An anti-stress immune enhancer containing sea cucumber gut and a preparation method and application thereof

Abstract

The present application relates to the field of aquaculture immunological preparation technology, and more particularly to a sea cucumber gut-derived anti-stress immune enhancer and its preparation method and application, which comprises sea cucumber gut powder, Szechuan lovage rhizome extract, immature bitter orange fruit extract, nutritional adjuvant and functional bacteria agent. The present application uses sea cucumber gut as raw material, combines specific enzymatic pretreatment and directional fermentation process to fully release various bioactive components such as sea cucumber gut peptide, polysaccharide and saponin, and realizes time-sequential slow release of active ingredients in the gut of Apostichopus japonicus by preparing double-layer coated microparticles with good stability. The present application can effectively regulate the diversity and composition structure of intestinal microbial community of Apostichopus japonicus, improve the functional state of immune organs, enhance the proliferation ability of macrophages and the immune response level of spleen cells, and strengthen the intestinal physical barrier function, thereby playing the roles of immune regulation and physiological protection during the key periods of wintering low temperature and summer dormancy high temperature of Apostichopus japonicus, and laying a solid foundation for the anti-stress effect of subsequent preparations.

Description

Technical Field

[0001] This invention relates to the field of aquaculture immune preparations technology, and in particular to a sea cucumber intestinal-derived anti-stress immune enhancer, its preparation method, and its application. Background Technology

[0002] Sea cucumber (Ashy sea cucumber) is an important specialty aquaculture species in the northern coastal areas, with high economic value and a large-scale industry. However, in actual aquaculture production, the high-temperature period when the water temperature reaches or exceeds 23°C in summer (summer dormancy) and the low-temperature period when the water temperature is below or equal to 5°C during the ice melt in winter are critical periods for the outbreak and mortality of sea cucumber diseases, which seriously restricts the stable production and efficiency and large-scale development of the industry. High and low temperature environmental stress can directly lead to a decline in the immune defense function of sea cucumbers, damage to the intestinal mucosal barrier, and disorder of the intestinal flora structure, thereby inducing continuous stress damage. At the same time, high temperatures in summer are often accompanied by water quality deterioration, salinity fluctuations, and accumulation of harmful substances, while low temperatures in winter exacerbate nutrient consumption and metabolic decline. The superposition of multiple stress factors significantly increases the risk of aquaculture and economic losses.

[0003] Currently, most immune enhancers used in sea cucumber mimicry on the market use polysaccharides or conventional plant extracts as core raw materials, and generally suffer from the following technical defects: First, the production cost is high, and the product design is not optimized for extreme high and low temperature stress scenarios, resulting in insufficient environmental adaptability; Second, the active ingredients have poor stability during processing and storage, and low bioavailability in vivo; Third, the effects are relatively simple, mainly achieving basic immune regulation, making it difficult to accurately control specific stress diseases caused by temperature stress, and the application effect has obvious limitations.

[0004] Sea cucumber intestines, a major byproduct of sea cucumber processing, are widely available and inexpensive. They are rich in various functional components such as immunomodulatory polysaccharides, bioactive peptides, and taurine, possessing potential benefits including repairing intestinal damage, regulating gut microbiota balance, and enhancing non-specific immunity. They are a high-quality natural raw material for developing aquatic immunomodulatory agents. However, traditional processing techniques achieve an extraction and utilization rate of less than 5% for the active ingredients in sea cucumber intestines, and the lack of standardized and controllable production processes results in poor stability of the active components, hindering their high-value industrial utilization.

[0005] To overcome the common shortcomings of existing technologies, such as low raw material utilization, immature processes, and insufficient targeted anti-stress measures, this invention focuses on the high-value resource utilization of sea cucumber intestinal byproducts. Through a combination of specific enzymatic pretreatment and directional fermentation, inert components such as macromolecular proteins and complex polysaccharides in sea cucumber intestines are efficiently cleaved into highly active ingredients that are easily absorbed by the intestines of sea cucumber cultivars, including small-molecule active peptides, functional oligosaccharides, and free amino acids. Simultaneously, it innovatively combines natural plant extracts such as Ligusticum chuanxiong and Citrus aurantium with functional nutritional supplements, and uses circulating fluidized bed granulation and encapsulation technology to stabilize the active ingredients. The resulting immune enhancer can be precisely adapted to high and low temperature stress aquaculture environments, possessing both intestinal repair and immune regulation effects. It can effectively solve the industry problem of high incidence of diseases caused by extreme temperature stress in sea cucumber cultivars, filling the technological gap in dedicated anti-stress immune preparations, and providing important technical support for loss reduction, efficiency improvement, and green sustainable development in northern sea cucumber cultivars farming. Summary of the Invention

