the microencapsulated microalgae astaxanthin antibiotic alternative prepared in Example 1 with the basal diet.

[0125] (3) Mixing steps: Weigh each component of the basal diet according to the formula and put it into a twin-shaft paddle mixer (capacity 3000 kg); set the mixer speed to 45 r / min and mix the basal diet for 15 min; evenly sprinkle 6 kg of antibiotic alternative additive on the surface of the basal diet. Continue mixing for 25 min to ensure that the antibiotic alternative additive is fully and evenly mixed with the feed. Take samples from different positions (top, middle, bottom and four corners) of the mixer every 8 min, for a total of 9 samples. Measure the microalgae astaxanthin content in each sample. A coefficient of variation <3% indicates that the mixing is uniform.

[0126] (4) Quality control: After mixing, the content of microalgae astaxanthin in the final feed was measured to be 54.6 ppm and the total number of viable bacteria was 9.0×105 CFU / g, which met the expected design requirements.

[0127] (5) Packaging and storage: Pack the mixed feed into moisture-proof woven bags, 50 kg per bag, and seal. Store in a cool, dry place (temperature ≤25℃, relative humidity ≤65%), avoiding direct sunlight. The feed should be used within 30 days of preparation.

[0128] A comparative feeding trial of weaned piglets was conducted in a large-scale pig farm with an annual output of 10,000 piglets. Healthy weaned piglets were selected and grouped according to initial weight, litter size, and sex balance. The control group was fed a conventional basal diet, while the experimental group received 0.3% of the microencapsulated microalgae astaxanthin antibiotic alternative of this invention in their basal diet. The piglets were fed continuously for 28 days with free access to feed and ample clean drinking water. During the trial, piglet weight, feed intake, feed conversion ratio, and diarrhea were recorded. At the end of the trial, serum samples were collected to measure indicators such as IgG, IgA, SOD, and MDA to evaluate the application effect of the product of this invention under farm conditions.

[0129] like Figure 6 (A represents piglet weight, B represents diarrhea rate, C represents survival rate, D represents average daily weight gain, E represents average daily feed intake, and F represents feed conversion ratio.) As shown, compared with the control group, the experimental group showed an increasing trend in piglet weight, survival rate, average daily weight gain, and average daily feed intake, while the diarrhea rate and feed conversion ratio showed a decreasing trend. This indicates that the microencapsulated microalgae astaxanthin antibiotic alternative of this invention can improve the growth performance and health status of weaned piglets.

[0130] like Figure 7 As shown, A represents serum IgG content in piglets, B represents serum IgA content, C represents serum SOD activity, and D represents serum MDA content. Compared with the control group, the experimental group showed significantly increased serum IgG and IgA levels, significantly enhanced SOD activity, and significantly decreased MDA content, with statistically significant differences. The results indicate that the microencapsulated microalgae astaxanthin antibiotic alternative of this invention can improve the immune level and antioxidant capacity of weaned piglets, reduce oxidative damage caused by weaning stress, and thus help improve the health status of piglets.

[0131] Application Example 2 Ninety-six 28-day-old weaned piglets (Large White × Landrace crossbreed, weighing 7.5 ± 0.8 kg, in good health) were randomly divided into three groups of 32 piglets each. Control group (CON): fed with basal diet Antibiotic group (ANT): basal diet + 75 ppm chlortetracycline + 20 ppm bacitracin zinc Experimental group (DMA): The mixed feed prepared in Example 1 (containing 0.3% antibiotic alternative additives) Prepared feed was used to feed 28-day-old weaned piglets with free access to feed and a sufficient supply of clean drinking water. Feeding continued for 21 days, and the growth performance, health status, and diarrhea status of the piglets were observed. During the experiment, the piglets' weight, feed intake, average daily gain (ADG), average daily feed intake (ADFI), and feed conversion ratio (F / G) were calculated. Diarrhea, coat development, and behavioral activity were observed. On day 22, 12 piglets were randomly selected from each group, and blood and fecal samples were collected after a 12-hour fast. Part of the blood samples were used for routine blood tests, and the other part was used to separate serum. Biochemical reagent kits were used to detect serum liver and kidney function indicators, antioxidant enzyme activity, and digestive enzyme activity. ELISA kits were used to detect the levels of serum immunoglobulins (IgG, IgA, IgM) and inflammatory factors (IL-6, IL-1β, TNF-α). Fecal samples were used to analyze the intestinal flora structure. The abundance of major bacterial groups (Lactobacillus, Bifidobacterium, Escherichia coli, and Salmonella) was determined using real-time quantitative PCR. All experimental data were statistically analyzed using SPSS 26.0 software, and are expressed as mean ± standard deviation (mean ± SD). One-way ANOVA was used to process the data, and Duncan's multiple comparison test was used to examine the significance of differences between groups. P < 0.05 indicated a significant difference, and P < 0.01 indicated an extremely significant difference.

