Sea cucumber feed as well as preparation method and application thereof

Abstract

The invention discloses a sea cucumber feed as well as a preparation method and application thereof, and belongs to the technical field of sea cucumber feed preparation. According to the technical scheme, every 100 g of feed comprises 40 g of sea mud, 15-25 g of gulfweed powder, 5-15 g of enzymolysis gulfweed liquid and 1 g of multi-vitamin multi-mineral premix. The dosage of the enzymolysis gulfweed liquid is calculated according to the dry weight of the gulfweed, and the enzymolysis gulfweed liquid is obtained by performing enzymolysis on the gulfweed through algin degrading enzyme, pectinase and cellulase. The sea cucumber feed is applied to the aspect of sea cucumber feeding, solves the problem that the existing sea cucumber feed still cannot well promote accumulation of sea cucumber saponin and fucose in the body wall of the sea cucumber so as not to improve the nutritional value of the sea cucumber, and can effectively promote accumulation of sea cucumber saponin and fucose in the body wall of the sea cucumber so as to improve the nutritional value of the sea cucumber.

Description

Technical Field

[0001] This invention belongs to the field of sea cucumber feed preparation technology, and particularly relates to a sea cucumber feed, its preparation method and application. Background Technology

[0002] Sea cucumber is a traditional Chinese nutritional and health product. Due to its nutritional value, health benefits, and medicinal effects, market demand is constantly increasing, and the scale of aquaculture is continuously expanding (the main farmed species being the spiny sea cucumber). Apostichopus japonicus The demand for sea cucumber feed is increasing. With the scarcity of Sargassum resources, large brown algae such as Sargassum fusiforme and kelp have become important raw materials for sea cucumber feed. Sargassum fusiforme, as a major raw material for sea cucumber feed, has a large yield and is rich in carbohydrates, minerals, vitamins, free amino acids, and fatty acids. It not only provides abundant carbohydrates but is also a major source of protein. However, Sargassum fusiforme also contains non-starch polysaccharides such as fucoidan, cellulose, hemicellulose, β-glucan, and pectin, which are difficult for endogenous enzymes secreted by animals to degrade, thus limiting its digestibility and absorption efficiency to some extent. Therefore, pre-treating the polysaccharide components of Sargassum fusiforme to degrade them is an effective way to improve its utilization rate and reduce adverse effects.

[0003] Currently, the main processes for degrading large brown algae include chemical degradation, enzymatic degradation, physical degradation, and microbial fermentation. Compared with physical and chemical degradation methods, enzymatic degradation has advantages such as mild reaction conditions and high specificity. Enzymatic degradation uses specific glycosidases or non-specific enzymes to specifically cleave a glycosidic bond in the polysaccharide, breaking it down into smaller, more easily absorbed nutrients, reducing digesta viscosity, improving feed utilization, and thus improving the growth performance of farmed animals. Chinese patent CN119120622A discloses an enzymatic method for efficiently preparing seaweed oligosaccharides from Sargassum fusiforme. Through the combined action of alginate-degrading enzymes with pectinase and cellulase, a one-step deep enzymatic hydrolysis of Sargassum fusiforme is achieved, degrading Sargassum polysaccharides into oligosaccharides.

[0004] However, existing sea cucumber feeds still cannot effectively promote the accumulation of sea cucumber saponins and fucose in the body wall of sea cucumbers, thereby improving the nutritional value of sea cucumbers. Summary of the Invention

[0005] In view of the shortcomings of the existing technology, the technical problem to be solved by the present invention is that the existing sea cucumber feed still cannot effectively promote the accumulation of sea cucumber saponins and fucose in the body wall of sea cucumber, thereby improving the nutritional value of sea cucumber. The present invention proposes a sea cucumber feed that can effectively promote the accumulation of sea cucumber saponins and fucose in the body wall of sea cucumber, thereby improving the nutritional value of sea cucumber, its preparation method and application.

[0006] To solve the aforementioned technical problem, the technical solution adopted by the present invention is as follows: This invention provides a sea cucumber feed that promotes growth, enhances immunity, and improves nutritional value. Per 100g of feed, it includes 40g of sea mud, 15-25g of Sargassum powder, 5-15g of enzymatically hydrolyzed Sargassum liquid, and 1g of multivitamin and mineral premix. The amount of enzymatically hydrolyzed Sargassum liquid is based on the dry weight of Sargassum. The enzymatically hydrolyzed Sargassum liquid is obtained by enzymatically hydrolyzing Sargassum using alginate degrading enzyme, pectinase, and cellulase.

