Fermented algal bacterial preparation for fast-growing f1 generation population cultivation of nereis solea

Abstract

This invention discloses a fermented algae preparation for the rapid F1 generation population cultivation of *Ulva monocyclic aromatica*, relating to the field of aquatic seedling cultivation engineering technology. The preparation includes algae-derived solids overlapping to form intergranular gaps, with the fermented bacteria and algae-derived soluble components distributed within these gaps. By setting the feed particles in a layered structure of a core and an outer layer, the outer layer softens in seawater before the core layer, while the core layer maintains its granular structure in seawater. This coordinates the substrate contact state after particle placement and the structural retention state before feeding. Furthermore, the overlapping of algae-derived solids in the core layer to form intergranular gaps, and the distribution of fermented bacteria and algae-derived soluble components within these gaps, helps reduce the premature loss of effective components before feeding.

Description

Technical Field

[0001] This invention relates to the field of aquatic seedling cultivation engineering technology, and in particular to a fermented algae preparation for the rapid F1 generation population cultivation of *Ulva monocyclic aromatica*. Background Technology

[0002] Ulva unicinctus larvae are benthic feeders, and their cultivation is usually carried out in seawater with sediment. The feed needs to sink to the surface of the sediment and adapt to the larvae's feeding behavior. Existing algal feeds, fermented feeds, and probiotic preparations have been used to improve the nutrient supply of larvae and the microecology of the water. These preparations are mostly applied in powder, paste, or ordinary granule form to provide algal nutrients, fermented bacteria, and their metabolites to the cultivation system. As the cultivation of fast-growing F1 generation Ulva unicinctus populations requires more continuous feeding and uniform growth, sediment-type and granular feed preparations suitable for seawater with sediment still need further optimization.

[0003] However, existing algae-based feeds, fermented feeds, or probiotic preparations are mostly in powder, paste, or ordinary homogeneous granules. The distribution of their components inside and outside the granules is relatively small. After entering seawater with bottom sediment, it is difficult to form a layered state where the outer surface absorbs water and softens first, while the core still maintains the granular structure. For the benthic feeding process of *Ulva unicornu* fry, if the granules soften too quickly, it will easily affect their stay on the bottom surface and the retention of components before feeding. If the overall structure of the granules is too stable, it will not be conducive to dispersion in the digestive tract environment after feeding. Therefore, existing ordinary feed granules are difficult to coordinate the relationship between the bottom sediment contact state in seawater and the core dispersion state after feeding. Summary of the Invention

[0004] This invention provides a fermented algae preparation for the cultivation of a fast-growing F1 generation population of Ulva monocyclic urticaria, comprising layered feed particles; The layered feed pellets are used to be placed into seawater seedling cultivation water with bottom sediment; The layered bait particles include a core layer and an outer layer covering the outside of the core layer; The core layer contains algal solids, fermented cells, extracellular polysaccharides, and algal soluble components. The algal source solids overlap to form interparticle gaps, and the fermented cells and the algal source soluble components are distributed within the interparticle gaps. The outer layer contains finely broken algal solids, extracellular polysaccharides, and algal soluble components; The D50 particle size of the fine algal solids is 0.20-0.40 times that of the algal solids in the core layer; The content of extracellular polysaccharide dry matter in the outer layer is 1.60-2.20 times that in the core layer; The dry basis content of algal soluble components in the outer layer is 1.40-1.80 times that of the dry basis content of algal soluble components in the core layer; The outer layer absorbs water and softens in seawater before the core layer, while the core layer maintains its granular structure in seawater and disperses in the digestive tract environment of the *Ulva unicornu* seedlings.

[0005] As a preferred embodiment of the fermented algae preparation for the cultivation of the rapid-growing F1 generation population of *Ulva monocyclic urticaria* according to the present invention, wherein: the algal source solids contain algal cell wall fragments; The algal solids in the core layer contain flaky fragments, and the gaps between the particles are located between adjacent flaky fragments.

[0006] As a preferred embodiment of the fermented algae preparation for the rapid F1 generation population cultivation of *Ulva monocyclicis* as described in this invention, the algae-derived solids are formed by enzymatic hydrolysis and fermentation of *Chlorella vulgaris* powder and kelp enzymatic hydrolysis residue, *Chlorella vulgaris* powder and wakame seaweed enzymatic hydrolysis residue, or *Spirulina* powder and kelp enzymatic hydrolysis residue.

[0007] As a preferred embodiment of the fermented algae preparation for the cultivation of the rapid F1 generation population of *Ulva monocyclic urticaria* according to the present invention, wherein: the fine algal solids in the outer layer are attached to the outer side of the core layer, and the extracellular polysaccharide is distributed between adjacent fine algal solids.

[0008] As a preferred embodiment of the fermented algae preparation for the rapid F1 generation population cultivation of *Ulva monocyclic urticaria* as described in this invention, wherein the algae-derived soluble component contains algae-derived soluble protein.

[0009] As a preferred embodiment of the fermented algae preparation for the rapid F1 generation population cultivation of *Ulva monocyclic urticaria* according to the present invention, wherein: the fermented bacteria are attached to the surface of the algal source solids in the core layer and distributed in the interparticle spaces.

[0010] As a preferred embodiment of the fermented algae preparation for the rapid F1 generation population cultivation of *Ulva monocyclic aromatica* according to the present invention, wherein the apparent density of the stratified feed particles is greater than the density of the seawater used for seedling cultivation.

[0011] As a preferred embodiment of the fermented algae preparation for the cultivation of the rapid F1 generation population of *Ulva monocyclic urticaria* according to the present invention, wherein: the outer layer absorbs water to form a moist outer surface that contacts the substrate particles.

[0012] As a preferred embodiment of the fermented algae preparation for the rapid F1 generation population cultivation of *Ulva monocyclicis* according to the present invention, the core layer, after being dispersed in the digestive tract environment of *Ulva monocyclicis* seedlings, forms a dispersion containing the fermented bacteria, fragments of the algal solids, and algal soluble components.

[0013] As a preferred embodiment of the fermented algae preparation for the cultivation of the rapid-growing F1 generation population of *Ulva monocyclic urticaria* according to the present invention, wherein the fermented bacterial cell is *Lactobacillus plantarum* SC256.

[0014] The beneficial effects of this invention are as follows: By setting the feed pellets into a layered structure of a core layer and an outer layer, the outer layer absorbs water and softens in seawater before the core layer, while the core layer maintains its pellet structure in seawater. This coordinates the contact state with the substrate after pellet placement and the structural retention state before feeding. The combination of fine algal solids, extracellular polysaccharides, and algal soluble components in the outer layer facilitates the formation of a moist outer surface for the pellets after water absorption, improving the problem of unstable retention of ordinary homogeneous pellets on the substrate surface. The algal solids in the core layer overlap to form intergranular gaps, and the fermented bacteria and algal soluble components are distributed within these gaps, which helps reduce the premature loss of effective components before feeding. After the pellets are ingested by *Ulva monocyclic* larvae, the core layer can disperse in the digestive tract environment, forming a dispersion containing fermented bacteria, algal solid fragments, and algal soluble components. This ensures that the stability of substrate feeding and the dispersion and release process after feeding are coordinated. Attached Figure Description

[0015] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the following description of the embodiments will be briefly introduced. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0016] Figure 1 This is a schematic diagram showing the dispersion of the stratified bait particles in the digestive tract environment after ingestion, as shown in Example 1.

[0017] Figure 2 This is a schematic diagram of the layered bait particle structure in Example 1. Detailed Implementation

[0018] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

[0019] In this invention, the rapid-growing F1 generation population cultivation refers to a cultivation method that evaluates the early growth and population uniformity of seedlings under seawater seedling cultivation conditions with bottom sediment by using the 14-day body length growth rate, 14-day weight gain rate, 14-day survival rate, and final body length variation coefficient.

[0020] In this invention, the overlapping of algal source solids refers to the contact state between adjacent algal source solids in the core layer cross-sectional image, where there is edge contact, local overlap or cross support, and the adjacent algal source solids do not form a continuous dense block structure. In a 200x optical microscope image or a 1000x scanning electron microscope image, when the projected edges of two or more algal solids are connected or partially overlapped, and a measurable gap is retained in their adjacent areas, it is counted as a single overlapping structure. The interparticle gaps are formed by the void regions between overlapping algal solids. When the interparticle gaps are measured in both frozen and undried wet samples, the interparticle gap area is included.

[0021] Example 1, referring to Figure 1 and Figure 2 In this embodiment, the layered bait pellets refer to granular fermented algae and bacteria preparations with a core layer and an outer layer; Algal solids refer to algal cell fragments, algal cell wall remnants, and algal residues that remain in solid form after enzymatic hydrolysis and fermentation of algal raw materials. Algal soluble components refer to soluble algal proteins, short algal peptides, free amino acids, and algal oligosaccharides that enter the soluble phase after enzymatic hydrolysis and fermentation of algal raw materials; The soluble components of algae in the outer layer are conducive to water absorption and softening, while in the core layer they are conducive to the delivery of particles and the release of usable nutrients after ingestion. Extracellular polysaccharides refer to polysaccharide substances released outside the cells during lactic acid bacteria fermentation; In the core layer, extracellular polysaccharides mainly serve to connect algal solids and fermentation cells. In the outer layer, they mainly work with fine algal solids and algal soluble components to form an outer layer structure that has the ability to wet after absorbing water. D50 particle size was determined using a laser particle size analyzer and represents the particle size corresponding to 50% of the cumulative volume distribution in the sample. The artificial seawater was prepared using commercially available artificial seawater salt, with a salinity adjusted to 30‰, a pH adjusted to 8.0, and a test temperature set at 23℃. The initial softening time of the outer layer was determined with the aid of a texture analyzer. It was used to evaluate the time point when the outer layer of the stratified bait pellets absorbed water and softened after entering the water. It was used together with the core layer integrity rate to determine whether the pellets formed a state where the outer layer softened first while the core layer still maintained the pellet structure. The layered bait particles were immersed in the artificial seawater, and samples were taken every 30 seconds to measure the compressive hardness of the outer layer of the particles. When the outer layer compressive hardness drops to 70% of the initial outer layer compressive hardness and a continuous wet layer forms on the outer surface of the particles, it is recorded as the outer layer initial softening time. A continuous wetting layer refers to a circumferential length of the outer surface of a particle covered by a continuous hydration layer in a 20x stereomicroscope image, which accounts for no less than 80% of the particle's outer contour circumference. The texture analyzer uses a flat-bottomed probe, a testing speed of 1.0 mm / s, and a compression displacement of 20% of the average particle size. Core integrity is used to evaluate how well the core layer retains particles in seawater. After soaking the layered bait particles in the artificial seawater for 10 minutes, the particles that still retain the core particle morphology are recovered using a 0.5 mm sieve. Use 30 mL of artificial seawater to rinse the particles on the screen along the edge of the screen to remove the softened outer layer; Collect the particles that still have the core layer particle shape on the sieve, drain the water, and weigh them; Core integrity rate is the proportion of the wet mass of the recovered core layer particles to the wet mass of the core layer in the added particles. Chlorella powder and kelp enzymatic hydrolysis residue were selected as algal source materials, with a dry basis mass ratio of 65:35 between chlorella powder and kelp enzymatic hydrolysis residue. Artificial seawater with a salinity of 30‰ was added to the algal source material to adjust the dry basis mass concentration of the solution to 13.0%, and the pH was adjusted to 6.2 with sodium bicarbonate. Then add neutral protease and cellulase. The amount of neutral protease added is 0.30% of the dry weight of the algal raw material, and the amount of cellulase added is 0.25% of the dry weight of the algal raw material. Incubate at 52℃ for 2 hours for enzymatic hydrolysis. After enzymatic hydrolysis, the enzyme was inactivated by incubation at 90℃ for 10 minutes and then cooled to 32℃ to obtain algal enzymatic hydrolysate.

