Bacillus utilizing grease and application thereof

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to bacillus utilizing grease and application thereof. The bacillus Sdh10 is found for the first time, the strain has good biological safety, triglyceride in fish oil can be utilized, and free fatty acid can be generated. In a zebra fish model, the constant value of the bacillus Sdh10 is beneficial to the absorption of the zebra fish to grease, can promote the synthesis of lipid in the zebra fish body, provides additional fatty acid for the zebra fish, and is beneficial to the growth of the zebra fish under a high-fat condition. The strain can be massively added into a water body or feed, can promote digestion and absorption of fish on grease, provides additional fatty acid for the fish, and relieves the inhibition effect of a high-fat condition on fish growth.

Description

Technical Field

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

[0002] Fish is an important source of food and nutrition for humans, providing high-quality protein. Artificial breeding has become the main source of fish supply. Although the breeding and management technologies of various fish are very mature, how to further reduce the occurrence of various diseases in the breeding process, improve the utilization rate of feed, and improve the quality of breeding objects are still the focus of aquaculture.

[0003] Most farmed fish have high demands for nutrients such as fat and protein. During the growth of farmed objects, feed containing high fat and protein must be fed. The protein in fish feed mainly comes from high-quality protein feed raw materials such as fish meal and soybean meal. The limited fishery resources have caused the price of fish meal to continue to rise. Although plant proteins such as soybean meal are relatively cheap and are commonly used as substitutes for fish meal, their nutritional value and digestibility are relatively low, and they cannot fully meet the nutritional needs of fish. In order to meet the nutritional needs of farmed objects, a large amount of fish meal must be added to the feed, which is also the main reason for the increase in fish farming costs. The fat in the feed can be converted into protein required for fish growth. In order to reduce the cost of farming, in production practice, the method of increasing the proportion of fat in the feed is often adopted to save protein and ensure the nutritional needs of farmed objects, while reducing the cost of farming. However, excessive fat intake may cause excessive fat accumulation in the body of farmed objects, leading to liver damage, affecting the metabolism and detoxification ability of fish, and adversely affecting the health of fish. In addition, excessive fat intake may also lead to reduced immune function of fish, reduced feed utilization, decreased growth rate, and affect the quality of fish meat. How to meet the nutritional needs of fish, reduce breeding costs and ensure the healthy and rapid growth of breeding objects is a key issue in the current field of artificial fish breeding.

[0004] As a beneficial microorganism, probiotics are increasingly used in the field of aquaculture. At present, common probiotics in the field of aquaculture mainly include a variety of lactic acid bacteria, bifidobacteria, bacillus, yeast and photosynthetic bacteria. Practice has shown that the application of probiotics in aquaculture has shown many advantages. The use of probiotics can improve the environment of aquaculture waters, enhance the immunity of aquaculture objects, improve intestinal health and promote the absorption and utilization of nutrients, improve growth efficiency, and reduce the use of antibiotics and other drugs in the aquaculture process, reducing pollution to the environment. With the development of aquaculture, the advantages of the application of probiotics in the field of aquaculture are more prominent. However, the effects of probiotics may also vary in different aquaculture environments and different aquaculture objects, and there is a lack of broad-spectrum and effective probiotic strains. In addition, the existing probiotics are mostly used to improve the environment of aquaculture waters and improve the immunity of aquaculture objects. There are few reports on probiotics that can be used to improve aquaculture objects, especially fish fat metabolism. 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 purpose of the fifth aspect of the present invention is 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 name of the Bacillus is Sd_h10, and its taxonomic name is Bacillus thuringiensis Sd_h10. It was deposited in the China Center for Type Culture Collection (located at Wuhan University, Wuhan, China) on October 31, 2024, and the deposit number is 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 the activities of amylase, casein, lipase, gelatinase and protease, but does not have the activities of urease and lecithinase.

[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 of the first aspect of the present invention into a culture medium and culturing the culture medium to obtain the culture medium.

[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 12-48h; preferably, the culture temperature is 28-30°C.

[0021] The 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 includes one or more of glycerol, benzoate, sorbate, polyglycol, and Tween.

[0029] Preferably, the bacterial agent is in the form of at least one of a liquid, a powder or a 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 Bacillus of the first aspect of the present invention and / or the culture of the second aspect of the present invention are used as active ingredients to obtain the bacterial agent.

[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 may lead to a series of metabolic diseases.