[0007] In some embodiments, the amount of enzymatically hydrolyzed Sargassum fusiforme solution used is 10g.

[0008] The aforementioned technical solution employs industrially produced, highly active alginate-degrading enzymes, cellulases, and pectinases to specifically and deeply enzymatically hydrolyze Sargassum. The hydrolysate then partially replaces the raw Sargassum powder. The resulting oligosaccharides from deep enzymatic hydrolysis enhance the sea cucumber's immunity, alter its metabolism, and promote the accumulation of active ingredients in the sea cucumber's body wall. Specifically, the enzymatic pre-digestion of Sargassum using a complex enzyme system, primarily alginate-degrading enzymes, not only promotes the release of nutrients but also generates a large amount of oligosaccharides with immunomodulatory and metabolic regulatory functions, thereby improving the health and nutritional value of farmed sea cucumbers.

[0009] In some embodiments, the multivitamin and mineral premix comprises: thiamine 25mg, riboflavin 45mg, pyridoxine hydrochloride 20mg, vitamin B12 0.1mg, vitamin K3 10mg, inositol 800mg, calcium pantothenate 60mg, nicotinamide 200mg, folic acid 20mg, biotin 1.2mg, vitamin A acetate 32mg, vitamin D3 0.5mg, α-tocopherol 120mg, ethoxyquinoline 150mg, MgSO4•H2O 4000mg, MnSO4•H2O 50mg, KI 1mg, CoCl2 1mg, CuSO4•5H2O 20mg, FeSO4•H2O 260mg, ZnSO4•H2O 150mg, and Na2SeO3 0.5mg. The multivitamin and mineral premix with the above composition is beneficial for the balanced digestion and absorption of sea cucumbers, and can improve the utilization rate of feed nutrients and energy.

[0010] In some embodiments, the sea cucumber feed also includes 5g soybean meal, 3g fish meal, 5g shrimp meal, 14.6g wheat flour, 1g squid paste, and 0.4g binder.

[0011] In some embodiments, per 100g of feed, sea cucumber feed consists of 40g of sea mud, 15-25g of Sargassum powder, 5-15g of enzymatically hydrolyzed Sargassum liquid, 1g of multivitamin and mineral premix, 5g of soybean meal, 3g of fish meal, 5g of shrimp meal, 14.6g of wheat flour, 1g of squid paste, and 0.4g of binder.

[0012] Another aspect of the present invention provides a method for preparing sea cucumber feed according to any of the above technical solutions, wherein the raw materials are mixed and water is added, and the feed is made into pellets with a particle size of 3 mm using a feed pelleting machine, dried in an oven at 60°C until the moisture content is 10%, and then sealed and stored at -20°C.

[0013] In some embodiments, the enzymatic hydrolysis of Sargassum fusiforme is obtained by the following method: Sargassum fusiforme ultrafine powder pulverized to 300 mesh and alginate degrading enzyme, pectinase, and cellulase are placed in an Erlenmeyer flask and mixed. Buffer solution is added and mixed. The flask is then placed in a water bath pre-set to 47°C for incubation and enzymatic hydrolysis. During the enzymatic hydrolysis process, the enzymatic hydrolysis system is continuously mixed with a rotor. After the enzymatic hydrolysis is completed, the water bath temperature is immediately adjusted to 100°C and reacted for 10 minutes to inactivate the enzymes in the reaction system. The flask is then removed and allowed to cool naturally to room temperature.

[0014] This invention also provides the application of the above-mentioned sea cucumber feed in sea cucumber farming, including: feeding the sea cucumber feed twice a day, with the morning feeding accounting for 30% of the daily feed amount and the afternoon feeding accounting for 70% of the daily feed amount; the total feed amount accounts for 1%-2% of the sea cucumber's body weight. During the farming period, the water temperature is 13-17℃, the salinity is 30‰, the seawater pH is 7.8, and the dissolved oxygen is not less than 6mg / L.

[0015] The present invention also provides the application of the above-mentioned sea cucumber feed in improving the nutritional value of sea cucumbers.