[0022] The extracellular polysaccharide-producing lactic acid bacteria used is Lactobacillus plantarum SC256, which is classified and named Lactiplantibacillus plantarum SC256. This strain has been deposited at the China Center for Type Culture Collection, located at Wuhan University, China, on October 26, 2023, with accession number CCTCC NO: M20232050. Lactobacillus plantarum SC256 was inoculated into MRS liquid medium and activated at 30°C for 18 h to obtain an activated bacterial solution with a viable count of 1.6 × 10⁻⁶. 8 CFU / mL; In this embodiment, the extracellular polysaccharide-producing lactic acid bacteria refers to lactic acid bacteria that can produce extracellular polysaccharides during the fermentation of algal enzymatic hydrolysate. The extracellular polysaccharides participate in the connection between the algal solids in the core layer and the outer layer, and participate in the formation of a moist contact structure after the outer layer absorbs water. Inoculate the activated bacterial solution at 4.0% of the wet weight of the algae-derived enzymatic hydrolysate, and let it ferment at 30℃ for 28 hours. The final pH of the fermentation was 4.36. After centrifuging the fermentation broth to remove the cells and solids, the supernatant was collected. Three volumes of anhydrous ethanol were added to the supernatant, and the mixture was allowed to stand at 4°C for 12 hours. The precipitate was collected by centrifugation, and after redissolving the precipitate, the protein was removed by trichloroacetic acid. The extracellular polysaccharide content was then determined by the phenol-sulfuric acid method. The concentration of extracellular polysaccharides in the fermentation broth was found to be 0.82 g / L. After fermentation, the wet algae and bacteria material is screened through a 60-mesh sieve, and the wet material on the sieve is collected as the core material. The core material contains algal solids, fermented cells, extracellular polysaccharides, and algal soluble components that have been screened and retained. The D50 particle size of algal solids in the core material was determined to be 131.8 μm by laser particle size analyzer. The core material was adjusted to a moisture content of 45.2% and extruded through an extrusion sieve with a 1.0 mm aperture. The extrusion screw speed was 24 r / min. The extruded strips are rolled in a disc for 3 minutes at a speed of 150 r / min to obtain wet core particles.

[0023] The undersize portion of the fermented algae wet material was then processed twice by a colloid mill and passed through a 160-mesh sieve to obtain an outer wet material containing finely broken algal solids, extracellular polysaccharides, and algal soluble components. Finely crushed algal source solids refer to algal source solid components with a particle size smaller than that of the core layer algal source solids after being crushed, ground, or sieved; When fine algal solids are located in the outer layer, their surface area is large, which can form an outer layer structure that is easy to absorb water with extracellular polysaccharides and algal soluble components, so that the outer layer absorbs water and softens in seawater before the core layer. The solid content of the outer wet material is adjusted to 18.0%, and it is sprayed on the outside of the core wet particles in two stages. After each spraying, it is dried in a forced-air dryer at 32°C for 15 minutes before the next spraying is carried out. The outer dry weight accounts for 23.5% of the total dry weight of the stratified feed pellets; After spraying, the pellets are dried at 35°C until the moisture content of the pellets is 18.6%, resulting in layered bait pellets. The dried, layered bait particles were frozen and sectioned, and the core layer cross-section was observed using optical microscopy. The observation results showed that the algal solids in the core layer were in the form of flakes or irregular fragments, and intergranular gaps were formed between adjacent algal solids. After Gram staining of the core section, it was observed that the fermentation cells were attached to the surface of the algal solids and distributed in the interparticle spaces. This result confirms that the algal solids, fermentation cells, extracellular polysaccharides, and algal soluble components in the core layer do not exist in a dense block form, but rather form a core layer structure with intergranular gaps. According to the test, the core layer algal solids D50 particle size of the layered bait particles obtained in Example 1 was 131.8 μm, and the outer layer fine algal solids D50 particle size was 33.6 μm. The dry weight content of extracellular polysaccharides in the core layer was 4.6%, and the dry weight content of extracellular polysaccharides in the outer layer was 8.8%. The dry basis content of soluble components from algae in the core layer was 13.2%, and the dry basis content of soluble components from algae in the outer layer was 21.7%. In the layered feed pellets obtained in Example 1, the D50 particle size of the fine algal solids in the outer layer is smaller than that of the algal solids in the core layer, the dry basis content of the extracellular polysaccharides in the outer layer is higher than that in the core layer, and the dry basis content of the soluble algal components in the outer layer is higher than that in the core layer. To investigate the effects of layered structure, outer layer particle size, outer layer extracellular polysaccharide enrichment, outer layer algal-derived soluble component enrichment, and extracellular polysaccharide-producing lactic acid bacteria on particle properties, comparative examples 1 to 5 were set up: Comparative Example 1 uses the same fermented algae wet material to produce non-stratified homogeneous granules; Comparative Example 2 kept the core layer, outer layer, and component distribution unchanged, only ensuring that the D50 particle size of the algal source solids in the outer layer was not less than the D50 particle size of the algal source solids in the core layer; Comparative Example 3 maintains the outer layer particle size and the dry basis content of the outer layer algal soluble components close to those of Example 1, and ensures that the dry basis content of the outer layer extracellular polysaccharides is not higher than that of the core layer. Comparative Example 4 maintains the outer layer particle size and outer layer extracellular polysaccharide dry basis content close to those of Example 1, and ensures that the dry basis content of the algal-derived soluble components in the outer layer is not higher than that in the core layer; Comparative Example 5 used lactic acid bacteria with low extracellular polysaccharide production capacity to replace extracellular polysaccharide-producing lactic acid bacteria, and the other preparation conditions were the same as in Example 1. In Comparative Example 2, the outer layer wet material was not treated with a colloid mill, and the wet material obtained after sieving with a 60-mesh sieve was used to adjust the outer layer wet material so that the D50 particle size of the outer layer algal source solids was not less than the D50 particle size of the core layer algal source solids. The proportion of the outer dry basis mass to the total dry basis mass of the particles, the number of spraying times, the drying temperature, and the final moisture content were all the same as in Example 1; In Comparative Example 3, after centrifugation, some of the supernatant containing extracellular polysaccharides was removed from the outer wet material, and then artificial seawater with a salinity of 30‰ was added to make up to the same solid content as in Example 1, so that the dry basis content of extracellular polysaccharides in the outer layer was not higher than that in the core layer. The outer algal source solids D50 particle size, outer dry basis mass ratio, number of spraying times and drying conditions were the same as in Example 1; In Comparative Example 4, the outer wet material was washed with artificial seawater and then centrifuged to remove some of the algal soluble components. The solid content was then adjusted to the same level as in Example 1, so that the dry basis content of the algal soluble components in the outer layer was not higher than that in the core layer. The outer algal solids D50 particle size, outer extracellular polysaccharide dry basis content, outer dry basis mass ratio, number of spraying times and drying conditions were the same as in Example 1; Comparative Example 5 used Lactobacillus plantarum LP23 instead of Lactobacillus plantarum SC256; Lactobacillus plantarum LP23 has been deposited at the China Center for Type Culture Collection, with accession number CCTCC NO: M20231552; Lactobacillus plantarum LP23 was inoculated into MRS liquid medium and activated at 30℃ for 18 h to obtain an activated bacterial solution. The viable count of the activated bacterial solution was adjusted to 1.6 × 10⁻⁶. 8 CFU / mL; The algal enzymatic hydrolysate was inoculated with the same amount as in Example 1 and fermented at 30°C for 28 hours. The concentration of extracellular polysaccharides in the fermentation broth was measured to be 0.31 g / L. Except for replacing the lactic acid bacteria strain, the algal source material, enzymatic hydrolysis conditions, fermentation temperature, fermentation time, sieving method, core layer granulation method, outer layer spraying method and drying conditions of Comparative Example 5 are the same as those of Example 1. The seabed sediment retention rate was determined using an artificial seawater system with seabed sediment. The substrate was a mixture of fine sand and mud in a dry weight ratio of 7:3. After being sterilized at high temperature, it was laid at the bottom of the test container with a thickness of 2.0 cm. The artificial seawater was 10 cm deep. Substrate surface layer refers to the surface of the substrate and the area within 2 mm above it; The stratified bait pellets were placed into artificial seawater. After the pellets settled to the bottom surface, air was introduced into the bottom of the test container through an air stone at a rate of 0.20 L / min, and the disturbance was continued for 20 min. After the disturbance ends, the particles that are still located in the substrate surface area are recovered, and the substrate surface retention rate is calculated. The digestive tract environment was prepared using the supernatant of a homogenate of the digestive tract of *Ulva monocranium* seedlings; Take the digestive contents of the same batch of *Ulva unicornu* seedlings, add artificial seawater with a salinity of 30‰ at a mass-to-volume ratio of 1g:4mL, homogenize in an ice bath, centrifuge at 8000r / min for 10min at 4℃, and collect the supernatant as digestion solution. The stratified feed particles were placed in a digestive solution and shaken at 80 r / min for 60 min at 23℃. The core layer dispersion rate and the release rate of algal soluble components were then measured. The core layer dispersion rate is calculated as the proportion of the dry mass of the core layer that does not maintain the core layer particle morphology after treatment to the dry mass of the core layer in the added particles. The release rate of algal soluble components is calculated as the proportion of the total amount of algal soluble proteins, short peptides, free amino acids and oligosaccharides released into the liquid phase to the total amount of the corresponding components in the particles; Among them, algal soluble proteins were determined by the Coomassie brilliant blue method, short peptides were determined by the trichloroacetic acid soluble nitrogen conversion method, free amino acids were determined by the ninhydrin method, and oligosaccharides were determined by the phenol-sulfuric acid method. The test results are shown in Table 1; Table 1: Layer structure of stratified feed pellets, softening of the outer layer after water introduction, retention of the core layer, retention of bottom sediment, digestion and release, and early cultivation results. Main differences Layering, refinement of the outer layer, enrichment of extracellular polysaccharides and soluble components in the outer layer, using extracellular polysaccharide-producing lactic acid bacteria. Non-stratified homogeneous particles The outer layer D50 is not less than the core layer D50 Extracellular polysaccharides were not enriched in the outer layer. The soluble components of the outer algal source were not enriched. Lactic acid bacteria with low extracellular polysaccharide production capacity Core layer D50 / μm 131.8 — 130.5 132.1 131.4 130.9 Outer layer D50 / μm 33.6 — 139.4 34.9 34.1 35.1 Core layer extracellular polysaccharides / % 4.6 — 4.7 4.8 4.7 3.2 extracellular polysaccharides / % 8.8 — 8.6 4.7 8.7 5.5 Core layer algal soluble components / % 13.2 15 13 13.4 13.3 12.6 Soluble components of outer algae / % 21.7 15 21.4 21.2 13.1 20.5 Homogeneous granules, total soluble components of algae / % — 15.0 — — — — Homogeneous particles total extracellular polysaccharide / % — 5.1 — — — — Outer layer initial softening time / min 2.8 — 7.6 5.1 4.8 5.2 10-minute core integrity rate / % 88.4 — 85.1 87.2 87.6 86.3 Substrate surface retention rate after 20 minutes of disturbance / % 76.8 45.9 56.7 61 64 62.1 Percentage of soluble components entering the water body within 30 minutes / % 17.4 28.3 20.8 22.1 23 21.2 60min core layer dispersion rate / % 72.6 55.4 68.1 69 68.4 64.7 60min soluble component release rate / % 70.8 57.6 66.9 66.2 64.7 63.8 14-day weight gain rate / % 36.4 30.8 32.6 32.5 32.8 33 Final body length coefficient of variation / % 9.7 12.8 11.9 12 11.8 11.6 As shown in Table 1, in Example 1, the outer layer D50 particle size is lower than that of the core layer, the dry basis content of extracellular polysaccharides in the outer layer is higher than that in the core layer, and the dry basis content of algal soluble components in the outer layer is higher than that in the core layer. Comparative Example 1 is a non-layered homogeneous particle that does not distinguish between the core and outer layers. Its overall algal soluble components and overall extracellular polysaccharide content are used to represent the overall composition of the homogeneous particle. The above results are used to illustrate that the particles obtained in Example 1 have a layered structure that is different from that of non-layered homogeneous particles; In Example 1, the initial softening time of the outer layer was 2.8 min, and the core layer integrity rate was 88.4% after 10 min, indicating that when the outer layer first absorbs water and softens in seawater, the core layer still maintains its granular structure. In Comparative Example 2, after the outer layer was not refined, the initial softening time of the outer layer was extended to 7.6 min, and the retention rate of the substrate surface layer decreased to 56.7%. After the enrichment of extracellular polysaccharides and soluble components from algae in the outer layer were removed in Comparative Examples 3 and 4, respectively, the retention rate and digestion release rate of the substrate surface both decreased. In Comparative Example 5, after using lactic acid bacteria with low extracellular polysaccharide production capacity, the extracellular polysaccharide content of the outer layer decreased, the softening time of the outer layer was prolonged, and the substrate surface retention rate and digestion release rate were lower than those in Example 1. Under the conditions of this embodiment, the layered bait particles obtained in Example 1 simultaneously possess a layered structure in which the particle size of the fine algal solids in the outer layer is lower than that in the core layer, the dry basis content of the extracellular polysaccharides in the outer layer is higher than that in the core layer, and the dry basis content of the soluble algal components in the outer layer is higher than that in the core layer. Under the conditions of this embodiment, the above detection results can characterize the state of the stratified bait particles in seawater, where the outer layer first absorbs water and softens, the core layer maintains the particle structure, and the particles are dispersed and released in the digestive tract environment.

