Bacillus lentus and application thereof in aquaculture

Abstract

The invention relates to the technical field of functional microorganism screening and application, and particularly provides bacillus lentus and application of the bacillus lentus in aquaculture. The preservation number of the bacillus lentus is CCTCC NO: M20241882, and the bacillus lentus has very strong protease producing capacity, can efficiently inhibit various pathogenic bacteria, is widely applied to aquatic feed, can obviously improve the growth performance and immunity of aquatic animals, increase the activity of intestinal digestive enzyme and improve the feed utilization rate, and has wide application prospects.

Description

Technical Field

[0001] The invention relates to the technical field of functional microorganism screening and application, and in particular to a strain of Bacillus lentus and application thereof in aquaculture. Background Art

[0002] With the development of modern intensive and large-scale aquaculture, stressors have increased, resulting in a decline in the immune resistance of aquatic animals and greatly increased susceptibility to diseases. People usually use antibiotics and chemical synthetic drugs to prevent and treat aquatic animal diseases, but the negative effects of long-term and large-scale use of antibiotics have become increasingly apparent: not only do they destroy the normal intestinal flora of aquatic animals, causing intestinal microecological imbalance, leading to increased susceptibility to pathogenic microorganisms and bacterial resistance, but they also remain in the body, causing adverse reactions such as allergies, and causing pollution to the entire ecological environment.

[0003] Probiotics colonize in the intestines of aquatic animal hosts, accelerate the host's absorption and utilization of nutrients in their bodies, improve the host's immunity and the balance of intestinal flora structure. Unlike antibiotics, probiotics do not have resistance problems and do not pollute the environment. Relevant studies have shown that probiotics can promote digestion and nutrient absorption in aquatic animals, thereby increasing their growth rate. They can also prevent the attachment of pathogenic bacteria by competing for sites on the intestinal mucosa, thereby reducing the occurrence of diseases. They can also decompose harmful substances in water, such as ammonia and nitrite, reduce the concentration of toxic substances in water, and thus improve water quality. This can not only reduce toxicity to aquatic animals, but also reduce pollution to the environment. In general, they are a safe and environmentally friendly microbial preparation that can be used for a long time without affecting the safety of animals and product quality.

[0004] Based on the above advantages, screening out probiotics that can be efficiently used in aquaculture is of great significance for the green, healthy and sustainable development of aquaculture. Summary of the invention

[0005] The purpose of the present invention is to provide a strain of Bacillus lentus and its application in aquaculture. The Bacillus lentus is screened from fresh squid viscera, has strong environmental tolerance, can inhibit a variety of pathogens, helps reduce the incidence of diseases, improves the growth performance and immunity of aquatic animals, increases breeding benefits, and has broad application prospects.

[0006] In order to achieve the above object, the present invention adopts the following technical solution:

[0007] On the one hand, the present invention provides a strain of Bacillus lentus, named Bacillus lentus SK001, which was deposited in the China Type Culture Collection of Wuhan University, Wuhan, China on September 2, 2024, and its deposit number is CCTCC NO: M20241882.

[0008] In one aspect, the present invention provides the use of the Bacillus lentus in preparing aquatic feed.

[0009] The present invention also provides the use of the Bacillus lentus in preparing biological products with the function of inhibiting pathogenic bacteria.

[0010] The pathogenic bacteria are selected from any one or more of Staphylococcus aureus, Escherichia coli, Bacillus cereus, Vibrio parahaemolyticus, Aeromonas hydrophila, Pseudomonas fluorescens, and Escherichia coli.

[0011] The invention also provides application of the Bacillus lentus in the production of protease.

[0012] The present invention also provides a microbial preparation comprising the Bacillus lentus.

[0013] The microbial preparation also includes any one or more of Bacillus licheniformis, Bacillus subtilis, Bacillus coagulans, Bacillus pumilus, Bacillus laterosporus, Saccharomyces cerevisiae, Bifidobacterium, Enterococcus faecium, Lactobacillus acidophilus, Enterococcus faecalis, Pediococcus acidilactici, and Clostridium butyricum.

[0014] The microbial preparation contains no less than 10 8 CFU / g.

[0015] The invention also provides application of the microbial preparation in preparing aquatic feed.

[0016] The Bacillus lentus SK001 provided by the present invention has a strong tolerance to the digestive tract environment. After being treated in artificial gastric juice and artificial intestinal juice for 3 hours, its survival rate is still as high as over 85%. It can enter the intestine through the stomach better, exert its probiotic effect, and achieve unexpected technical effects.

