A strain of Bacillus subtilis L25 and its application

Abstract

The present invention belongs to the technical field of probiotic additives for aquatic feed, and specifically relates to a strain of Bacillus subtilis L25 and its application. The strain was deposited in the China Center for Type Culture Collection on March 8, 2024, with the deposit number CCTCC NO: M2024428. The present invention provides a strain of Bacillus subtilis L25, which is isolated from the intestine of large yellow croaker; the probiotic effect of Bacillus subtilis L25 isolated from the intestine of large yellow croaker in the feed of large yellow croaker juveniles was verified by in vivo experiments. The Bacillus subtilis L25 can improve the intestinal health of large yellow croaker juveniles by improving the digestion and absorption capacity of the intestine of large yellow croaker juveniles and promoting their intestinal development, thereby improving their growth performance.

Description

Technical Field

[0001] The invention belongs to the technical field of aquatic feed probiotic additives, and particularly relates to a strain of Bacillus subtilis L25 and an application thereof. Background Art

[0002] Juvenile fish are in the early stage of fish growth and development. Their intestinal structure is not fully developed, digestive enzyme activity is low, and intestinal flora has not yet been fully established. During this period, the intestines of juvenile fish are easily stimulated by the external environment, which can easily lead to intestinal damage, and then cause problems such as high mortality and high morbidity. These challenges seriously hinder the sustainable development of seed breeding. At this critical stage, it is necessary to take effective measures to promote the healthy development of the intestines of juvenile fish, improve the activity of digestive enzymes, and strengthen the establishment of the immune system, thereby reducing mortality and morbidity, and creating more favorable conditions for the sustainable development of aquaculture.

[0003] Large yellow croaker is the fish with the largest marine aquaculture output in my country, and the success of artificial breeding has made large-scale farming possible. However, the imperfect development of the digestive system of juvenile fish is one of the important factors leading to high mortality, which seriously limits the healthy and sustainable development of large yellow croaker aquaculture. The intestinal microbiota plays a vital role in the development and maturation of the intestine. The early colonization of the intestinal microbiota contributes to the health of the host by promoting the establishment of intestinal barrier function and the maturation of the immune system. Therefore, improving the intestinal health of juvenile fish by regulating the intestinal flora is of great significance for seed cultivation. Summary of the invention

[0004] In order to overcome the shortcomings of the prior art, the present invention aims to provide a strain of Bacillus subtilis L25 and applications thereof.

[0005] The technical problem to be solved by the present invention is to provide a strain of Bacillus subtilis L25 and its application. After application, the strain of the present invention can enhance the intestinal digestion and absorption capacity of juvenile large yellow croaker and promote its intestinal development, thereby improving its intestinal health and further improving its growth performance.

[0006] The purpose of the present invention is achieved by at least one of the following technical solutions.

[0007] The present invention provides a Bacillus altitudinis L25 (Bacillus altitudinis L25), which was deposited in the China Center for Type Culture Collection on March 8, 2024, with an address of No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, and a preservation number of CCTCC NO: M2024428.

[0008] Furthermore, the 16S rDNA and gyrB sequences of the Bacillus subtilis L25 are shown in SEQ NO.1 and SEQ NO.2, respectively.

[0009] The invention discloses an application of the Bacillus subtilis L25 provided by the invention in preparing feed for juvenile large yellow croaker.

[0010] The invention provides a feed for juvenile yellow croaker containing Bacillus subtilis L25, wherein the number of viable bacteria of Bacillus subtilis L25 is 1×10 6 ~1×10 8 cfu / g feed.

[0011] The invention discloses an application of the Bacillus subtilis L25 provided by the invention in preparing a microecological preparation for improving the intestinal health of juvenile large yellow croaker.

[0012] The invention discloses an application of the Bacillus subtilis L25 provided by the invention in preparing a microecological preparation for improving the growth performance of large yellow croaker.

[0013] The invention provides a microecological preparation containing Bacillus subtilis L25.

[0014] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0015] The invention provides a highland bacillus L25, which is isolated from the intestine of large yellow croaker; the probiotic effect of the highland bacillus L25 isolated from the intestine of large yellow croaker in the feed of large yellow croaker fry is verified through in vivo experiments. The highland bacillus L25 can promote the intestinal development of large yellow croaker fry, improve its intestinal health, and thus improve its growth performance by improving the digestion and absorption capacity of the intestine of large yellow croaker fry. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a graph showing the effect of Bacillus subtilis L25 on the survival rate of juvenile large yellow croaker.

