A Bacillus strain for utilizing oil and fat and its application

Abstract

The present invention belongs to the field of microbial technology, and specifically relates to a Bacillus strain that utilizes oil and fat and its application. The present invention discovered for the first time a Bacillus strain Sd_h10, which has good biosafety, can utilize triglycerides in fish oil, and produce free fatty acids. In a zebrafish model, the constant value of Bacillus Sd_h10 is beneficial to the zebrafish's absorption of oil and fat, can promote the synthesis of lipids in the zebrafish body, provide the zebrafish with additional fatty acids, and promote the growth of fish under high-fat conditions. The strain can be added in large quantities to water or feed, can promote the fish's digestion and absorption of oil and fat, provide the fish with additional fatty acids, and alleviate the inhibitory effect of high-fat conditions on fish growth.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a bacillus strain that utilizes oil and fat and application thereof. Background Art

[0002] Fish are an important source of food and nutrition for humans, providing high-quality protein. Artificial aquaculture has become a major source of fish supply. Although aquaculture and management techniques for various fish species are highly mature, reducing the incidence of various diseases during the aquaculture process, improving feed utilization, and enhancing the quality of the aquacultured animals remain key concerns in aquaculture.

[0003] Most farmed fish have high requirements for nutrients such as fat and protein. During their growth, farmed fish must be fed a diet high in fat and protein. Protein in fish feed primarily comes from high-quality protein feed ingredients such as fishmeal and soybean meal. Limited fishery resources have led to rising fishmeal prices. While plant proteins such as soybean meal, while relatively inexpensive and a common alternative, are relatively low in nutritional value and digestibility, they do not fully meet the nutritional needs of fish. To meet the nutritional needs of farmed fish, large amounts of fishmeal must be added to feed, a major contributor to rising fish farming costs. Fat in feed can be converted into protein for fish growth. To reduce farming costs, increasing the fat content in feed is often employed to conserve protein while meeting the nutritional needs of farmed fish and reducing costs. However, excessive fat intake can lead to excessive fat accumulation in farmed fish, impairing liver function, and impairing their metabolism and detoxification abilities, negatively impacting their health. Furthermore, excessive fat intake can also weaken fish immune function, reduce feed utilization, slow growth, and compromise fish meat quality. How to meet the nutritional needs of fish, reduce breeding costs and ensure the healthy and rapid growth of breeding objects is currently a key issue in the field of artificial fish breeding.

[0004] As beneficial microorganisms, probiotics are increasingly being used in aquaculture. Currently, common probiotics in aquaculture include various lactic acid bacteria, bifidobacteria, bacillus, yeasts, and photosynthetic bacteria. Practice has demonstrated that the use of probiotics in aquaculture offers numerous advantages. Probiotics can improve the aquaculture water environment, enhance the immunity of aquacultured animals, improve intestinal health, promote nutrient absorption and utilization, and increase growth efficiency. They can also reduce the use of antibiotics and other medications during the aquaculture process, thereby reducing environmental pollution. With the development of the aquaculture industry, the advantages of probiotics in aquaculture have become increasingly prominent. However, the effectiveness of probiotics can vary across different aquaculture environments and species, and there is a lack of broadly effective probiotic strains. Furthermore, currently available probiotics are primarily used to improve the aquaculture water environment and enhance the immunity of aquacultured animals. Reports on probiotics that can improve fat metabolism in aquacultured animals, particularly fish, are limited. Summary of the Invention

[0005] The first aspect of the present invention aims to provide a Bacillus strain.

[0006] The second aspect of the present invention aims to provide a culture.

[0007] The third aspect of the present invention aims to provide a method for preparing the culture of the second aspect of the present invention.

[0008] The fourth aspect of the present invention aims to provide a bacterial agent.

[0009] The fifth aspect of the present invention aims to provide a method for preparing the bacterial agent of the fourth aspect of the present invention.

[0010] The sixth aspect of the present invention aims to provide applications of the Bacillus of the first aspect, the culture of the second aspect, and the bacterial agent of the fourth aspect.

[0011] The seventh aspect of the present invention aims to provide a product.

[0012] The eighth aspect of the present invention aims to provide a method.

[0013] In order to achieve the above-mentioned purpose of the present invention, the technical solution adopted by the present invention is:

[0014] The Bacillus is named Sd_h10, and its taxonomic name is Bacillus thuringiensis Sd_h10. It was deposited in the China Center for Type Culture Collection (located in Wuhan University, Wuhan, China) on October 31, 2024, with a deposit number of CCTCC NO: M 20242406.