[0049] Metabolic abnormalities: Due to nutritional imbalance or excess, fish may experience metabolic abnormalities, manifested 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 adjuster, 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 feed 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 first discovered a strain of Bacillus Sd_h10, which has good biosafety, can utilize triglycerides in fish oil, and produce free fatty acids. In a zebrafish model, the fixed value of Bacillus Sd_h10 is beneficial to the absorption of oil by zebrafish, can promote the synthesis of lipids in zebrafish, provide additional fatty acids for zebrafish, and is beneficial to the growth of fish under high-fat conditions. The strain can be added to water or feed in large quantities, can promote the digestion and absorption of oil by fish, provide additional fatty acids for fish, and alleviate the inhibitory effect 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, wherein:

[0060] Figure 1The identification result 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 determination of the extracellular enzyme activity of Bacillus Sd_h10 in Example 1 of the present invention; wherein AG is the test results of amylase, tyrosine, lipase, gelatinase, lecithinase, urease and protease activities in turn.

[0062] Figure 3 Figure 3 is the result of the oil utilization ability of Bacillus Sd_h10; AC is the growth curve of Bacillus Sd_h10 in different concentrations of fish oil, soybean oil and paraffin; 3D-E are the test results of triglyceride and free fatty acid content respectively; F is the determination result 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 is the Oil Red O (ORO) staining result of Bacillus Sd_h10; DE is the relative area and average optical density 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 is the relative weight growth rate and relative length growth rate of zebrafish, respectively. DETAILED DESCRIPTION

[0064] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments 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 those skilled in the art can obtain them through conventional purchase.

[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 fully ground under sterile conditions, and the intestinal grinding liquid was spread on TSB agar plates, incubated at 28°C for 18 hours, and then streaked. After multiple streaking separations, 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 the 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 in the air, and culture and observe continuously 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 sp. 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 papers used included penicillin (10U), ampicillin (10μg), ceftazidime (30μg), cefazolin (30μg), cefoperazone (30μg), cephalexin (30μg), piperacillin (100μg), ceftriaxone (30μg), cefuroxime sodium (30μg), lincomycin (2μg), polymyxin B (300IU), erythromycin (15μg), tetracycline (30μg), imidazocycline (30μg), doxycycline (30μg), gentamicin (10μg), amikacin (30μg), streptomycin (10μg), vancomycin (30μg), and kanamycin (30μg).

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

[0078] Table 1

[0079]

[0080] 4) Extracellular enzyme activity assay

[0081] 100 μL of Bacillus Sd_h10 bacterial solution was applied to a tryptic soy agar plate covered with cellophane. After culturing 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 12000 rpm for 30 minutes, the supernatant was collected and freeze-dried for 48 hours. The freeze-dried product was collected to obtain a crude extract of extracellular products of Bacillus Sd_h10. The crude extract was completely dissolved with PBS, sterilized by filtration through a 0.22 μm filter membrane, and stored at -20°C. 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 respectively and punched. 20 μL of crude extracellular product extract was added to each well and cultured at 28°C for 12 hours.

[0082] The results are as follows Figure 2 As shown, AG tested the activity of amylase, caseinase, lipase, gelatinase, lecithinase, urease and protease in turn. The results showed that the extracellular product of Bacillus Sd_h10 had the activities of amylase, caseinase, lipase, gelatinase and protease, but not urease and lecithinase.

[0083] 3. Evolutionary tree construction and biochemical identification

[0084] 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), and 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: 6). NO:6) Amplify the gyrB gene of 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 D, Bacillus Sd_h10 is most closely related to Bacillus thuringiensis JJ1216. The biochemical identification kit for Bacillus cereus (Guangdong Huankai Microbiological Technology Co., Ltd., GB4789.14) was used to perform biochemical identification of Bacillus Sd_h10 with reference to the instructions. The results of biochemical identification are shown in Table 2. According to the instructions, it can be considered that Bacillus Sd_h10 is not Bacillus cereus. Bacillus Sd_h10 can be considered to be a strain of Bacillus thuringiensis, so the strain is named Bacillus thuringiensis Sd_h10.

[0085] The sequence determined by Sd_h10 16S rDNA universal primer is as follows:

[0086] TGGGTGTGCTATACATGCAAGTCGAGCGAATGGATTGAGAGCTTGCTCTCAAGAAG

[0087] TTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCCATAAGACTGGGATAACTCC

[0088] GGGAAACCGGGGCTAATACCGGATAACATTTTGAACCGCATGGTTCGAAATTGAAAGGC

[0089] GGCTTCGGCTGTCACTTATGGATGGACCCGCGTCGCATTAGCTAGTTGGTGAGGTAACG

[0090] GCTCACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACT

[0091] GAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGA

[0092] AAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGCTTTCGGGTCGTAAAACTCTGT

