A type of aquatic feed yeast and its application in aquaculture

Abstract

This invention discloses a type of aquatic feed yeast, *Rhodotorula buergerianum*, and its application in aquaculture, relating to the field of microbial technology, specifically to a type of *Rhodotorula buergerianum* Ho7-3 (… Rhodotorula toruloides Ho7-3 can be applied in aquaculture, and it can enhance the activity of intestinal digestive enzymes, immune response and antioxidant defense system in fish, thus significantly improving the growth performance of farmed fish.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a type of aquatic feed yeast, Rhodotorula buergerianum, and its application in aquaculture. Background Technology

[0002] Probiotics, as a class of microorganisms that benefit host health, exert their effects primarily through mechanisms such as regulating gut microbiota homeostasis, enhancing immune responses, and improving nutrient metabolism, and are therefore widely used in human and livestock farming. Currently, exogenous probiotics suitable for terrestrial hosts (such as lactic acid bacteria, Lactobacillus rhamnosus, Bifidobacterium, Bacillus, and yeast) have been frequently used in aquaculture for economically important fish farming. However, recent research has revealed potential risks of these non-native probiotics in aquatic animals, including the development of colonization resistance, induction of intestinal tissue damage in fish, the risk of feeding cessation, alteration of fish nutrient metabolism, overactivation of immune responses, and induction of gut microbiota imbalance. A typical case comes from the human-derived star strain Lactobacillus rhamnosus GG (LGG): although it exhibits excellent intestinal protection and pathogen resistance in mammals, its fimbriae protein SpaC directly causes intestinal damage in zebrafish by inducing pyroptosis of intestinal epithelial cells and gut microbiota dysbiosis. This finding highlights the host-specific safety risks in the cross-species application of probiotics. Therefore, when applying non-native probiotics in aquaculture, the safety and efficacy of the strains must be evaluated first. Enhancing the discovery of native aquatic probiotics and promoting the standardized application of feed probiotics will be the core strategy to avoid negative effects caused by non-native probiotics.

[0003] Yeast, rich in nutrients such as protein, essential amino acids, vitamins, and growth factors, and possessing both safety and palatability advantages, has become a key component of aquatic feed additives. Marine yeast can also secrete various extracellular enzymes and killer factors, such as cellulase, alkaline protease, and amylase, further enhancing its value as a feed additive. Functionally, yeast can not only partially replace fishmeal to address protein shortages but also improve the digestibility and absorption efficiency of fish and enhance immunity: its enzyme system effectively degrades large molecular substrates such as starch, cellulose, and protein, converting them into easily absorbed small molecules; under anaerobic conditions, yeast breaks down sugars into ethanol through glycolysis, a metabolite that inhibits the proliferation of pathogens and optimizes the intestinal flora structure through competitive exclusion; simultaneously, unique cell wall components (such as β-glucan and mannan oligosaccharides) directly activate non-specific immune pathways, promoting the development of immune organs and enhancing the body's disease resistance. In recent years, with the rapid expansion of my country's aquaculture industry, yeast preparations have been widely used as aquatic feed additives, and their multi-functional characteristics provide crucial support for sustainable green aquaculture.

[0004] Therefore, developing a multi-functional yeast preparation that can be applied to aquaculture has become a research hotspot in this field in recent years. Summary of the Invention

[0005] This invention relates to a type of aquatic feed yeast, Rhodotorula buergerianum, and its application in aquaculture. It possesses strong antioxidant activity, high protein content, and high essential amino acid content, and exhibits significant colonization ability in the intestinal environment of fish. It can effectively promote the growth performance of aquatic animals, reduce the feed conversion ratio, enhance the body's immune response and antioxidant level, thereby improving disease resistance.

[0006] On the one hand, the present invention provides a Rhodotorula buergerianum Ho7-3, which is deposited at the China Center for Type Culture Collection with accession number CCTCC NO: M 20252122.