[0016] The present invention also provides the application of the above-mentioned sea cucumber feed in promoting the accumulation of sea cucumber saponins and fucose in the body wall of sea cucumber.

[0017] The present invention also provides the application of the above-mentioned sea cucumber feed in promoting the function of phagocytic cells in the intestinal tract of sea cucumber by improving AKP activity.

[0018] The present invention also provides the application of the above-mentioned sea cucumber feed in increasing the content of Firmicutes and Verrucous bacteria in the intestinal tract of sea cucumbers.

[0019] The present invention also provides the application of the above-mentioned sea cucumber feed in increasing the abundance of Rhodobulbaceae and Akkermansiaceae, and decreasing the abundance of Vibrioceae bacteria.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention provides a sea cucumber feed. By optimizing the sea cucumber feed formula and using enzymatically hydrolyzed Sargassum liquid to replace part of the Sargassum powder, this feed not only promotes sea cucumber growth and improves immunity, but also increases the content of the main active ingredients in the sea cucumber body wall, namely sea cucumber saponins and fucose, thereby improving the nutritional value of the sea cucumber. Attached Figure Description

[0021] Figure 1 This is a schematic diagram of a 100x magnification slice of the intestinal tissue of sea cucumber fed with the control group feed provided in an embodiment of the present invention; Figure 2 This is a schematic diagram of a 400x magnification slice of the intestinal tissue of sea cucumber fed with the control group feed provided in an embodiment of the present invention. Figure 3 A schematic diagram of a 100x magnification slice of sea cucumber intestinal tissue fed with D1 feed (using enzymatically hydrolyzed Sargassum to replace 5% Sargassum powder) provided in an embodiment of the present invention. Figure 4 A schematic diagram of a 400x magnification slice of sea cucumber intestinal tissue fed with D1 feed (using enzymatically hydrolyzed Sargassum to replace 5% Sargassum powder) provided in an embodiment of the present invention. Figure 5 A schematic diagram of a 100x magnification slice of sea cucumber intestinal tissue fed with D2 feed (using enzymatically hydrolyzed Sargassum to replace 10% Sargassum powder) provided in an embodiment of the present invention. Figure 6 A schematic diagram of a 400x magnification slice of sea cucumber intestinal tissue fed with D2 feed (using enzymatically hydrolyzed Sargassum to replace 10% Sargassum powder) provided in an embodiment of the present invention. Figure 7 A schematic diagram of a 100x magnification slice of sea cucumber intestinal tissue fed with D3 feed (using enzymatically hydrolyzed Sargassum to replace 15% Sargassum powder) provided in an embodiment of the present invention. Figure 8 A schematic diagram of a 400x magnification slice of sea cucumber intestinal tissue fed with D3 feed (using enzymatically hydrolyzed Sargassum to replace 15% Sargassum powder) provided in an embodiment of the present invention. Figure 9 This is a schematic diagram illustrating the effect of adding enzymatically hydrolyzed Sargassum to D2 feed on the phylum level of dominant intestinal flora in sea cucumbers, as provided in an embodiment of the present invention. Figure 10 This is a schematic diagram illustrating the effect of adding enzymatically hydrolyzed Sargassum to D2 feed on the family level of dominant intestinal flora in sea cucumbers, as provided in an embodiment of the present invention. Figure 11 This is a schematic diagram illustrating the effect of adding enzymatically hydrolyzed Sargassum to the D2 feed provided in this embodiment of the invention on the genus level of the dominant intestinal flora of sea cucumbers.

[0022] To provide a clearer and more detailed description of the sea cucumber feed, its preparation method, and its application provided in the embodiments of the present invention, the following description will be based on specific embodiments.

[0023] Example 1. Preparation of sea cucumber feed Preparation of Sargassum enzymatic hydrolysate: Sargassum was enzymatically hydrolyzed using the technology reported in the invention patent (2024115260700). The main steps are as follows: 50g of Sargassum ultrafine powder (crushed to 300 mesh and dried), along with alginate degrading enzyme (25000U), pectinase (250000U), and cellulase (16000U), were weighed and placed in a 2L Erlenmeyer flask and mixed thoroughly. 500mL of buffer solution (citric acid-disodium hydrogen phosphate buffer, pH 5.5) was added, mixed thoroughly, and placed in a water bath preheated to 47℃ for 8 hours of enzymatic hydrolysis. During the enzymatic hydrolysis, the system was continuously mixed with a rotor. After the enzymatic hydrolysis was completed, the water bath temperature was immediately adjusted to 100℃ and reacted for 10 minutes to inactivate the enzymes in the reaction system. The system was then removed and allowed to cool naturally to room temperature.