[0015] The Bacillus Sd_h10 is a Gram-positive bacterium and has a certain sensitivity to all antibiotics except cephalexin; Bacillus Sd_h10 is slightly sensitive to antibiotics such as penicillin, ceftazidime, lincomycin, etc., and is very sensitive to antibiotics such as cefoperazone, imidazocycline, kanamycin, etc.

[0016] The extracellular product of the Bacillus Sd_h10 has amylase, casein, lipase, gelatinase and protease activities, but does not have urease and lecithinase activities.

[0017] The second aspect of the present invention provides a culture obtained by culturing the Bacillus according to the first aspect of the present invention.

[0018] The third aspect of the present invention provides a method for preparing the culture of the second aspect of the present invention, comprising inoculating the Bacillus sp. of the first aspect of the present invention into a culture medium and culturing the culture to obtain the culture.

[0019] Preferably, the culture medium comprises at least one of broth agar medium and tryptic soy broth medium.

[0020] In some embodiments of the present invention, the culture condition is culturing at 28-37°C for 12-48 hours; preferably, the culture temperature is 28-30°C.

[0021] A fourth aspect of the present invention provides a bacterial agent comprising the Bacillus of the first aspect of the present invention and / or the culture of the second aspect of the present invention.

[0022] Preferably, the bacterial agent is a solid bacterial agent or a liquid bacterial agent.

[0023] Preferably, the solid bacterial agent comprises a carrier.

[0024] Preferably, the carrier comprises at least one of a mineral material and a plant material.

[0025] Further preferably, the mineral material comprises at least one of activated carbon, clay, peat soil, vermiculite, talc, kaolin, montmorillonite, white carbon, zeolite, silica, and diatomaceous earth.

[0026] Further preferably, the plant material comprises at least one of corn flour, soybean flour and wheat bran.

[0027] Preferably, the liquid bacterial agent includes a protective agent.

[0028] Preferably, the protective agent comprises one or more of glycerol, benzoate, sorbate, polyethylene glycol, and Tween.

[0029] Preferably, the bacterial agent is in the form of at least one of liquid, powder or granule.

[0030] The fifth aspect of the present invention provides a method for preparing the bacterial agent of the fourth aspect of the present invention, wherein the bacterial agent is obtained by using the Bacillus of the first aspect of the present invention and / or the culture of the second aspect as active ingredients.

[0031] A sixth aspect of the present invention provides the use of any one of (a1) to (a3) ​​in at least one of (b1) to (b2):

[0032] (a1) the Bacillus described in the first aspect of the present invention;

[0033] (a2) the culture according to the second aspect of the present invention;

[0034] (a3) the bacterial agent according to the fourth aspect of the present invention;

[0035] (b1) preparing products that promote oil absorption;

[0036] (b2) preparing feed additives.

[0037] A seventh aspect of the present invention provides a product comprising at least one of (a1) to (a3):

[0038] (a1) the Bacillus described in the first aspect of the present invention;

[0039] (a2) the culture according to the second aspect of the present invention;

[0040] (a3) The bacterial agent according to the fourth aspect of the present invention.

[0041] Preferably, the product comprises feed;

[0042] Preferably, the feed comprises fish feed.

[0043] Preferably, the feed is used for raising fish in a high-fat environment; or for alleviating related physiological abnormalities in fish caused by high fat, such as a decrease in body length and weight growth rate.

[0044] The physiological abnormalities related to fat metabolism include:

[0045] Fatty liver: It is a common nutritional metabolic disease in fish, mainly referring to the physiological phenomenon of excessive fat accumulation in the liver of fish.

[0046] Hepatobiliary syndrome: The phenomenon of fish liver and gallbladder lesions caused by factors such as high breeding density, deterioration of breeding water environment, excessive feeding, feed deterioration, and nutritional imbalance.

[0047] Lipid metabolism disorders: In a high-fat environment, fish may experience lipid metabolism disorders, leading to abnormal accumulation of fat in non-fat tissue areas.

[0048] Overnutrition: In a high-fat environment, fish may suffer from overnutrition due to excessive energy intake, which in turn may lead to a series of metabolic diseases.