[0017] Figure 2a This is a graph showing the effect of Bacillus subtilis L25 on the weight of juvenile yellow croaker during growth performance.

[0018] Figure 2b This is a graph showing the effect of Bacillus subtilis L25 on the specific growth rate of juvenile yellow croaker.

[0019] Figure 3a This is a graph showing the effect of Bacillus subtilis L25 on the amylase activity in the intestinal digestion and absorption function of juvenile large yellow croaker.

[0020] Figure 3b This is a graph showing the effect of Bacillus subtilis L25 on trypsin activity in the intestinal digestion and absorption function of juvenile large yellow croaker.

[0021] Figure 3cThis is a graph showing the effect of Bacillus subtilis L25 on the lipase activity in the intestinal digestion and absorption function of juvenile large yellow croaker.

[0022] Figure 3d This is a graph showing the effect of Bacillus subtilis L25 on the ATPase activity in the intestinal digestion and absorption function of juvenile large yellow croaker.

[0023] Figure 4a This is a graph showing the effects of Bacillus subtilis L25 on leucine aminopeptidase related to intestinal development in juvenile large yellow croaker.

[0024] Figure 4b This is a graph showing the effects of Bacillus subtilis L25 on alkaline phosphatase related to intestinal development of juvenile large yellow croaker.

[0025] Figure 4c This is a graph showing the effect of Bacillus subtilis L25 on the expression of OCD genes related to intestinal development in juvenile large yellow croaker.

[0026] Figure 4d This is a graph showing the effect of Bacillus subtilis L25 on the expression of PCNA gene related to intestinal development of juvenile large yellow croaker.

[0027] Figure 5 Schematic diagram of the 16S rDNA gene evolutionary tree of Bacillus subtilis L25.

[0028] Figure 6 Schematic diagram of the evolutionary tree of the gyrB gene of Bacillus subtilis L25. DETAILED DESCRIPTION

[0029] The specific implementation of the present invention is further described below in conjunction with examples, but the implementation and protection of the present invention are not limited thereto. It should be noted that if there are processes that are not particularly described in detail below, they can be implemented or understood by those skilled in the art with reference to the prior art. If the manufacturer of the reagents or instruments used is not indicated, they are deemed to be conventional products that can be purchased commercially.

[0030] Example 1

[0031] Example 1 Isolation and molecular identification of Bacillus subtilis L25.

[0032] Collect fresh intestinal samples of large yellow croaker under sterile conditions, squeeze out the intestinal contents, and rinse the intestine with sterile PBS. Place the intestinal sample in a sterile homogenizer and add an equal amount of sterile PBS for homogenization. Use sterile PBS to dilute the obtained intestinal homogenate at a ratio of 1:10, take 100μl of the intestinal homogenate of each concentration gradient, and evenly spread it on LB nutrient agar solid medium, culture at 28℃, and after a single colony grows on the plate, use an inoculation loop to pick a single colony and streak it to purify the strain. Pick the purified single colony into LB broth medium and culture it for 18h at 28℃ and a shaker speed of 200rpm. The bacterial solution was taken, and after the bacterial genomic DNA was extracted, the 16S rDNA sequence of the strain was amplified using the 16S rDNA universal primers 27F (AGAGTTTGATCCTGGCTCAG, as shown in SEQ ID NO.3) and 1492R (GGTTACCTTGTTACGACTT, as shown in SEQ ID NO.4), and the gyrB gene primers gyrB-F (GAAGTCATCATGACCGTTCTGCAYGCNGGNGGNAARTTYGA, as shown in SEQ ID NO.5) and gyrB-R (AGCAGGGTACGGATGTGCGAGCCRTCNACRTCNGCRTCNGTCAT, as shown in SEQ ID NO.6). The PCR reaction system: PremixSTAR Max DNA Polymerase 12.5μl, DNA template 2μl, ddH2O 8.5μl, Forward primer 1μl, Reverse primer 1μl. PCR program: 98℃10s, 55℃15s, 72℃1.5min, 35 cycles; 72℃10min. The PCR product was sent to Shanghai Sangon Biotech Co., Ltd. for sequencing, and the obtained 16S rDNA and gyrB sequences were compared on the NCBI website and preliminarily identified as Bacillus altitudinis, which was numbered L25.

