LU503123B1

LU503123B1 - Bacillus subtilis dk-03 and use thereof

Info

Publication number: LU503123B1
Application number: LU503123A
Authority: LU
Inventors: Yan Li; Guiqin Liu; Qingshan Ma; Changfa Wang; Yonghui Wang
Original assignee: Liaocheng Univ
Priority date: 2022-12-01
Filing date: 2022-12-01
Publication date: 2024-06-03

Abstract

The present disclosure provides a Bacillus subtilis DK-03 and use thereof. The B. subtilis DK-03 has been deposited at China Center for Type Culture Collection (CCTCC) on January 20, 2021 under accession number M2021018. The B. subtilis DK-03 provided by the present disclosure has excellent enzyme-producing and antibacterial properties and high safety, which can be used as a microbial inoculant or a feed additive to improve the balance of intestinal flora in donkey foals, improve an immune function of a donkey foal, promote the digestion and absorption of nutrients in a donkey foal feed, increase the growth performance of the donkey foal, and help improve economic benefits of donkey farms.

Description

BACILLUS SUBTILIS DK-03 AND USE THEREOF 1009168

TECHNICAL FIELD

[0001] The present disclosure belongs to the technical field of microorganisms, and particularly relates to a Bacillus subtilis DK-03 and use thereof.

BACKGROUND ART

[0002] Donkey industry 1s a special animal husbandry in China and a key anti-poverty livelihood industry. Healthy farming of donkey foals is a prerequisite for the development of donkey industry.

At the stage of donkey foal, the diversity and richness of donkey gut microbiota in the gut are low, their functions are single, intestinal lipase, amylase, and other endogenous enzymes are secreted insufficiently; moreover, influenced by weaning and feed changing stress and pathogenic microorganisms like Escherichia coli, alteration of intestinal flora, indigestion, and decreased production performance occur easily. It has been found that lactic acid bacteria preparation can antagonize pathogenic microorganisms, regulate the balance of intestinal flora, promote the digestion and absorption of nutrients, and boost immunity, so as to achieve the objectives of regulating the intestinal flora, improving the intestinal health and increasing the production performance.

[0003] A lot of studies have shown that B. subtilis, as an important probiotic strain, can maintain the balance of intestinal flora, inhibit the multiplication of pathogens, reduce the endotoxin level, boost immunity and antioxidant levels, and further improve the intestinal health and growth performance of donkey foals.

[0004] However, studies and reports of the use of B. subtilis in donkey foal feeds are limited, specific application dosage and the influence on the growth performance of donkey foals should be studied further.

SUMMARY

[0005] In view of this, the present disclosure aims to set forth a B. subtilis DK-03 and use thereof, so as to investigate the B. subtilis and use thereof in the field of donkey foal husbandry.

[0006] To achieve the above objectives, the technical solutions of the present disclosure are realized as follows:

[0007] The present disclosure provides a B. subtilis DK-03, which has been deposited at China

Center for Type Culture Collection (CCTCC), Wuhan University, Bayi Road, Hongshan District,

Wuhan, Hubei Province on January 20, 2021 under accession number M2021018. The B. subtilis 1

DK-03 was isolated from donkey intestinal contents by the applicants. The strain has the following 903129 biological characteristics: the colony is flat, dry, and mat on the surface, opaque, and white; the colony has an irregular edge, belongs to Gram-positive cocci, and grows under aerobic conditions at an optimum growth temperature of 37°C. The B. subtilis DK-03 in the present disclosure can further produces amylase, lipase, cellulase, and the like, and have a strong inhibitory effect on

Staphylococcus aureus and a certain tolerance to the pH value of the living environment. The strain has a 16S rRNA sequence shown in SEQ ID No: 1. As shown in FIG. 3, by aligning to the sequence of Genbank, the result shows a 99% similarity to the 16S rRNA sequence of B. subtilis. Through bacterial morphology, physiological and biochemical characteristics, suitable growth conditions, and sequencing identification results, the strain DK-03 is determined as B. subtilis.

[0008] The present disclosure provides a microbial inoculant, including the foregoing B. subtilis

DK-03. In some implementations, the microbial inoculant only includes the foregoing B. subtilis

DK-03; in other implementations, the microbial inoculant further includes other microbial species, which are used in combination with the foregoing B. subtilis DK-03 as active substances of the microbial inoculant.

[0009] The present disclosure provides use of the foregoing B. subtilis DK-03 or the foregoing microbial inoculant in the field of donkey foal feed additives.