[0007] Rhodotorula rubrum Ho7-3 ( Rhodotorula toruloides Ho7-3 is a microorganism belonging to the genus Rhodozyma, invented by the inventor from the oysterfish (Hexagrammos ogora). Hexagrammos otakii It was isolated and screened from the intestinal environment of ) and officially named after being identified by the inventor. Rhodotorula toruloides Ho7-3. Molecular biological identification revealed that the ITS sequence of *Rhodotorula circophylla* Ho7-3, as shown in SEQ ID NO.1, indicates that it is a novel *Rhodotorula circophylla* strain.

[0008] Rhodotorula glutinis Ho7-3 can colonize the intestinal environment of fish, helping to improve fish growth performance, reduce feed conversion ratio, enhance immune response and antioxidant levels, thereby improving disease resistance. In in vitro tests, Rhodotorula glutinis Ho7-3 exhibited high scavenging rates of DPPH free radicals, hydroxyl radicals, and superoxide anions, as well as anti-lipid peroxidation capacity and reducing power, demonstrating significant antioxidant activity. Furthermore, in vitro experiments confirmed that the antioxidant capacity of intact Rhodotorula glutinis Ho7-3 cells was significantly superior to that of intracellular lysate supernatant and heat-inactivated cells, proving that live Rhodotorula glutinis Ho7-3 is a key factor in achieving its antioxidant effect.

[0009] Rhodotorula buergerianum Ho7-3 contains a variety of amino acids that can meet the essential amino acid requirements of fish. It can be used as a high-quality protein source feed, improve fish growth performance, enhance immunity and improve digestive function, thereby improving aquaculture efficiency.

[0010] In another aspect, the present invention provides a microbial agent comprising Rhodotorula rubra Ho7-3, wherein Rhodotorula rubra Ho7-3 is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20252122.

[0011] Based on the effects of Rhodotorula buergerianum Ho7-3 after colonization in the intestinal environment of fish, another aspect of the present invention provides the application of Rhodotorula buergerianum Ho7-3 or the above-mentioned bacterial agent in aquaculture.

[0012] Aquatic animals include, but are not limited to, fish, crustaceans, mollusks, and shellfish. In some embodiments, the aquatic animals are fish. By promoting the colonization of Rhodotorula glutinis Ho7-3 in the intestinal environment of fish, the antioxidant, growth-promoting, immunity-enhancing, and digestive-improving effects of Rhodotorula glutinis Ho7-3 are achieved.

[0013] There are many methods for colonizing the intestinal environment of aquatic animals. Besides feeding, the colonization of *Rhodotorula flocculationiensis* Ho7-3 in the aquatic animal intestine can also be promoted by regulating the aquatic environment. As a non-limiting embodiment, this invention provides the application of *Rhodotorula flocculationiensis* Ho7-3 or the aforementioned inoculum as a feed additive. Using *Rhodotorula flocculationiensis* Ho7-3 as a feed additive not only promotes its colonization in the aquatic animal intestine but also fully utilizes its ability to supplement the nutrition of aquatic animals, thereby improving their growth performance.

[0014] In some embodiments, the feed additive is a fish feed additive.

[0015] Those skilled in the art will understand that there are various methods for integrating microbial additives, especially yeast, into feed, and these methods are not limited to a single specific process. Some conventional methods include, but are not limited to, surface spraying, mixing and adding, post-granulation adsorption, and integration into fermented feed. As a non-limiting example, the *Rhodotorula glutinis* Ho7-3 of this invention can be added to fish feed pellets via surface spraying, as follows:

[0016] - Prepare a suspension of Rhodotorula glutinis Ho7-3;

[0017] - Use a sterile spraying system to evenly inoculate the suspended bacterial solution onto the surface of the feed pellets.

[0018] In vivo experiments on fish have shown that Rhizopus cylindrica Ho7-3 can increase the activity of enzymes such as trypsin and amylase in the fish intestine. Therefore, another aspect of the present invention provides the use of Rhizopus cylindrica Ho7-3 or the above-mentioned bacterial agent to enhance the activity of digestive enzymes in the fish intestine, or its use in the preparation of compositions for enhancing the activity of digestive enzymes in the fish intestine.