[0017] The Bacillus lentus SK001 provided by the present invention can effectively inhibit multiple pathogens such as Staphylococcus aureus, Escherichia coli, Bacillus cereus, Vibrio parahaemolyticus, Aeromonas hydrophila, Pseudomonas fluorescens, Escherichia coli, etc., especially has the strongest antibacterial effect on Staphylococcus aureus and Escherichia coli, and the diameter of the antibacterial zone exceeds 22mm, which is significantly better than the commercially available Bacillus lentus. The self-aggregation and hydrophobicity of the strain are as high as 43.1% and 56.1% respectively, both higher than the commercially available Bacillus lentus, which is conducive to its better colonization in the intestinal tract of aquatic organisms.

[0018] The Bacillus lentus SK001 can produce a high amount of protease, and the protease activity in the fermentation supernatant reaches 389.1 U / mL, which is significantly higher than that of the commercially available Bacillus lentus in the control group, achieving an unexpected technical effect.

[0019] The Bacillus lentus SK001 can be widely used as a feed additive in aquaculture, and can significantly improve the growth performance and immunity of aquatic animals, increase the activity of intestinal digestive enzymes, and improve feed utilization. Compared with the control group, the weight gain rate of the freshwater grouper in the probiotic group fed with Bacillus lentus SK001 bacterial powder increased by 70.8% (P<0.05), the specific growth rate increased by 52.8% (P<0.05), the total superoxide dismutase and glutathione peroxidase activities in the serum of the freshwater grouper increased by 24.2% and 13.5%, respectively, and the catalase and malondialdehyde contents decreased by 126.7% and 9.5%, respectively, achieving unexpected technical effects.

[0020] The Bacillus lentus SK001 can better induce the production of immune factors, actively participate in the body's immune defense, and thus improve the body's immune defense. Compared with the control group, the survival rate, weight gain rate, and specific growth rate of the probiotic group of shrimp fed with Bacillus lentus SK001 powder increased by 25.3%, 21.2%, and 12.9%, respectively, and the feed coefficient decreased by 18.5%; and the hepatopancreatic lysozyme activity of the probiotic group increased by 42.0%, achieving unexpected technical effects.

[0021] The Bacillus lentus SK001 provided by the present invention can be used alone, or can be mixed with probiotics such as Bacillus licheniformis, Bacillus subtilis, Bacillus coagulans, Bacillus brevis, Bacillus laterosporus, Saccharomyces cerevisiae, Bifidobacterium, Enterococcus faecium, Lactobacillus acidophilus, Enterococcus faecalis, Pediococcus acidilactici, Clostridium butyricum, etc. and applied in aquatic feed, which is helpful to increase the economic benefits of breeding and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the colony morphology of Bacillus lentus SK001. DETAILED DESCRIPTION

[0023] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0024] The culture medium selected in the embodiments of the present invention is listed below, which is only for illustration and is not intended to limit the present invention.

[0025] LB medium: 5 g yeast extract, 10 g tryptone, 10 g sodium chloride, distilled water, dilute to 1000 mL. pH 7.0. Add 2% agar to the solid medium.

[0026] Isolation medium: tryptone 12.0 g, yeast extract 8.0 g, potassium dihydrogen phosphate 2.0 g, magnesium sulfate 0.1 g, agar powder 20.0 g, deionized water 1000 mL, pH 7.0±0.2.

[0027] Casein medium: Add 20 mL of 0.1 mol / L NaOH solution to 4 g of casein and dissolve in a water bath. Weigh 1.07 g of disodium hydrogen phosphate heptahydrate and 0.36 g of potassium dihydrogen phosphate, dissolve them in distilled water, mix with the casein solution, and adjust the volume to 1000 mL, pH 7.5. Add 2% agar to the solid medium.

[0028] Nutrient agar medium: peptone 10.0, beef powder 3.0, sodium chloride 5.0, agar 15.0, pH 7.3±0.1 (25℃).