[0006] This invention addresses prominent technical problems in the cultivation of sea cucumber (Stichopus japonicus) such as reduced stress resistance, frequent stress-related diseases, and low survival rates caused by extreme environmental stresses like high temperatures in summer and low temperatures during winter. It provides a sea cucumber intestinal immune enhancer adapted to high and low temperature stress environments and its preparation method. By optimizing the compound formulation system and combining it with proprietary fermentation activation and encapsulation molding core processes, this invention can effectively repair intestinal barrier damage in sea cucumbers, strengthen the body's non-specific immune function, and significantly improve the stress resistance and survival rate of sea cucumbers under extreme temperature cultivation conditions.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a sea cucumber intestinal-derived anti-stress immune enhancer, comprising the following raw materials in parts by weight: 80-90 parts sea cucumber intestinal powder, 3-8 parts Ligusticum chuanxiong extract, 3-6 parts Citrus aurantium extract, 2-5 parts nutritional supplements, and 2-5 parts functional probiotics.

[0008] Preferably, the nutritional supplement is composed of the following components in weight percentage: β-glucan 0.8-1.2%, methionine selenium 0.04-0.06%, vitamin C 0.8-1.2%, guanidinoacetic acid 0.4-0.6%, anti-enteritis protein 1.8-2.2%, and sea mud 95-96%.

[0009] Preferably, the functional microbial agent is a mixture of Bacillus subtilis and Lactobacillus plantarum in a mass ratio of 1:1.5 to 2.5.

[0010] Preferably, the preparation method of the sea cucumber intestine powder is as follows: after cleaning and desalting the sea cucumber intestine, enzymatic hydrolysis is carried out at a temperature of 45~55℃ and a pH of 7.5~8.5 for 3~5 h, enzyme is inactivated by boiling water bath for 8~12 min, freeze-drying and grinding through an 80~120 mesh sieve to obtain the product.

[0011] Preferably, the Ligusticum chuanxiong extract and Citrus aurantium extract are both commercially available, with the Ligusticum chuanxiong extract containing ≥10% effective substances and the Citrus aurantium extract containing ≥10% effective substances.

[0012] The β-glucan, yeast selenium, guanidinoacetic acid, and anti-enteritis protein are all commercially available. The effective substance content of β-glucan is ≥90%, the effective substance content of yeast selenium is ≥98%, the effective substance content of guanidinoacetic acid is ≥98%, and the potency of anti-enteritis protein is ≥8000 times.

[0013] The VC mentioned is commercially available coated VC with a particle size of 60 mesh or higher.

[0014] The Bacillus subtilis and Lactobacillus plantarum were commercially available, with an effective live bacteria content of ≥10 billion CFU / g.

[0015] The chitosan and sodium tripolyphosphate are commercially available. The chitosan has a deacetylation degree of ≥95% and the sodium tripolyphosphate has a particle size of ≥80 mesh.

[0016] This invention provides a method for preparing the aforementioned sea cucumber gut-derived anti-stress immune enhancer, comprising the following steps: (1) Mix sea cucumber rice noodle rolls, chuanxiong extract, citrus aurantium extract, and nutritional supplements to obtain mixed raw materials; (2) Mix the raw materials obtained in step (1) with brown sugar, add distilled water, and obtain the fermentation substrate; (3) Inoculate the functional microbial agent into LB liquid medium (containing 3% sea salt), and activate it by shaking culture at 28℃ and 180 r / min for 5-12 h. Inoculate the substrate at an amount of 2-5% of the substrate mass, mix well, and carry out fermentation using a two-stage fermentation process. (4) After fermentation, cool down to 20~25℃, pass through a 20~40 mesh sieve, and dry at a low temperature of 40~55℃ with forced air until the moisture content is 15%~20% to obtain the pretreated material; (5) The pretreated material obtained in step (4) is encapsulated and shaped by circulating fluidized granulation.

[0017] Preferably, the mass ratio of the mixed raw materials to brown sugar in step (2) is 100~200:1; the mass ratio of the mixed raw materials to water is 1:0.4~0.6.

[0018] Preferably, the two-stage fermentation process in step (3) is as follows: First stage: Ferment at 25~28℃ under anaerobic conditions for 20~28h, control dissolved oxygen at 0.3~0.5 mg / L, and induce the strain to produce protease (enzyme activity ≥120 U / mL). The second stage involves heating the temperature to 30-35℃ and fermenting in a micro-aerobic environment with dissolved oxygen of 1-2 mg / L for 30-40 hours, stirring every 5-7 hours for 8-12 minutes at a speed of 40-60 r / min to induce the release of polysaccharides and polypeptides from the sea cucumber intestine powder.