[0024] Example 2 Based on the layered feed particles obtained in Example 1, this embodiment further examines the morphology of algal solids in the core layer, particle spacing, attachment status of fine algal solids in the outer layer, content of soluble algal protein, and distribution of fermented cells in the core layer. In this embodiment, algal cell wall fragments refer to algal source solid fragments that retain the cell wall boundary in a 1000x scanning electron microscope image, wherein the length of the cell wall boundary accounts for no less than 50% of the projected outer perimeter of the fragment; Flake-like fragments refer to algal solids with a major axis / minor axis ratio of not less than 2 and a projected area of ​​not less than 50 μm² in the cross-sectional image of the core layer; Intergranular gaps refer to the void regions located between adjacent flaky fragments in a cross-section of the core layer; When this void region is measured in both frozen section samples and undried wet section samples, the interparticle area is included. Algal soluble protein refers to the protein components that can be extracted by 0.05 mol / L, pH 7.0 phosphate buffer after enzymatic hydrolysis and fermentation of algal raw materials, and determined by the Coomassie brilliant blue method; Intergranular bacteria refer to lactic acid bacteria located in the interstitial region between adjacent algal solids in the core section stained image; The layered bait pellets of Example 2 were prepared according to the method of Example 1; The core material is the wet material obtained by screening the fermented algae and bacteria wet material in Example 1 through a 60-mesh sieve. The core material is not subjected to colloid milling treatment. The moisture content is adjusted to 45.5%. It is extruded through an extrusion sieve plate with a pore size of 1.0 mm and the extrusion screw speed is 24 r / min. The extruded strips were rolled in a disc for 3 minutes at a speed of 150 r / min to obtain wet core particles. The outer layer material is taken from the sieve portion of the same batch of fermented algae and bacteria wet material, processed twice by a colloid mill and passed through a 160-mesh sieve; The outer layer material is sprayed onto the outside of the core layer wet particles in two stages, and after each spraying, it is dried at 32°C for 15 minutes by forced air drying. After spraying, the pellets are dried at 35°C until the moisture content of the pellets is 18.6%, resulting in layered bait pellets. The fermentation cells used were Lactobacillus plantarum SC256 from Example 1. After fermentation, the cells were not collected by centrifugation and then added externally, but instead entered the core material along with the wet material of the fermenting algae. Set up comparative examples 6 to 9; Comparative Example 6 used the same algal source material, enzymatic hydrolysis conditions and fermentation conditions as Example 2, but the core material was treated with a colloid mill three times before granulation, and then the moisture content was adjusted to 45.5% before being extruded into granules, in order to reduce the retention ratio of algal cell wall fragments and flaky fragments in the core layer. Comparative Example 7 used the same core layer, outer layer particle size, and spraying method as Example 2; After centrifuging the outer wet material at 8000 r / min for 10 min, the solids were collected. The supernatant was added with 3 times the volume of anhydrous ethanol and allowed to stand at 4℃ for 12 h. The polysaccharide precipitate was obtained by centrifugation to remove the ethanol precipitate. The supernatant after removing polysaccharide precipitate was added back to the outer solids, and the content of the outer dry basis was adjusted to the same as in Example 2 with the same batch of algal soluble components, thereby reducing the dry basis content of the outer extracellular polysaccharide. Comparative Example 8 uses the same core and outer solid structure as Example 2; The outer soluble components were treated with a 10 kDa ultrafiltration membrane, and the permeate was collected and concentrated to the same dry basis weight as in Example 2, so that the dry basis weight content of the outer algal soluble protein was adjusted to 1.4%. After the above treatment, the total dry basis weight of the soluble components in the outer layer was the same as in Example 2, and the dry basis weight of the algal soluble protein in the outer layer was 1.4%. Comparative Example 9 used the same algal source, enzymatic hydrolysis conditions, and granulation conditions as Example 2; After fermentation, the lactic acid bacteria cells were collected by centrifugation at 6000 r / min for 10 min, washed once with sterile artificial seawater and resuspended. The viable count was then calculated according to the MRS plate count results to be the same as that of the core material in Example 2. The core material was then added, mixed for 8 min, and the moisture content was adjusted to 45.5% and extruded into granules. During testing, 30 particles were randomly selected from each treatment. Frozen sections were prepared using a cryostat, with a section thickness of 50 μm. Undried wet section samples were embedded in 2% agarose and then sectioned, with a section thickness of 100 μm; Three fields of view were randomly selected from the core layer section of each particle, and images were acquired using a 200x optical microscope and a 1000x scanning electron microscope. The area ratio and quantity ratio were calculated using image analysis software. The area ratio of algal cell wall fragments is the proportion of the area of ​​fragments that meet the criteria for algal cell wall fragments to the total observed area of ​​the core solids. The proportion of sheet-like fragments is the ratio of the number of algal solids that meet the criteria for sheet-like fragments to the total number of algal solids observed in the core layer. The intergranular area ratio is the ratio of the void area between adjacent flaky fragments to the total area of ​​the core layer cross-section. The overlap rate of algal source solids was calculated based on the cross-sectional image of the core layer; For each treatment, 30 particles were randomly selected, and for each particle, 3 fields of view were randomly selected from the core layer section. In 200x optical microscope images, structures in which the projected edges of two or more algal solids meet or partially overlap, and retain measurable gaps in adjacent areas, are called overlapping structures. The algal solids overlap rate is the proportion of the number of algal solids participating in the overlap structure to the total number of algal solids observed in the core section. The method for determining the adhesion rate of the outer solids is as follows: Weigh 1.00 g of particles on a dry basis and place them in a 250 mL conical flask containing 100 mL of artificial seawater with a salinity of 30‰. Shake the flask at 60 r / min for 5 min on a horizontal shaker. After shaking, the particles were recovered using a 0.15mm sieve. The detached solids in the artificial seawater were collected, dried, and weighed. The adhesion rate of the outer solids layer is calculated using the following formula: Outer solids adhesion rate = (mass of dry outer layer coating - mass of detached dry outer layer coating) / mass of dry outer layer coating × 100%; The extracellular polysaccharides of the outer layer were observed using the alcian blue staining method; Unstained blank controls were set up for the outer layer sections in the same batch; the outer layer sections to be stained were placed in 1% aloxine blue staining solution for 10 min, rinsed 3 times with distilled water, and images were acquired under a 200x optical microscope. Alixin blue staining is used to show regions of enrichment of acidic or negatively charged polysaccharides in the outer layer. Extracellular polysaccharide-linked regions are defined as the continuous blue regions that appear in the stained sample relative to the unstained blank control. The continuous blue region located between adjacent fine algal solids and appearing relative to the unstained blank control is counted as the extracellular polysaccharide linking region. The proportion of the outer extracellular polysaccharide linking region is the ratio of the area of ​​the extracellular polysaccharide linking region to the outer observation area. Algal soluble proteins were extracted using 0.05 mol / L, pH 7.0 phosphate buffer. The dry weight of the sample was 1 g to 20 mL of buffer. The sample was shaken at 25 °C and 120 r / min for 30 min, then centrifuged at 8000 r / min for 10 min. The supernatant was collected and determined using the Coomassie Brilliant Blue method. The method for determining the adhesion rate of fermentation cells is as follows: Weigh 1.00 g of particles with a dry basis, place them in 50 mL of sterile physiological saline, shake at 60 r / min for 2 min, discard the washing liquid, and then determine the number of cells retained on the core solids. The proportion of bacterial cells retained on the core solids to the total number of bacterial cells in the particles is called the fermentation cell adhesion rate. The bacterial count was performed using MRS plate counting. The location of fermentation cells was observed using Gram staining. Cells located on the surface of algal solids and in the interstitial areas of adjacent algal solids in the core section were counted separately. The proportion of bacteria within the intergranular space is the ratio of the number of bacteria located within the intergranular space to the total number of bacteria observed in the core section. Table 2: Results of algal solids morphology, outer layer attachment structure, and fermentation cell distribution in stratified feed particles Main differences The core layer retains algal cell wall fragments and sheet-like fragments; the outer layer contains extracellular polysaccharide junctions; it contains algal-derived soluble proteins; the bacterial cells enter the core layer along with the fermented wet substrate. Over-refinement of core material The content of extracellular polysaccharides in the outer layer is reduced. The content of soluble protein from outer algae is reduced. The bacterial cells were centrifuged and then mixed into the core layer. Percentage of cell wall fragments in the core layer algae / % 41.8 18.6 40.7 39.9 40.5 Percentage of core layer sheet fragments / % 36.2 12.4 35.5 34.8 35.1 Core layer interparticle area ratio / % 22.6 9.8 21.9 21.5 20.7 Core layer algal source solids overlap rate / % 58.6 24.3 56.8 55.9 54.7 Outer solids adhesion rate / % 84.3 80.5 63.4 78.6 81.4 Percentage of extracellular polysaccharide-linked regions in outer layer / % 46.9 44.1 21.8 42.8 43.5 Algal soluble protein dry basis content / % 6.7 6.3 6.5 1.4 6.4 Core layer fermentation cell adhesion rate / % 69.4 52.8 67.1 66.5 31.6 Percentage of bacteria in interparticle spaces / % 38.7 21.6 37.4 36.8 17.9 Percentage of bacteria on the surface of the core solids / % 44.5 36.9 43.1 42.6 23.8 As shown in Table 2, in Example 2, the proportion of core layer algal cell wall fragments was 41.8%, the proportion of core layer plate-like fragments was 36.2%, and the proportion of core layer intergranular space area was 22.6%. Comparative Example 6 only applied excessive refining to the core material, reducing the above three indicators to 18.6%, 12.4%, and 9.8%, respectively. In Example 2, the outer solids adhesion rate was 84.3%, and the proportion of the outer extracellular polysaccharide-linked region was 46.9%. In Comparative Example 7, after only reducing the content of extracellular polysaccharides in the outer layer, the outer solids adhesion rate was 63.4%, and the proportion of the extracellular polysaccharide-connected region was 21.8%. Example 2: The dry basis content of algal soluble protein was 6.7%; Comparative Example 8 adjusted the dry weight content of the soluble protein from the outer algae to 1.4%, while keeping the structure of the solids in the remaining core and outer layers consistent. In Example 2, the cell adhesion rate of the core layer fermentation was 69.4%, the proportion of cells in the interparticle spaces was 38.7%, and the proportion of cells on the surface of the core layer solids was 44.5%. Comparative Example 9 used a method of centrifuging the bacterial cells before mixing them into the core layer. The bacterial cell adhesion rate in the core layer, the proportion of bacterial cells in the interparticle gaps, and the proportion of bacterial cells on the surface of the solid material in the core layer were 31.6%, 17.9%, and 23.8%, respectively. The above-mentioned fermentation cell adhesion rate, cell proportion in interparticle gaps, and cell proportion on the surface of core solids are used together to indicate the retention position of fermentation cells in the core layer; In Example 2, the overlap rate of algal-derived solids in the core layer was 58.6%, and the interparticle area ratio of the core layer was 22.6%. In Comparative Example 6, after excessive refinement of the core layer material, the overlap rate of algal-derived solids in the core layer decreased to 24.3%, and the interparticle area ratio of the core layer decreased to 9.8%. The above results are used to represent the overlap state of algal solids in the core layer and the corresponding interparticle gap formation. In Example 2, the algal cell wall fragments, sheet-like fragments, outer extracellular polysaccharide connection regions, algal soluble proteins, and the distribution of bacteria in the core layer correspond to the core skeleton, outer attachment, nutrient release, and bacterial retention structures of the stratified feed particles, respectively.