[0019] Furthermore, in vivo experiments on fish have demonstrated that Rhodotorula buergerianum Ho7-3 can increase the levels of immunoglobulin M (IgM), complement 3 (C3), and complement 4 (C4) in the blood of fish, and activate the activities of acid phosphatase (ACP) and alkaline phosphatase (AKP). Therefore, another aspect of the present invention provides the use of Rhodotorula buergerianum Ho7-3 or the above-mentioned agents for enhancing the immune response of fish, or for use in the preparation of compositions for enhancing the immune response of fish.

[0020] In addition, based on the high scavenging rate of Rhizopus cylindrica Ho7-3 against DPPH free radicals, hydroxyl free radicals, and superoxide anions, as well as its anti-lipid peroxidation ability and reducing power, another aspect of the present invention provides the use of Rhizopus cylindrica Ho7-3 or the above-mentioned bacterial agent for optimizing the antioxidant status of fish, or its use in the preparation of compositions for optimizing the antioxidant status of fish.

[0021] On the other hand, the present invention provides a method for raising aquatic animals, including feeding aquatic animals feed containing Rhodotorula glutinis Ho7-3.

[0022] The present invention has the following advantages and effects:

[0023] 1. The Rhodotorula buergerianum Ho7-3 provided by this invention has excellent antioxidant capacity. After colonizing the intestines of fish, it can help fish remove oxidants such as DPPH free radicals, hydroxyl free radicals, and superoxide anions present in their intestines, thereby improving the health level of farmed fish and thus achieving the effect of increasing aquaculture yield and aquatic product quality.

[0024] 2. The Rhodotorula buergerianum Ho7-3 provided by this invention has an excellent ability to enhance the activity of digestive enzymes. After colonizing the intestines of fish, it can help farmed fish digest and absorb nutrients, thereby improving the quality of aquatic products.

[0025] 3. The Ho7-3 rhodotorula spp. provided by this invention can significantly improve the immune response of fish. After colonizing the intestines of fish, it can enhance the fish's resistance to diseases and improve the health level of farmed fish, thereby achieving the effect of increasing aquaculture yield and aquatic product quality.

[0026] 4. The Rhodotorula buergerianum Ho7-3 provided by this invention is isolated and screened from the intestinal environment of fish. It has excellent intestinal tolerance and no biofilm formation ability. It is friendly to the fish intestine and does not damage the intestinal environment of fish. It can be used as a probiotic for fish. Attached Figure Description

[0027] Figure 1 This is a colony morphology diagram of strain Ho7-3.

[0028] Figure 2This is a cell morphology diagram of strain Ho7-3.

[0029] Figure 3 A phylogenetic tree for strain Ho7-3 constructed based on its ITS sequence.

[0030] Figure 4 The graph shows the DPPH free radical scavenging rate results in Example 2.

[0031] Figure 5 The graph shows the lipid peroxidation inhibition rate results in Example 2.

[0032] Figure 6 The graph shows the hydroxyl radical scavenging rate results in Example 2.

[0033] Figure 7 This is a diagram showing the reduction force results in Example 2.

[0034] Figure 8 The graph shows the superoxide anion scavenging rate results in Example 2.

[0035] Figure 9 The graph shows the sum of the five antioxidant capacities in Example 2.

[0036] Figure 10 This is a photograph of the biofilm formation capacity test in Example 3.

[0037] Figure 11 The graph shows the growth performance test results in Example 5.

[0038] Figure 12 The graph shows the results of transaminase activity assay in Example 5.

[0039] Figure 13 This is a graph showing the serum immunogenicity assay results from Example 5.

[0040] Figure 14 The graph shows the results of the antioxidant capacity measurement in Example 5.

[0041] Figure 15 The graph shows the results of the digestive enzyme activity assay in Example 5. Detailed Implementation

[0042] To make the objectives, features, and advantages of this invention more apparent and understandable, the invention will be further described in detail below with reference to embodiments and accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without inventive effort are within the scope of protection of the appended claims.

[0043] Unless otherwise specified, all experimental reagents and materials used in this invention are commercially available.