[0024] Experimental feed preparation: A basic feed was prepared using Sargassum powder, marine mud, multivitamin and mineral premix, soybean meal, and fish meal. Enzymatically hydrolyzed Sargassum powder was used to replace the powder in this formula at dry weights of 5%, 10%, and 15%, and thoroughly mixed with the dry ingredients. After adding an appropriate amount of moisture, the feed was pelleted into 3mm pellets using an EL260 feed pelleting machine. The pellets were dried in a 60℃ oven until the moisture content was approximately 10%, and then stored in a sealed container at -20℃. The composition of the four experimental feeds is shown in Table 1. All dry ingredients were weighed according to the feed formula proportions and then pelleted using a pelleting machine.

[0025] Table 1. Composition of the experimental diet (dry matter basis)

[0026] The above-mentioned multivitamin and mineral premix (mg / kg) includes: thiamine 25mg, riboflavin 45mg, pyridoxine hydrochloride 20mg, vitamin B12 0.1mg, vitamin K3 10mg, inositol 800mg, calcium pantothenate 60mg, nicotinamide 200mg, folic acid 20mg, biotin 1.2mg, vitamin A acetate 32mg, vitamin D3 0.5mg, α-tocopherol 120mg, ethoxyquinoline 150mg, MgSO4•H2O 4000mg, MnSO4•H2O 50mg, KI 1mg, CoCl2 1mg, CuSO4•5H2O 20mg, FeSO4•H2O 260mg, ZnSO4•H2O 150mg, and Na2SeO3 0.5mg.

[0027] 2. Adding deeply enzymatically hydrolyzed Sargassum to feed can promote the growth of sea cucumbers. Sixty hundred healthy sea cucumbers of uniform size, with an initial weight of 12.48 ± 0.16 g, were randomly selected and randomly assigned to 12 culture tanks (300 L each). The experiment was divided into four groups, with three replicates per group and 50 sea cucumbers per replicate. The control group was fed ordinary feed supplemented with 30% ultrafine Sargassum powder. The experimental groups (D1, D2, and D3) were fed Sargassum liquid instead of raw Sargassum powder at ratios of 5%, 10%, and 15%, respectively, for 60 days. Apparent feeding was conducted twice a day (8:00 AM and 6:00 PM), with 30% of the daily feed given in the morning and 70% in the afternoon, and the total feed amount accounting for 1%-2% of the sea cucumber's body weight. During the culture period, the water temperature was 13-17℃, the salinity was 30‰, the seawater pH was 7.8, and the dissolved oxygen was not less than 6 mg / L.

[0028] After the experiment, the growth indicators of the sea cucumbers were measured, including body weight, specific growth rate, weight gain rate, feed conversion ratio, and intestinal wall ratio. The calculation methods are as follows: Weight gain rate (WGR,%) = 100 × (Wt) W0) / W0 Specific growth rate (SGR, % / d) = 100 × (lnWt) lnW0) / t Intestinal wall ratio (IBR, %) = 100 × Wi / Ww Feed conversion ratio = Average weight gain × Sea cucumber weight (total sea cucumber body weight) / Feed consumption Wherein: Wt is the final body mass of sea cucumber (FBW) (g), W0 is the initial body mass of sea cucumber (IBW) (g), t is the number of days in the culture experiment (d), Wi is the intestinal mass of sea cucumber (g), and Ww is the body wall mass of sea cucumber (g).

[0029] Table 2 shows that adding enzymatically hydrolyzed Sargassum extract to the feed increased final body weight (FBW), weight gain (WGR), and specific growth rate (SGR), with no significant differences between the two groups (D2 and D3) with higher addition levels. FBW, WGR, SGR, and intestinal wall ratio (IBR) in groups D2 and D3 were significantly higher than those in the control group (P < 0.05). Specifically, the weight gain rate (WGR) in group D2 increased by 34.7%, and the specific growth rate (SGR) increased by 32.3%. The feed conversion ratio (FCR) in group D2 decreased by 26.3%.

[0030] Table 2. Effects of adding enzymatically hydrolyzed Sargassum fusiforme to feed on sea cucumber growth.