[0025] Example 3 In this embodiment, the layered bait particles prepared in Example 1 are used as the test object to test the apparent density, sedimentation behavior, and surface state of the layered bait particles after absorbing water and contacting the bottom sediment particles. Apparent density refers to the ratio of the mass of a stratified bait pellet to its outer contour volume in the state before it is placed in the feed. The outer contour volume of the particles was determined using the liquid paraffin displacement method. Liquid paraffin is insoluble in water and does not cause the feed pellets to absorb water and swell, and is used to determine the outer contour volume of the pellets before they are released. The density of the seawater used for seedling cultivation was determined using artificial seawater with a salinity of 30‰, a pH of 8.0, and a temperature of 23℃. The artificial seawater was prepared from commercially available artificial seawater salt, and its density was measured using a hydrometer. The increase in water absorption on the particle surface refers to the ratio of the increase in mass of the particles after immersion in artificial seawater to the dry mass of the particles. The surface contact performance after water absorption was evaluated by the increase in water absorption on the particle surface, the contact angle of the surface material during tableting, the contact amount of the substrate particles, and the amount of substrate particles retained on the surface after vibration. The contact angle of the tablet is used to compare the differences in hydrophilicity of the surface material of each treated particle after water absorption. The formation of the wetted outer surface is evaluated in combination with the increase in water absorption on the particle surface, the amount of contact with the substrate particles, and the amount of substrate particles retained on the surface after vibration. The layered bait pellets used in Example 3 were prepared according to the method in Example 1, with the core layer pelleting, outer layer coating, and drying conditions being the same as in Example 1; The moisture content of the particles was determined by drying at 105℃ to constant mass method. The average particle size was determined using an image method. 100 particles were randomly selected from each treatment, and the maximum projected diameter was measured and the average value was taken. The average particle size of the layered bait pellets obtained in Example 3 was 0.96 mm, and the particle moisture content was 18.6%. Set up comparative examples 10 to 13; Comparative Example 10 was used to prepare low apparent density particles. Its algal source material, fermentation cells, core material, outer material, outer dry basis ratio, drying temperature and final moisture content were the same as those in Example 3. The extrusion screw speed was 12 r / min and the rounding time was 1 min during granulation. Comparative Example 11 used the same core layer composition, outer layer composition, outer layer dry basis ratio, spraying method, and drying temperature as Example 3. After spraying, it was further dried at 35°C until the particle moisture content was 6.8%. Comparative Example 12 used the same core layer composition, outer layer composition, outer layer dry basis ratio, and spraying method as Example 3. After spraying, it was dried at 60°C, and the final moisture content was controlled to be 18.4%. Comparative Example 13 used the same fermented algae wet material, the same endpoint moisture content, and the same average particle size control conditions as Example 3, but without setting a core layer and an outer layer. The material was mixed and then directly extruded and spherical to obtain non-layered particles. For the apparent density determination, 2.00 g of the particles before application were weighed for each treatment, placed in a 25 mL specific gravity bottle, and liquid paraffin was added to the mark and air bubbles were removed. The volume of particle replacement is calculated using the following formula: Volume of particle replacement = Difference between the mass of liquid paraffin in the blank specific gravity bottle and the mass of liquid paraffin in the specific gravity bottle after adding particles / Density of liquid paraffin at 23℃; Before the test, the same batch of particles were placed in liquid paraffin for 60 seconds. No measurable change in particle mass was observed, confirming that the liquid paraffin did not enter the particle interior and form a mass increment within the test time. Take another specific gravity bottle and add only liquid paraffin to the mark to serve as a blank; Each treatment was measured three times, and the average value was taken. Apparent density is calculated as the ratio of the mass of the particles before delivery to the volume of particles that were replaced. The density of artificial seawater was measured to be 1.021 g / cm³ at 23°C. During the settling performance test, the particles were placed in a transparent cylindrical container filled with artificial seawater at a depth of 20cm, and the bottom of the container was covered with a 2.0cm thick layer of fine sand and mud mixture. The fine sand is sea sand that passes through a 20-mesh sieve and remains on a 100-mesh sieve, and the mud is sea mud that passes through a 100-mesh sieve. The dry weight ratio of the fine sand to the mud is 7:3. The bottom sediment moisture content was adjusted to 18.0% using artificial seawater, and the bottom sediment moisture content was calculated based on the difference between wet and dry weight. The dry weight of the particles was 1.00 g for each treatment, and the time required for the particles to enter the bottom sediment surface area was recorded. For treatments where the sediment does not fully enter the bottom surface area within 300 seconds, the settling time should be recorded as greater than 300 seconds. The substrate surface area is the substrate surface and the area within 2 mm above it; The 5-minute bottom surface reach rate is calculated as the proportion of the dry mass of particles located in the bottom surface area within 5 minutes to the total dry mass of the applied particles. When determining the increase in water absorption on the surface of the particles, weigh 1.00 g of dry weight of the particles, soak them in 100 mL of artificial seawater for 3 min, recover the particles through a 0.15 mm sieve, let them stand to drain for 30 s, and then weigh them. The increase in water absorption on the particle surface is calculated as the ratio of the increase in particle mass after soaking to the dry basis mass of the added particles; The contact angle was measured using a tablet made from the surface material. Take the surface material of the particles corresponding to each treatment, press it under 10MPa pressure for 30s, and make it into a disc with a diameter of 10mm and a thickness of 1mm. For non-layered particles, scrape material from the outer surface of the particles, with a scraping depth not exceeding 10% of the particle radius, and compress them into tablets under the same conditions; After soaking each tablet in artificial seawater for 3 minutes, remove it, let it stand and drain for 30 seconds, place it on the sample stage of the contact angle measuring instrument, add 2 μL of artificial seawater to the surface of the tablet, collect the droplet image at 5 seconds and calculate the contact angle; When measuring the contact amount of substrate particles, particles with a dry basis mass of 1.00g and after absorbing water for 3 minutes are evenly spread on the surface of a fine sand and mud mixture substrate with a moisture content of 18.0%, covered with a circular sieve with a diameter of 50mm, and a weight with a mass of 50g is applied to the sieve for 10s. After removing the weights and sieves, use tweezers to remove the particles, collect the substrate particles attached to the outer surface of the particles, and dry them at 60°C to constant weight. Calculate the substrate particle contact amount based on the dry weight of substrate particles corresponding to each gram of dry particles. When determining the amount of sediment particles retained on the surface after shaking for 5 min, the particles after the sediment particle contact amount measurement was completed were placed in a 250 mL Erlenmeyer flask containing 100 mL of artificial seawater and shaken at 60 r / min for 5 min on a horizontal shaker. After the oscillation is complete, the particles are removed, and the substrate particles still attached to the outer surface of the particles are collected. They are dried at 60°C to constant weight, and the weight is calculated based on the dry weight of the substrate particles corresponding to each gram of dry basis particles. The test results are shown in Table 3; Table 3: Apparent density and surface contact properties of layered bait pellets after water absorption Main differences Layered granules, dried at 35℃ to a moisture content of 18.6%. Low apparent density particles, outer layer composition and final moisture content are the same as in Example 3. Layered granules, dried at 35℃ to a moisture content of 6.8%. Layered granules, dried at 60℃ to a moisture content of 18.4%. Non-stratified particles, with final moisture content and average particle size controlled according to Example 3. Particle moisture content / % 18.6 18.9 6.8 18.4 18.4 <![CDATA[Apparent density / g·cm -3 > 1.164 1.008 1.158 1.153 1.143 <![CDATA[Artificial seawater density / g·cm -3 > 1.021 1.021 1.021 1.021 1.021 Settling time at a water depth of 20cm / s 38.6 Greater than 300 40.7 42.4 40.9 5-minute substrate surface reach rate / % 92.4 39.6 90.1 88.6 88.7 <![CDATA[3min Particle surface water absorption increment / g·g -1 > 0.48 0.45 0.29 0.23 0.27 Contact angle of tablet after 3 minutes of water absorption / ° 37.8 39.5 58.4 69.1 64.2 <![CDATA[Substrate particle contact amount / mg·g -1 > 186.5 172.4 112.6 88.9 96.8 <![CDATA[Amount of surface sediment particles retained after 5 minutes of oscillation / mg·g -1 > 121.6 106.3 62.5 40.8 45.5 As shown in Table 3, under the conditions of artificial seawater with a salinity of 30‰, pH of 8.0, and temperature of 23℃, the density of artificial seawater is 1.021 g / cm³. The apparent density of Example 3 was 1.164 g / cm³, the settling time at a water depth of 20 cm was 38.6 s, and the bottom surface reach rate was 92.4% after 5 minutes. The outer layer composition and endpoint water content of Comparative Example 10 were the same as those of Example 3. The apparent density was 1.008 g / cm³, which was lower than that of artificial seawater. The settling time at a water depth of 20 cm was recorded as greater than 300 s, and the bottom surface reach rate was 39.6% at 5 min. Under the test conditions of this embodiment, when the apparent density of the particles is higher than that of artificial seawater, the proportion of particles entering the bottom surface area within 5 minutes is relatively high. The apparent densities of Example 3 and Comparative Example 13 were both higher than those of artificial seawater, and their 5-minute seabed surface reach rates were 92.4% and 88.7%, respectively. Comparative Example 13, without a core layer and an outer layer, showed a 3-minute increase in water absorption on the particle surface of 0.27 g / g, a tablet contact angle of 64.2° after 3 minutes of water absorption, a substrate particle contact amount of 96.8 mg / g, and a substrate particle retention amount of 45.5 mg / g after 5 minutes of shaking. The corresponding indicators for Example 3 were 0.48 g / g, 37.8°, 186.5 mg / g, and 121.6 mg / g, respectively. Under the condition that all particles can enter the surface area of ​​the substrate, the particle surface water absorption increment, tablet contact angle, substrate particle contact amount and substrate particle retention after oscillation in Example 3 are all better than those in Comparative Example 13, indicating that the layered outer layer structure is related to the surface contact performance of the particles after water absorption. Comparative Example 11 had the same drying temperature as Example 3, but the final moisture content decreased from 18.6% to 6.8%; its 3-minute particle surface water absorption increment was 0.29 g / g, the tablet contact angle after 3 minutes of water absorption was 58.4°, the substrate particle contact amount was 112.6 mg / g, and the surface substrate particle retention amount after 5 minutes of shaking was 62.5 mg / g. The endpoint moisture content of Comparative Example 12 was close to that of Example 3, but the drying temperature was adjusted from 35°C to 60°C. The surface water absorption increment of the particles in 3 minutes was 0.23 g / g, the contact angle of the tablet after 3 minutes of water absorption was 69.1°, the contact amount of the substrate particles was 88.9 mg / g, and the amount of substrate particles retained on the surface after 5 minutes of shaking was 40.8 mg / g. Under the test conditions of this embodiment, after the endpoint moisture content decreased or the drying temperature increased, the amount of substrate particles in contact with the particles after absorbing water and the amount of substrate particles retained on the surface after oscillation were both lower than in Example 3.