[0132] 1. Changes in growth performance of piglets after weaning The results are shown in Table 1. Compared with the control group, the experimental group showed a highly significant increase in average daily weight gain and average daily feed intake (P<0.01), and a highly significant decrease in feed conversion ratio (P<0.01). There was no significant difference between the antibiotic group and the experimental group (P>0.05). This indicates that adding microencapsulated microalgae astaxanthin to the diet has a significant promoting effect on the growth performance of weaned piglets, and its antibiotic-alternative effect is comparable to that of antibiotics.

[0133] Table 1. Changes in growth performance of piglets after weaning Note: Compared with the control group, **: P<0.01.

[0134] 2. Diarrhea in piglets after weaning Diarrhea incidence (%) = (Number of diarrheal heads × Number of days with diarrhea) / (Number of heads × Number of days in the trial) × 100%. Diarrhea index = Sum of fecal scores / (Number of heads × Number of days in the trial).

[0135] The results are shown in Table 2. Compared with the control group, the diarrhea rate of piglets in the experimental group was significantly reduced (P<0.01), and the diarrhea index was significantly reduced (P<0.05). The antibiotic group showed comparable results to the experimental group. This indicates that adding microencapsulated microalgae astaxanthin as an antibiotic alternative to the diet can significantly reduce the incidence of diarrhea in weaned piglets and improve intestinal health.

[0136] Table 2. Diarrhea in piglets after weaning Note: Compared with the control group, *: P<0.05, **: P<0.01. Diarrhea index = Sum of fecal scores / (Number of subjects × Number of days).

[0137] 3. Serum immunoglobulin levels in piglets after weaning Table 3 shows that, compared with the control group, the levels of IgG, IgA, and IgM in the serum of piglets in the experimental group were significantly increased (P<0.01). Specifically, IgG levels increased by 95.65%, IgA by 98.18%, and IgM by 76.32%. The immunoglobulin levels in the antibiotic group were also significantly increased, but slightly lower than those in the experimental group. These results indicate that microencapsulated microalgae astaxanthin as an antibiotic alternative can significantly enhance the immune function of piglets after weaning.

[0138] Table 3 Serum immunoglobulin levels in piglets after weaning Note: Compared with the control group, **: P<0.01.

[0139] 4. Antioxidant indicators in piglets after weaning Table 4 shows that, compared with the control group, the activity of superoxide dismutase (SOD) in the serum of piglets in the experimental group was significantly increased (P<0.01), with an increase of 59.05%; the activity of glutathione peroxidase (GSH~Px) was significantly increased (P<0.05); and the content of malondialdehyde (MDA) was significantly decreased (P<0.01), with a decrease of 45.58%. These results indicate that microencapsulated microalgae astaxanthin as an antioxidant additive can significantly improve the antioxidant capacity of piglets after weaning stress and reduce oxidative stress damage.

[0140] Table 4 Antioxidant Indicators of Piglets After Weaning Note: Compared with the control group, *: P<0.05, **: P<0.01.

[0141] 5. Results of inflammatory factor detection in piglets after weaning Table 5 shows that, compared with the control group, the levels of interleukin-6 (IL-6), interleukin-1β (IL-1β), and tumor necrosis factor-α (TNF-α) in the serum of piglets in the experimental group were all significantly reduced (P<0.01). The results indicate that adding microencapsulated microalgae astaxanthin supplements to the diet can significantly alleviate the inflammatory response caused by weaning stress in piglets and reduce the levels of inflammatory factors.

[0142] Table 5 Results of inflammatory factor detection in piglets after weaning Note: Compared with the control group, **: P<0.01.

[0143] 6. Intestinal flora structure of piglets after weaning Table 6 shows that, compared with the control group, the experimental group exhibited a highly significant increase in the number of beneficial bacteria (Lactobacillus, Bifidobacterium) and a highly significant decrease in the number of harmful bacteria (Escherichia coli, Salmonella) in the piglets' intestines (P<0.01). Similar effects were observed in the antibiotic group. These results indicate that the microencapsulated microalgae astaxanthin antibiotic alternative can effectively regulate the intestinal flora structure of piglets, promote the colonization of beneficial bacteria, inhibit the growth of harmful bacteria, and improve the intestinal microecological balance.

[0144] Table 6. Intestinal flora structure of piglets after weaning Note: Compared with the control group, **: P<0.01.