[0044] Example 1: Isolation, screening and identification of Rhodotorula glutinis Ho7-3

[0045] 1.1 Separation and Screening

[0046] Anhydrous ethanol was used to treat *Hexagrammos otakii* ( Hexagrammos otakii After surface disinfection, the *Hexagrammos oyster* was dissected and its intestinal tissue was separated in a biosafety cabinet. The intestinal tissue was homogenized in a sterile grinder, and 100 μL of the homogenate was evenly spread onto YPD solid medium plates containing double antibiotics (chloramphenicol 100 μg / mL + ampicillin 100 μg / mL). The plates were incubated at 28°C for 72 h. After incubation, single yeast colonies grew on the medium. Strains with the following characteristics were selected:

[0047] On culture plates, colonies are round, pink, about 2-3 mm in diameter, with a moist, opaque surface, smooth edges without fuzz, and no raised wrinkles in the center. Under a light microscope, the cells are mainly spherical or oval in shape, reproduce by budding, and lack hyphae and pseudohyphae. (Specific details are as follows...) Figure 1 and Figure 2 As shown.

[0048] Among them, the Otaki hexagram was purchased from Aoshan Bay Wharf in Qingdao.

[0049] 1.2 Identification

[0050] The selected strain was identified by ITS sequence analysis, and its ITS sequence is shown in SEQ ID NO.1, confirming that the strain is *Rhodotorula buergerianum*. Rhodotorula toruloides It was named Rhodotorula circophylla Ho7-3, and its phylogenetic tree is as follows: Figure 3 As shown.

[0051] Example 2 In vitro antioxidant test

[0052] 2.1 Reagents and Materials

[0053] YPD liquid culture medium, PBS buffer, DPPH kit, hydroxyl radical kit, superoxide anion kit, linoleic acid emulsion, 0.01% FeSO4 solution, 0.2 mmol / L H2O2, TCA, TBA, BHT solution, chloroform, potassium ferricyanide solution, trichloroacetic acid, ferric chloride solution.

[0054] 2.2 Instruments

[0055] Water bath, constant temperature shaker, benchtop centrifuge, multi-functional microplate reader.

[0056] 2.3 Samples to be tested and grouping

[0057] The Ho7-3 strain was divided into three groups: live bacteria group, intracellular contents group, and heat-inactivated cells group.

[0058] 2.4 Experimental Methods

[0059] Single colonies were picked from the YPD solid medium in Example 1 and inoculated into 50 mL of YPD liquid medium. After incubation at 28°C and 180 rpm for 24 h with shaking, the bacterial cells were collected by centrifugation, washed with PBS, and resuspended to 10 mL. 6 CFU / mL concentration. The bacterial suspension was divided into three equal parts: live bacteria group, intracellular substance group, and heat-inactivated group. The intracellular substance group was autoclaved, centrifuged, and the supernatant was collected; the heat-inactivated group was boiled at 100℃ for 30 min; the live bacteria group was not treated. The DPPH radical scavenging activity, hydroxyl radical scavenging activity, and superoxide anion scavenging activity of each group of samples were tested using a DPPH kit, a hydroxyl radical scavenging kit, and a superoxide anion scavenging kit, respectively.

[0060] The lipid peroxidation inhibition rate of each sample was determined using the following method:

[0061] Add 0.5 mL of sample, 0.5 mL of PBS, 1 mL of linoleic acid emulsion, 0.2 mL of 0.01% FeSO4 solution, and 0.2 mmol / L H2O2 to each test tube. Set up three replicates for each sample, with PBS serving as a blank control. Incubate at 37°C in the dark for 12 h. After the reaction, add 0.2 mL of 4% TCA, 2 mL of 0.8% TBA, and 0.2 mL of 0.4% BHT solution to each test tube. Incubate at 100°C for 30 min, then rapidly cool in ice water. Add 2.5 mL of chloroform, mix thoroughly, centrifuge at 12000 rpm for 5 min, and measure the absorbance at 532 nm using the supernatant, zeroing the sample with PBS.