[0026] Example 4 In this embodiment, the layered feed pellets prepared in Example 1 were used as the test object to detect the composition of the dispersion formed after the core layer was dispersed in the digestive tract environment of the F1 generation of *Ulva unicornu* seedlings. In this embodiment, the digestive tract environment refers to the in vitro treatment conditions formed by the supernatant of the digestive tract tissue homogenate of the F1 generation of *Ulva uniflora* seedlings, artificial seawater, and constant temperature shaking treatment, which are used to evaluate the dispersion state of the particle core layer under digestive tract conditions after feeding. The dispersion refers to the solid-liquid mixture of the test particles after they have been processed in the digestive tract environment and passed through a 0.5 mm sieve. It includes algal soluble components in the liquid phase, algal solid fragments in a suspended or settled state, and fermentation cells released with the test particles. A 0.5mm sieve is used to distinguish between undispersed particles that still retain the core particle shape and dispersed fragments formed after processing. In Example 1, the average particle size of the wet particles in the core layer is greater than 0.5 mm, and the portion that passes through a 0.5 mm sieve is counted as the dispersion. Algal solid fragments refer to the algal solid residues obtained after centrifugation of the dispersion, and their particle size is determined by a laser particle size analyzer. Algal-derived soluble components include algal-derived soluble proteins, short peptides, free amino acids, and oligosaccharides; The layered bait pellets used in Example 4 were prepared according to the method in Example 1; To reduce the interference of the outer layer material on the detection of the core layer dispersion composition, the stratified bait particles were soaked in artificial seawater with a salinity of 30‰, pH of 8.0 and temperature of 23℃ for 3 minutes. The particles were then recovered through a 0.5mm sieve, and the particles on the sieve were rinsed along the edge of the sieve with 30mL of artificial seawater to remove the softened outer layer material. All treatments used the same pre-soaking and rinsing conditions, and the particles on the sieve after the outer layer was removed were used to evaluate the dispersion of the core layer in the digestive tract environment. The outer layer removal step is only used as a pretreatment before detection and is not used as an actual feeding step. The rinsing solution after outer layer removal is not included in the dispersion detection results of this embodiment. For Comparative Example 14, the pre-soaking and rinsing conditions are used only as the same pretreatment for testing and do not indicate that it has a core or outer layer structure. After this step, the particles on the sieve retain the core layer particle morphology, and the dry basis mass of the core layer test particles is calculated based on the moisture content of the same batch of particles. The outer layer pre-soaking time adopts the treatment condition of 3 minutes of particle water absorption in Example 3; The digestive fluid was prepared as follows: 60 seedlings of the same batch of *Urechis monocyclicis* F1 generation were taken. The average body length of the seedlings was 19.2 mm. They were not fed for 6 hours before sampling and their bodies were rinsed with artificial seawater with a salinity of 30‰. When separating digestive tract tissues, the contents of the digestive tract are retained and not sterilized. Add artificial seawater with a salinity of 30‰ at a ratio of 1g:4mL to the weight of digestive tract tissue and volume of artificial seawater, and homogenize for 2 minutes under ice bath conditions; The homogenate was centrifuged at 8000 r / min for 10 min at 4℃, and the supernatant was collected as the digestive tract homogenate supernatant. The supernatant of the digestive tract homogenate was mixed with artificial seawater with a salinity of 30‰ at a volume ratio of 1:4 to obtain the digestive fluid; The digestive solution has a pH of 7.4 and should be used within 2 hours of preparation. Take the core layer test particles and add 1.00 g of dry weight to 20 mL of digestion solution. Shake at 80 r / min for 60 min at 23 °C. After processing, the system was separated by passing it through a 0.5 mm sieve. The particles retained on the 0.5 mm sieve were dried at 60 °C to constant weight and used as the dry weight of the undispersed test particles. The solid-liquid mixture passes through a sieve as a dispersion; The particle dispersion rate is calculated using the following formula: Particle dispersion rate = Dry mass of dispersed test particles / Dry mass of test particles added × 100%; The dry mass of the dispersed test particles is obtained by subtracting the dry mass of the undispersed test particles from the dry mass of the test particles that were added. For particles with a core layer structure, the particle dispersion rate is used to represent the core layer dispersion rate. For non-stratified homogeneous particles, the particle dispersion rate is measured to represent the particle dispersion rate. The fermentation cells in the dispersion were detected using two methods; When no test particles are added to the same batch of digestion solution, it serves as a blank control. MRS plate counting is performed under the same dilution, coating, and culture conditions as the dispersion sample. The amount of culturable fermentable cells in the dispersion was calculated by subtracting the background lactic acid bacteria count in the blank control from the MRS plate count results of the treatment group with added test particles. Take a sample containing a mixture of dispersion liquid phase and suspended solids, dilute it tenfold, spread it on MRS agar plates, incubate it at 30℃ for 48h, and count the number of culturable lactic acid bacteria. The amount of culturable fermentable cells in Table 4 is the result after deducting the background lactic acid bacteria of the blank control in the same batch of digestion solution. Another dispersion was Gram-stained and observed under a 1000x optical microscope. 30 fields of view were randomly collected for each treatment, and the number of bacteria with lactic acid bacteria morphology was counted. MRS plate count is used to determine the number of culturable lactic acid bacteria in a dispersion. Microscopic counting is used to observe the presence of lactic acid bacteria morphology in dispersions and their co-distribution with algal solid fragments; it does not replace culturable cell counting. The method for determining algal solid fragments in the dispersion is as follows: take the dispersion and centrifuge at 5000 r / min for 5 min, collect the precipitate, wash it once with artificial seawater with a salinity of 30‰, and then determine the dry mass and D50 particle size of the fragments. The method for determining the algal soluble components in the dispersion is as follows: take the supernatant after centrifugation and determine the contents of algal soluble protein, short peptides, free amino acids and oligosaccharides respectively; Algal soluble proteins were determined using the Coomassie Brilliant Blue method; short peptides were determined using the trichloroacetic acid soluble nitrogen conversion method; and free amino acids were determined using the ninhydrin method. Oligosaccharides were determined using the phenol-sulfuric acid method; The dry mass of the dispersion includes algal solid fragments, algal soluble components released into the liquid phase, extracellular polysaccharides, inorganic salts, and fermentation cell dry matter; The total release of algal soluble components in Table 4 is the sum of detectable algal soluble proteins, short peptides, free amino acids, and oligosaccharides. Set up comparative examples 14 to 17; Comparative Example 14 used the same fermented algae wet material and endpoint moisture content as Example 4, but did not set a core layer and an outer layer. After the material was mixed, it was directly extruded and rolled into rounds to obtain non-layered homogeneous particles. Comparative Example 15 used the same core layer material and outer layer material as Example 4, but the wet particles of the core layer were pressed by a 0.40MPa plate for 30s before the outer layer was sprayed. After pressing, the moisture content of the core layer wet particles is adjusted to 45.2%, and then the outer layer is sprayed to obtain core layer compacted particles. Comparative Example 16 used the same core solids and fermentation cells as Example 4. Before granulation, the core material was washed twice with artificial seawater with a salinity of 30‰ at a material-to-liquid ratio of 1g:5mL, each time with shaking at 100r / min for 2min, and then the solids were recovered through a 0.5mm sieve. Before granulation, the number of fermentation cells in the core material was detected, and the same batch of washed cell suspension was added to make the number of fermentation cells in the core material of Comparative Example 16 close to the number of fermentation cells in the core material of Example 4. Then, artificial seawater with a salinity of 30‰ was used to adjust the water content to 45.2% before granulation, which was used to reduce the content of soluble components from algae in the core layer; Comparative Example 17 used the same algal source material and fermentation cells as Example 4. The core material was treated with a colloid mill three times and passed through a 160-mesh sieve before granulation. Before granulation, the supernatant from the same batch of fermentation was added to make the content of algal soluble components in the core material of Comparative Example 17 consistent with that in the core material of Example 4, in order to reduce the particle size of algal solid fragments in the core. Table 4: Composition of dispersions formed by test particles in the digestive tract environment Main differences Layered particle core layer, preparation conditions in Example 1 Non-stratified homogeneous particles Core layer compacted particles Reduced soluble content in the core layer Excessive refinement of core solids 60-minute test particle dispersion rate / % 73.4 56.8 48.5 69.1 76.2 <![CDATA[Dry mass of dispersion / mg·g -1 Test particle dry basis]]> 728.6 561.4 482.7 684.5 755.8 <![CDATA[Cultivable fermented bacterial count / log CFU·g -1 Test particle dry basis]]> 6.2 5.71 5.54 6.05 6.14 <![CDATA[Microscopic count of bacterial biomass / 10 5 CFU·mL -1 dispersion]]> 4.8 3.2 2.7 4.4 4.6 <![CDATA[Dry mass of algae-derived solid fragments / mg·g -1 Test particle dry basis]]> 286.4 214.7 196.5 271.8 304.9 Algal solid fragments D50 / μm 42.8 65.4 78.1 45.6 18.7 <![CDATA[Algae-derived soluble protein release amount / mg·g -1 Test particle dry basis]]> 31.6 24.9 21.8 12.7 29.4 <![CDATA[Short peptide release amount / mg·g -1 Test particle dry basis]]> 46.8 36.5 31.9 18.4 43.1 <![CDATA[Free amino acid release amount / mg·g -1 Test particle dry basis]]> 11.2 8.4 7.6 5.1 10.7 <![CDATA[Oligosaccharide release amount / mg·g -1 Test particle dry basis]]> 39.5 31.2 27.5 16.8 37.9 <![CDATA[Total release of soluble components from algae source / mg·g -1 Test particle dry basis]]> 129.1 101 88.8 53 121.1 In Table 4, after 60 min of digestion, the particle dispersion rate of Example 4 was 73.4%, and the dry weight of the dispersion was 728.6 mg / g of dry test particles. Culturable fermentable cells were detected in the dispersion at a quantity of 6.20 log CFU / g dry basis of test particles; The bacterial count under microscopic examination was 4.8 × 10⁻⁶. 5 CFU / mL dispersion; Algal solid fragments were detected in the dispersion, with a dry weight of 286.4 mg / g test particle dry basis and a D50 particle size of 42.8 μm; Algal soluble proteins, short peptides, free amino acids and oligosaccharides were detected in the liquid phase, and the total release of algal soluble components was 129.1 mg / g dry basis of test particles; The above-mentioned test items correspond to fermentation cells, algal solid fragments, and algal soluble components in the dispersion, respectively. Compared with Example 4, Comparative Example 14 consists of non-layered homogeneous particles. The particle dispersion rate, dry mass of dispersion, amount of culturable fermentable cells, dry mass of algal solid fragments, and total release of algal soluble components were all lower than those of Example 4 after 60 min testing. After core-layer compaction, Comparative Example 15 showed a particle dispersion rate of 48.5% after 60 minutes, a dry mass of dispersion of 482.7 mg / g dry particle weight, a culturable fermentable cell mass of 5.54 log CFU / g dry particle weight, and a total release of algal soluble components of 88.8 mg / g dry particle weight, all of which were lower than those in Example 4. The above results show that core compaction reduces the amount of dispersion formed after digestion. After reducing the soluble components in the core layer, Comparative Example 16 showed that the particle dispersion rate was 69.1% after 60 min, the dry weight of algal solid fragments was 271.8 mg / g dry weight of test particles, and the amount of culturable fermentable cells was 6.05 log CFU / g dry weight of test particles. The total release of algal-derived soluble components was 53.0 mg / g dry basis of the test particles, which was lower than that in Example 4; This set of data is used to distinguish between two test objects: particle dispersion and the release of soluble components from algae. In Comparative Example 17, the particle dispersion rate was 76.2% after 60 min, and the dry weight of algal solid fragments was 304.9 mg / g of test particle dry basis. However, the D50 of algal solid fragments was 18.7 μm, which was lower than 42.8 μm in Example 4. This set of data indicates that the pretreatment method of the core solids affects the particle size of algal solid fragments in the dispersion; Example 4 shows that the dispersion formed under the same digestion conditions contains fermentation cells, algal solid fragments, and algal soluble components.