[0031] Note: D1 uses enzymatically hydrolyzed Sargassum to replace 5% Sargassum powder; D2 uses enzymatically hydrolyzed Sargassum to replace 10% Sargassum powder; D3 uses enzymatically hydrolyzed Sargassum to replace 15% Sargassum powder. In the same row, identical superscript letters or the absence of a letter indicates no significant difference. P >0.05), different superscript letters represent significant differences ( P <0.05).

[0032] 3. Adding deeply enzymatically hydrolyzed Sargassum to feed can significantly increase the content of sea cucumber saponins and fucose in the body wall of sea cucumbers. The sea cucumber farming experiment was conducted as described above. After the farming was completed, the body walls of the sea cucumbers were collected for nutritional analysis. Sea cucumber saponins and fucose are currently recognized as beneficial components of sea cucumbers, and their functions in antibacterial and immune-activating effects have been confirmed by numerous studies. This technique focused on the changes in the content of these two components. The protein, fat, moisture, and dry matter content of the sea cucumber body wall were determined using the method in Chinese National Standard GB 5009.5-2016. The sea cucumber saponin content of the sea cucumber body wall was determined using the method in National Standard GB / T 33108-2016. The monosaccharide composition of the enzymatic hydrolysate was determined using liquid chromatography. The detection method is as follows: Take the sample and hydrolyze it with trifluoroacetic acid solution at 110 ℃ for 2 h. Adjust the pH to 7 with sodium hydroxide and add water to a final volume of 25 ml. Take 1 ml of this solution and add 1 mL of 0.3 mol / L sodium hydroxide solution, then add 1 mL of 0.5 mol / L PMP methanol solution and incubate at 70 ℃ for 70 min. After cooling, add 1 mL of 0.3 mol / L acetic acid solution and add water to a final volume of 10 ml. Take 2 mL of the derivatized solution into a 10 mL stoppered test tube, extract with chloroform, and filter the upper layer through an organic filter membrane for detection. A Shimadzu high-performance liquid chromatograph was used. The chromatographic column was a C18 column (4.6 × 250 mm, 5 μm); column temperature was 40 ℃; detector was an SPD-20A UV detector; wavelength was 254 nm; flow rate was 1.0 ml / min; mobile phase A was 50 mmol / L KH₂PO₄; mobile phase B was acetonitrile; injection volume was 25 μL.

[0033] The results are shown in Table 3. When 5-15% of Sargassum powder was replaced with enzymatically hydrolyzed Sargassum in the feed, the content of sea cucumber saponins in the body wall increased by 23.6-27.1%, and the content of fucose increased by 25-40%. The main active ingredients in the sea cucumber body wall were significantly improved.

[0034] Table 3. Effects of adding enzymatically hydrolyzed Sargassum to feed on the nutrient composition of sea cucumber body wall.

[0035] 4. Adding deeply enzymatically hydrolyzed Sargassum to sea cucumber feed can significantly improve its immune and antioxidant properties. The aquaculture experiment was conducted as described above. After aquaculture, the intestines of sea cucumbers were collected, and the activities of catalase (CAT), superoxide dismutase (SOD), malondialdehyde (MDA), total nitric oxide (T-NOS), acid phosphatase (ACP), and alkaline phosphatase (AKP) were measured using a kit produced by Nanjing Jiancheng Bioengineering Institute. The results are shown in Table 4. Deep enzymatic hydrolysis of Sargassum significantly improved the immune and antioxidant indicators of sea cucumbers. The best performing experimental group was group D2, with a Sargassum hydrolysate replacement volume of 10%. SOD activity increased by 36.9%, CAT activity increased by 39.4%, MDA content decreased by 52.1%, total NOS activity increased by 25.5%, and AKP activity increased by 124.5%. The fucoidan produced by the enzymatic hydrolysis of brown algae itself has strong antioxidant activity and can significantly improve the antioxidant performance of fed sea cucumbers. AKP mainly participates in promoting the phagocytosis of pathogens by phagocytes, indicating that enzymatic hydrolysis of Sargassum promotes the function of phagocytes in the sea cucumber intestine.

[0036] Table 4. Effects of enzymatic hydrolysis of Sargassum on the intestinal antioxidant and immune properties of sea cucumber.