[0019] Preferably, the circulating fluidized granulation method in step (5) is as follows: the inlet air temperature of the circulating fluidized equipment is set to 55-65℃ and the circulating air speed is controlled at 1.2-1.8 m / s. The pretreated material is fed in to fluidize it. Sodium alginate (effective substance content ≥98%) is selected as the binder substrate. A homogeneous binder liquid with 18-22% solids is prepared and sprayed onto the fluidized powder until the particle size is stable at 0.4-0.8 mm. After granulation, the inlet air temperature is adjusted to 45-55℃ and the circulating dryer is dried for 15-25 min until the moisture content is ≤8%. After drying, the material is classified by a vibrating screen to remove fine powder below 0.3 mm and ultra-large particles above 1.2 mm to obtain inner core particles.

[0020] Preferably, chitosan and sodium tripolyphosphate are compounded at a mass ratio of 1.8-2.2:1, and beeswax is added to prepare a composite coating solution with a solid content of 18-22%. This solution is sprayed onto the surface of the inner core particles, and combined with circulating drying to reduce the particle moisture content to below 7%, controlling the coating weight gain to 5-6%. Furthermore, 0.1-1% soybean oil by weight of the total product is added to the coating solution to adjust the coating permeability coefficient. This invention provides the application of the aforementioned sea cucumber intestinal-derived anti-stress immune enhancer, which is used as a feed additive in sea cucumber farming feed at a dosage of 5-20 kg per ton of sea cucumber feed.

[0021] This invention addresses the common shortcomings of traditional immune enhancers, such as low utilization rate of active ingredients, poor targeting, and insufficient environmental adaptability. Using sea cucumber rice noodle rolls (a byproduct of sea cucumber processing) as the core matrix, and adhering to the concept of food and medicine sharing the same origin, this invention combines extracts of two natural plants, Ligusticum chuanxiong and Citrus aurantium, with functional nutritional supplements. Through targeted and controllable enzymatic pretreatment and deep fermentation with a proprietary functional microbial agent, combined with circulating fluidized bed granulation and encapsulation technology, a unified technical system integrating formulation and process is formed, resulting in a specialized immune enhancer with high stability and bioavailability.

[0022] The core technical solution of this invention includes: after enzymatic hydrolysis and activation of sea cucumber intestines, they are mixed with extracts of Ligusticum chuanxiong, Citrus aurantium, and nutritional excipients, and then inoculated with Bacillus subtilis and Lactobacillus plantarum for two-stage fermentation (first anaerobic enzyme production, then microaerobic conversion). Based on this, a double-layer gradient coating structure is adopted, using sodium alginate as the inner layer bonding film-forming substrate and a chitosan and sodium tripolyphosphate composite system as the outer layer coating substrate to construct stable double-layer coated microparticles. Combined with a circulating fluidized bed granulation process, the fermentation active products are integrated and embedded for shaping. The inner sodium alginate membrane can quickly encapsulate fermentation active components and form a dense particle core, effectively locking in internal active substances and avoiding activity loss during processing. The outer chitosan-sodium tripolyphosphate composite coating further optimizes the membrane pore structure and barrier properties. Relying on the synergistic advantages of the double-layer coating system, combined with the technical characteristics of low-temperature granulation, uniform film formation, and uniform material heating in the circulating fluidized bed process, long-term stable encapsulation of core functional components such as probiotics and active peptides can be achieved. By precisely controlling the permeability of the outer membrane, the time-dependent slow-release of active ingredients in the sea cucumber intestine can be controlled, effectively improving the storage stability and intestinal bioavailability of fermented products, achieving a slow-release and synergistic feeding effect. The finished product obtained through this process can maintain its intact form in water for at least 4 hours, effectively preventing premature dissolution of components. After entering the sea cucumber intestine, it adapts to a pH environment of 6.5-7.5, achieving a stable slow release for 4-6 hours, increasing the bioavailability of active ingredients by 30%-50%, fundamentally solving the problems of high loss of active ingredients and short action cycle in traditional products.

[0023] Pond culture trials have shown that administering this preparation before the critical periods of overwintering and summer dormancy in sea cucumbers can significantly activate immune cell activity, induce efficient expression of immune factors, repair intestinal barrier function, enhance the body's tolerance to high and low temperature stress, greatly reduce the incidence of stress-related diseases and mortality, and improve the survival rate of cultured sea cucumbers. Simultaneously, this product can also regulate amino acid metabolism, promote collagen synthesis in the body wall, and improve the nutritional quality and commercial flavor of sea cucumbers. This invention combines the dual effects of immune enhancement and quality improvement, providing practical and feasible technical support for stable production, reduced losses, and improved quality in large-scale sea cucumber farming under extreme temperature conditions, and has broad prospects for industrial application.

