Preparation method of a river snail extract and application thereof in preparation of aquatic feed

Abstract

The application provides a preparation method of an Anodonta woodiana extract and application of the Anodonta woodiana extract in preparation of aquatic feed, and relates to the technical field of aquatic feed. The Anodonta woodiana extract is prepared by using the Anodonta woodiana muscle as raw material and by using the biological enzymolysis technology to direct hydrolysis of macromolecular protein into small-molecule functional peptide and free amino acid, so that umami amino acid and other flavoring substances are fully released. A plant lactobacillus Y-9 fermentation broken wall liquid is also provided, and the plant lactobacillus Y-9 fermentation broken wall liquid is rich in probiotic metabolites and bacterial active fragments. The Anodonta woodiana extract and the plant lactobacillus Y-9 fermentation broken wall liquid are jointly applied to the aquatic feed, and the two have a synergistic effect. The synergistic effect is significant in terms of the foraging effect, growth performance, feed utilization rate, immune index and survival rate, and the Anodonta woodiana extract and the plant lactobacillus Y-9 fermentation broken wall are a new efficient, natural and safe solution for aquatic functional feed additives.

Description

Technical Field

[0001] This invention relates to the field of aquatic feed technology, and in particular to a method for preparing an extract from the snail *Heliotropium indicum* and its application in the preparation of aquatic feed. Background Technology

[0002] Aquaculture is a pillar industry of my country's fishery economy. With the promotion of intensive aquaculture models and the rapid development of the aquatic feed industry, the demand for functional feed additives is becoming increasingly urgent. High-quality functional feed additives not only need to provide a balanced nutritional composition, but also should possess bioactive functions such as attracting appetite, promoting growth, and enhancing immunity. Currently, commercially available aquatic feed functional additives mainly include chemically synthesized amino acids (such as methionine and lysine), hydrolysates of terrestrial animal proteins (such as fishmeal hydrolysates and plasma protein powder), microbial fermentation products (such as yeast hydrolysates), and plant extracts (such as traditional Chinese medicine extracts). Chemically synthesized amino acids have low bioavailability, and excessive addition increases aquaculture costs and nitrogen emissions; fishmeal resources are becoming increasingly scarce, and prices continue to rise; plant proteins contain anti-nutritional factors, limiting their digestibility and absorption in aquatic animals. Therefore, developing new and high-quality raw material sources for aquatic functional feed additives is of great significance.

[0003] my country has abundant freshwater resources, and gastropod snails are distributed in various freshwater areas. They are usually the dominant group of benthic animals in lakes, providing excellent natural food for economically important fish such as grass carp, and playing an important role in the lake's nutrient cycle. (The text then abruptly shifts to a seemingly unrelated topic: *Hemiberlesia lataniae*.) Rivularia auriculata *Viviparus* is a freshwater snail found only in relatively clear lakes and rivers in Hunan, Hubei, Guizhou, Jiangxi, and Guangdong provinces of my country. It belongs to the phylum Mollusca, class Gastropoda, subclass Prosobranchia, order Gastropoda, family Viviparidae, and genus *Viviparus*. Individuals are relatively small, with an exceptionally thick shell containing four whorls. The spire is low-set, and the whorls are large, occupying the majority of the shell. The aperture is large, oval, with a thick, everted inner lip. Current research on *Viviparus* is extremely limited, primarily focusing on its population distribution, reproductive system, and morphological aspects of the digestive system. Research on its nutritional value and potential utilization remains largely unexplored.

[0004] On the other hand, the application of probiotics in aquaculture is becoming increasingly widespread. Conventional probiotic preparations, in the form of live bacteria, are easily used in the pelleting process of aquatic feed. However, due to high temperatures and extrusion shear forces, a large number of live bacteria are prone to death, resulting in a significant reduction in the number of effective live bacteria reaching the intestines of aquatic animals.

[0005] There are no existing reports on the application of combining spirulina enzymatic hydrolysis extract with Lactobacillus plantarum fermentation cell wall disruption liquid in aquatic feed. Summary of the Invention

[0006] Therefore, this invention proposes a method for preparing an extract from the snail *Heliotropium indicum* and its application in the preparation of aquatic feed.