[0049] Metabolic abnormalities: Due to nutritional imbalance or excess, fish may experience metabolic abnormalities, which manifest as abnormal changes in blood sugar and blood lipid levels.

[0050] Preferably, the product may further include other active ingredients.

[0051] Preferably, the other active ingredients include but are not limited to other conventional drugs that are beneficial to fat metabolism.

[0052] Preferably, the product may further include pharmaceutically acceptable excipients.

[0053] Preferably, the pharmaceutically acceptable excipients include at least one of a solvent, a colorant, a stabilizer, a preservative, a pH regulator, a buffer, a surfactant, a foaming agent, a defoaming agent, a thickener, and a carrier.

[0054] An eighth aspect of the present invention provides a method for raising fish, comprising the following steps:

[0055] The product according to the seventh aspect of the present invention is used to raise fish.

[0056] Preferably, the fish includes at least one of black carp, grass carp, silver carp, bighead carp, carp, crucian carp, bighead carp, bream, sturgeon, eel, grouper, large yellow croaker, flounder, sea bass, golden pomfret, and zebrafish.

[0057] The beneficial effects of the present invention are:

[0058] The present invention discovered a strain of Bacillus sp. Sd_h10 for the first time. This strain has excellent biosafety and can utilize triglycerides in fish oil and produce free fatty acids. In a zebrafish model, colonization of Bacillus sp. Sd_h10 facilitates the fish's oil absorption and lipid synthesis, providing additional fatty acids and promoting their growth under high-fat conditions. This strain can be added in large quantities to water or feed to promote fish digestion and absorption of oil, providing additional fatty acids and alleviating the inhibitory effects of high-fat conditions on fish growth. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] The present invention will be further described below with reference to the accompanying drawings and embodiments, in which:

[0060] Figure 1The identification results of Bacillus Sd_h10 in Example 1 of the present invention; wherein A is the colony morphology; B is the Gram staining result; C is the hemolytic ability test result; and D is the phylogenetic tree construction result.

[0061] Figure 2 This is the extracellular enzyme activity determination of Bacillus Sd_h10 in Example 1 of the present invention; wherein AG is the result of amylase, tyrosine kinase, lipase, gelatinase, lecithinase, urease and protease activity detection in sequence.

[0062] Figure 3 The results show that Bacillus Sd_h10 can utilize oil. AC shows the growth curves of Bacillus Sd_h10 in different concentrations of fish oil, soybean oil and paraffin. DE shows the detection results of triglyceride and free fatty acid contents respectively. F shows the determination results of triglyceride and free fatty acid in carbon-free culture medium.

[0063] Figure 4 The results of the effects of Bacillus Sd_h10 on zebrafish; A is the safety test result of Bacillus Sd_h10; BC are the Oil Red O (ORO) staining results of Bacillus Sd_h10; DE are the relative areas and average optical densities of the two zebrafish feeding groups after Oil Red staining; F is the expression of lipid metabolism-related genes in the two zebrafish feeding groups; GH are the relative weight growth rate and relative body length growth rate of zebrafish, respectively. DETAILED DESCRIPTION

[0064] The following will clearly and completely describe the concept and technical effects of the present invention in conjunction with the embodiments to fully understand the purpose, features and effects of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0065] The reagents and materials not specifically described in the following examples are all conventional materials on the market and can be obtained by those skilled in the art through conventional procurement.

[0066] Example 1 Isolation and Identification of Bacillus Sd_h10

[0067] 1. Bacterial isolation

[0068] The oil-utilizing strain, Bacillus Sd_h10, was isolated from the intestine of greater amberjack. The intestine of greater amberjack was thoroughly ground under sterile conditions, and the intestinal grinding solution was spread on TSB agar plates. The plates were incubated at 28°C for 18 hours and then streaked. After multiple streaking, milky white, irregularly shaped colonies were obtained, such as Figure 1 As shown in A.

[0069] 2. Identification of bacterial physiological characteristics

[0070] 1) Gram staining

[0071] Gram staining results showed that Bacillus Sd_h10 was a Gram-positive bacterium ( Figure 1 Middle B).

[0072] 2) Hemolytic ability assay

[0073] Use an Oxford cup to punch holes on a blood agar plate, add 20 μL of sterile tryptic soy broth medium (negative control), 20 μL of Streptococcus pneumoniae cultured for 12 hours (positive control), and 20 μL of Bacillus Sd_h10 cultured for 12 hours into the air, and continue culturing and observing at 28°C for 48 hours.