[0062] Lipid peroxidation inhibition rate = (1-A) 532 (Sample) / A 532 (Blank) × 100%

[0063] The reducing power of each sample was determined using the following method:

[0064] Take 0.5 mL of each sample solution and place it in a test tube. Add 1 mL of PBS buffer solution and 1 mL of 1% potassium ferricyanide solution sequentially. After mixing, react in a 50 °C water bath for 30 min. Then, quickly add 1 mL of 10% trichloroacetic acid to the reaction solution, mix thoroughly, and centrifuge at 5000 rpm for 10 min. Take 500 μL of the supernatant, add 500 μL of distilled water and 100 μL of 0.1% ferric chloride solution, and incubate the reaction system in the dark for 20 min. Use PBS instead of the sample as a blank control, and measure the absorbance of the mixture at 700 nm using a spectrophotometer.

[0065] 2.5 Experimental Results

[0066] The results of DPPH free radical scavenging rate are shown in Figure 4 The results of lipid peroxidation inhibition rate are shown in [the table below]. Figure 5 The results of hydroxyl radical scavenging rate are shown in [the table below]. Figure 6 The reducing power results are shown in Figure 7 The results of superoxide anion scavenging rate are shown in [the table below]. Figure 8 The sum of the five antioxidant capacities is shown in [the original text]. Figure 9 .

[0067] The results showed that Ho7-3 possesses broad-spectrum antioxidant activity. Its live bacteria group, intracellular substance group, and heat-inactivated cell group all exhibited certain levels of free radical scavenging ability, lipid peroxidation inhibition ability, and reducing power. In the DPPH free radical scavenging activity index, the scavenging rates of all three groups were at a high level, namely 90.34% (live bacteria group), 88.46% (intracytoplasmic substance group), and 84.04% (heat-inactivated cell group). The reducing power levels of the three groups were also similar, with the live bacteria group showing the highest reducing power. Regarding lipid peroxidation inhibition rate, the live bacteria group showed the highest inhibition activity at 25.59%, followed by the intracellular substance group, while the heat-inactivated cell group showed the lowest. In terms of hydroxyl free radical scavenging ability, the live bacteria group had a scavenging rate as high as 89.28%, the intracellular substance group 34.86%, and the heat-inactivated cell group only 11.11%.

[0068] In summary, a comprehensive evaluation of the total antioxidant capacity of Ho7-3 shows a clear gradient relationship between the antioxidant activities of the live bacteria group, the intracellular substance group, and the heat-inactivated cell group. The live bacteria group has the highest antioxidant activity, followed by the intracellular substance group, and the heat-inactivated cell group has the lowest.

[0069] Example 3: In vitro gut activity test

[0070] 3.1 Reagents and Materials

[0071] PBS buffer (pH 7.4), toluene, xylene, potassium nitrate solution (0.1 mol / L), simulated saliva, simulated gastric juice, simulated intestinal juice, and YPD medium.

[0072] Simulated saliva components: 0.22 g / L CaCl2, 6.2 g / L NaCl, 2.2 g / L KCl, 1.2 g / L NaHCO3 and 100 mg / L lysozyme, pH 6.5.

[0073] Simulated gastric juice components: 0.9% NaCl, 3 g / L pepsin, pH 2.5.

[0074] The simulated intestinal fluid composition consisted of 1 g / L trypsin, 3 g / L bile extract, 6.5 g / L NaCl, 0.835 g / L KCl, 0.22 g / L CaCl2, and 1.386 g / L NaHCO3, with a pH of 7.0.

[0075] 3.2 Instruments

[0076] Incubator, shaker, benchtop centrifuge, multi-functional microplate reader.

[0077] 3.3 Sample to be tested

[0078] Ho7-3 live bacterial cells.

[0079] 3.4 Experimental Methods

[0080] 3.4.1 Cell Surface Hydrophobicity (CSH) Analysis

[0081] Toluene and xylene were used as solvent systems to detect the hydrophobicity of the samples in toluene and xylene solvents, respectively. First, the sample was suspended in 10 mL of potassium nitrate solution, and its absorbance (A0) was measured at 600 nm. Then, 3 mL of toluene or xylene was added to the sample, vortexed for 30 s, and allowed to stand for 60 min. The change in absorbance of the aqueous phase (A0) was then measured. F And calculate CSH. The formula for calculating CSH is as follows:

[0082] CSH(%) =[1-(A F / A0)] ×100%

[0083] Where A0 is the absorbance of the aqueous phase before the reaction, A F The absorbance of the aqueous phase after the reaction is given.