[0007] The technical solution of this invention is implemented as follows: A method for preparing an extract from *Heliotropium indicum*, the specific preparation method comprising: S1. Remove the shell from the fresh ear snails, separate the muscle tissue, wash, drain, and homogenize to obtain ear snail muscle homogenate. S2. Add water to the homogenate of the ear snail muscle, adjust the pH to 6.0-8.5, add protease for enzymatic hydrolysis, and obtain the hydrolysate. S3. The enzyme hydrolysate is heated to inactivate the enzyme, cooled, centrifuged, and the supernatant is collected, concentrated, and dried to obtain the Hericium erinaceus extract.

[0008] Furthermore, in step S2, the amount of water added is 5-20 times the volume of the ear snail muscle homogenate.

[0009] Furthermore, in step S2, the amount of protease added is 0.1%-5% of the protein mass in the ear snail muscle homogenate; the protease is selected from one or more of papain, trypsin, neutral protease and alkaline protease.

[0010] Furthermore, the protease is a complex enzyme of papain and trypsin, with a mass ratio of 1:0.5-2.

[0011] Furthermore, in step S2, the enzymatic hydrolysis temperature is 40-55℃, the pH is 7.0-7.5, and the enzymatic hydrolysis time is 4-6h.

[0012] The extract of *Heliotropium indicum* obtained by this invention is a light yellow to light brown powder with a protein content of 55%-75%, a free amino acid content of 15%-35%, a small molecule peptide content of 20%-45%, a moisture content of ≤8%, and an ash content of ≤10%.

[0013] An application of an extract from *Heliotropium indicum* prepared by a method for preparing *Heliotropium indicum* extract in the preparation of aquatic feed.

[0014] Furthermore, the aquatic feed contains the *Heliotropium indicum* extract and *Lactobacillus plantarum* fermentation cell-wall breaking liquid.

[0015] Furthermore, the amount of the *Eriocheir sinensis* extract added to aquatic feed is 0.5wt%-3wt%.

[0016] Furthermore, the amount of the Lactobacillus plantarum fermentation cell wall-breaking liquid added to aquatic feed is 0.5wt%-3wt%.

[0017] Furthermore, the *Lactobacillus plantarum* fermentation cell-wall breaking broth is made by fermenting *Lactobacillus plantarum* Y... 9. Inoculate into MRS liquid medium and incubate statically at 36℃-38℃ for 12-20 h. Then, inoculate 3%-5% into fermentation medium and ferment at 36℃-38℃, pH 6.0±0.1, and a rotation speed of 100-200 rpm for 20-24 h to obtain the fermentation broth with a viable cell count greater than 2.5×10⁹ cells / day. 10 The fermentation broth was centrifuged at 3-5℃ and 7000-9000 rpm for 10-20 min to collect the cells. The bacterial sludge was resuspended in physiological saline and circulated 2-4 times at 750-850 bar to break the cell walls. The cell wall-broken bacterial suspension and the supernatant obtained by centrifugation were combined to obtain the Lactobacillus plantarum fermentation cell wall-broken broth, which was stored at 4℃ for later use.

[0018] Furthermore, the fermentation medium formula is as follows: glucose 45g / L, yeast extract 12g / L, rapeseed peptone 28g / L, ammonium citrate 4g / L, dipotassium hydrogen phosphate 3g / L, magnesium sulfate 0.2g / L, and manganese sulfate 0.04g / L.

[0019] Compared with the prior art, the beneficial effects of the present invention are: 1. The extract from the ear snail of this invention possesses both feeding-inducing and nutritional functions. Through a specific enzymatic hydrolysis process, it fully releases and enriches the natural umami amino acids (aspartic acid, glutamic acid, glycine, alanine, tyrosine, phenylalanine) and functional amino acids (lysine, proline, valine, threonine, etc.) in the ear snail muscle. Among these, the umami amino acids have a strong stimulating effect on the olfactory and gustatory chemoreceptors of aquatic animals, shortening the feeding latency period and significantly increasing feed intake and feed utilization. The functional amino acids can be directly absorbed and utilized by aquatic animals, participating in protein synthesis, promoting body protein deposition and muscle growth, and increasing weight gain.