[0074] The results are as follows Figure 1 As shown in C, the negative control showed no hemolysis, the positive control showed α-hemolysis, and Bacillus Sd_h10 did not produce an obvious hemolytic ring on the blood agar medium, so Bacillus Sd_h10 was γ-hemolytic.

[0075] 3) Bacterial drug sensitivity test

[0076] The sensitivity of Bacillus Sd_h10 to various antibiotics was determined by disk diffusion method. 100 μl of 1×10 8 The bacterial solution was spread on LB agar plates. After overnight culture, the presence of inhibition zones was observed and their diameters were recorded. The antibiotic susceptibility strips used included penicillin (10 U), ampicillin (10 μg), ceftazidime (30 μg), cefazolin (30 μg), cefoperazone (30 μg), cephalexin (30 μg), piperacillin (100 μg), ceftriaxone (30 μg), cefuroxime sodium (30 μg), lincomycin (2 μg), polymyxin B (300 IU), erythromycin (15 μg), tetracycline (30 μg), imidazocycline (30 μg), doxycycline (30 μg), gentamicin (10 μg), amikacin (30 μg), streptomycin (10 μg), vancomycin (30 μg), and kanamycin (30 μg).

[0077] The results are shown in Table 1: Bacillus Sd_h10 was sensitive to all antibiotics except cephalexin, slightly sensitive to penicillin, ceftazidime, lincomycin and other antibiotics, and very sensitive to cefoperazone, imidazocycline, kanamycin and other antibiotics.

[0078] Table 1

[0079]

[0080]

[0081] 4) Extracellular enzyme activity assay

[0082] 100 μL of Bacillus Sd_h10 bacterial suspension was spread onto a tryptic soy agar plate covered with cellophane. After incubation at 28°C for 48 hours, the cellophane was rinsed with sterile PBS and transferred to a 1.5 mL EP tube. After centrifugation at 12,000 rpm for 30 minutes, the supernatant was collected and freeze-dried for 48 hours. The lyophilized product was collected to obtain a crude extracellular product extract of Bacillus Sd_h10. The crude extract was completely dissolved in PBS, sterilized by filtration through a 0.22 μm filter, and stored at -20°C. Separate agar plates containing 1% gelatin, 1% casein, 1% starch, 1% Tween 80, 2% urea (with 0.5% phenol red indicator), 8% skim milk, and egg yolk (2.5%) were prepared and well-welled. 20 μL of the crude extracellular product extract was added to each well and incubated at 28°C for 12 hours.

[0083] The results are as follows Figure 2 As shown in the figure, the activity of amylase, tyrosine kinase, lipase, gelatinase, lecithinase, urease and protease was tested in AG. The results showed that the extracellular product of Bacillus sp. Sd_h10 had amylase, tyrosine kinase, lipase, gelatinase and protease activities, but did not have urease and lecithinase activities.

[0084] 3. Evolutionary tree construction and biochemical identification

[0085] After DNA was extracted from the purified bacteria, the 16S rDNA of the bacteria was amplified using primers 27F (5′-AGAGTTTCATCTGGCTCAG-3′, SEQ ID NO: 1) and 1492R (5′-GGTTACCTTGTTACGACTT-3′, SEQ ID NO: 2). The gyrA gene of the bacteria was amplified using primers gyrA-F (5′-GCGTCTGCAACGTTTAACTGG-3′, SEQ ID NO: 3) and gyrA-R (5′-TGTCGCTACCTCTTGCTCATC-3′, SEQ ID NO: 4). The gyrB gene was amplified using primers gyrB-F (5′-TTGRCGGHRGYGGHTATAAAGT-3′, SEQ ID NO: 5) and gyrB-R (5′-TCCDCCSTCAGARTCWCCCTC-3′, SEQ ID NO: NO:6) Amplify the gyrB gene of the bacteria according to the following procedure: preheat at 94°C for 5 minutes; 35 cycles of 94°C for 30 seconds, 55°C for 30 seconds, and 72°C for 30 seconds; and finally extend at 72°C for 10 minutes. The amplified product was sent to BGI (China) for sequencing, and the sequencing results were compared on NCBI (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi). The strain sequence with the highest similarity was downloaded from GenBank. After gene concatenation, a phylogenetic tree was constructed using the neighbor-joining method using MEGA-X 10.2.2 software (https: / / www.megasoftware.net / ). The constructed phylogenetic tree is shown in Figure 2. Figure 1 As shown in Figure D, Bacillus Sd_h10 is most closely related to Bacillus thuringiensis JJ1216. Biochemical identification of Bacillus Sd_h10 was performed using the Bacillus cereus biochemical identification kit (Guangdong Huankai Microbiology Technology Co., Ltd., GB4789.14) according to the instructions. The biochemical identification results are shown in Table 2. Based on the instructions, it is believed that Bacillus Sd_h10 is not Bacillus cereus. Therefore, Bacillus Sd_h10 is considered a Bacillus thuringiensis strain and was named Bacillus thuringiensis Sd_h10.