[0024] Compared with the prior art, the present invention has the following beneficial effects: ① This invention uses sea cucumber intestines as raw material, combining specific enzymatic pretreatment and directional fermentation processes to fully release various bioactive components such as sea cucumber intestinal peptides, polysaccharides, and saponins. Furthermore, by preparing stable, double-layered coated microparticles, the active ingredients are released sequentially and sustainably within the intestines of the sea cucumber replica. This effectively regulates the diversity and composition of the sea cucumber replica's intestinal microbiota, improves the functional state of immune organs, enhances macrophage proliferation and spleen cell immune response, and strengthens the intestinal physical barrier function. Therefore, it exerts immunomodulatory and physiological protective effects during the critical periods of wintering at low temperatures and summer dormancy at high temperatures in the sea cucumber replica, laying a solid foundation for the anti-stress efficacy of subsequent formulations.

[0025] ② This invention breaks through the limitations of single-raw material applications, innovatively combining sea cucumber gut-derived active substances with extracts of Ligusticum chuanxiong and Citrus aurantium, optimizing the formula based on the concept of food and medicine sharing the same origin. Both Ligusticum chuanxiong and Citrus aurantium are mature natural Chinese herbal medicines, rich in volatile oils, flavonoids, organic acids, and other active components, exhibiting clear efficacy in regulating physiological functions, alleviating stress damage, and enhancing defense capabilities. When used synergistically with sea cucumber gut-derived active substances, they form a complementary mechanism: the sea cucumber gut-derived active substances primarily focus on activating the immune system and enhancing disease resistance, while the extracts of Ligusticum chuanxiong and Citrus aurantium focus on harmonizing metabolic functions and alleviating stress responses caused by environmental stress. The synergistic effect of both comprehensively strengthens the resilience of sea cucumber from two dimensions: immune activation and stress relief, resulting in a significantly better overall effect than single-component formulations.

[0026] ③ The nutritional adjuvant of this invention is scientifically designed to exert a dual regulatory effect on the physiological metabolism and stress resistance of the sea cucumber. On the one hand, the nutritional adjuvant participates in the synthesis and metabolism of intercellular matrix components such as collagen and mucopolysaccharides in the sea cucumber, maintaining the integrity of connective tissue structure and functional homeostasis. It also promotes the accumulation of collagen in the body wall by regulating amino acid metabolic pathways, thereby improving the nutritional quality and flavor of the sea cucumber. On the other hand, this adjuvant can effectively regulate the body's immune response level, promote the synthesis of antibodies, and enhance the sea cucumber's resistance to environmental stress and pathogenic microorganisms. Simultaneously, the rational formulation of the nutritional adjuvant can also improve the growth rate and survival rate of the sea cucumber, further boosting the overall economic benefits of aquaculture.

[0027] ④ This invention employs a proprietary directional fermentation process to bioactivate the composite raw materials. During fermentation, various active secondary metabolites, such as bacteriocins, chitinases, and glucanases, are produced. These active ingredients further activate the immune cells of the sea cucumber, promote the secretion and expression of immune-related factors, and enhance the body's stress resistance and environmental tolerance during winter and summer dormancy cycles. Simultaneously, the fermentation process effectively improves the degradation and transformation efficiency of inert macromolecular components in the sea cucumber intestine, increasing the extraction and utilization rate of active ingredients and providing process assurance for the efficacy stability and practical effectiveness of the final formulation. Attached Figure Description

[0028] Figure 1 Effects of different levels of immune enhancers on the height of intestinal folds in sea cucumbers (1-5 represent D0-D4 respectively).

[0029] Figure 2 Effects of different levels of immune enhancers on the intestinal tissue structure of sea cucumbers. Detailed Implementation

[0030] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0031] Example 1

[0032] The sea cucumber intestinal-derived anti-stress immune enhancer in this embodiment is composed of the following raw materials in parts by weight: 85 parts sea cucumber intestinal powder, 6 parts Ligusticum chuanxiong extract, 4 parts Citrus aurantium extract, 5 parts nutritional supplements, and 3 parts functional bacteria agent.

[0033] The nutritional supplement is composed of the following components by weight percentage: 1.0% β-glucan, 0.05% methionine selenium, 1.0% vitamin C, 0.5% guanidinoacetic acid, 2.0% anti-enteritis protein, and 95.45% sea mud. The anti-enteritis protein has a potency of 10,000 times; the vitamin C is encapsulated vitamin C with a particle size ≥60 mesh.

[0034] The functional microbial agent is composed of Bacillus subtilis and Lactobacillus plantarum mixed at a mass ratio of 1:2, with the effective live bacteria content of both bacteria being ≥10 billion CFU / g.

[0035] The preparation method of the sea cucumber intestine powder is as follows: after cleaning and desalting the sea cucumber intestine, enzymatic hydrolysis is carried out at a temperature of 50℃ and pH 8.0 for 4 hours, enzyme is inactivated by boiling water bath for 10 minutes, freeze-drying and grinding is carried out through a 100-mesh sieve to obtain the product.