[0029] Example 1 Isolation, screening and identification of strains

[0030] 1. Isolation and screening of strains

[0031] Fresh squid viscera were purchased from Manyuwan Farmers Market in Licang District, Qingdao. After the squid viscera were beaten into a pulp and filtered through four layers of gauze to remove the sediment, 10 ml of the filtrate was placed in a conical flask, 90 ml of sterile water was added, and then heated in an 80°C water bath for 10 minutes to eliminate non-Bacillus bacteria. Under a sterile environment, 10 ml of the cooled squid viscera juice was added to 90 ml of LB liquid culture medium and oscillated at 37°C for 24 hours. The mixture was diluted to 10 with physiological saline. -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 . Pipette 20 μL of sample solution of different dilutions onto casein solid agar culture plates, spread evenly, let stand for 30 minutes, turn the plates upside down, and culture at 37°C. Inoculate three plates for each dilution. Observe the plates daily to see if there are colonies, and preliminarily judge the protease production capacity of the strains based on the size of the transparent circle.

[0032] The colony with the largest transparent circle was inoculated on a new casein culture plate, and the plate was streaked repeatedly until pure colonies were obtained. Finally, the pure colonies were inoculated on the slant culture medium and stored at 4°C after cultivation.

[0033] The strain with the strongest protease production ability obtained from the above screening was named SK001.

[0034] 2. Identification of strains

[0035] (1) Colony biological identification

[0036] The colony morphology of strain SK001 on nutrient agar medium is as follows Figure 1 As shown, the colony is 1-2mm in diameter, round, with neat edges, opaque, grayish white on the front, convex in the middle, smooth, bright on the surface, moist in texture, and easy to pick up.

[0037] (2) Physiological and biochemical identification results

[0038] According to the Manual of Identification of Common Bacteria Systems and the Bergey Manual of Identification of Bacteria, the obtained strains were subjected to physiological and biochemical identification, involving specific indicators such as indole test, hydrogen sulfide test, acetylmethylcarbinol test (VP test), nitrate reduction, propionate utilization, D-mannitol fermentation, and 7% sodium chloride growth. Based on the above results, the strain SK001 can be preliminarily judged to be Bacillus lentus.

[0039] Table 1 Physiological and biochemical characteristics of Bacillus lentus SK001

[0040]

[0041] Note: "+" indicates positive, "-" indicates negative

[0042] (3) Molecular identification

[0043] The obtained strain was extracted using a bacterial genomic DNA extraction kit, and the universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-TACGGCTACCTTGTTACGACTT-3') were used for PCR amplification. The amplified product was added to a 1.5% agarose gel and analyzed by electrophoresis. After the detection of successful amplification, the PCR product was sent to a third-party testing company for sequence determination. The sequencing results were compared for homology using the BLAST tool in NCBI to determine the species classification of the strain. The NCBI comparison results showed that the screened SK001 strain had more than 99.3% similarity with Bacillus subtilis.

[0044] Based on the morphological characteristics, physiological and biochemical characteristics and molecular identification results of the SK001 strain, the applicant determined that the SK001 strain was Bacillus lentus, named it Bacillus lentus SK001 (Bacillus lentusSK001), and it was deposited in the China Type Culture Collection of Wuhan University, Wuhan, China on September 2, 2024, and its preservation number is CCTCC NO: M20241882.

[0045] Example 2 Safety Analysis of Bacillus lentus SK001

[0046] 1. Hemolytic test

[0047] Use a sterile inoculation loop to take the bacterial lawn of Bacillus in the exponential growth period, streak it on Columbia sheep blood plate, incubate it in a constant temperature incubator at 37℃ for 24 hours, and observe whether there is a hemolytic ring around the colony. Use Staphylococcus aureus as a control.

[0048] The results of the hemolytic test showed that there was an obvious hemolytic ring around the Staphylococcus aureus bacterial lawn, while the strain SK001 did not produce an obvious transparent hemolytic ring and had no hemolytic phenomenon.

[0049] 2. Antibiotic resistance test

[0050] Activate the isolated strains to make the OD600 value of the strains reach 1, take 1mL of bacterial solution, centrifuge at 4°C, resuspend the bacteria with PBS buffer, take 100μL of the resuspended bacterial solution and evenly spread it on the LB solid culture medium, use sterilized tweezers to clamp the antibiotic sensitive paper and place it on the surface of the plate coated with the bacterial solution, seal the plate and place it in a constant temperature incubator for 24h to observe whether an inhibition zone is produced. The specific results are shown in Table 2.

[0051] Table 2 Antibiotic sensitivity test of Bacillus lentus SK001

[0052]

[0053]

[0054] Note: S is sensitive; I is intermediate; R is resistant.