[0020] 2. In this invention, the fermentation broth of Lactobacillus plantarum Y-9 is centrifuged to collect the bacterial cells, and then subjected to high-pressure cell disruption. The disrupted bacterial suspension is then combined with the fermentation supernatant for use. This process preserves the intracellular active components and fermentation metabolites of the bacterial cells, avoiding the loss of active substances caused by discarding the fermentation supernatant in conventional postbiotic preparation. The resulting product has more comprehensive functions.

[0021] 3. This invention uses the muscle of the freshwater shellfish *Rhizoctonia solani* as raw material, and utilizes bio-enzymatic hydrolysis technology to directionally hydrolyze large molecular proteins into small molecular functional peptides and free amino acids, fully releasing umami amino acids and other flavor substances to obtain *Rhizoctonia solani* extract. Simultaneously, it provides *Lactobacillus plantarum* Y-9 fermentation cell-wall-breaking liquid, which is rich in probiotic metabolites and active bacterial fragments. The combined application of *Rhizoctonia solani* extract and *Lactobacillus plantarum* fermentation cell-wall-breaking liquid to aquatic feed exhibits synergistic effects, demonstrating significant synergistic effects in terms of appetite stimulation, growth performance, feed utilization, immune indicators, and survival rate. This provides a highly efficient, natural, and safe new solution for functional feed additives in aquaculture. Detailed Implementation

[0022] To better understand the technical content of this invention, specific embodiments are provided below to further illustrate the invention.

[0023] Unless otherwise specified, the experimental methods used in the embodiments of this invention are all conventional methods.

[0024] Unless otherwise specified, all materials and reagents used in the embodiments of this invention are commercially available.

[0025] The *Lactobacillus plantarum* HEW-A490 strain of this invention is a commercially available strain with accession number CGMCC NO.12554, originating from the China General Microbiological Culture Collection Center.

[0026] The snails used in this invention are sourced from the Yuanjiang River basin in Changde City, Hunan Province.

[0027] Example 1 Take 50 kg of fresh, live river snails and keep them for 3-4 days to purge the mud and sand. Change the water once during this period and ensure continuous oxygenation. After removing them, clean the shells to remove any debris and dry the surface with filter paper. Crush the shells with pliers and use tweezers and a dissecting needle to separate the muscle tissue (foot muscles and mantle muscles) from the shell and internal organs. Wash the muscle tissue with distilled water and dry it with filter paper.

[0028] 1. Common nutritional components include crude moisture content, crude fat content, crude protein content, and crude ash content. Crude moisture content was determined using the direct drying method, according to GB / T 6435-2014; crude ash content was determined using the high-temperature ignition method, according to GB / T 6438-2007; crude protein content was determined using the Kjeldahl method, according to GB / T 6432-2018; and crude fat content was determined using a Soxhlet extractor, according to GB / T 6433-2006. The common nutritional components of the ear snail muscle are shown in Table 1.

[0029] Table 1. Routine nutritional composition of the muscle of the ear snail (by wet weight, %)

[0030] As shown in Table 1, the crude protein content of the muscle of the ear snail is higher than that of other common shellfish such as river clams, triangular sail mussels and hard clams, while the crude fat content is lower than that of most freshwater economic fish, exhibiting the nutritional characteristics of high protein and low fat.

[0031] 2. The muscle was crushed using a high-speed homogenizer and then freeze-dried and stored in an ultra-low temperature freezer at -70℃ for the determination of 25 amino acids.

[0032] Amino acid assay derivatization process: Take 50 μL of standard mixture, 50 μL of test sample, and 50 μL of protein precipitant. Mix well and centrifuge at 13200 rpm for 4 min. Take 10 μL of supernatant, add 50 μL of labeling buffer, mix well, and briefly incubate. Add 20 μL of derivatization solution, mix well, briefly incubate, and derivatize at 55℃ for 15 min. After derivatization, cool the sample in a refrigerator, mix well, briefly incubate, and take 50 μL for analysis.

[0033] Nineteen amino acids were detected in the muscle of the ear snail, and the results are shown in Table 2.

[0034] The umami amino acids include six types: aspartic acid, glutamic acid, glycine, alanine, tyrosine, and phenylalanine. The essential amino acids include ten types: histidine, threonine, arginine, isoleucine, leucine, valine, phenylalanine, tryptophan, lysine, and methionine.