[0093] TGTTAGGGAAGAACAAGTGCTAGTTGAATAAGCTGGCACCTTGACGGTACCTAACCAGA

[0094] AAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCC

[0095] GGAATTATTGGGCGTAAAGCGCGCGCAGGTGGTTTCTTAAGTCTGATGTGAAAGCCCAC

[0096] GGCTCAACCGTGGAGGGTCATTGGAAACTGGGAGACTTGAGTGCAGAAGAGGAAAGT

[0097] GGAATTCCATGTGTAGCGGTGAAATGCGTAGAGATATGGAGGAACACCAGTGGCGAAGG

[0098] CGACTTTCTGGTCTGTAACTGACACTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATT

[0099] AGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGAGGGTTTCCGCC

[0100] CTTTAGTGCTGAAGTTAACGCATTAAGCACTCCGCCTGGGGAGTACGGCCGCAAGGCTG

[0101] AAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGGAGCATGTGGTTTATTTCG

[0102] AAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGAAAACCCTAGAGATAGGGC

[0103] TTCTCCTTCGGGAGCAGAGTGACAGGTGGTGCATGGCTTGTCGTCAGCCTCCGTGTCGT

[0104] GAAGATGTTGGGTAAAGTCCCGCCAACGAAGCGCAACCCCTTGGATCTTTAGTTGCC

[0105] (SEQ ID NO:7).

[0106] The sequence measured by Sd_h10 gyrA-F / R primers is as follows:

[0107] GGGCCAATCGAGCAGATATCAAGACTTAATGAAATTAATTGCTGAATTAAAAGCGAT

[0108] TTTAGCAGATGAAGAGAAAGTTCTTGAGATTATTCGTGAAGAATTAACAGAAGTAAAAG

[0109] AGCGTTTCAATGATAAGAGACGAACAGAAATTACAATTGGCGGTATGGAATTTATTGAAG

[0110] ATGAAGATTTAATTCCTGAGCAAAACATTGCGATTACGTTAACTCATAACGGTTATATTAA

[0111] GAGGTTACCAGCTTCTACGTACAAAACACAGAACCGTGGGGGACGTGGTGTACAAGGA

[0112] ATGGGTACGAATGATGATGACTTTGTTGAACACTTATTAACAACGTCTACTCATGATCATA

[0113] TTTTATTCTTTACAAACAAGGGTAAAGTATACCGTACGAAAGGATATGAAATCCCAGAGT

[0114] ATAGTCGTACTGCAAAAGGGCTACCAATTATTAACCTATTAGGGGTAGATAAGGGTGAGT

[0115] GGATCAATGCCATTATTCCAATTCGTGAATTTGGTGACGACCAGTTCTTATTCTTTACAAC

[0116] GAAACAAGGTATTTCTAAGAGAACGCCACTTTCATCATTTGCAAATATACGTACAAACGG

[0117] TTTAATTGCAATTTCGCTTCGTGAAGAAGATGAAGTGATTTCTGTACGTTTAACATCTGG

[0118] CGATAAAGATATTATCGTAGGGACAAGCAACGGTATGTTAATTCGCTTCAATGAACAAGA

[0119] TGTACGTTCTATGGGCCGTAATGCAGCTGGTGTAAAGCTATTACATTAGGCGAAGAGGA

[0120] CCAAGTAGTAGGTATGGAAATTGTTGAAGAAGATACGAATGTTTTAATTGTAACGAAAA

[0121] ACGGGTATGGTAAGCGTACTCCAGTTGAAGAGTATCGACTACAAAGCCGTGGTGGTAAA

[0122] GGTCTTAAGACTTGTAACATTACAGATAAAAACGGTAAGTTAGTAGCGGTTAAGTCTGTA

[0123] ACAGGTGAAGAAGACATCATGCTAATTACAGCCGCAGGCGTTATTATTCGTATGCCAGTT

[0124] GATCAAATCTCTCAAATGGGACGTAATACACAAGGGTCTCAATGT(SEQ ID NO:8).

[0125] The sequence measured by Sd_h10 gyrB-F / R primers is as follows:

[0126] CGCATGCATGCGAGTGTGCATCTGTTGTAATGCCTTATCACAGAATTAGAAGTATTTG

[0127] TACATCGTGATGGCAAAATCCATTACCAAAAATACGAAAGAGGTATTCCGGTTGCAGATT

[0128] TAAAAGTCATCGGTGATACAGATAAGACTGGAACAATAACTCGCTTTAAACCGGATCCA

[0129] GAAATTTTTAAAGAGACGACAGAATATGAATTCGATACGCTAGCGACTCGTATGCGTGAG

[0130] TTGGCGTTTTTAAATCGTAATATTAAATTAACAATTGAAGATAAACGTGAACATAAGCAA

[0131] AAGAAAGAGTTCCACTATGAAGGTGGAATTAAATCATATGTTGAACATTTAAATCGTTCA

[0132] AAACAACCAATTCATGAAGAGCCTGTATATGTAGAAGGTTCAAAAGATGGTATTCAAGTT

[0133] GAAGTTGCGCTTCAATATAACGAAGGATATACAAATCATATTTACTCATTTACAAATAATAT

[0134] TCATACGTATGAAGGTGGTACACATGAGGTAGGATTTAAAACTGCCTTAACACGTGTGAT

[0135] TAACGATTATGGTCGTAAAAATAACATTTTAAAAGATGCGGATAGTAATTTGACTGGTGA

[0136] AGATGTTCGTGAAGGTTTAACAGCAATCGTGTCAATTAAACATCCAAATCCACAATTTGA

[0137] AGGGCAAACGAAGACGAAACTTGGAAATAGTGAAGCGAGAACGATTACGGAGTCAGTA

[0138] TTCTCTGAGGCTTTTGAAAAATTCTTACTGGAAAATCCCAATGTTGCACGTAAGGTTGTA

[0139] GATAAAGGGACGATGGCAGCACGTGCGCGTGTAGCAGCTAAAAAGGCTCGTGAGCTAA

[0140] CTCGCCGAAAGAGTGCTTTAGAAGTTTCAAGTTTACCAGGGAAATTGGCAGATTGTCCT

[0141] TCTAAAGATCCCCCT (SEQ ID NO:9).

[0142] Table 2 Biochemical identification results of Bacillus cereus

[0143]

[0144] 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 at Wuhan University, Wuhan, China) on October 31, 2024, and the preservation number is CCTCC NO: M 20242406.

[0145] Example 2 The ability of Bacillus Sd_h10 to utilize oil

[0146] 1) Growth curve determination

[0147] After Bacillus Sd_h10 was cultured overnight at 28°C, the bacterial culture was adjusted to OD600 = 0.3. Subsequently, it was inoculated into tryptic soy broth (TSB) supplemented with (2%, 4%, 6%, 8%, 10%, 12%, 14%, 16%, V / V) fish oil, soybean oil and paraffin at a ratio of 10 V / V%, and cultured with shaking at 28°C and 240 rpm. The growth curve of the bacteria was then measured.

[0148] Figure 3 AC are the growth curves of Bacillus Sd_h10 in different concentrations of fish oil, soybean oil and paraffin respectively. It can be found that the growth rate and final concentration of Bacillus Sd_h10 in fish oil are significantly better than those in soybean oil and paraffin. It can be considered that Bacillus Sd_h10 can utilize fish oil for growth.

[0149] 2) Determination of triglyceride and free fatty acid content in TSB

[0150] According to the growth trend of bacteria, Bacillus Sd_h10 was cultured in TSB containing 2%, 8% and 16% fish oil (V / V). The culture medium was taken out at 30min and 36h, respectively, and allowed to stand for 30min. 1mL of the bottom culture medium was aspirated and centrifuged under sterile conditions (4℃, 5000rpm, 10min). 600μL of the middle liquid was extracted and the changes in triglyceride and free fatty acid content in the bacterial liquid were detected using Amplex Red Triglyceride Assay Kit and Amplex Red Free Fatty Acid Assay Kit (Biyuntian Biotechnology Co., Ltd.).

[0151] Figure 3 DE in the middle are the test results of triglyceride and free fatty acid content, respectively. In TSB, the triglyceride content in the bacterial culture medium with different concentrations of fish oil added showed a significant decrease after 36 hours of culture. As for free fatty acids, after 36 hours of culture, the free fatty acid content of the 2% fish oil group increased slightly, while the free fatty acid content in the bacterial culture medium with 8% and 16% fish oil added was significantly reduced. These results show that Bacillus Sd_h10 can utilize triglycerides in the culture medium and produce free fatty acids. When it grows to a certain stage, Bacillus Sd_h10 may also utilize the produced free fatty acids.

[0152] 3) Determination of triglycerides and free fatty acids in culture medium with fish oil as carbon source only

[0153] 2% (V / V) fish oil was added as a carbon source to a basic medium without a carbon source (prepared in a ratio of 41 g diammonium hydrogen phosphate, 1 g sodium chloride, 0.2 g magnesium sulfate heptahydrate, and 0.5 g potassium dihydrogen phosphate per liter of double distilled water), without adding any other carbon source. According to the experimental methods described in 1) and 2) above, samples were taken after culturing the Bacillus Sd_h10 strain for 30 minutes, 12 hours, 14 hours, 16 hours, and 18 hours, respectively, to detect the content of triglycerides and free fatty acids in the culture medium.