[0033] Bacillus subtilis L25 was deposited in the China Center for Type Culture Collection on March 8, 2024, with the deposit number CCTCC NO: M2024428, and the 16S rDNA sequence (as shown in SEQ ID NO.1):

[0034] CGGGGGGGGTGCTATACATGCAGTCGAGCGGACAGAAGGGAGCTTGCTCCCGGATGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGAGCTAATACCGGATAGTTCCTTGAACCGCATGGTTCAAGGATGAAAGACGGTTTCGGCTGTCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTGCAAGAGTAACTGCTTGCACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGAAACTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAACCCTAGAGATAGGGCTTTCCCTTCGGGGACAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTTAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACAGAACAAAGGGCTGCGAGACCGCAAGGTTTAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGCAACACCCGAAGTCGGTGAGGTAACCTTTATGGAGCCAGCCCCGCCA

[0035] Phylogenetic tree of 16S rDNA gene (as Figure 5 shown).

[0036] gyrB sequence (as shown in SEQ ID NO.2):

[0037] CCGGGGCAGCGGTTATAAGTATCTGGCGGTCTGCATGGTGTAGGGGCATCTGTTGTTAATGCGTTATCTACGACCTTAGACGTGACCGTATATCGTGATGGGAAAATTCATTATCAACAATTCAAACGCGGTGTTCCAGTTGGAGATTTAGAGGTCATTGGTGAAACAGATGTAACAGGGACAACCACTCATTTTGTGCCAGATCCAGAAATTTTCACTGAAACCATTGAATTTGATTACGACACACTTGCTAACCGTGTACGTGAGTTAGCTTTCTTAACAAAAGGCGTCAACATCATCATTGAAGACTTACGTGAAGGCAAAGAGCGAAGAAACGAATACTGCTATGAAGGCGGTATTAAGAGCTATGTAGAACATTTGAATCGCTCGAAGGAAGTCGTTCATGAAGAACCAGTTTACATCGAGGGTGAAAAAGACGGAATCACCGTTGAAGTTGCACTGCAATACAACGATTCCTATACAAGCAATATTTATTCTTTCGCCAATAACATCAACACATATGAAGGTGGATCACACGAAGCTGGCTTTAAAACCGGTCTGACGCGTGTCATCAATGATTATGCTCGTAAAAATGGCG TATTCAAAGATGGTGACTCGAATTTGAGCGGAGAAGATGTACGAGAAGGCTTAACAGCCATTATCTCTATCAAACATCCAGACCCTCAATTCGAAGGACAAACGAAGACAAAGCTCGGTAACTCAGAAGCAAGAACCATTACCGACTCCCTCTTCTCCGAAGCACTTGAGAAATTCCTCTTAGAGAACCCTGATG CTGCAAAGAAAATTGTGGAGAAAGGTGTGATGGCAGCTCGTGCAAGAATGGCTGCCAAAAAGGCACGTGAGCTGACAAGACGTAAAAGTGCACTGGAAGTCTCTAGTTTGCCTGGGAAACTGGCAGACTGTTCTTCTAAAGATCCTTCCATCTCTGAGCTTTATATCGTAGAGGGAGATTCTGCGGGCGGATCTG CTAAGCAAGGTCGTGATCGACATTTCCAAGCGATCTTACCGTTAAGAGGGAAGATCCTAAACGTTGAAAAGGCCCGACTAGATAAAATTTTATCCAACAACGAGGTTCGTTCAATGATTACAGCGCTAGGGACTGGAATCGGAGAAGACTTCACTTTAGAGAAAGCACGTATCACAATCGCTGTGATGCCTAGCCC

[0038] Reference for gyrB gene evolutionary tree Figure 6 shown.