[0035] Table 2. Amino acid composition and content of the muscle of the ear snail (mg / g dry weight)

[0036] Table 2 shows that the muscle of the ear snail is rich in amino acids, containing all 10 essential amino acids, with umami amino acids accounting for as much as 39.04% of the total amino acids. Alanine is the most abundant amino acid in the muscle, followed by lysine and proline. Alanine is not only a umami amino acid but also plays an important role in energy supply, muscle synthesis and repair, improving athletic performance, enhancing immune function, and promoting brain function. Lysine is an important essential amino acid that directly participates in protein synthesis and plays a vital role in animal growth and maintaining nervous system function; it is also known as a growth amino acid. Proline has physiological functions such as alleviating oxidative stress, participating in energy metabolism, regulating immunity, promoting protein synthesis, and improving animal growth and reproductive performance.

[0037] 3. Place the muscle sample stored at -20℃ in a glass petri dish and dry it at 105℃ until constant weight. Weigh the dried sample (approximately 0.2g) and place it in a 100ml PTFE beaker. Add 12ml of concentrated nitric acid and digest on a 95℃ hot plate for 2 hours. Cool to room temperature, add 4ml of hydrogen peroxide, heat on a 95℃ hot plate for 30 minutes, cool to room temperature, and dilute to a 50ml glass volumetric flask with ultrapure water. Filter the solution through a 0.45µm syringe filter into a 60ml PET bottle and store at 4℃ until analysis.

[0038] The trace element content of the muscle of the ear snail is shown in Table 3.

[0039] Table 3. Trace element content in the muscle of the ear snail (mg / kg dry weight)

[0040] As shown in Table 3, the highest Zn content (183.55 mg / kg) was found in the muscle of the ear snail, followed by Fe (96.68 mg / kg), Cu (30.58 mg / kg), Mn (15.03 mg / kg) and Se (0.82 mg / kg). Zn is a component and cofactor of many enzymes in animals, playing a crucial role in growth, development, and immune function; Fe is a component of hemoglobin and an activator of peroxidase, catalase, hydroxylase, and flavonoids, and in the ear snail, it may function more as a cofactor or activator of enzymes; Cu exists in many redox enzymes, participates in various biological processes, and has functions such as bactericidal, growth-promoting, and antioxidant properties, and is an essential element for maintaining the normal physiological function of the cardiovascular system; Mn is an essential trace element for the human body, existing as a cofactor of many enzymes, and is a component of many redox enzymes such as peroxidase and catalase, and has a strong immune-activating ability, playing an important role in development, reproduction, metabolism, nerve function, and antioxidation; Se is an essential component of glutathione peroxidase and has antioxidant functions.

[0041] 4. Grind the muscle into a fine powder, weigh an appropriate amount of sample, and add distilled water to homogenize. Take 1 ml of the homogenate and add 2 ml of n-hexane to a glass test tube, vortex for 2 min, let stand for 1 min, collect the supernatant, repeat 3 times, and combine the supernatants. Dry the extract with nitrogen gas. Reconstitute with 1 ml of acetonitrile / isopropanol (4:6) (containing 500 ng / ml chloramphenicol as internal standard), and then use high performance liquid chromatography-tandem mass spectrometry to detect the fatty acid composition and content of the soft tissue of *Triplophysa rhamnoides*. The fatty acid composition of *Triplophysa rhamnoides* muscle is shown in Table 4.

[0042] Table 4 Fatty acid composition of ear snail muscle (% total fatty acids)

[0043] Note: SFA stands for saturated fatty acids, MUFA stands for monounsaturated fatty acids, and PUFA stands for polyunsaturated fatty acids.

[0044] As shown in Table 4, the muscle of the ear snail is rich in fatty acids. The proportion of polyunsaturated fatty acids in the muscle is 45.36%, monounsaturated fatty acids are 14.13%, and saturated fatty acids are 40.51%, indicating an excellent fatty acid composition. Among them, arachidonic acid (C20:4) content is the highest, and the EPA+DHA content is 7.89%, which is significantly higher than that of many other freshwater aquatic products.

[0045] The more double bonds a PUFA has, the higher its degree of unsaturation, and the higher its nutritional value. PUFAs include the n-3, n-6, and n-9 series, with n-3 and n-6 ​​being the most bioactive.