[0145] in conclusion The results of this test case indicate that: Microencapsulated microalgae astaxanthin antibiotics significantly improved the growth performance of weaned piglets, increasing average daily weight gain by 32.7% and reducing feed conversion ratio by 8.9%. The diarrhea rate was reduced by 70.1%, and microencapsulated microalgae astaxanthin significantly improved the intestinal health of piglets; Serum immunoglobulin levels were significantly increased (IgG increased by 95.65%, IgA increased by 98.18%), and microencapsulated microalgae astaxanthin enhanced immune function; Significantly enhanced antioxidant capacity (SOD activity increased by 59.05%, MDA content decreased by 45.58%), microencapsulated microalgal astaxanthin alleviates oxidative stress; The levels of inflammatory factors were significantly reduced (IL-6, IL-1β, and TNF-α decreased by more than 50%), and microencapsulated microalgal astaxanthin alleviated the inflammatory response; The gut microbiota structure was significantly improved, with an increase in beneficial bacteria and a decrease in harmful bacteria; The efficacy of the alternative to antibiotics is comparable to that of the antibiotic group, and it has the advantage of reducing the risk of drug resistance and drug residues.

[0146] In conclusion, microencapsulated microalgae astaxanthin antibiotic alternatives can alleviate weaning stress in piglets through antioxidant, immunomodulatory, and gut microbiota improvement mechanisms, and can be considered as a technical option for antibiotic alternatives.

[0147] The microencapsulated microalgae astaxanthin antibiotic alternative and its preparation method provided by this invention successfully solve the problems of stability and bioavailability of microalgae astaxanthin and probiotics in application through scientific formulation design, innovative double-layer microencapsulation process, and advanced active ingredient processing technology. The product can effectively alleviate weaning stress in piglets, improve growth performance and health through synergistic effects via multiple pathways, demonstrating a clear antibiotic alternative effect. Furthermore, this invention does not introduce antibiotic-based growth promoters, making it suitable for the development of feed additives for weaned piglets and possessing significant application value.

[0148] The above description is only a preferred embodiment of the present invention. It should be noted that those skilled in the art can make several improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A microencapsulated microalgae astaxanthin antibiotic alternative, characterized in that, It includes the following components by weight: 30-45 parts of microencapsulated complex, 0.3-0.5 parts of antioxidant, 0.8-1.2 parts of immune enhancer, 3-3.5 parts of prebiotic, and 55-65 parts of carrier.

2. The microencapsulated microalgae astaxanthin alternative to antibiotics according to claim 1, characterized in that, The microencapsulated complex was prepared by the following method: S1. Mix sodium alginate solution, microalgae astaxanthin extract and probiotic concentrate evenly to obtain a mixture; S2. The mixture is dripped into a calcium chloride solution for cross-linking and curing to obtain primary microcapsules; S3. The primary microcapsules are immersed in a chitosan solution for coating, and then filtered, washed and dried to obtain the microencapsulated complex.

3. The microencapsulated microalgae astaxanthin alternative to antibiotics according to claim 2, characterized in that, The mass ratio of sodium alginate solution, microalgae astaxanthin extract, and probiotic concentrate in S1 is 100:10:

12.

4. The microencapsulated microalgae astaxanthin alternative to antibiotics according to claim 3, characterized in that, The sodium alginate solution has a mass concentration of 2.5-3.5%.

5. The microencapsulated microalgae astaxanthin alternative to antibiotics according to claim 3, characterized in that, The probiotic concentrate in S1 is composed of *Lactobacillus plantarum*, *Bacillus subtilis*, and *Clostridium butyricum* in a live bacteria ratio of 1:1:0.8; the live bacteria count of the probiotic concentrate is 1.5 × 10⁻⁶. 10 CFU / mL.

6. The microencapsulated microalgae astaxanthin alternative to antibiotics according to claim 3, characterized in that, In step S2, a multi-hole nozzle or vibrating dripping device with an aperture of 0.3~0.5 mm is used to drip the mixture into a calcium chloride solution with a mass concentration of 2.0~2.5% at a rate of 30~40 drops / min through a single hole, and the nozzle is 8~12 cm away from the liquid surface.

7. The microencapsulated microalgae astaxanthin alternative to antibiotics according to claim 3, characterized in that, The chitosan solution in S3 has a mass concentration of 0.8-1.2% and a pH of 5.0-5.

5.

8. The microencapsulated microalgae astaxanthin alternative to antibiotics according to claim 3, characterized in that, The coating time in S3 is 12 minutes.

9. The microencapsulated microalgae astaxanthin alternative to antibiotics according to claim 1, characterized in that, The antioxidant is selected from one or more of vitamin E, tea polyphenols, and rosemary extract; The immune enhancer is selected from one or more of β-glucan, astragalus polysaccharide, and yeast cell wall; The prebiotics are selected from one or more of fructooligosaccharides, xylooligosaccharides, and mannan oligosaccharides; The carrier is selected from one or more of wheat bran powder, rice bran powder, and corn powder.

10. The method for preparing the microencapsulated microalgae astaxanthin antibiotic alternative according to any one of claims 1 to 9, characterized in that, Includes the following steps: Weigh the raw materials according to the stated mass fractions; After uniformly mixing the microencapsulated complex with antioxidants, immune enhancers, prebiotics and carriers, the mixture is dried to obtain the microencapsulated microalgae astaxanthin alternative to antibiotics.

Images