[0086] The sequence determined by the Sd_h10 16S rDNA universal primer is as follows:

[0087] TGGGTGTGCTATACATGCAAGTCGAGCGAATGGATTGAGAGCTTGCTCTCAAGAAG

[0088] TTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCCATAAGACTGGGATAACTCC

[0089] GGGAAACCGGGGCTAATACCGGATAACATTTTGAACCGCATGGTTCGAAATTGAAAGGC

[0090] GGCTTCGGCTGTCACTTATGGATGGACCCGCGTCGCATTAGCTAGTTGGTGAGGTAACG

[0091] GCTCACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACT

[0092] GAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGA

[0093] AAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGCTTTCGGGTCGTAAAACTCTGT

[0094] TGTTAGGGAAGAACAAGTGCTAGTTGAATAAGCTGGCACCTTGACGGTACCTAACCAGA

[0095] AAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCC

[0096] GGAATTATTGGGCGTAAAGCGCGCGCAGGTGGTTTCTTAAGTCTGATGTGAAAGCCCAC

[0097] GGCTCAACCGTGGAGGGTCATTGGAAACTGGGAGACTTGAGTGCAGAAGAGGAAAGT

[0098] GGAATTCCATGTGTAGCGGTGAAATGCGTAGAGATATGGAGGAACACCAGTGGCGAAGG

[0099] CGACTTTCTGGTCTGTAACTGACACTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATT

[0100] AGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGAGGGTTTCCGCC

[0101] CTTTAGTGCTGAAGTTAACGCATTAAGCACTCCGCCTGGGGAGTACGGCCGCAAGGCTG

[0102] AAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGGAGCATGTGGTTTATTTCG

[0103] AAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGAAAACCCTAGAGATAGGGC

[0104] TTCTCCTTCGGGAGCAGAGTGACAGGTGGTGCATGGCTTGTCGTCAGCCTCCGTGTCGT

[0105] GAAGATGTTGGGTAAAGTCCCGCCAACGAAGCGCAACCCCTTGGATCTTTAGTTGCC

[0106] (SEQ ID NO:7).

[0107] The sequence measured by Sd_h10 gyrA-F / R primers is as follows:

[0108] GGGCCAATCGAGCAGATATCAAGACTTAATGAAATTAATTGCTGAATTAAAAGCGAT

[0109] TTTAGCAGATGAAGAGAAAGTTCTTGAGATTATTCGTGAAGAATTAACAGAAGTAAAAG

[0110] AGCGTTTCAATGATAAGAGACGAACAGAAATTACAATTGGCGGTATGGAATTTATTGAAG

[0111] ATGAAGATTTAATTCCTGAGCAAAACATTGCGATTACGTTAACTCATAACGGTTATATTAA

[0112] GAGGTTACCAGCTTCTACGTACAAAACACAGAACCGTGGGGGACGTGGTGTACAAGGA

[0113] ATGGGTACGAATGATGATGACTTTGTTGAACACTTATTAACAACGTCTACTCATGATCATA

[0114] TTTTATTCTTTACAAACAAGGGTAAAGTATACCGTACGAAAGGATATGAAATCCCAGAGT

[0115] ATAGTCGTACTGCAAAAGGGCTACCAATTATTAACCTATTAGGGGTAGATAAGGGTGAGT

[0116] GGATCAATGCCATTATTCCAATTCGTGAATTTGGTGACGACCAGTTCTTATTCTTTACAAC

[0117] GAAACAAGGTATTTCTAAGAGAACGCCACTTTCATCATTTGCAAATATACGTACAAACGG

[0118] TTTAATTGCAATTTCGCTTCGTGAAGAAGATGAAGTGATTTCTGTACGTTTAACATCTGG

[0119] CGATAAAGATATTATCGTAGGGACAAGCAACGGTATGTTAATTCGCTTCAATGAACAAGA

[0120] TGTACGTTCTATGGGCCGTAATGCAGCTGGTGTAAAGCTATTACATTAGGCGAAGAGGA

[0121] CCAAGTAGTAGGTATGGAAATTGTTGAAGAAGATACGAATGTTTTAATTGTAACGAAAA

[0122] ACGGGTATGGTAAGCGTACTCCAGTTGAAGAGTATCGACTACAAAGCCGTGGTGGTAAA

[0123] GGTCTTAAGACTTGTAACATTACAGATAAAAACGGTAAGTTAGTAGCGGTTAAGTCTGTA

[0124] ACAGGTGAAGAAGACATCATGCTAATTACAGCCGCAGGCGTTATTATTCGTATGCCAGTT

[0125] GATCAAATCTCTCAAATGGGACGTAATACACAAGGGTCTCAATGT(SEQ ID NO:8).

[0126] The sequence measured by the Sd_h10 gyrB-F / R primers is as follows:

[0127] CGCATGCATGCGAGTGTGCATCTGTTGTAATGCCTTATCACAGAATTAGAAGTATTTG

[0128] TACATCGTGATGGCAAAATCCATTACCAAAAATACGAAAGAGGTATTCCGGTTGCAGATT

[0129] TAAAAGTCATCGGTGATACAGATAAGACTGGAACAATAACTCGCTTTAAACCGGATCCA

[0130] GAAATTTTTAAAGAGACGACAGAATATGAATTCGATACGCTAGCGACTCGTATGCGTGAG

[0131] TTGGCGTTTTTAAATCGTAATATTAAATTAACAATTGAAGATAAACGTGAACATAAGCAA

[0132] AAGAAAGAGTTCCACTATGAAGGTGGAATTAAATCATATGTTGAACATTTAAATCGTTCA

[0133] AAACAACCAATTCATGAAGAGCCTGTATATGTAGAAGGTTCAAAAGATGGTATTCAAGTT

[0134] GAAGTTGCGCTTCAATATAACGAAGGATATACAAATCATATTTACTCATTTACAAATAATAT

[0135] TCATACGTATGAAGGTGGTACACATGAGGTAGGATTTAAAACTGCCTTAACACGTGTGAT

[0136] TAACGATTATGGTCGTAAAAATAACATTTTAAAAGATGCGGATAGTAATTTGACTGGTGA

[0137] AGATGTTCGTGAAGGTTTAACAGCAATCGTGTCAATTAAACATCCAAATCCACAATTTGA

[0138] AGGGCAAACGAAGACGAAACTTGGAAATAGTGAAGCGAGAACGATTACGGAGTCAGTA

[0139] TTCTCTGAGGCTTTTGAAAAATTCTTACTGGAAAATCCCAATGTTGCACGTAAGGTTGTA

[0140] GATAAAGGGACGATGGCAGCACGTGCGCGTGTAGCAGCTAAAAAGGCTCGTGAGCTAA

[0141] CTCGCCGAAAGAGTGCTTTAGAAGTTTCAAGTTTACCAGGGAAATTGGCAGATTGTCCT

[0142] TCTAAAGATCCCCCT (SEQ ID NO:9).

[0143] Table 2 Biochemical identification results of Bacillus cereus

[0144]

[0145] The Bacillus thuringiensis is named Sd_h10, and its taxonomic name is Bacillus thuringiensis Sd_h10. It was deposited in the China Center for Type Culture Collection (located in Wuhan University, Wuhan, China) on October 31, 2024, with the preservation number CCTCCNO: M 20242406.

[0146] Example 2: Oil and fat utilization ability of Bacillus Sd_h10

[0147] 1) Growth curve determination

[0148] After culturing Bacillus sp. Sd_h10 overnight at 28°C, the bacterial culture was adjusted to an OD600 of 0.3. Subsequently, the culture was inoculated into tryptic soy broth (TSB) supplemented with fish oil, soybean oil, and paraffin (2%, 4%, 6%, 8%, 10%, 12%, 14%, and 16%, v / v) at a ratio of 10% v / v. The culture was shaken at 28°C and 240 rpm. Bacterial growth curves were then measured.