[0046] Example 2 - Preparation of Hericium erinaceus extract S1. Remove the shell from the fresh live ear snails, separate the muscle tissue, weigh 5.0 kg of muscle tissue, wash, drain, homogenize, and obtain ear snail muscle homogenate. S2. Add deionized water to the homogenate of the muscle of the snail *Sinocyclocheilus* to a total volume of 50 L, adjust the pH to 7.2, add 12 g of compound enzyme (12.0% as determined by Kjeldahl nitrogen determination method) at 2% of the protein content in the homogenate. The compound enzyme is papain and trypsin in a 1:1 mass ratio. In a constant temperature water bath at 50 °C, stir at 100 rpm for 5 h to obtain the enzymatic hydrolysate. S3. Heat the enzymatic hydrolysate to 95℃ and hold for 15 min to inactivate the enzyme. After cooling to room temperature, centrifuge at 6000 rpm for 15 min and collect 46 L of supernatant. Discard the precipitate and concentrate the supernatant to 8 L under vacuum at 60℃ and -0.09 MPa. Dry the supernatant to obtain 405 g of *Helicobacter pylori* extract. The yield is 8.1% based on muscle wet weight.

[0047] The obtained *Heliotropium indicum* extract was a light yellow powder. Analysis showed that it contained 61.5% protein, 26.8% free amino acids, 31.2% small molecule peptides (molecular weight <5000 Da), 5.1% moisture, and 7.3% ash. Umami amino acids accounted for 45.9% of the total amino acids, essential amino acids accounted for 38.8%, and peptides with a molecular weight <3000 Da accounted for 74.6% of the total peptides.

[0048] Example 3 - Preparation of Lactobacillus plantarum Y-9 fermentation cell wall disruption broth (1) Lactobacillus plantarum Y 9. Inoculate into MRS liquid medium and incubate at 37°C for 18 hours to obtain seed culture; (2) The seed culture was inoculated into the fermentation medium at a rate of 4% (v / v). The fermentation medium formula was: glucose 45 g / L, yeast extract 12 g / L, rapeseed peptone 28 g / L, ammonium citrate 4 g / L, dipotassium hydrogen phosphate 3 g / L, magnesium sulfate 0.2 g / L, and manganese sulfate 0.04 g / L. The fermentation conditions were: fermentation at 37℃, pH 6.0±0.1, and a rotation speed of 150 rpm for 22 h to obtain the fermentation broth with a viable cell count greater than 2.5 × 10⁻⁶. 10 CFU / mL; (3) Centrifuge the fermentation broth at 4℃ and 8000 rpm for 15 min, collect the supernatant and bacterial sludge respectively, store the supernatant at 4℃ for later use, resuspend the bacterial sludge with physiological saline, and circulate it 3 times at 800 bar to break the cell wall. Combine the cell wall-broken bacterial suspension and the supernatant obtained by centrifugation to obtain the fermentation cell wall-broken liquid of Lactobacillus plantarum, and store it at 4℃ for later use.

[0049] Example 4 - Application effect of the compound of *Heliotropium indicum* extract and *Lactobacillus plantarum* fermentation cell wall disruption liquid in grass carp feed (1) Preparation of basic feed: A basic feed (32.0% crude protein and 6.0% crude fat) was prepared using 17 parts fishmeal, 23 parts soybean meal, 10 parts rapeseed meal, 8 parts cottonseed meal, 34 parts wheat flour, 1.8 parts fish oil, 1.8 parts soybean oil, 2.2 parts vitamin premix, and 2.2 parts mineral premix as raw materials. The following treatment groups were set up based on the basic feed: Control group: Basic feed, with no other additives; Experimental Group A: 1.5 wt% of the Hericium erinaceus extract prepared in Example 2 (equal amount to fish meal) was added to the basal feed. Experimental Group B: 1.5 wt% of the fermented cell wall-breaking liquid of Lactobacillus plantarum prepared in Example 3 was added to the basic feed (mixed evenly with the feed ingredients in liquid form, and the amount added was calculated based on dry matter). Experimental Group C: The basic feed was simultaneously supplemented with 1.5 wt% of the Hericium erinaceus extract prepared in Example 2 and 1.5 wt% of the Lactobacillus plantarum fermentation cell wall breaking liquid prepared in Example 3 (equal amount to fish meal, the fermentation cell wall breaking liquid was added in liquid form, and the amount added was calculated based on dry matter).