[0039] Example 2 Physiological and biochemical identification of Bacillus subtilis L25

[0040] The strain L25 is Gram-positive, rod-shaped, and the single colony on the LB nutrient agar solid medium is light yellow and round. The physiological and biochemical indicators of the strain L25 are determined by the API bacterial identification system. The strain L25 can produce acid using glycerol, L-arabinose, D-ribose, D-xylose, D-galactose, D-glucose, D-fructose, D-mannose, D-mannitol, methyl-α-D-pyranose mannoside, methyl-α-D-pyranose glucopyranoside, N-acetyl-glucosamine, amygdalin, arbutin, ferric citrate of esculin, salicin, D-cellobiose, D-sucrose, D-trehalose and D-tagatose. The main physiological and biochemical characteristics are shown in Table 1. Combining the morphological, physiological and biochemical results and the above-mentioned 16S rDNA and gyrB gene evolution tree results, it can be confirmed that the strain L25 of the present invention is Bacillus altitudinis.

[0041] Table 1 Physiological and biochemical characteristics of strain L25

[0042]

[0043]

[0044] +: positive, -: negative

[0045] Example 3 Preparation of Large Yellow Croaker Juvenile Feed Containing the Highland Bacillus L25

[0046] The highland Bacillus L25 strain stored at -80°C was taken out, and the bacterial liquid was streaked and inoculated on LB nutrient agar solid medium, and revived and cultured at 28°C. After a single colony grew out, a single colony was picked and inoculated into 150ml LB broth medium for 72h (220rpm, 28°C). During the culture period, the bacterial liquid was taken in a clean bench for Gram staining observation. When the spore production rate was greater than 90%, the culture was stopped, 2ml of the bacterial liquid was taken and placed in a water bath at 85°C for 5min, and the viable bacteria were counted on the LB nutrient agar solid medium after gradient dilution. Finally, all the bacterial liquids were collected and placed in a water bath at 85°C for 5min, and the bacterial liquids were centrifuged (6000g, 10min, 4°C) according to the results of the plate count, so as to obtain highland Bacillus L25 mud containing different numbers of spores, which were stored at 4°C in the dark.

[0047] The basal feed for juvenile yellow croaker used in the embodiment of the present invention contains the following components in percentage by mass (as shown in Table 2 below): 45.00% white fish meal, 22.00% krill meal, 3.50% yeast extract, 3.00% squid viscera powder, 5.00% high-gluten flour, 3% α-starch, 2.00% sodium alginate, 6.50% fish oil, 0.20% choline chloride, 5.00% soybean lecithin, 0.20% L-ascorbic acid-2-phosphate, 2.00% monocalcium phosphate, 1.50% multivitamin, 1.00% multimineral, 0.05% mildew inhibitor and 0.05% antioxidant. The above raw materials were all screened with a 120-mesh sieve, and then mixed in a step-by-step order. The highland Bacillus L25 sludge containing different spore numbers was added to an equal amount of sterile ultrapure water to dilute it into a suspension containing different spore numbers. The suspension containing different spore numbers was added to the basic feed respectively and mixed to make the content added to the feed 1×10 6 ~1×10 8 cfu / g. The control group feed was made by adding equal volume of sterile ultrapure water to the basic feed. Finally, the feed was extruded into thin strips using an axial single-screw spherical extruder, and then the feed was made into micro-granular feed with a particle size of up to 1mm using a spheronizer. The prepared micro-granular feed was air-dried indoors and stored in a refrigerator at 4°C.

[0048] Table 2 Basic feed formula

[0049]

[0050] 1 Complex vitamins (mg / kg): retinal acetate, 32; α-tocopherol, 240; menadione, 10; vitamin B1, 25; pyridoxine hydrochloride, 20; vitamin B12, 10; riboflavin, 45; pantothenic acid, 60; vitamin D3, 5; folic acid, 20; niacin, 200; biotin, 60; inositol, 800; microcrystalline cellulose, 13473.

[0051] 2 Composite minerals: MgSO4·7H2O, 1200; FeSO4·H2O, 80; ZnSO4·H2O, 50; CuSO4·5H2O, 10; MnSO4·H2O, 45; CoCl2·6H2O, 50; Na2SeO3, 20; H2CaIO4, 60; zeolite powder, 13485.