[0055] From the results in Table 2, it can be seen that strain SK001 is sensitive to common antibiotics such as ampicillin, furazolidone, chloramphenicol, tetracycline, penicillin G, gentamicin, etc., and has good biosafety. Example 3 Analysis of the probiotic properties of Bacillus lentus SK001

[0056] 1. Antibacterial properties of Bacillus lentus

[0057] Centrifuge the 18-20h Bacillus nutrient broth culture at 12000r / min for 5min, and place the supernatant in a 4℃ refrigerator for later use. 6 100 μL of indicator bacteria solution with a concentration of 1×10 CFU / mL was evenly spread on the LB solid plate, and an Oxford cup was placed. 8 200 μL of the fermentation supernatant of Bacillus lentus with CFU / mL was cultured at 37°C for 24 hours to observe whether an inhibition zone was produced and measure the size of the inhibition zone. At the same time, commercially available Bacillus lentus was used as a control.

[0058] Table 3 Antibacterial activity of Bacillus lentus SK001

[0059]

[0060] From the data in Table 3, it can be seen that the Bacillus lentus SK001 provided by the present invention can effectively inhibit various pathogens such as Staphylococcus aureus, Escherichia coli, Bacillus cereus, Vibrio parahaemolyticus, Aeromonas hydrophila, Pseudomonas fluorescens, Escherichia coli, etc., especially the strongest antibacterial effect on Staphylococcus aureus and Escherichia coli, with the diameter of the inhibition zone exceeding 22 mm, which is significantly better than the commercially available Bacillus lentus.

[0061] 2. Self-aggregation and hydrophobicity of Bacillus lentus

[0062] The self-aggregation and surface hydrophobicity of probiotics are important factors that reflect the strength of their adhesion ability and whether they can colonize in the intestines and play a probiotic role. The greater the hydrophobicity of the cell surface, the stronger its adhesion. The self-aggregation of probiotics refers to the phenomenon that the bacteria aggregate into clusters due to large-scale and rapid reproduction during the cultivation process. The stronger the surface hydrophobicity and self-aggregation, the stronger its intestinal adhesion.

[0063] Inoculate Bacillus in LB liquid medium and culture at 37°C for 18 hours. Centrifuge the cultured bacterial suspension at 4°C and 12000r / min for 5 minutes to collect the bacterial cells, and wash them twice with sterile saline to remove the residual liquid in the culture medium. Adjust the OD600 of the bacterial suspension to 0.5 (A 0 ). Take 2 mL of bacterial suspension and let it stand at room temperature for 3 h, take 200 μL of the upper bacterial liquid, and measure the OD value (A) at 600 nm. 1 ); Take another 2 mL of bacterial suspension and mix it with an equal volume of xylene for 2 min, let it stand for 30 min, and measure the OD (A) of the aqueous phase at 600 nm. 2 ). Physiological saline was used as blank.

[0064] The calculation formula of self-aggregation and hydrophobicity is:

[0065] Self-aggregation (%) = (1-A 1 / A0 )×100%.

[0066] Hydrophobicity (%) = [(A 0 -A 2 ) / A 0 ]×100%.

[0067] Where: A 0 is the OD value of the initial bacterial solution at 600nm; A1 is the OD value of the supernatant at 600nm after standing for 3h; A 2 It is the OD value of the aqueous phase at 600nm after mixing with xylene.

[0068] Table 4 Self-aggregation and hydrophobicity of Bacillus lentus SK001

[0069]

[0070] As can be seen from Table 4, the self-aggregation and hydrophobicity of Bacillus lentus SK001 are as high as 43.1% and 56.1%, respectively, which are higher than those of commercially available strains, thus facilitating its better colonization in the intestinal tract of aquatic organisms.

[0071] Example 4 Tolerance test of Bacillus lentus SK001 in simulated artificial stomach and intestinal fluid

[0072] Prepare artificial gastric juice according to the method of Chinese Pharmacopoeia: take 16.4mL of dilute hydrochloric acid, add 800mL of water and 10g of pepsin, shake well, and dilute to 1000mL with water; dilute hydrochloric acid is 234mL of concentrated hydrochloric acid diluted to 1000mL with water to obtain 9.5% to 10.5% dilute hydrochloric acid. Artificial intestinal juice: take 6.8g of potassium dihydrogen phosphate, add 500mL of water to dissolve it, adjust the pH value to 6.8 with 0.1mol / L sodium hydroxide solution, take 10g of pancreatic enzyme, add appropriate amount of water to dissolve it, mix the two liquids, and dilute to 1000mL with water.