[0027] Example 5 Based on the preparation method in Example 1, this embodiment further examines the ratio of the D50 particle size of the outer layer fine algal solids to the D50 particle size of the core layer algal solids, the ratio of the dry basis content of the outer layer extracellular polysaccharides to the dry basis content of the core layer extracellular polysaccharides, and the ratio of the dry basis content of the outer layer algal soluble components to the dry basis content of the core layer algal soluble components. These findings affect the initial softening time of the outer layer, the integrity rate of the core layer, the retention rate of the bottom sediment surface, and the core layer dispersion rate and the release rate of the algal soluble components under simulated digestion conditions of the stratified feed particles in seawater. In this embodiment, the outer layer D50 ratio is the ratio of the D50 particle size of the outer layer fine algal source solids to the D50 particle size of the core layer algal source solids. The ratio of extracellular polysaccharides in the outer layer is the ratio of the dry weight content of extracellular polysaccharides in the outer layer to the dry weight content of extracellular polysaccharides in the core layer. The proportion of soluble components in the outer layer is the ratio of the dry basis content of soluble components from algae in the outer layer to the dry basis content of soluble components from algae in the core layer. The algal source material, enzymatic hydrolysis conditions, Lactobacillus plantarum SC256 activation conditions, fermentation conditions, core layer granulation method, outer layer spraying method, drying endpoint moisture content, substrate system, and digestion treatment conditions used in Example 5 are all the same as those in Example 1. By adjusting the number of times the outer wet material is processed by colloid milling, the remixing ratio of the outer wet material to the supernatant of the same batch of fermentation, and the remixing amount of extracellular polysaccharide precipitate in the outer wet material, layered feed particles with different layer difference ratios were prepared. Set up Examples 5-1, 5-2, and Comparative Examples 18 and 19; Among them, Example 5-1 is used to verify the low boundary of the three sets of layer difference ratios, Example 1 is used as the midpoint within the range of the three sets of layer difference ratios, and Example 5-2 is used to verify the high boundary of the three sets of layer difference ratios. Comparative Example 18 was used to verify the particle performance when the layer difference ratio was below the lower limit, and Comparative Example 19 was used to verify the particle performance when the layer difference ratio was above the upper limit. The moisture content of the particles in each treatment was controlled at 18.0%-19.0%; In Example 5-1, the D50 particle size of the algal-derived solids in the core layer was 132.0 μm, the D50 particle size of the fine algal-derived solids in the outer layer was 26.4 μm, and the D50 ratio of the outer layer was 0.20. The dry basis content of extracellular polysaccharides in the core layer was 4.7%, the dry basis content of extracellular polysaccharides in the outer layer was 7.5%, and the proportion of extracellular polysaccharides in the outer layer was 1.60. The dry basis content of soluble components from algae in the core layer was 13.5%, and the dry basis content of soluble components from algae in the outer layer was 18.9%, with the proportion of soluble components in the outer layer being 1.40. In Example 5-2, the D50 particle size of the algal source solids in the core layer was 130.8 μm, the D50 particle size of the fine algal source solids in the outer layer was 52.3 μm, and the D50 ratio of the outer layer was 0.40. The dry basis content of extracellular polysaccharides in the core layer was 4.5%, the dry basis content of extracellular polysaccharides in the outer layer was 9.9%, and the proportion of extracellular polysaccharides in the outer layer was 2.20. The dry basis content of soluble components from algae in the core layer was 13.1%, and the dry basis content of soluble components from algae in the outer layer was 23.6%, with a soluble component ratio of 1.80 in the outer layer. In Comparative Example 18, the D50 particle size of algal solids in the core layer was 131.2 μm, the D50 particle size of algal solids in the outer layer was 72.2 μm, and the outer layer D50 ratio was 0.55; the dry basis content of extracellular polysaccharides in the core layer was 4.6%, the dry basis content of extracellular polysaccharides in the outer layer was 5.5%, and the outer layer extracellular polysaccharide ratio was 1.20. The dry basis content of soluble components from algae in the core layer was 13.3%, and the dry basis content of soluble components from algae in the outer layer was 15.3%, with the proportion of soluble components in the outer layer being 1.15. In Comparative Example 19, the D50 particle size of algal solids in the core layer was 132.5 μm, the D50 particle size of fine algal solids in the outer layer was 15.9 μm, and the D50 ratio of the outer layer was 0.12. The dry weight content of extracellular polysaccharides in the core layer was 4.6%, the dry weight content of extracellular polysaccharides in the outer layer was 12.0%, and the proportion of extracellular polysaccharides in the outer layer was 2.61. The dry basis content of soluble components from algae in the core layer was 13.0%, and the dry basis content of soluble components from algae in the outer layer was 27.3%, with the proportion of soluble components in the outer layer being 2.10. The outer layer initial softening time, core layer integrity rate at 10 min, bottom surface retention rate after 20 min of disturbance, proportion of soluble components entering the water body at 30 min, particle dispersion rate at 60 min, and soluble component release rate at 60 min were all determined according to the method in Example 1. The test results are shown in Table 5; Table 5: Boundary Verification Results of Layered Bait Particle Difference Ratio Main differences Three groups of low-boundary proportions Midpoint of the three sets of proportions Three groups of high-boundary proportions The floor difference ratio is lower than the lower limit The floor difference ratio is higher than the upper limit. Core layer algal solids D50 / μm 132 131.8 130.8 131.2 132.5 Outer layer of fine algal solids D50 / μm 26.4 33.6 52.3 72.2 15.9 Outer layer D50 ratio 0.2 0.255 0.4 0.55 0.12 Core layer extracellular polysaccharide dry weight content / % 4.7 4.6 4.5 4.6 4.6 Exopolysaccharide dry weight content of outer layer / % 7.5 8.8 9.9 5.5 12 Outer cell extracellular polysaccharide ratio 1.6 1.91 2.2 1.2 2.61 Core layer algal soluble component dry basis content / % 13.5 13.2 13.1 13.3 13 Soluble content of outer algae (dry basis) / % 18.9 21.7 23.6 15.3 27.3 Proportion of soluble components in the outer layer 1.4 1.64 1.8 1.15 2.1 Outer layer initial softening time / min 3.4 2.8 3.1 7.2 2.1 10-minute core integrity rate / % 86.2 88.4 84.9 82.6 71.4 Substrate surface retention rate after 20 min of disturbance / % 72.5 76.8 73.6 55.4 60.7 Percentage of soluble components entering the water body within 30 minutes / % 18.6 17.4 20.5 25.8 34.6 60-minute test particle dispersion rate / % 69.8 72.6 70.4 61.5 75.8 60min soluble component release rate / % 66.1 70.8 68.5 58.2 77.4 As shown in Table 5, the outer layer D50 ratios of Examples 5-1, 1, and 5-2 were 0.20, 0.255, and 0.40, respectively; the outer layer extracellular polysaccharide ratios were 1.60, 1.91, and 2.20, respectively; and the outer layer soluble component ratios were 1.40, 1.64, and 1.80, respectively. All three processes are within the range of the aforementioned layer difference ratio; Within the specified layer difference ratio range, the initial softening times of the outer layer in Examples 5-1, 1, and 5-2 were 3.4 min, 2.8 min, and 3.1 min, respectively; the core layer integrity rates at 10 min were 86.2%, 88.4%, and 84.9%, respectively; and the substrate surface retention rates after 20 min of disturbance were 72.5%, 76.8%, and 73.6%, respectively. Under the test conditions of this embodiment, when the ratio of the three layers is within the above range, the stratified bait particles can form a state in seawater where the outer layer first absorbs water and softens, while the core layer maintains the particle structure and remains on the bottom surface. The particle dispersion rates of Examples 5-1, 1, and 5-2 after 60 min of digestion were 69.8%, 72.6%, and 70.4%, respectively, and the soluble component release rates after 60 min were 66.1%, 70.8%, and 68.5%, respectively. Under the test conditions of this embodiment, when the ratio of the three groups of layer differences is within the above range, the core layer can be dispersed after digestion and release algal soluble components; In Comparative Example 18, the proportion of outer layer D50 was 0.55, the proportion of outer layer extracellular polysaccharide was 1.20, and the proportion of outer layer soluble components was 1.15, all of which were below the boundary requirements of the corresponding proportion ranges. Compared with Examples 5-1, 1 and 5-2, the initial softening time of the outer layer in Comparative Example 18 was extended to 7.2 min, the retention rate of the substrate surface decreased to 55.4% after 20 min of disturbance, and the release rate of soluble components decreased to 58.2% after 60 min. When the layer difference ratio is lower than the above range, the results of outer layer water absorption softening and bottom surface retention are lower than the range required for treatment. In Comparative Example 19, the proportion of outer layer D50 was 0.12, the proportion of outer layer extracellular polysaccharide was 2.61, and the proportion of outer layer soluble components was 2.10, all of which were higher than the boundary requirements of the corresponding proportion ranges. The initial softening time of the outer layer in Comparative Example 19 was 2.1 min, but the core layer integrity rate dropped to 71.4% after 10 min, the proportion of soluble components entering the water increased to 34.6% after 30 min, and the bottom surface retention rate was 60.7% after 20 min of disturbance. When the layer difference ratio is higher than the above range, the proportion of the outer layer absorbing water too quickly and the proportion of soluble components entering the water body prematurely increases, and the particle retention result of the core layer in seawater is lower than the range. The results of this embodiment show that when the outer layer D50 ratio is 0.20-0.40, the outer layer extracellular polysaccharide ratio is 1.60-2.20, and the outer layer soluble component ratio is 1.40-1.80, the detection results of the stratified bait particles in seawater, including the outer layer absorbing water and softening, the core layer maintaining the particle structure, the bottom surface layer retaining the particles, and the dispersion and release in the digestive environment, are all within the repeatable range.