[0154] The results are as follows Figure 3 As shown in Figure F, in the culture with fish oil as the carbon source, triglycerides decreased significantly after 12 hours of culture, and there was no significant change thereafter. Free fatty acids continued to increase within 14 hours after culture, and then gradually decreased and approached the initial level. This result further shows that Bacillus Sd_h10 can utilize triglycerides in fish oil and produce free fatty acids.

[0155] Example 3 Safety Determination of Bacillus Sd_h10

[0156] 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 concentrations 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 control (CK). The fish were cultured continuously at 28°C and observed for 10 days.

[0157] The results are as follows Figure 4 As shown in A, there was no death of zebrafish in the control group, and there was no death of zebrafish in water bodies with different bacterial concentrations, indicating that Bacillus Sd_h10 has no harm to zebrafish within a certain range.

[0158] Example 4 Effect of Bacillus Sd_h10 colonization on zebrafish fry

[0159] 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 the two groups of zebrafish. Zebrafish were collected 5 hours later for Oil Red O (ORO) staining and gene expression analysis using 2^ -ΔΔCt The relative expression of genes was calculated by the method. dgat2 is mainly involved in the conversion of diacylglycerol (DAG) into triglyceride (TG), which is the last step of fat synthesis; fabp4a can transport fatty acids from the cell membrane to the mitochondria for oxidative decomposition, or to the endoplasmic reticulum for triglyceride synthesis; fasn gene encodes fatty acid synthase; acaca catalyzes fatty acid synthesis. The primers of each gene are shown in Table 3 below.

[0160] Table 3 Quantitative analysis genes and their primers

[0161]

[0162]

[0163] Oil red O (ORO) staining results are as follows Figure 4 As shown, Figure 4 BC are the staining results of group A and group N respectively. Figure 4 DE in the middle are the relative area and average optical density after oil red staining of the two zebrafish feeding groups. It can be intuitively found that the lipid droplets in the gastrointestinal tract of group A zebrafish are more densely distributed and darker in color; the lipid droplets in the gastrointestinal tract of group N zebrafish are fewer and lighter in color.

[0164] The 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 fixed value of Bacillus Sd_h10 is beneficial to the absorption of oil by zebrafish.

[0165] 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 dgat2 and fabp4a related to triglyceride synthesis in group A zebrafish was significantly upregulated, while the expression of genes fasn and acaca related to fatty acid synthesis in group A zebrafish was significantly downregulated. The changes in the expression of these genes indicate that the addition of Bacillus Sd_h10 can promote the synthesis of lipids in zebrafish and provide additional fatty acids for zebrafish.

[0166] Example 5 Effect of Bacillus Sd_h10 colonization on zebrafish body length and weight

[0167] Twenty-four zebrafish aged 120 days 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 keep the bacterial concentration at 1×10 4 cfu / mL. The feeding experiment was carried out for 14 days. The length and weight of the fish were measured on the 1st and 15th days. The relative weight growth rate and relative body length growth rate of zebrafish were then calculated.

[0168] Figure 4 GH in the middle are the relative weight growth rate and relative length growth rate of zebrafish. Compared with feeding normal feed, the length and weight growth rate of zebrafish fed with 2% fish oil feed decreased significantly, which shows that high-fat feed has an inhibitory effect on the growth of zebrafish, and adding a certain concentration of Bacillus Sd_h10 in the water can effectively alleviate this inhibitory effect. These results show that Bacillus Sd_h10 is beneficial to the growth of fish 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. It was deposited in the China Center for Type Culture Collection on October 31, 2024, and the deposit number is CCTCC NO: M 20242406.

2. A culture obtained by culturing the Bacillus according to claim 1.

3. The method for preparing the culture according to claim 2, comprising the following steps: The bacillus according to claim 1 is inoculated into a culture medium and cultured to obtain.

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

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

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

7. Use of any one of (a1) to (a3) ​​in at least one of (b1) to (b2); (a1) the Bacillus according to claim 1; (a2) the culture of claim 2; (a3) the bacterial agent according to claim 4 or 5; (b1) preparing products that promote lipid metabolism; (b2) preparing feed additives.

8. A product comprising at least one of (a1) to (a3): (a1) the Bacillus according to claim 1; (a2) the culture of claim 2; (a3) The bacterial agent according to claim 4 or 5.

9. The product according to claim 8, characterized in that: The products include feed; Preferably, the feed comprises fish feed.

10. A method for raising fish, comprising the following steps: Using the product according to any one of claims 8 to 9 to feed fish.

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