[0050] The feed for each treatment group was made into isonitrogenous and isoenergetic feed, which was crushed, sieved, mixed, pelleted (particle size 2.5 mm), and dried before use.

[0051] (2) Experimental fish and feeding management: 480 healthy grass carp with an initial weight of 50.0g±2.5g were randomly divided into 4 groups, with 3 replicates per group and 40 fish per replicate. They were raised in an indoor recirculating aquaculture system (breeding tank volume 300 L), with a water temperature of 26±2℃, dissolved oxygen ≥6 mg / L, pH 7.5±0.1, and ammonia nitrogen <0.05 mg / L. They were fed three times a day (8:00, 13:00, 18:00) at a daily feed amount of 3%-5% of the fish's body weight. The feed amount was adjusted by weighing every 2 weeks. The experimental period was 8 weeks.

[0052] (3) The growth performance results are shown in Table 5.

[0053] Table 5. Effects of *Heliotropium indicum* extract and *Lactobacillus plantarum* fermentation broth on growth performance and feed utilization of grass carp.

[0054] As shown in Table 5, there is a synergistic effect between the *Heliotropium indicum* extract and the *Lactobacillus plantarum* fermentation cell wall disruption liquid of the present invention. The small molecule peptides and flavor amino acids in the *Heliotropium indicum* extract improve the palatability and digestibility of feed, providing a highly efficient nutrient substrate for aquatic animals. At the same time, the organic acids in the *Lactobacillus plantarum* fermentation cell wall disruption liquid lower the intestinal pH, further activating the activity of intestinal digestive enzymes and improving the digestibility and utilization of nutrients. The immune-active substances enhance the immune defense capabilities of fish, reduce the energy consumption for maintaining immunity, and allow more nutrient energy to be used for body protein deposition and growth.

[0055] (4) Serum immune indicators: After the experiment, blood was collected from the tail vein of 9 fish in each group and serum immune indicators were measured. The results are shown in Table 6.

[0056] Table 6. Effects of *Heliotropium indicum* extract and *Lactobacillus plantarum* fermentation broth on serum immune indicators of grass carp.

[0057] Table 6 shows that the serum lysozyme and SOD activities of grass carp in each supplemented group were significantly higher than those in the control group, while the MDA content was significantly lower. Among them, experimental group C showed the most outstanding immune enhancement effect, with all immune indicators superior to the two single-supplemented groups. The immune enhancement mechanisms of the two components are complementary, and their combined use achieves a more comprehensive immune regulation effect.

[0058] Example 5 - Application effect of the combination of *Heliotropium indicum* extract and *Lactobacillus plantarum* fermentation cell wall disruption liquid in *Litopenaeus vannamei* feed (1) Preparation of basic feed: A basic feed (40.0% crude protein, 8.0% crude fat) was prepared using 33 parts fishmeal, 17 parts soybean meal, 14 parts peanut meal, 23.5 parts wheat flour, 5 parts shrimp shell powder, 3 parts fish oil, 1.5 parts soybean lecithin, 1.5 parts vitamin premix, and 1.5 parts mineral premix as raw materials. The following treatment groups were set up based on the basic feed: Control group: basal feed; Experimental group: 1.0 wt% of the Hericium erinaceus extract prepared in Example 2 and 1.0 wt% of the Lactobacillus plantarum fermentation cell wall breaking liquid prepared in Example 3 were added to the basic feed (equal amount to fish meal, and the amount of fermentation cell wall breaking liquid added was calculated based on dry matter).

[0059] The feed is made into isonitrogenous and isoenergetic feed, which is then crushed, sieved (80 mesh), mixed, pelleted (1.5 mm in diameter), and dried for later use.

[0060] (2) Experimental shrimp and rearing management: 300 healthy Litopenaeus vannamei shrimp with an initial weight of 1.00g±0.10g were randomly divided into 2 groups, with 3 replicates per group and 50 shrimp per replicate. They were reared in an indoor recirculating aquaculture system (200L tank volume) at a water temperature of 28±1℃, salinity of 15‰, dissolved oxygen ≥5 mg / L, and pH of 8.0±0.2. They were fed 4 times a day (7:00, 12:00, 17:00, 21:00) at a daily feed amount of 5%-8% of the shrimp's body weight. The experimental period was 6 weeks.