[0028] Example 6 This embodiment is used to verify whether the layered feed particles prepared by different combinations of algal source solids can form the same type of layer difference structure as in Example 1, and maintain the corresponding particle behavior in the marine environment and the digestive tract environment of *Ulva unicornu* seedlings. Except for the algal source combination that was specifically adjusted in this embodiment, the dry basis mass concentration of algal source raw materials, enzymatic hydrolysis conditions, activation conditions of Lactobacillus plantarum SC256, inoculum amount, fermentation temperature, fermentation time, core layer granulation method, outer layer spraying method, drying endpoint moisture content, outer layer initial softening time, core layer integrity rate, bottom surface retention rate, proportion of soluble components entering the water body, and digestion treatment conditions are all the same as in Example 1. This embodiment includes Example 6-1, Example 6-2, and Comparative Example 20; Example 6-1 uses Chlorella powder and Undaria pinnatifida enzymatic hydrolysis residue as algal source materials, with a dry basis mass ratio of Chlorella powder to Undaria pinnatifida enzymatic hydrolysis residue of 65:35; Example 6-2 uses spirulina powder and kelp enzymatic hydrolysis residue as algal source materials, with a dry basis mass ratio of spirulina powder to kelp enzymatic hydrolysis residue of 65:35; Comparative Example 20 used seaweed powder and seaweed enzymatic hydrolysis residue as algal source materials, with a dry basis mass ratio of seaweed powder to seaweed enzymatic hydrolysis residue of 65:35. Chlorella powder, spirulina powder, and laver powder are all used after passing through an 80-mesh sieve. The enzymatic hydrolysis residues of wakame seaweed, kelp, and laver are all solid residues obtained after the corresponding algae are washed, crushed, enzymatically hydrolyzed, and separated into solid and liquid forms. The enzymatic hydrolysis was performed under the same neutral protease and cellulase treatment conditions as in Example 1. The solid residue collected after solid-liquid separation was dried at 60°C until the moisture content was less than 10% before use. Each treatment was adjusted to the dry basis mass concentration of the liquid according to the method in Example 1. Neutral protease and cellulase were added for enzymatic hydrolysis. After enzyme inactivation, the liquid was cooled to 32°C and inoculated with activated Lactobacillus plantarum SC256 bacterial solution. The liquid was then allowed to ferment at 30°C for 28 hours. After fermentation, the fermented algae wet material is screened through a 60-mesh sieve, and the wet material on the sieve is used as the core material. The undersize portion is processed by a colloid mill and then passed through a 160-mesh sieve as the outer wet material. The core granulation, outer layer coating, and drying conditions were the same as in Example 1; The following parameters were measured for each treatment: core layer algal solids D50 particle size, outer layer fine algal solids D50 particle size, core and outer layer extracellular polysaccharide dry basis content, core and outer layer algal soluble component dry basis content, outer layer initial softening time, core layer integrity rate at 10 min, substrate surface retention rate after 20 min of disturbance, core layer dispersion rate at 60 min, and soluble component release rate at 60 min. The outer layer D50 ratio is calculated as the ratio of the D50 particle size of the outer layer fine algal source solids to the D50 particle size of the core layer algal source solids. The proportion of extracellular polysaccharides in the outer layer is calculated as the ratio of the dry basis content of extracellular polysaccharides in the outer layer to the dry basis content of extracellular polysaccharides in the core layer. The proportion of soluble components in the outer layer is calculated as the ratio of the dry basis content of soluble components from the outer layer algae to the dry basis content of soluble components from the core layer algae. The test results are shown in Table 6; Table 6: Stratification structure and digestion release results of stratified feed particles prepared with different algal source solids combinations Main differences Chlorella powder + kelp enzymatic hydrolysis residue Chlorella powder + wakame enzyme hydrolysate residue Spirulina powder + kelp enzymatic hydrolysis residue seaweed powder + seaweed enzyme hydrolysate Core layer algal solids D50 / μm 131.8 128.6 124.3 92.5 Outer layer of fine algal solids D50 / μm 33.6 35.1 36.8 48.7 Outer layer D50 ratio 0.255 0.273 0.296 0.526 Core layer extracellular polysaccharide dry weight content / % 4.6 4.5 4.3 4.1 Exopolysaccharide dry weight content of outer layer / % 8.8 8.4 8.1 5.4 Outer cell extracellular polysaccharide ratio 1.91 1.87 1.88 1.32 Core layer algal soluble component dry basis content / % 13.2 12.8 14.1 10.7 Soluble content of outer algae (dry basis) / % 21.7 20.6 22.9 13.8 Proportion of soluble components in the outer layer 1.64 1.61 1.62 1.29 Outer layer initial softening time / min 2.8 3 3.2 6.8 10-minute core integrity rate / % 88.4 86.9 85.7 78.3 Substrate surface retention rate after 20 min of disturbance / % 76.8 73.9 72.6 54.8 60min core layer dispersion rate / % 72.6 70.8 69.5 58.4 60min soluble component release rate / % 70.8 67.9 68.6 55.7 As shown in Table 6, when Chlorella powder and Undaria pinnatifida enzymatic hydrolysis residue were combined in Example 6-1, the outer layer D50 ratio was 0.273, the outer layer extracellular polysaccharide ratio was 1.87, and the outer layer soluble component ratio was 1.61, all of which were within the range of layer difference ratios defined in claim 1. In Example 6-1, the initial softening time of the outer layer was 3.0 min, the core layer integrity rate was 86.9% after 10 min, the substrate surface retention rate was 73.9% after 20 min of disturbance, the core layer dispersion rate was 70.8% after 60 min, and the soluble component release rate was 67.9% after 60 min. In Example 6-2, when Spirulina powder and kelp enzymatic hydrolysis residue were combined, the outer layer D50 ratio was 0.296, the outer layer extracellular polysaccharide ratio was 1.88, and the outer layer soluble component ratio was 1.62, all of which were within the layer difference ratio range defined by this invention. In Example 6-2, the initial softening time of the outer layer was 3.2 min, the core layer integrity rate was 85.7% after 10 min, the substrate surface retention rate was 72.6% after 20 min of disturbance, the core layer dispersion rate was 69.5% after 60 min, and the soluble component release rate was 68.6% after 60 min. Examples 1, 6-1, and 6-2 used different combinations of algal solids, with the outer layer D50 ratio all in the range of 0.20-0.40, the outer layer extracellular polysaccharide ratio all in the range of 1.60-2.20, and the outer layer soluble component ratio all in the range of 1.40-1.80. The corresponding outer layer initial softening time was no more than 3.2 min, the core layer integrity rate was no less than 85.7% at 10 min, and the core layer dispersion rate was no less than 69.5% at 60 min. The above results show that the combination of Chlorella powder and kelp enzymatic hydrolysis residue, the combination of Chlorella powder and wakame enzymatic hydrolysis residue, and the combination of Spirulina powder and kelp enzymatic hydrolysis residue can all form layered feed particles that meet the layer difference ratio requirements. When Comparative Example 20 used a combination of laver powder and laver enzymatic hydrolysis residue, the outer layer D50 ratio was 0.526, the outer layer extracellular polysaccharide ratio was 1.32, and the outer layer soluble component ratio was 1.29, all of which did not fall within the layer difference ratio range defined by this invention. The initial softening time of the outer layer of Comparative Example 20 was 6.8 min; the core layer integrity rate was 78.3% after 10 min; the substrate surface retention rate was 54.8% after 20 min of disturbance; the core layer dispersion rate was 58.4% after 60 min; and the soluble component release rate was 55.7% after 60 min. Different combinations of algal source solids have an impact on the formation of stratification structure. Not any combination of algal sources can form stratified feed particles that meet the D50 ratio, extracellular polysaccharide ratio and soluble component ratio. The results of this embodiment show that, under the same conditions of enzymatic hydrolysis, fermentation, core layer granulation, outer layer spraying and drying, the combination of Chlorella powder and Undaria pinnatifida enzymatic hydrolysis residue and the combination of Spirulina powder and Kelp enzymatic hydrolysis residue can both form the same type of core layer and outer layer layer difference structure as in Example 1. The proportions of outer layer D50, outer layer extracellular polysaccharide, and outer layer soluble components of the corresponding particles were all within the set range, and the results obtained in the detection of outer layer initial softening time, core layer integrity rate, substrate surface retention rate, core layer dispersion rate, and soluble component release rate were the same as those in Example 1. Under the conditions of this embodiment, the combination of laver powder and laver enzymatic hydrolysis residue did not form stratified feed particles within the specified layer difference ratio range.