[0037] Note: D1 uses enzymatically hydrolyzed Sargassum instead of 5% Sargassum powder; D2 uses enzymatically hydrolyzed Sargassum instead of 10% Sargassum powder; D3 uses enzymatically hydrolyzed Sargassum instead of 15% Sargassum powder. In the same row, the same superscript letter or no letter indicates no significant difference (P>0.05), and different superscript letters indicate significant difference (P<0.05).

[0038] 5. Adding enzymatically hydrolyzed Sargassum extract to feed can significantly improve the intestinal health of sea cucumbers. The aquaculture experiment was conducted as described above. After the aquaculture was completed, intestinal tissue sections of sea cucumbers were observed and the composition of the intestinal microbiota was analyzed.

[0039] Observation of intestinal tissue sections: Sea cucumber intestines were fixed in 4% paraformaldehyde solution for 24 hours. The intestinal tissue was removed from the fixative, appropriately trimmed in a fume hood, and dehydrated with 75%-100% ethanol in progressively increasing concentrations. The tissue was embedded in paraffin, cut into 4μm thick sections, stained with hematoxylin and eosin, and photographed under a HistoCore AUTOCUT microscope (Germany). ImagePro Plus 6.0 software was used to measure intestinal villus width (VW), villus height (VH), and muscle layer thickness (MT). The results showed that sea cucumbers fed with enzymatically hydrolyzed Sargassum fusiforme showed increased villus width, decreased villus length, and more compact cells, indicating promotion of intestinal cell proliferation (e.g., Figure 1-8 ).

[0040] Analysis of the gut microbiota of sea cucumbers: Intestinal tissues were collected from the intestines of three sea cucumbers in the experimental group and group D2. Total DNA was extracted from the samples, and PCR amplification was performed using the following primers for the V3-V4 region of bacterial 16S rDNA: F: 5 -ACTCCTACGGGAGGCAGCA-3 ; 806R: 5 -GGACTACHVGGGTWTCTAAT-3

[0041] After purification, quantification, and homogenization, the products were sequenced using the Illumina Novaseq sequencing platform. The raw data underwent filtering, noise reduction, assembly, and chimera removal to obtain valid sequences, and representative sequences were annotated and tree-built. Operational taxonomic units (OTUs) were clustered using Usearch software with a similarity threshold of 97.0%. Diversity analysis of the sample sequences was performed using QIIME2 software (version 2024.5). Results showed that adding enzymatically hydrolyzed Sargassum to the feed significantly improved the intestinal microbial diversity of sea cucumbers. 931 unique OTUs were identified in the gut of the control group, while 1960 unique OTUs were identified in the experimental group, for a total of 225 OTUs. The Chao 1 index, Shannon index, and Simpson index of the experimental group were all higher than those of the control group (Table 5), indicating higher microbial diversity.

[0042] Analysis of the dominant gut microbiota of sea cucumbers yielded the following results: Figure 9 , 10As shown in Figure 11, at the phylum level, enzymatic hydrolysis of Sargassum increased the content of Firmicutes and Verrucomicrobiota bacteria in the sea cucumber gut. Firmicutes have functions such as maintaining the intestinal barrier, inhibiting harmful bacteria, and promoting nutrient absorption, while Verrucomicrobiota microorganisms have strong polysaccharide degradation capabilities and intestinal mucosal repair functions. Proteobacteria, mainly composed of Gram-negative bacteria, contain a variety of pathogens, and their relative abundance reduced the potential disease risk in aquatic animals. At the family level, enzymatic hydrolysis of Sargassum increased the abundance of Rhodobacteraceae and Akkermansiaceae, and the relative abundance of Rhodobacteraceae and Akkermansiaceae can improve the growth efficiency of aquatic animals. The addition of enzymatic hydrolyzed Sargassum reduced the abundance of Vibrionaceae bacteria. As a common pathogen in aquatic animals, the reduced abundance of Vibrionaceae helps to reduce inflammation and infection in aquatic animals. At the genus level, the addition of Sargassum enzymatic hydrolysate significantly increased the activity of Rhodopseudomonas spp. ( Rubritalea ) and *Phytophthora* genus ( Lutimonas Relative abundance. *Rhodopseudomonas* species possess the ability to produce carotenoids and squalene. Carotenoids and squalene have been reported to have antioxidant and antibacterial activities. *Sphaeromonas* is a halophilic marine bacterium that can alleviate high-salt stress damage in sea cucumbers. Enzymatic hydrolysis of *Sargassum fusiforme* significantly reduced the abundance of common aquatic pathogens such as *Vibrio*. Vibrio Bacterial abundance. The results of this study show that enzymatic hydrolysis of Sargassum can directly inhibit the growth and adhesion of Vibrio, and can also optimize the intestinal microecological structure by promoting the proliferation of probiotics, thus forming a barrier against pathogenic bacteria.