[0036] This embodiment describes a method for preparing a sea cucumber gut-derived anti-stress immune enhancer, comprising the following steps: (1) Mix sea cucumber rice noodle rolls, chuanxiong extract, citrus aurantium extract, and nutritional supplements to obtain mixed raw materials; (2) Mix the mixed raw materials obtained in step (1) with brown sugar at a mass ratio of 100:1, add distilled water to adjust the material-to-water ratio to 1:0.5, and obtain the fermentation substrate; (3) Inoculate the functional microbial agent into the fermentation substrate (the inoculation amount is 3% based on the total mass of the raw materials, but in this example, 3 parts of functional microbial agent have already been added by mass, so mix them directly and evenly), and carry out fermentation using a two-stage fermentation process: First stage: Ferment at 28℃ under anaerobic conditions for 24 hours, controlling the dissolved oxygen level at 0.3~0.5 mg / L; Second stage: Heat to 32℃ and ferment in a micro-aerobic environment with dissolved oxygen of 1~2 mg / L for 36 h, stirring once every 6 h for 10 min each time at a speed of 50 r / min. (4) After fermentation, the temperature is rapidly reduced to 25°C, passed through a 40-mesh sieve, and dried at 40°C with a low-temperature forced air drying until the moisture content is 20% to obtain the pretreated material; (5) The active components of the fermentation substrate are encapsulated and shaped using a circulating fluidized bed granulation method. The specific steps are as follows: ① Pretreatment of materials First, equipment operation condition debugging and material fluidization pretreatment were carried out. The inlet air temperature of the circulating fluidization equipment was set to 65℃ and the circulating air velocity was stably controlled at 1.8 m / s. After the parameters stabilized, the pretreated material that had passed the pretreatment was introduced. The material continuously suspends and boils inside the cavity, forming a uniform and stable fluidized material layer, providing stable operating conditions for subsequent uniform granulation and continuous encapsulation.

[0037] ② Granulation of inner core

[0038] Food-grade sodium alginate was selected as the binder substrate. A homogeneous binder solution with 22% solids was prepared by fully dissolving the atomized liquid in warm water. This solution was then uniformly sprayed from top to bottom using a high-pressure atomizing spray gun at a pressure of 0.35 MPa. The fermented powder in a fluidized state continuously absorbed the atomized droplets, gradually agglomerating and forming a dense structure. Spraying continued until the particle size stabilized within the 0.4-0.8 mm range, completing the inner layer granulation. After granulation, equipment parameters were adjusted for low-temperature drying and shaping. The hot air circulation velocity was appropriately increased, and the inlet air temperature was lowered to 55℃. Continuous circulation drying was performed for 25 minutes to reduce the particle moisture content to below 8%. This process effectively solidifies the particle structure, improves mechanical strength, and prevents breakage and pulverization caused by subsequent outer coating erosion. After drying, the material was graded by a vibrating screen, removing fine powder smaller than 0.3 mm and oversized particles larger than 1.2 mm. Qualified particles entered the coating process, while all fine powder was returned to the fluidized bed for regranulation, achieving zero-loss production.

[0039] ③ Outer layer sustained-release coating

[0040] The outer coating system uses a 2:1 mass ratio of chitosan and sodium tripolyphosphate, with a small amount of beeswax added to regulate the membrane pore structure. A composite coating solution with 22% solids is prepared and sprayed uniformly at a constant atomization pressure of 0.35 MPa. Combined with circulating drying, the particle moisture content is reduced to below 7%, and the coating weight gain is controlled at 6%. This process adjusts the membrane permeability by adding 0.1-1% soybean oil (by weight of the product), enabling the product to stably release probiotics, active peptides, and other functional components within the sea cucumber's intestines over 2-6 hours, matching the digestive and absorption characteristics of sea cucumbers.

[0041] ④ Packaging

[0042] Finally, the finished product is sealed, packaged, and stored. The finished pellets are promptly fed into automated vacuum packaging equipment and vacuum-sealed using aluminum foil barrier bags. The relative humidity of the packaging environment is controlled below 45% to effectively isolate moisture and oxygen, preventing moisture absorption, oxidation, and degradation of live bacteria. The finished product is stored in a cool, dark place, ensuring that the live probiotic retention rate is no less than 70% of the initial value within a 6-month shelf life, guaranteeing product quality and feeding stability.

[0043] Example 2

[0044] The sea cucumber intestinal-derived anti-stress immune enhancer in this embodiment is composed of the following raw materials in parts by weight: 80 parts sea cucumber intestinal powder, 8 parts chuanxiong extract, 6 parts zhi shi extract, 5 parts nutritional supplements, and 4 parts functional bacteria agent.