[0084] 3.4.2 Continuous simulated gastrointestinal digestion test

[0085] First, the sample was placed in simulated saliva and incubated at 37°C with shaking for 5 minutes. After centrifugation, it was transferred to simulated gastric juice for 2 hours of digestion and incubation, and finally transferred to simulated intestinal juice for 4 hours of digestion and incubation. The final bacterial survival rate was calculated using the plate count method. The formula for calculating the bacterial survival rate is as follows:

[0086] Strain survival rate (%) = (N t / N0)×100%

[0087] Where N0 is the initial viable count, N t The viable count is t.

[0088] 3.4.3 Biofilm formation capacity test

[0089] Using the 96-well plate method, bacterial suspension (10 6 CFU / mL was inoculated into YPD medium and incubated at 28°C for 48 h. The presence or absence of biofilm formation was then observed.

[0090] 3.5 Experimental Results

[0091] The results showed that Ho7-3 had CSH values ​​of 21.27% and 29.71% in toluene and xylene solvent tests, respectively. This low hydrophobicity facilitates binding to hydrophilic intestinal mucosa, thereby improving the colonization efficiency of the strain. Furthermore, in continuous simulated gastrointestinal digestion experiments, the final survival rate of Ho7-3 was 22.74%, demonstrating excellent intestinal tolerance; biofilm formation ability results are shown below. Figure 10 The results showed that Ho7-3 had no biofilm formation ability and met the safety standards for probiotics.

[0092] Example 4: Nutritional Evaluation of Protein and Amino Acids

[0093] The crude protein content of strain Ho7-3 was determined by the Kjeldahl method (GB5009.5-2016), and the results showed that the crude protein content of strain Ho7-3 reached 72.57% of the dry weight.

[0094] After hydrolyzing strain Ho7-3 with hydrochloric acid, the amino acids in strain Ho7-3 were quantitatively analyzed using a fully automated amino acid analyzer. The results showed that its specific composition was threonine (1.31 g / 100g DW), valine (1.82 g / 100g DW), methionine (0.11 g / 100g DW), isoleucine (1.23 g / 100g DW), leucine (2.31 g / 100g DW), phenylalanine (1.31 g / 100g DW), lysine (2.04 g / 100g DW), histidine (0.67 g / 100g DW), and arginine (2.27 g / 100g DW). Based on the essential amino acid requirements of fish, the essential amino acid index (EIA) was calculated. The EAAI (Effective Acids and Proteins) value was 127.07 (a baseline value of 100 indicates that the strain fully meets the requirements of the target animal), indicating that the strain can meet the essential amino acid requirements of fish and has the potential to be developed as a high-quality protein source for feed.

[0095] EAAI calculation formula:

[0096] ;

[0097] Where aa n The content of the nth essential amino acid in the strain (g / 100g protein), AA n is the corresponding amino acid requirement for fish (g / 100g protein), and n is the number of essential amino acid types.

[0098] Example 5: In vivo fish testing

[0099] 5.1 Reagents and Materials

[0100] YPD culture medium, basal feed, PBS, physiological saline, aspartate aminotransferase (AST) kit G0424W, alanine aminotransferase (ALT) kit G0423W, blood collection tubes, IgM enzyme-linked immunosorbent assay (ELISA) kit MM-33677O2, C3 ELISA kit MM-33667O2, C4 ELISA kit MM-1690O2, AKP activity assay kit, ACP activity assay kit, SOD-WST-8 activity assay kit, CAT kit, GSH-Px kit, GSH content kit, ROS content kit, MDA content kit, trypsin kit, α-amylase (starch-iodine colorimetric method) activity assay kit, lipase activity kit.

[0101] turbot( Scophthalmus maximus The juvenile fish were purchased from a turbot farm in Haiyang, Yantai City, Shandong Province.