[0073] The concentration was 1×10 9 CFU / mL of Bacillus lentus suspension was inoculated into simulated gastric juice, simulated intestinal juice and 0.9% saline, respectively, with an inoculation amount of 10%, and the saline group was used as the control group. The suspension was incubated at 37℃ on a shaker (180r / min). The suspension was diluted 10 times with sterile saline at 0, 1, 2, and 3 hours of culture, and the plate colony count was performed. Each dilution was repeated 3 times. The number of viable bacteria at 0h was used as a control to calculate the survival rate under each treatment condition.

[0074] Table 5 Tolerance of Bacillus lentus SK001 to simulated artificial gastric and intestinal fluids

[0075]

[0076] From the results in Table 5, it can be seen that compared with the commercially available Bacillus lentus, the Bacillus lentus SK001 provided by the present invention has a stronger tolerance to the digestive tract environment. After being treated in artificial gastric juice and artificial intestinal juice for 3 hours, the survival rate of the strain is still as high as more than 85%, achieving an unexpected technical effect, and can better pass through the stomach into the intestines to play its probiotic role.

[0077] Example 5 Determination of protease production capacity of Bacillus lentus SK001

[0078] After activation, Bacillus lentus SK001 was inoculated into LB liquid culture medium and cultured at 37°C for 36 hours; the fermentation broth was centrifuged at 4°C and 12000r / min for 5 minutes to collect the supernatant, and the enzyme activity of the protease in the supernatant was determined using the Folin phenol reagent method.

[0079] Table 6 Protease activity of Bacillus lentus SK001

[0080] strain Protease (U / mL) Bacillus lentus 25.7±17.9 Bacillus lentus SK001 389.1±20.7

[0081] From the data in Table 6, it can be seen that the Bacillus lentus SK001 provided by the present invention can produce a high amount of protease, and the protease activity in the fermentation supernatant reaches 389.1 U / mL, which is significantly higher than that of the commercially available Bacillus lentus in the control group.

[0082] Example 6 Application of Bacillus lentus SK001 in freshwater grouper farming

[0083] 1. Preparation of bacterial powder

[0084] The Bacillus lentus SK001 was activated, expanded, and fermented in liquid form, centrifuged, concentrated, and freeze-dried to produce a culture medium with a live bacterial content of about 10 10 CFU / g of bacterial powder.

[0085] 2. Experimental Design

[0086] Freshwater grouper with an average weight of about 60g was selected, and a control group and a probiotic group were set up. The control group was fed with basic feed, and the probiotic group was fed with the above-mentioned Bacillus lentus SK001 powder at a weight ratio of 5‰ on the basis of the basic feed, and the mixture was evenly mixed before feeding. Three parallels were set for each group, with 40 freshwater groupers in each parallel, and the water temperature was controlled at 22-25℃. The feeding time was 9:00 and 17:00 respectively. The feeding should be appropriate at 9:00. After 0.5h, the feeding situation of the fish should be observed and recorded. The feeding amount was adjusted appropriately according to the feeding situation. The remaining feed was removed 0.5h after feeding. The bottom of the breeding barrel was sucked every morning and evening, and 1 / 3 of the breeding water was replaced. The replaced water was disinfected, aerated, and tap water of the same temperature. Feeding and breeding for 28d.

[0087] On the 28th day of the experiment, 3 freshwater groupers were selected from each barrel, weighed, and measured in length, and the weight gain rate, specific growth rate, survival rate, fatness, liver-body ratio, organ-body ratio, and feed coefficient were calculated. The fish were starved for 24 hours the day before blood collection, and blood was collected from the tail vein. The blood samples were centrifuged at 4°C and 4000r / min for 10 minutes, and the supernatant was taken to determine the antioxidant index, which was determined using the Nanjing Jiancheng Bioengineering Research Institute kit. The results are shown in Tables 7-9.