[0045] The nutritional supplement is composed of the following components by weight percentage: 1.2% β-glucan, 0.06% methionine selenium, 1.2% vitamin C, 0.6% guanidinoacetic acid, 2.2% anti-enteritis protein, and 94.74% sea mud. The anti-enteritis protein has a potency of 12,000 times; the vitamin C is encapsulated vitamin C with a particle size ≥60 mesh.

[0046] The functional microbial agent is composed of Bacillus subtilis and Lactobacillus plantarum mixed at a mass ratio of 1:2.5, with the effective live bacteria content of both bacteria being ≥10 billion CFU / g.

[0047] The preparation method of the sea cucumber intestine powder is as follows: after cleaning and desalting the sea cucumber intestine, enzymatic hydrolysis is carried out at a temperature of 55℃ and pH 8.5 for 5 hours, enzyme is inactivated by boiling water bath for 12 minutes, freeze-drying and grinding is carried out through a 120-mesh sieve to obtain the product.

[0048] This embodiment describes a method for preparing a sea cucumber gut-derived anti-stress immune enhancer, comprising the following steps: (1) Mix sea cucumber rice noodle rolls, chuanxiong extract, citrus aurantium extract, and nutritional supplements to obtain mixed raw materials; (2) Mix the mixed raw materials obtained in step (1) with brown sugar at a mass ratio of 200:1, add distilled water to adjust the material-to-water ratio to 1:0.6, and obtain the fermentation substrate; (3) Inoculate the functional microbial agent into the fermentation substrate (add 4 parts by weight), and carry out fermentation using a two-stage fermentation process: First stage: Ferment at 28℃ under anaerobic conditions for 24 hours, controlling the dissolved oxygen level at 0.3~0.5 mg / L; Second stage: Heat to 35℃ and ferment in a micro-aerobic environment with dissolved oxygen of 1~2 mg / L for 36 h, stirring once every 6 h for 10 min each time, at a speed of 50 r / min; (4) After fermentation, the temperature is rapidly reduced to 25°C, passed through a 40-mesh sieve, and dried at 50°C with a low-temperature forced air drying process until the moisture content is 15% to obtain the pretreated material. (5) The active components of the fermentation substrate are encapsulated and shaped using a circulating fluidized bed granulation method. The specific steps are as follows: ① Pretreatment of materials First, equipment operation condition debugging and material fluidization pretreatment were carried out. The inlet air temperature of the circulating fluidization equipment was set to 60℃ and the circulating air velocity was stably controlled at 1.5 m / s. After the parameters stabilized, the pretreated material that had passed the pretreatment was introduced. The material continuously suspends and boils inside the cavity, forming a uniform and stable fluidized material layer, providing stable operating conditions for subsequent uniform granulation and continuous encapsulation.

[0049] ② Granulation of inner core

[0050] Food-grade sodium alginate was selected as the binder substrate. A homogeneous binder solution with 20% solids was prepared by fully dissolving the atomized powder in warm water. This solution was then sprayed evenly from top to bottom using a high-pressure atomizing spray gun at a pressure of 0.35 MPa. The fermented powder in a fluidized state continuously absorbed the atomized droplets, gradually agglomerating and forming a dense structure. Spraying continued until the particle size stabilized within the 0.4-0.8 mm range, completing the inner layer granulation. After granulation, equipment parameters were adjusted for low-temperature drying and shaping. The hot air circulation velocity was appropriately increased, and the inlet air temperature was lowered to 50°C. Continuous circulating drying was performed for 20 minutes to reduce the particle moisture content to below 8%. This process effectively solidifies the particle structure, improves mechanical strength, and prevents breakage and pulverization caused by subsequent outer coating erosion. After drying, the material was graded by a vibrating screen, removing fine powder smaller than 0.3 mm and oversized particles larger than 1.2 mm. Qualified particles entered the coating process, while all fine powder was returned to the fluidized bed for regranulation, achieving zero-loss production.

[0051] ③ Outer layer sustained-release coating

[0052] The outer coating system uses a 2:1 mass ratio of chitosan and sodium tripolyphosphate, with a small amount of beeswax added to regulate the membrane pore structure. A composite coating solution with 20% solids is prepared and sprayed uniformly at a constant atomization pressure of 0.25-0.35 MPa, combined with circulating drying to reduce the particle moisture content to below 7%, with the coating weight gain controlled at 5-6%. This process adjusts the membrane permeability coefficient by adding 0.1-1% soybean oil (by weight of the product), enabling the product to stably release probiotics, active peptides, and other functional components within the sea cucumber's intestines over 2-6 hours, matching the digestive and absorption characteristics of sea cucumbers.

[0053] ④ Packaging

[0054] Finally, the finished product is sealed, packaged, and stored. The finished pellets are promptly fed into automated vacuum packaging equipment and vacuum-sealed using aluminum foil barrier bags. The relative humidity of the packaging environment is controlled below 45% to effectively isolate moisture and oxygen, preventing moisture absorption, oxidation, and degradation of live bacteria. The finished product is stored in a cool, dark place, ensuring that the live probiotic retention rate is no less than 70% of the initial value within a 6-month shelf life, guaranteeing product quality and feeding stability.