[0043] Table 5. Alpha diversity analysis of sea cucumber gut microbiota

Claims

1. A sea cucumber feed, characterized in that, The feed consists of 40g of marine mud, 15-25g of Sargassum powder, 5-15g of enzymatically hydrolyzed Sargassum liquid, and 1g of multivitamin and mineral premix per 100g of feed. The amount of enzymatically hydrolyzed Sargassum liquid is based on the dry weight of Sargassum. The enzymatically hydrolyzed Sargassum liquid is obtained by hydrolyzing Sargassum with alginate degrading enzyme, pectinase, and cellulase.

2. The sea cucumber feed according to claim 1, characterized in that, The multi-dimensional and multi-mineral premix comprises: thiamine 25mg, riboflavin 45mg, pyridoxine hydrochloride 20mg, vitamin B12 0.1mg, vitamin K3 10mg, inositol 800mg, calcium pantothenate 60mg, nicotinamide 200mg, folic acid 20mg, biotin 1.2mg, vitamin A acetate 32mg, vitamin D3 0.5mg, α-tocopherol 120mg, ethoxyquinoline 150mg, MgSO4•H2O 4000mg, MnSO4•H2O 50mg, KI 1mg, CoCl2 1mg, CuSO4•5H2O 20mg, FeSO4•H2O 260mg, ZnSO4•H2O 150mg, and Na2SeO3 0.5mg.

3. The sea cucumber feed according to claim 1, characterized in that, The sea cucumber feed also includes 5g soybean meal, 3g fish meal, 5g shrimp meal, 14.6g wheat flour, 1g squid paste, and 0.4g binder.

4. The sea cucumber feed according to claim 1, characterized in that, The sea cucumber feed consists of 40g of sea mud, 15-25g of Sargassum powder, 5-15g of enzymatically hydrolyzed Sargassum liquid, 1g of multivitamin and mineral premix, 5g of soybean meal, 3g of fish meal, 5g of shrimp meal, 14.6g of wheat flour, 1g of squid paste, and 0.4g of binder per 100g of feed.

5. The method for preparing sea cucumber feed according to any one of claims 1-4, characterized in that, After mixing the raw materials, add water and use a feed pelleting machine to make pellets with a particle size of 3mm. Dry them in a 60℃ oven until the moisture content is 10%, and store them in a sealed container at -20℃.

6. The method for preparing sea cucumber feed according to claim 5, characterized in that, The enzymatic hydrolysis solution of Sargassum fusiforme was obtained by the following method: The dried Sargassum fusiforme powder, pulverized to 300 mesh, along with alginate degrading enzyme, pectinase, and cellulase, were placed in an Erlenmeyer flask and mixed thoroughly. Buffer solution was added and mixed again. The flask was then placed in a water bath preheated to 47°C for enzymatic hydrolysis. During the hydrolysis process, the system was continuously mixed with a rotor. After the hydrolysis was completed, the water bath temperature was immediately adjusted to 100°C and reacted for 10 minutes to inactivate the enzymes in the reaction system. The flask was then removed and allowed to cool naturally to room temperature.

7. The application of the sea cucumber feed according to any one of claims 1-4 in sea cucumber farming, characterized in that, include: The sea cucumbers were fed the aforementioned feed twice a day, with 30% of the daily feed being given in the morning and 70% in the afternoon. The total amount of feed should be 1%-2% of the sea cucumber's body weight.

8. The application according to claim 7, characterized in that, During the aquaculture period, the water temperature should be 13-17℃, the salinity 30‰, the seawater pH 7.8, and the dissolved oxygen 6mg / L.

9. The application of the sea cucumber feed according to any one of claims 1-4 in improving the nutritional value of sea cucumbers.

10. The use of the sea cucumber feed according to any one of claims 1-4 in promoting the accumulation of sea cucumber saponins and fucose in the body wall of sea cucumber.

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