[0088] Table 7 Effects of Bacillus lentus SK001 on the growth performance of freshwater grouper

[0089] Group Control group Probiotics group Weight gain rate / % <![CDATA[57.61±0.15 a ]]> <![CDATA[98.42±0.74 b ]]> Specific growth rate / (% / d) <![CDATA[2.31±0.36 a ]]> <![CDATA[3.53±0.41 b ]]> Liver to body ratio / % 1.53±0.18 1.64±0.31 Viscera to body ratio / % 3.32±0.26 3.73±0.22 <![CDATA[Condition factor / (g / cm 3 )]]> 1.25±0.29 1.42±0.27 Feed coefficient 1.62±0.24 1.13±0.24 Survival rate / % 95 100

[0090] Table 8 Effects of Bacillus lentus SK001 on the immune ability of freshwater grouper

[0091] index Control group Probiotics group Total superoxide dismutase / (U / mL) <![CDATA[55.42±0.91 a ]]> <![CDATA[68.83±1.32 a ]]> Catalase / (U / mL) 9.75±0.22 <![CDATA[4.30±0.69 b ]]> Malondialdehyde / (μmol / L) <![CDATA[140.27±1.06 a ]]> <![CDATA[128.05±2.44 b ]]> Glutathione peroxidase GSH / (μmol / L) <![CDATA[103.53±3.41 a ]]> <![CDATA[117.50±3.42 b ]]>

[0092] Table 9 Effects of Bacillus lentus SK001 on serum liver function indicators of freshwater grouper

[0093] index Control group Probiotics group Aspartate aminotransferase / (U / L) <![CDATA[21.32±1.47 a ]]> <![CDATA[15.73±0.54 b ]]> Alanine aminotransferase / (U / L) <![CDATA[21.07±1.22 a ]]> <![CDATA[15.62±1.42 a ]]>

[0094] From the data in Table 7, it can be seen that compared with the control group, the weight gain rate of freshwater grouper in the probiotic group fed with Bacillus lentus SK001 powder increased by 70.8% (P<0.05), the specific growth rate increased by 52.8% (P<0.05), and the growth performance was significantly improved.

[0095] From the data in Table 8, it can be seen that compared with the control group, the activities of total superoxide dismutase and glutathione peroxidase in the serum of freshwater grouper in the probiotic group fed with Bacillus lentus SK001 powder increased by 24.2% and 13.5%, respectively, and the contents of catalase and malondialdehyde decreased by 126.7% and 9.5%, respectively.

[0096] As shown in Table 9, compared with the control group, the levels of aspartate aminotransferase and alanine aminotransferase in the serum of freshwater grouper in the probiotic group fed with Bacillus lentus SK001 powder decreased by 35.5% and 34.9%, respectively.

[0097] The above results show that the Bacillus lentus SK001 provided by the present invention can significantly improve the antioxidant capacity and immune level in the body of freshwater grouper, reduce liver damage in aquatic animals, and effectively promote the growth of farmed animals.

[0098] Example 7 Application of Bacillus lentus SK001 in the cultivation of Litopenaeus vannamei

[0099] 1. Preparation of bacterial powder

[0100] The Bacillus lentus SK001 was activated, expanded, and fermented in liquid form, centrifuged, concentrated, and freeze-dried to produce a culture medium with a live bacterial content of about 10 10 CFU / g of bacterial powder.

[0101] 2. Experimental design

[0102] The experiment set up a control group and a probiotic group. The control group was fed with basic feed. The probiotic group added the above-mentioned Bacillus lentus SK001 powder to the basic feed at a weight ratio of 3‰, mixed evenly and fed. Three parallels were set up in each group, with 50 shrimps in each parallel. The breeding experiment lasted for 8 weeks, and the daily feeding amount was 3% of the total weight of the shrimp. The shrimps were fed twice a day, and the shrimps were weighed every 2 weeks to adjust the daily feeding amount. The dry matter weight of the feed fed to the control group and the probiotic group was kept consistent. The breeding water was replaced once a week, and the water change amount was 1 / 4 of the total water volume. During the experiment, the dissolved oxygen was ≥8.3mg / l, the temperature was 25-29℃, the salinity was 6-7‰, and the pH was 7.5-7.9. After the breeding experiment, the shrimps were counted and weighed, and fasted for one day before weighing. Its survival rate, specific growth rate, weight gain rate, etc. were calculated. Hepatopancreatic T-AOC, superoxide dismutase, and lysozyme were detected using the Nanjing Jiancheng Bioengineering Institute kit. The specific results are shown in Tables 10 and 11.