[0055] Experimental Example 1

[0056] The immune enhancer prepared in Example 2 was used in a pond culture trial of sea cucumbers before their summer dormancy. Test site: Dongying Base, Shandong Provincial Institute of Marine Resources and Environment Trial period: April 18, 2025 to June 18, 2025 Experimental grouping and management: All sea cucumbers used were locally farmed. Sixty hundred healthy sea cucumbers with an average weight of 69.80±1.35g were selected and divided into 5 groups, with 3 replicates per group and 40 sea cucumbers per replicate. The control group (D0 group) was fed a basic experimental feed (10% crude protein), while the other four groups were supplemented with 0.5% (D1), 1.0% (D2), 1.5% (D3), and 2% (D4) sea cucumber intestinal immune enhancer, respectively. The culture period was 8 weeks, using a pond-cage culture model, with feeding once every other day until full.

[0057] Growth indicators and routine nutritional components: Initial and final body weights were recorded before and after the start of the breeding experiment to calculate the weight gain rate.

[0058] Weight gain rate (%) = 100 × (final weight - initial weight) / initial weight

[0059] After drying and grinding the whole ginseng, routine components were tested: crude protein (CP) content was determined by the Kjeldahl method, crude fat (CL) content was determined by Soxhlet extraction (petroleum ether as solvent), and crude ash (Ash) content was determined by high-temperature ignition method.

[0060] Intestinal tissue enzyme activity: The total antioxidant capacity (T-AOC), catalase (CAT) activity, and digestive enzyme activities such as amylase and protease in intestinal tissue were detected using the Nanjing Jiancheng reagent kit.

[0061] Preparation and observation of intestinal tissue sections: After dehydration, embedding, and sectioning, the intestinal tissue was stained with H&E. The state of the intestinal tissue was observed under a 4×10 magnification microscope, and the length of the intestinal villi was measured.

[0062] After 8 weeks of culture, the results showed that: As shown in Table 1, compared with group D0, the groups with 1% and 2% immune enhancer in the feed had significantly higher final body weights of sea cucumbers.P < 0.05; the weight gain rate showed an initial upward trend followed by a downward trend, with each supplemented group significantly higher than the control group ( P < 0.05. The crude protein and crude ash content of the whole ginseng in groups D1-D4 were significantly higher than those in the control group ( P < 0.05%, while crude fat was significantly lower than the control group ( P <0.05).

[0063] Table 1. Effects of different levels of immune enhancers on sea cucumber growth and body composition.

[0064] As shown in Table 2, the activities of trypsin, amylase, total antioxidant enzymes, and catalase in the sea cucumber intestine all showed a trend of first increasing and then decreasing, and the activities of each supplemented group were significantly higher than those of the control group. P < 0.05).

[0065] Table 2. Effects of different levels of immune enhancers on the activity of gut-associated enzymes in sea cucumbers.

[0066] Depend on Figure 1 As shown in Table 2, the intestinal structure of each experimental group was intact and without lesions. Compared with group D0, the intestinal villus length of groups D1-D4 was significantly increased. P < 0.05); Group D4 reached the highest value.

[0067] A series of test results show that the product processed using the circulating fluidized bed double-layer gradient encapsulation and shaping process possesses excellent water stability and intestinal targeted sustained-release performance. After being introduced into aquaculture water, the product maintains its intact form for at least 4 hours, effectively preventing premature dissolution of active ingredients. Once inside the sea cucumber, it adapts to the physiological environment of the intestinal pH 6.5–7.5, allowing for a stable and continuous release of various active ingredients within 2–6 hours. This process fundamentally reduces the loss of active substances in water, prolongs the action period of active ingredients in the sea cucumber's intestines, and ultimately increases the bioavailability of active ingredients by 30%–50%.

[0068] This invention provides a sea cucumber gut-derived anti-stress immune enhancer, which can be used as a special feed additive for sea cucumbers during overwintering and summer dormancy. Actual pond aquaculture trials further verified that the sea cucumber gut-derived anti-stress immune enhancer developed in this invention is safe to use, has stable effects, and is highly effective. Feeding this product before the critical overwintering and summer dormancy periods of sea cucumbers can effectively strengthen the immune foundation of the sea cucumbers, significantly improve their tolerance to extreme temperature stresses such as low winter temperatures and high summer temperatures, continuously maintain the physiological health and functional stability of the sea cucumber gut, greatly reduce mortality and losses caused by high and low temperature stress, and effectively improve the survival rate of sea cucumbers during overwintering and summer dormancy.