[0052] Example 4 Effect of application of Bacillus subtilis L25 on feed for juvenile large yellow croaker

[0053] 42,000 juvenile yellow croakers with an initial body weight of 9.44±0.48 mg were selected for the experiment and randomly divided into 4 groups, with three replicates in each group and 3,500 fish in each replicate, for a 30-day feeding and growth experiment; the culture water temperature was 23-26°C. The control group was fed with a control group feed (without the highland Bacillus L25), and the experimental group was fed with a basal feed supplemented with 1×10 6 ~1×10 8 cfu / g of Bacillus subtilis L25, and were fed 7 times a day, all at fullness, at 05:30, 08:30, 11:00, 13:30, 16:00, 18:30 and 23:00. The fish were weighed and counted at the beginning and end of the culture, and the specific growth rate and survival rate of juvenile yellow croaker were calculated. The experimental data were statistically analyzed using one-way analysis of variance in SPSS20.0, and Tukey's multiple comparisons were performed after significant differences, with a significance level of P<0.05. The experimental data obtained were expressed as mean ± standard error (mean ± SEM, n = 3).

[0054] The experimental results showed that with the increase of the amount of Bacillus subtilis L25 added, the survival rate of yellow croaker fry increased first and then decreased, but there was no significant difference among the groups (P>0.05) (refer to Figure 1 Add 1×10 8 The final body weight and specific growth rate of juvenile yellow croaker fed with the high-dose Bacillus L25 diet were significantly higher than those in the control group (P<0.05) (ref. Figure 2a and Figure 2b As shown in Figure 2), for the digestion and absorption function of juvenile yellow croaker, relative to amylase, 1×10 8 The addition of cfu / g Bacillus subtilis L25 significantly increased the trypsin activity in the intestine of large yellow croaker (refer to Figure 3a and Figure 3b In addition, with the increase of the addition of Bacillus subtilis L25, the intestinal lipase of juvenile yellow croaker increased, but there was no significant difference among the groups (P>0.05) (refer to Figure 3c As shown), at the same time, 1×10 7 ~1×10 8 The addition of Bacillus subtilis L25 significantly increased the Na content of the brush border on the intestine of large yellow croaker. + -K + -ATPase activity (P<0.05) (ref. Figure 3d As shown in Figure 2), for the intestinal development of large yellow croaker, 1×10 7 The activity of intestinal brush border leucine aminopeptidase in the treatment group with cfu / g Bacillus subtilis L25 was significantly higher than that in the control group (P<0.05) (ref. Figure 4aIn addition, with the increase of the addition of Bacillus subtilis L25, the activity of alkaline phosphatase in the brush border of the intestine of juvenile yellow croaker first increased and then decreased, but there was no significant difference among the groups (P>0.05) (refer to Figure 4b Compared with the control group, the 6 ~1×10 8 cfu / g Bacillus subtilisinus significantly increased the expression of intestinal development-related genes (ocd) in juvenile yellow croaker (P<0.05) (ref. Figure 4c At the same time, add 1×10 8 The expression level of intestinal development-related gene (pcna) in the treatment group with cfu / g Bacillus subtilis L25 was significantly higher than that in the control group (P<0.05) (ref. Figure 4d In summary, compared with the control group, the addition of Bacillus subtilis L25 significantly improved the digestion and absorption capacity of the intestine of juvenile large yellow croaker, promoted its intestinal development, improved its intestinal health, and thus improved its growth performance.

[0055] The above embodiments are only preferred implementation modes of the present invention and are only used to explain the present invention rather than to limit the present invention. Any changes, substitutions, modifications, etc. made by those skilled in the art without departing from the spirit of the present invention should fall within the scope of protection of the present invention.

Claims

1. A strain of Bacillus subtilis ( Bacillus altitudinis )L25, characterized in that The Bacillus subtilis L25 was deposited in the China Center for Type Culture Collection on March 8, 2024, with the address being No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, with the deposit number being CCTCC NO: M2024428.

2. Use of the Bacillus subtilis L25 according to claim 1 in preparing feed for juvenile large yellow croaker.

3. A feed for juvenile large yellow croaker containing the Bacillus subtilis L25 according to claim 1.

4. The juvenile yellow croaker feed according to claim 3, characterized in that The viable count of the Bacillus subtilis L25 is 1×10 6 ~1×10 8 cfu / g feed.

5. Use of the Bacillus subtilis L25 described in claim 1 in the preparation of a microecological preparation for improving the intestinal health of juvenile large yellow croaker.

6. Use of the Bacillus subtilis L25 described in claim 1 in the preparation of a microecological preparation for improving the growth performance of large yellow croaker.

7. A microecological preparation containing the Bacillus subtilis L25 according to claim 1.

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