[0102] 5.2 Instruments

[0103] Water bath, constant temperature incubator, constant temperature shaker, benchtop centrifuge, multi-functional microplate reader.

[0104] 5.3 Samples to be tested and grouping

[0105] Experimental group: basal feed + Ho7-3;

[0106] Control group: basal feed.

[0107] 5.4 Experimental Methods

[0108] Single colonies of strain Ho7-3 were inoculated into 50 mL of YPD medium (28℃, 180 rpm, 15 h) for activation. The bacterial culture was washed by centrifugation with sterile physiological saline (6000 rpm, 4℃, 5 min, repeated 3 times). Then, it was sprayed through a sterile spray system (pressure 0.2 MPa, 20 cm from the feed) to a final concentration of 10. 6CFU / g was uniformly inoculated onto the surface of the basal feed pellets, dried at 4℃ in the dark, and then sealed and stored in a moisture-proof container. Juvenile turbot with an initial body weight of 20.0±0.5 g were randomly divided into a control group and an experimental group, with three replicates per group (30 fish per replicate). They were cultured in a recirculating aquaculture system (water temperature 20±0.5℃, salinity 30‰, DO ≥ 6 mg / L). The experimental group was precisely fed basal feed sprayed with Ho7-3 strain at 08:00 and 18:00 daily, at 3% of the fish's body weight. The control group was precisely fed ordinary basal feed at 08:00 and 18:00 daily, at 3% of the fish's body weight. A cyclical feeding pattern of 5 consecutive days + 1 day of fasting was adopted, with a culture period of 8 weeks.

[0109] 5.4.1 Growth performance test

[0110] Weigh and measure body length (accurate to 0.01 g) after fasting for 24 hours at the beginning and end of the experiment.

[0111] Body length growth rate (%) = [(final body length - initial body length) / initial body length] × 100;

[0112] Weight gain rate (%) = [(final weight - initial weight) / initial weight] × 100;

[0113] Feed conversion ratio (FCR) = total feed intake (g) / total weight gain (g), with feed intake calibrated by accumulating daily feeding records.

[0114] 5.4.2 Transaminase Activity Assay

[0115] Liver tissue was collected, homogenized with pre-cooled PBS at a ratio of 1:9 (w / v), centrifuged at 5000×g for 10 min, and the supernatant was collected to obtain liver tissue homogenate.

[0116] Liver tissue homogenates were collected, and liver aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities were measured using the AST kit G0424W and ALT kit G0423W, respectively.

[0117] 5.4.3 Serum Immunoassay

[0118] Blood was collected from the fish tail vein using blood collection tubes. After standing at 4°C for 1 hour, the supernatant was collected by centrifugation at 3000×g for 15 minutes to prepare serum.

[0119] The activities of IgM, C3, C4, AKP and ACP in serum were measured using the IgM enzyme-linked immunosorbent assay kit MM-33677O2, the C3 enzyme-linked immunosorbent assay kit MM-33667O2, the C4 enzyme-linked immunosorbent assay kit MM-1690O2, the AKP activity assay kit and the ACP activity assay kit, respectively.

[0120] 5.4.4 Antioxidant capacity determination

[0121] The serum prepared above was used to determine the SOD content in the serum using the SOD-WST-8 activity assay kit.

[0122] The liver tissue homogenate prepared above was used to quantitatively determine the activities of CAT and GSH-Px, as well as the contents of GSH, ROS and MDA, using CAT kit, GSH-Px kit, GSH content kit, ROS content kit and MDA content kit, respectively.

[0123] 5.4.5 Digestive enzyme activity assay

[0124] Take intestinal contents and add 0.9% cold physiological saline at a ratio of 1:9 (w / v) to homogenize and obtain intestinal contents homogenate.

[0125] Intestinal contents were homogenized, and the activities of trypsin, amylase and lipase were determined using a trypsin kit, an α-amylase (starch-iodine colorimetric method) activity assay kit and a lipase activity assay kit, respectively.