[0069] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A sea cucumber gut-derived anti-stress immune enhancer, characterized in that, The ingredients consist of the following components by weight: 80-90 parts sea cucumber rice noodle roll, 3-8 parts chuanxiong extract, 3-6 parts zhi shi extract, 2-5 parts nutritional supplements, and 2-5 parts functional probiotics.

2. The sea cucumber gut-derived anti-stress immune enhancer according to claim 1, characterized in that, The nutritional supplement is composed of the following components by weight percentage: β-glucan 0.8-1.2%, methionine selenium 0.04-0.06%, vitamin C 0.8-1.2%, guanidinoacetic acid 0.4-0.6%, anti-enteritis protein 1.8-2.2%, and sea mud 95-96%.

3. The sea cucumber gut-derived anti-stress immune enhancer according to claim 2, characterized in that, The functional microbial agent is composed of Bacillus subtilis and Lactobacillus plantarum mixed in a mass ratio of 1:1.5~2.

5.

4. The sea cucumber gut-derived anti-stress immune enhancer according to claim 1, characterized in that, The preparation method of the sea cucumber intestine powder is as follows: after cleaning and desalting the sea cucumber intestine, it is enzymatically hydrolyzed with 1-3% alkaline protease at a temperature of 45-55℃ and pH 7.5-8.5 for 3-5 hours, the enzyme is inactivated by boiling water bath for 8-12 minutes, and then freeze-dried and ground through an 80-120 mesh sieve to obtain the product.

5. The sea cucumber gut-derived anti-stress immune enhancer according to claim 3, characterized in that, The potency of the anti-enteritis protein is 8000~12000 times; the vitamin C is coated vitamin C with a particle size ≥60 mesh; the effective live bacteria content of Bacillus subtilis and Lactobacillus plantarum is ≥10 billion CFU / g.

6. A method for preparing a sea cucumber intestinal-derived anti-stress immune enhancer as described in any one of claims 1 to 5, characterized in that, Includes the following steps: (1) Mix sea cucumber rice noodle rolls, chuanxiong extract, citrus aurantium extract, and nutritional supplements to obtain mixed raw materials; (2) Mix the raw materials obtained in step (1) with brown sugar, add water, and obtain the fermentation substrate; (3) Inoculate the functional microbial agent into the fermentation substrate, mix evenly, and carry out fermentation using a two-stage fermentation process; (4) After fermentation, cool down to 20~25℃, pass through a 20~40 mesh sieve, and dry at a low temperature of 40~55℃ with forced air until the moisture content is 15%~20% to obtain the pretreated material; (5) The pretreated material obtained in step (4) is encapsulated and shaped by circulating fluidized granulation.

7. The preparation method according to claim 6, characterized in that, In step (2), the mass ratio of the mixed raw materials to brown sugar is 100~200:1; the mass ratio of the mixed raw materials to water is 1:0.4~0.6; in step (3), the two-stage fermentation process is as follows: First stage: Ferment at 25~28℃ under anaerobic conditions for 20~28 hours, controlling dissolved oxygen at 0.3~0.5 mg / L; Second stage: Heat to 30~35℃ and ferment in a micro-aerobic environment with dissolved oxygen of 1~2 mg / L for 30~40 hours, stirring once every 5~7 hours for 8~12 minutes each time at a speed of 40~60 r / min.

8. The preparation method according to claim 6, characterized in that, The circulating fluidized granulation method described in step (5) is as follows: the inlet air temperature of the circulating fluidized equipment is set to 55-65℃ and the circulating air velocity is controlled at 1.2-1.8 m / s. The pretreated material is fed in to fluidize it. Sodium alginate is selected as the binder substrate, and a homogeneous binder liquid with 18-22% solids is prepared and sprayed onto the fluidized powder until the particle size is stable at 0.4-0.8 mm. After granulation, the inlet air temperature is adjusted to 45-55℃, and the circulating dryer is dried for 15-25 minutes until the moisture content is ≤8%. After drying, the material is classified by a vibrating screen to remove fine powder below 0.3 mm and ultra-large particles above 1.2 mm to obtain inner core particles.

9. The preparation method according to claim 8, characterized in that, A composite coating solution with 18-22% solids was prepared by mixing chitosan and sodium tripolyphosphate at a mass ratio of 1.8-2.2:1 and adding beeswax. This solution was sprayed onto the surface of the inner core particles and dried in a circulating manner to reduce the moisture content of the particles to below 7%. The weight gain of the coating was controlled at 5-6%. In addition, 0.1-1% soybean oil by weight of the total product was added to the coating solution to adjust the permeability coefficient of the coating.

10. The application of the sea cucumber intestinal-derived anti-stress immune enhancer according to any one of claims 1 to 5, characterized in that, The immune enhancer is used as a feed additive in sea cucumber farming feed, with an addition amount of 5-20 kg per ton of sea cucumber feed.

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