[0149] Figure 3 Figures A and B show the growth curves of Bacillus Sd_h10 in different concentrations of fish oil, soybean oil, and paraffin, respectively. The growth rate and final concentration of Bacillus Sd_h10 in fish oil were significantly better than those in soybean oil and paraffin. This suggests that Bacillus Sd_h10 can effectively utilize fish oil for growth.

[0150] 2) Determination of triglyceride and free fatty acid content in TSB

[0151] Based on bacterial growth trends, Bacillus sp. Sd_h10 was cultured in TSB supplemented with 2%, 8%, and 16% fish oil (v / v). The culture medium was removed at 30 and 36 hours, allowed to stand for 30 minutes, and 1 mL of the bottom layer of culture medium was aspirated. The culture medium was centrifuged under sterile conditions (4°C, 5000 rpm, 10 minutes), and 600 μL of the middle layer was extracted. Changes in triglyceride and free fatty acid content in the culture medium were measured using the Amplex Red Triglyceride Assay Kit and the Amplex Red Free Fatty Acid Assay Kit (Beyotime Biotechnology).

[0152] Figure 3 DE represents the results of triglyceride and free fatty acid content testing, respectively. In TSB, triglyceride content in bacterial cultures supplemented with different concentrations of fish oil showed a significant decrease after 36 hours of culture. Regarding free fatty acids, after 36 hours of culture, the free fatty acid content in the group supplemented with 2% fish oil increased slightly, while the free fatty acid content in the bacterial cultures supplemented with 8% and 16% fish oil decreased significantly. These results suggest that Bacillus Sd_h10 is able to utilize triglycerides in the culture medium and produce free fatty acids. At a certain stage of growth, Bacillus Sd_h10 may also utilize the produced free fatty acids.

[0153] 3) Determination of triglycerides and free fatty acids in culture medium containing only fish oil as a carbon source

[0154] A carbon-free minimal medium (prepared at a ratio of 41g diammonium phosphate (DAPO), 1g sodium chloride, 0.2g magnesium sulfate heptahydrate, and 0.5g potassium dihydrogen phosphate per liter of double-distilled water) was supplemented with 2% (v / v) fish oil as a carbon source. No other carbon sources were added. Following the experimental methods described in 1) and 2) above, samples were collected from the Bacillus Sd_h10 strain after culturing for 30 minutes, 12 hours, 14 hours, 16 hours, and 18 hours to measure triglyceride and free fatty acid levels.

[0155] The results are as follows Figure 3 As shown in Figure F, in cultures fed only with fish oil as a carbon source, triglycerides decreased significantly after 12 hours of culture and remained stable thereafter. Free fatty acids continued to increase for 14 hours after culture, then gradually decreased and approached their initial levels. This result further demonstrates that Bacillus Sd_h10 can utilize triglycerides in fish oil and produce free fatty acids.

[0156] Example 3 Safety Determination of Bacillus Sd_h10

[0157] 120 healthy zebrafish at 6 days post fertilization (dpf) were randomly divided into 6 groups, with 10 in each group. The five groups of zebrafish received 1 mL of different concentrations of Bacillus Sd_h10 bacterial solution, with the bacterial concentration in the water being 1×10 2 cfu / mL, 1×10 3 cfu / mL, 1×10 4 cfu / mL, 1×10 5 cfu / mL, 1×10 6 cfu / mL. One group received the same volume of saline as a control (CK). The fish were cultured continuously at 28°C and observed for 10 days.

[0158] The results are as follows Figure 4 As shown in A, no zebrafish died in the control group, and no zebrafish died in water bodies with different bacterial concentrations, indicating that Bacillus Sd_h10 has no harm to zebrafish within a certain range.

[0159] Example 4 Effect of Bacillus Sd_h10 Colonization on Zebrafish Juveniles

[0160] The same batch of zebrafish fertilized eggs were hatched under sterile conditions to obtain germ-free zebrafish. Zebrafish (6 dpf) were treated separately: Group A, egg yolk and Bacillus Sd_h10 (1×10 4cfu / mL); in group N, only egg yolk was added to the sterile zebrafish. After 24 hours of treatment as described above, sufficient amount of ground egg yolk was added to both groups of zebrafish. Zebrafish were collected 5 hours later for Oil Red O (ORO) staining and gene expression analysis using 2^ -ΔΔCt Relative gene expression was calculated using the dgat2 gene. dgat2 is primarily involved in the conversion of diacylglycerol (DAG) to triglyceride (TG), the final step in fat synthesis. fabp4a transports fatty acids from the cell membrane to the mitochondria for oxidative breakdown or to the endoplasmic reticulum for triglyceride synthesis. The fasn gene encodes fatty acid synthase. Acaca catalyzes fatty acid synthesis. The primers for each gene are shown in Table 3.