[0103] Table 10 Effects of Bacillus lentus SK001 on growth and survival of Litopenaeus vannamei

[0104] Control group Probiotics group Initial body weight / g 5.41±0.12 5.37±0.23 Final body weight / g 14.42±0.56 16.21±0.27 Survival rate / % <![CDATA[74.34±3.33 a ]]> <![CDATA[93.12±3.54 b ]]> Weight gain rate / % <![CDATA[166.54±20.25 a ]]> <![CDATA[201.86±15.92 b ]]> Specific growth rate / % <![CDATA[3.50±0.11 a ]]> <![CDATA[3.95±0.14 b ]]> Feed coefficient <![CDATA[1.73±0.21 a ]]> <![CDATA[1.41±0.35 b ]]>

[0105] Note: Different letters indicate significant differences (P<0.05).

[0106] Table 11 Effects of Bacillus lentus SK001 on immunity of Litopenaeus vannamei

[0107] Project (U / mg prot) Control group Probiotics group T-AOC 2.12±0.13 2.47±0.15 Superoxide dismutase 43.52±2.10 43.46±2.43 Lysozyme activity <![CDATA[101.30±7.67 b ]]> <![CDATA[143.82±5.32 a ]]>

[0108] Note: Different letters indicate significant differences (P<0.05).

[0109] From the results of Table 10 and Table 11, it can be seen that compared with the control group, the survival rate, weight gain rate, and specific growth rate of the probiotic group of shrimp fed with Bacillus lentus SK001 powder increased by 25.3%, 21.2%, and 12.9%, respectively, and the feed coefficient decreased by 18.5%; and the hepatopancreatic lysozyme activity of the probiotic group increased by 42.0%. This shows that the Bacillus lentus SK001 provided by the present invention can better induce the production of immune factors, actively participate in the body's immune defense, thereby improving the body's immune defense, and achieving unexpected technical effects.

[0110] In summary, the Bacillus lentus SK001 provided by the present invention has a strong ability to produce protease, has a strong tolerance to the digestive tract environment, can effectively inhibit Staphylococcus aureus, Escherichia coli, Bacillus cereus, Vibrio parahaemolyticus, Aeromonas hydrophila, Pseudomonas fluorescens, Escherichia coli and other pathogens, and reduce the incidence of aquatic animal diseases. Bacillus lentus SK001 can be used alone, or it can be compounded with probiotics such as Bacillus licheniformis, Bacillus subtilis, Bacillus coagulans, Bacillus brevis, Bacillus laterosporus, Saccharomyces cerevisiae, Bifidobacterium, Enterococcus faecium, Lactobacillus acidophilus, Enterococcus faecalis, Pediococcus acidilactici, Clostridium butyricum as a feed additive, which is widely used in aquaculture, can significantly improve the growth performance and immunity of aquatic animals, increase the activity of intestinal digestive enzymes, improve feed utilization, and help to improve the economic benefits of farmers, and has broad application prospects.

Claims

1. A Bacillus lentus, characterized in that The deposit number of the Bacillus lentus is CCTCC NO: M20241882.

2. Use of Bacillus lentus as claimed in claim 1 in preparing aquatic feed.

3. Use of the Bacillus lentus as claimed in claim 1 in the preparation of biological products having the function of inhibiting pathogenic bacteria.

4. The use according to claim 3, characterized in that The pathogenic bacteria are any one or more of Staphylococcus aureus, Escherichia coli, Bacillus cereus, Vibrio parahaemolyticus, Aeromonas hydrophila, Pseudomonas fluorescens, and Escherichia coli.

5. Use of Bacillus lentus as claimed in claim 1 in the production of protease.

6. A composite microbial preparation, characterized in that: The composite microbial preparation comprises the Bacillus lentus described in claim 1.

7. The composite microbial preparation according to claim 6, characterized in that: The composite microbial preparation further comprises any one or more combinations of Bacillus licheniformis, Bacillus subtilis, Bacillus coagulans, Bacillus pumilus, Bacillus laterosporus, Saccharomyces cerevisiae, Bifidobacterium, Enterococcus faecium, Lactobacillus acidophilus, Enterococcus faecalis, Pediococcus acidilactici, and Clostridium butyricum.

8. The composite microbial preparation according to claim 6, characterized in that The live bacteria amount of Bacillus lentus in the composite microbial preparation is at least 10 8 CFU / g.

9. Use of the composite microbial preparation according to any one of claims 6 to 8 in the preparation of aquatic feed.

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