[0029] Verification Example 1 This validation example is used to evaluate the effect of stratified feed pellets on the early population rearing results of F1 generation seedlings of Ulva monocranium under seawater seedling rearing conditions with bottom sediment; The F1 generation seedlings of *Ulva uniflora* used in the experiment were from the same batch of hatched seedlings from the same parent population. They were temporarily raised for 24 hours before the start of the experiment, and no fermented algae preparations were fed during the temporary raising period. The bottom of the test container was lined with a mixture of fine sand and mud, with a thickness of 2.0 cm and a dry mass ratio of fine sand to mud of 7:3. The artificial seawater had a salinity of 30‰, a pH of 8.0, and a water temperature of 23℃. Each treatment was set up in 3 replicates, and 120 seedlings were released into each replicate. The experimental setup included Example 1: layered feed pellet group, Comparative Example 1: non-layered homogeneous pellet group, Comparative Example 2: outer layer D50 not less than core layer D50 pellet group, Comparative Example 5: lactic acid bacteria pellet group with low extracellular polysaccharide production capacity, and conventional algae powder group. All treatments were fed the same dry-based feeding amount twice a day for 14 consecutive days. The conventional algae powder group was fed directly after mixing Chlorella powder and kelp enzymatic hydrolysis residue at the same dry basis mass ratio as in Example 1, without lactic acid bacteria fermentation or stratification into granules. During the experiment, 50% of the artificial seawater was replaced daily, and the bottom sediment was kept undisturbed. After the experiment, the number of surviving seedlings in each replicate was counted, and the 14-day survival rate was calculated. Thirty seedlings were randomly selected from each replicate to measure body length and wet weight, and the 14-day body length growth rate and 14-day weight gain rate were calculated. The coefficient of variation of final body length is calculated as the ratio of the standard deviation of final body length to the mean of final body length. The effective number of seedlings per unit substrate area is calculated based on the number of seedlings that survived at the end of the experiment and whose body length was not less than 80% of the average final body length of the treatment. The test results are shown in Table 7; Table 7: Cultivation effect of F1 generation seedlings of Ulva single-ringed ring after 14 days 14-day survival rate / % 91.8 84.6 87.2 88.1 82.9 14-day body length growth rate (%) 24.7 17.5 20.1 20.8 15.9 14-day weight gain rate / % 36.4 30.8 32.6 33 27.6 Final body length coefficient of variation / % 9.7 12.8 11.9 11.6 14.1 <![CDATA[Number of effective seedlings per unit substrate area / ind·m -2 > 1846 1542 1658 1684 1457 30-minute water turbidity / NTU 12.4 24.8 16.7 15.9 31.6 As shown in Table 7, under the same seedling source, substrate conditions and dry feed amount, the 14-day survival rate, 14-day body length growth rate, 14-day weight gain rate and effective seedling number per unit substrate area of ​​the stratified feed pellet group in Example 1 were higher than those of the non-stratified homogeneous pellet group and the conventional algae powder group. The coefficient of variation of final body length and 30-minute water turbidity were lower than those of the non-stratified homogeneous pellet group and the conventional algae powder group. Compared with Comparative Example 2, when the D50 of the outer layer of fine algal source solids was lower than that of the core layer of algal source solids, the growth rate of seedling body length and the number of effective seedlings per unit substrate area were higher in Example 1. The 14-day survival rate, 14-day weight gain rate, and coefficient of variation of final body length in Example 1 were 91.8%, 36.4%, and 9.7%, respectively, while the corresponding values ​​for Comparative Example 5 were 88.1%, 33.0%, and 11.6%, respectively. The above results indicate that stratified feed pellets can support the early population cultivation of F1 generation seedlings of *Ulva monocranium* under seawater seedling cultivation conditions with substrate.

[0030] Verification Example 2 This validation example is used to evaluate whether the intestinal contents of the F1 generation of *Ulva unicornu* seedlings, after ingesting stratified feed pellets, form a dispersion composition corresponding to the digestion treatment results of Example 4. The seedlings used in the experiment were the same as those in verification example 1, and the seedlings were fasted for 6 hours before the experiment; The animals were fed with layered feed pellets from Example 1, non-layered homogeneous pellets from Comparative Example 1, core-layer compacted pellets from Comparative Example 15, and core-layer soluble component reduced pellets from Comparative Example 16, respectively. All treatments were fed the same dry-based feeding amount. Samples were taken 2 hours after feeding, and 30 fish were randomly selected from each treatment to separate the intestinal contents. Intestinal contents were added to artificial seawater with a salinity of 30‰ at a mass-to-volume ratio of 1g:4mL, homogenized in an ice bath for 2 minutes, and then divided into three parts for testing. The first part was serially diluted tenfold and spread onto MRS agar plates, incubated at 30°C for 48 hours, and the number of culturable lactic acid bacteria was counted. The intestinal contents of seedlings from the same batch that were not fed were used as a baseline control. The count results of the baseline control were used to subtract the original lactic acid bacteria in the intestine. The second part involved centrifuging at 5000 r / min for 5 min, collecting the precipitate, and determining the dry mass and D50 particle size of the algal solid fragments. The third part involves taking the supernatant from centrifugation and determining the content of algal soluble proteins and short peptides using the Coomassie Brilliant Blue method and the trichloroacetic acid soluble nitrogen conversion method, respectively. The test results are shown in Table 8; Table 8: Composition of intestinal contents in F1 generation seedlings of *Ulva unicinctus* after feeding <![CDATA[Quantity of culturable lactic acid bacteria in intestine / log CFU·g -1 Intestinal contents]]> 5.86 5.21 5.08 5.71 <![CDATA[Dry mass of algae-derived solid fragments / mg·g -1 Intestinal content]]> 214.6 162.8 138.5 203.4 Algal solid fragments D50 / μm 44.1 67.2 75.6 46.3 <![CDATA[Algae-derived soluble protein / mg·g -1 Intestinal content]]> 24.8 18.6 16.9 10.5 <![CDATA[Short peptide / mg·g -1 Intestinal content]]> 36.7 27.4 24.1 15.6 Percentage of samples with detectable dispersions / % 86.7 63.3 56.7 80 As shown in Table 8, after feeding the layered feed pellets of Example 1, culturable lactic acid bacteria, algal solid fragments, algal soluble proteins and short peptides were detected in the intestinal contents of the F1 generation of *Ulva monocyclic* seedlings. Compared with Comparative Example 1 and Comparative Example 15, the gut microbiota cultured lactic acid bacteria, dry mass of algal solids fragments, content of algal soluble protein and short peptides in Example 1 group were higher, and the D50 of algal solids fragments was closer to the fragment particle size formed after digestion treatment in Example 4. The amount of culturable lactic acid bacteria in the gut and the dry weight of algal solid fragments in Comparative Example 16 were similar to those in Example 1, but the content of algal soluble protein and short peptides was lower than that in Example 1. The above results correspond to the detection results of the dispersion composition in the digestive tract environment in Example 4, indicating that after the stratified feed particles are ingested by the seedlings, fermented bacteria, algal solid fragments and algal soluble components can be detected in the intestinal contents.

Claims

1. A fermented algae preparation for the cultivation of a fast-growing F1 generation population of *Ulva monocyclic aromatica*, characterized in that, Including layered bait pellets; The layered feed pellets are used to be placed into seawater seedling cultivation water with bottom sediment; The layered bait particles include a core layer and an outer layer covering the outside of the core layer; The core layer contains algal solids, fermented cells, extracellular polysaccharides, and algal soluble components. The algal source solids overlap to form interparticle gaps, and the fermented cells and the algal source soluble components are distributed within the interparticle gaps. The outer layer contains finely broken algal solids, extracellular polysaccharides, and algal soluble components; The D50 particle size of the fine algal solids is 0.20-0.40 times that of the algal solids in the core layer; The content of extracellular polysaccharide dry matter in the outer layer is 1.60-2.20 times that in the core layer; The dry basis content of algal soluble components in the outer layer is 1.40-1.80 times that of the dry basis content of algal soluble components in the core layer; The outer layer absorbs water and softens in seawater before the core layer, while the core layer maintains its granular structure in seawater and disperses in the digestive tract environment of the *Ulva unicornu* seedlings.

2. The fermented algae preparation for cultivating the rapid-growing F1 generation population of *Ulva monocyclic aromatica* as described in claim 1, characterized in that: The algal-derived solids contain fragments of algal cell walls; The algal solids in the core layer contain flaky fragments, and the gaps between the particles are located between adjacent flaky fragments.

3. The fermented algae preparation for cultivating the rapid-growing F1 generation population of *Ulva monocyclic aromatica* as described in claim 1, characterized in that: The algal solids are formed by enzymatic hydrolysis and fermentation of Chlorella powder and kelp enzymatic hydrolysis residue, Chlorella powder and wakame enzymatic hydrolysis residue, or Spirulina powder and kelp enzymatic hydrolysis residue.

4. The fermented algae preparation for cultivating the rapid-growing F1 generation population of *Ulva monocyclic aromatica* as described in claim 1, characterized in that: The fine algal solids in the outer layer are attached to the outside of the core layer, and the extracellular polysaccharides are distributed between adjacent fine algal solids.

5. The fermented algae preparation for cultivating the rapid-growing F1 generation population of *Ulva monocyclic aromatica* as described in claim 1, characterized in that: The algal-derived soluble components contain algal-derived soluble proteins.

6. The fermented algae preparation for cultivating the rapid-growing F1 generation population of *Ulva monocyclic aromatica* as described in claim 1, characterized in that: The fermented bacteria are attached to the surface of the algal solids in the core layer and distributed in the interparticle spaces.

7. The fermented algae preparation for cultivating the rapid-growing F1 generation population of *Ulva monocyclic aromatica* as described in claim 1, characterized in that: The apparent density of the layered feed particles is greater than the density of the seawater used for seedling cultivation.

8. The fermented algae preparation for cultivating the rapid-growing F1 generation population of *Ulva monocyclic aromatica* as described in claim 1, characterized in that: The outer layer absorbs water and forms a moist outer surface that contacts the substrate particles.

9. The fermented algae preparation for cultivating the rapid-growing F1 generation population of *Ulva monocyclic aromatica* as described in claim 1, characterized in that: After the core layer is dispersed in the digestive tract environment of *Ulva monocyclic urticaria* seedlings, it forms a dispersion containing the fermented bacteria, fragments of the algal solids, and algal soluble components.

10. The fermented algae preparation for cultivating the rapid-growing F1 generation population of *Ulva monocyclic aromatica* as described in claim 1, characterized in that: The fermentation cell is Lactobacillus plantarum SC256.

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