[0161] Table 3 Quantitative analysis genes and their primers

[0162]

[0163]

[0164] Oil red O (ORO) staining results are as follows Figure 4 As shown, Figure 4 Middle BC are the staining results of group A and group N respectively. Figure 4 DE in the middle represents the relative area and mean optical density after Oil Red staining of the two zebrafish feeding groups. It can be seen intuitively that the lipid droplets in the gastrointestinal tract of group A zebrafish are more densely distributed and darker in color, while the lipid droplets in the gastrointestinal tract of group N zebrafish are fewer and lighter in color.

[0165] Data analysis results showed that the relative area and average optical density of oil red in the gastrointestinal tract of zebrafish in group A were significantly higher than those in group N. These results indicate that the colonization of Bacillus Sd_h10 is beneficial to the absorption of oil by zebrafish.

[0166] Figure 4 Figure F shows the expression of lipid metabolism-related genes in the two zebrafish feeding groups. Compared with the zebrafish in group N, the expression of genes related to triglyceride synthesis, dgat2 and fabp4a, was significantly upregulated in group A, while the expression of genes related to fatty acid synthesis, fasn and acaca, was significantly downregulated in group A. These changes in gene expression suggest that the addition of Bacillus Sd_h10 can promote lipid synthesis in zebrafish and provide them with additional fatty acids.

[0167] Example 5 Effect of Bacillus Sd_h10 Colonization on Zebrafish Body Length and Weight

[0168] Twenty-four zebrafish (120 days old) were divided into three groups. Group CK was fed with commercial feed, Group B was fed with commercial feed supplemented with 2% fish oil (m / m), and Group C was fed with commercial feed supplemented with 2% fish oil (m / m) and a bacterial suspension of Bacillus Sd_h10 was added to the feeding water to maintain the bacterial concentration at 1×10 4 cfu / mL. A 14-day feeding experiment was conducted. Fish length and weight were measured on days 1 and 15. The relative weight and length growth rates of the zebrafish were then calculated.

[0169] Figure 4 GH represents the relative weight growth rate and relative length growth rate of zebrafish, respectively. Compared with those fed a normal diet, zebrafish fed a diet supplemented with 2% fish oil showed a significant decrease in length and weight growth, indicating that a high-fat diet inhibits zebrafish growth. Adding a certain concentration of Bacillus Sd_h10 to the water can effectively alleviate this inhibitory effect. These results suggest that Bacillus Sd_h10 is beneficial for fish growth under high-fat conditions.

Claims

1. A Bacillus strain characterized by: The Bacillus is named Sd_h10, and its taxonomic name is Bacillus thuringiensis Sd_h10 was deposited in the China Center for Type Culture Collection on October 31, 2024, with the deposit number CCTCC NO: M 20242406.

2. A culture obtained by culturing the Bacillus according to claim 1; The culture medium includes at least one of broth agar medium and tryptic soy broth medium; The culture conditions are culturing at 28-37° C. for 12-48 hours.

3. A bacterial agent comprising the Bacillus according to claim 1 and / or the culture according to claim 2.

4. The microbial agent according to claim 3, characterized in that: The bacterial agent is a solid bacterial agent or a liquid bacterial agent.

5. A method for preparing the microbial agent according to claim 3 or 4, wherein the microbial agent is obtained by using the Bacillus according to claim 1 and / or the culture according to claim 2 as an active ingredient.

6. The application of any one of (a1) to (a3) ​​in at least one of (b1) to (b3); (a1) the Bacillus according to claim 1; (a2) the culture according to claim 2; (a3) the bacterial agent according to claim 3 or 4; (b1) Preparation of products for promoting the absorption of oils and fats by the intestines of fish; (b2) preparing products for promoting the synthesis of fish lipids; (b3) Preparation of products for supplying fatty acids to fish.

7. A product comprising at least one of (a1) to (a3): (a1) the Bacillus according to claim 1; (a2) the culture according to claim 2; (a3) the bacterial agent according to claim 3 or 4; The products include feed; The feed includes fish feed.

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