[0126] 5.5 Experimental Results

[0127] The results of the growth performance test are shown below. Figure 11 The results of the transaminase activity assay are shown in [the table below]. Figure 12 The results of serum immunogenicity assay are shown in [the table below]. Figure 13 The results of the antioxidant capacity test are shown in [the table below]. Figure 14 The results of the digestive enzyme activity assay are shown in [the table below]. Figure 15 .

[0128] The results showed that the growth rate of turbot in the experimental group was significantly higher than that in the control group by 70.36%. P <0.0001), the weight gain rate increased significantly by 182.80% ( P <0.0001), feed conversion ratio (FCR) decreased by 23.30%, and the activities of alanine aminotransferase (GPT) and aspartate aminotransferase (GOT) in the liver increased by 160.11% ( P <0.001) and 21.95%, indicating that Ho7-3 bacterial protein efficiently participates in amino acid metabolism and energy conversion processes, and synergistically promotes turbot protein synthesis and metabolism.

[0129] The serum immunoglobulin M (IgM), complement 3 (C3), and complement 4 (C4) levels in the experimental group of turbot were 15.88% higher than those in the control group. P <0.01), 59.33% P <0.001) and 8.83% ( P<0.05); the activities of acid phosphatase (ACP) and alkaline phosphatase (AKP) were significantly increased by 92.40% compared with the control group ( P <0.01) and 167.28% ( P <0.01), indicating that Ho7-3 bacteria can significantly enhance the immune response of turbot.

[0130] The serum superoxide dismutase (SOD) activity, liver catalase (CAT) activity, and liver glutathione peroxidase (GPx) activity in the experimental group of turbot increased by 184.74% compared with those in the control group. P The levels of reduced glutathione (GSH) were <0.01%, 20.40%, and 23.26%, respectively, with the total reduced glutathione (GSH) content increasing by 44.53% compared to the control group. P <0.05, the levels of reactive oxygen species (ROS) and malondialdehyde (MDA) decreased by 56.99% compared to the control group. P <0.05) and 41.25% ( P The result was <0.001, indicating that Ho7-3 bacteria exerted a significant antioxidant effect in turbot.

[0131] The intestinal trypsin and amylase activities of turbot in the experimental group were increased by 42.18% compared with those in the control group. P <0.01) and 31.25% ( P The value of Ho7-3 cells (<0.01) indicates that Ho7-3 cells can significantly improve the activity of digestive enzymes and thus improve the digestive function of turbot.

[0132] In summary, adding Ho7-3 strain to feed can significantly improve the growth performance of turbot juveniles by synergistically enhancing the activity of intestinal digestive enzymes, immune response, and antioxidant defense system.

[0133] In this invention, relational terms such as "first" and "second" are used merely to distinguish one entity or operation from another, without necessarily requiring or implying any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or terminal device that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or terminal device. Without further limitation, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or terminal device that includes said element.

[0134] Although specific embodiments of the invention have been described for illustrative purposes, various modifications or alterations can be made by those skilled in the art without departing from the spirit and scope of the invention. All such modifications or alterations should fall within the scope of the appended claims.

Claims

1. A type of aquatic feed Rhodotorula buergerianum ( Rhodotorula toruloides Ho7-3, characterized in that: The Rhodotorula buergerianum Ho7-3 is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20252122.

2. A microbial agent, characterized in that, This includes Rhodotorula buergerianum Ho7-3, which is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20252122.

3. The application of the Rhodotorula buergerianum Ho7-3 as described in claim 1 or the inoculum agent as described in claim 2 as a feed additive.

4. The application according to claim 3, characterized in that, The feed additive is a fish feed additive.

5. The use of the Rhodotorula buergerianum Ho7-3 of claim 1 or the inoculum of claim 2 in the preparation of a composition for enhancing the activity of intestinal digestive enzymes in fish.

6. The use of the Rhodotorula buergerianum Ho7-3 of claim 1 or the fungal agent of claim 2 in the preparation of a composition for enhancing the immune response of fish.

7. The use of the Rhodotorula buergerianum Ho7-3 of claim 1 or the fungal agent of claim 2 in the preparation of a composition for optimizing the antioxidant status of fish.

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