[0061] The results are shown in Table 8.

[0062] Table 8. Effects of the combination of *Heliotropium indicum* extract and *Lactobacillus plantarum* fermentation broth on *Litopenaeus vannamei*.

[0063] Table 8 shows that the combined addition of 1.0 wt% *Litopenaeus vannamei* extract and 1.0 wt% *Lactobacillus plantarum* fermentation cell-wall breaking liquid to the feed of Litopenaeus vannamei increased the weight gain rate by 32.1%, decreased the feed conversion ratio by 19.4%, and increased the survival rate by 14.1% compared to the control group. Litopenaeus vannamei has a relatively short intestine and a relatively simple digestive enzyme system, making the direct absorption of small molecule peptides and free amino acids particularly important for its growth. Simultaneously, the organic acids and bacteriocins in the *Lactobacillus plantarum* fermentation cell-wall breaking liquid have an antagonistic effect on common Vibrio pathogens in the shrimp's intestine, which is beneficial for maintaining intestinal health and improving survival rate.

[0064] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing an extract from *Heliotropium indicum*, characterized in that, Specific preparation methods include: S1. Remove the shell from the fresh ear snails, separate the muscle tissue, wash, drain, and homogenize to obtain ear snail muscle homogenate. S2. Add water to the homogenate of the ear snail muscle, adjust the pH to 6.0-8.5, add protease for enzymatic hydrolysis, and obtain the hydrolysate. S3. The enzyme hydrolysate is heated to inactivate the enzyme, cooled, centrifuged, and the supernatant is collected, concentrated, and dried to obtain the Hericium erinaceus extract.

2. The method for preparing an extract from *Heliotropium indicum* as described in claim 1, characterized in that, In step S2, the amount of water added is 5-20 times the volume of the ear snail muscle homogenate.

3. The method for preparing an extract from *Heliotropium indicum* as described in claim 1, characterized in that, In step S2, the amount of protease added is 0.1%-5% of the protein mass in the ear snail muscle homogenate; the protease is selected from one or more of papain, trypsin, neutral protease and alkaline protease.

4. The method for preparing an extract from *Heliotropium indicum* as described in claim 3, characterized in that, The protease is a complex enzyme of papain and trypsin, with a mass ratio of 1:0.5-2.

5. The method for preparing an extract from *Heliotropium indicum* as described in claim 1, characterized in that, In step S2, the enzymatic hydrolysis temperature is 40-55℃, the pH is 7.0-7.5, and the enzymatic hydrolysis time is 4-6h.

6. The use of the extract of *Heliotropium indicum* prepared by the method according to any one of claims 1-5 in the preparation of aquatic feed.

7. In the application described in claim 6, the characteristic is that, The aquatic feed contains the *Heliotropium indicum* extract and *Lactobacillus plantarum* fermentation cell wall disruption liquid.

8. In the application described in claim 7, the characteristic is that, The amount of the *Heliotropium indicum* extract added to aquatic feed is 0.5wt%-3wt%.

9. In the application described in claim 7, the characteristic is that, The amount of the fermented Lactobacillus plantarum cell wall-breaking liquid added to aquatic feed is 0.5wt%-3wt%.

10. In the application as described in claim 9, the characteristic is that, The *Lactobacillus plantarum* fermentation cell-wall breaking broth is made by fermenting *Lactobacillus plantarum* Y...

9. Inoculate into MRS liquid medium and incubate statically at 36℃-38℃ for 12-20 h. Then, inoculate 3%-5% into fermentation medium and ferment at 36℃-38℃, pH 6.0±0.1, and a rotation speed of 100-200 rpm for 20-24 h to obtain the fermentation broth with a viable cell count greater than 2.5×10⁹ cells / day. 10 The fermentation broth was centrifuged at 3-5℃ and 7000-9000 rpm for 10-20 min to collect the cells. The bacterial sludge was resuspended in physiological saline and circulated 2-4 times at 750-850 bar to break the cell walls. The cell wall-broken bacterial suspension and the supernatant obtained by centrifugation were combined to obtain the Lactobacillus plantarum fermentation cell wall-broken broth, which was stored at 4℃ for later use.

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