[0010] The present disclosure provides a donkey foal feed additive, including at least one of the foregoing B. subtilis DK-03 or the foregoing microbial inoculant. In some implementations, the foregoing B. subtilis DK-03 and the foregoing microbial inoculant are used alone, respectively; in some other implementations, the foregoing B. subtilis DK-03 and the foregoing microbial inoculant are used as a mixture; in some other implementations, besides the foregoing B. subtilis

DK-03 and the foregoing microbial inoculant, other additive components are further included.

[0011] The present disclosure provides use of the foregoing B. subtilis DK-03, the foregoing microbial inoculant, or the foregoing donkey foal feed additive in the field of donkey foal feed.

[0012] The present disclosure provides a donkey foal feed, including at least one of the B. subtilis

DK-03, the foregoing microbial inoculant, and the foregoing donkey foal feed additive. In some implementations, the foregoing B. subtilis DK-03, the foregoing microbial inoculant, and the foregoing donkey foal feed additive are used alone, respectively; in some other implementations, the foregoing B. subtilis DK-03, the foregoing microbial inoculant, and the foregoing donkey foal feed additive are used as a mixture; in some other implementations, besides the foregoing B. subtilis DK-03, the foregoing microbial inoculant, and the foregoing donkey foal feed additive, other donkey foal feed components are further included. Preferably, a viable count of the B. subtilis

DK-03 in the donkey foal feed may be 1 x 10"! CFU/kg. 2

[0013] The present disclosure provides use of the foregoing Bacillus subtilis DK-03, the 209123 foregoing microbial inoculant, the foregoing donkey foal feed additive, or the foregoing donkey foal feed in the preparation of at least one of the following products:

[0014] (1) a product for balancing intestinal flora structure in donkey foals;

[0015] (2) a product for improving an immune function of a donkey foal;

[0016] (3) a product for reducing disease incidence of donkey foals;

[0017] (4) a product for increasing digestion and absorption efficiencies of nutrients in a donkey foal feed; and

[0018] (5) a product for improving growth performance of a donkey foal.

[0019] Compared with the prior art, the B. subtilis DK-03 and the use thereof provided by the present disclosure have the following the advantages:

[0020] The B. subtilis DK-03 provided by the present disclosure has excellent enzyme-producing and antibacterial properties and high safety, which can be used as a microbial inoculant or a feed additive to improve the balance of intestinal flora in donkey foals, improve an immune function of a donkey foal, promote the digestion and absorption of nutrients in a donkey foal feed, increase the growth performance of the donkey foal, and help improve economic benefits of donkey farms.

BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The accompanying drawings constituting a part of the present disclosure are intended to provide a further understanding of the present disclosure; the exemplary examples of the present disclosure and descriptions thereof are intended to explain the present disclosure, and do not constitute an improper limitation of the present disclosure. In the accompanying drawings:

[0022] FIG. 1 shows a schematic diagram of colonies of the B. subtilis DK-03 described in the examples;

[0023] FIG. 2 shows a schematic diagram of Gram staining of the B. subtilis DK-03 described in the examples;

[0024] FIG. 3 shows a schematic diagram of a 16sDNA sequence alignment of the B. subtilis

DK-03 described in the examples;

[0025] FIG. 4 shows a schematic diagram of the hemolytic test of the B. subtilis DK-03 described in the examples;

[0026] FIG. 5 shows a schematic diagram of an analysis of amylase activity of the B. subtilis

DK-03 described in the examples;

[0027] FIG. 6 shows a schematic diagram of an analysis of cellulase activity of the B. subtilis

DK-03 described in the examples; 3

[0028] FIG. 7 shows a schematic diagram of an analysis of lipase activity of the B. subtilis DK- 903129 03 described in the examples;

[0029] FIG. 8 illustrates a comparison of average daily gains between probiotics group and control group described in the examples.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0030] Unless otherwise defined, the technical terms used in the following examples have the same meanings as commonly understood by those skilled in the art to which the present disclosure belongs. All experimental reagents used in the following examples are conventional biochemical reagents, unless otherwise specified; all experimental methods are conventional methods, unless otherwise specified.

[0031] The present disclosure will be described in detail below with reference to the examples and accompanying drawings.

[0032] Example 1 Screening of B. subtilis

[0033] A method for screening the B. subtilis provided by the present disclosure includes the following steps:

[0034] The strain was collected from healthy intestinal contents, fresh stools and surrounding soil from donkey farms in Yucheng and Chiping, Liaocheng City. The strain was heated in a 85°C water bath for 10 min and diluted to 10”; 100 uL each of 10 and 1077 diluted bacterial suspension was injected and inoculated on a nutrient agar plate, and cultured in an incubator at 37°C for 48 h.

A single colony was picked from the plate and subjected to Gram staining; Gram-positive bacteria were picked therefrom, and a single strain was purified and cultured until no infectious microbes were observed microscopically. The purified strain was stored in 30% (v/v) glycerol in a -80°C ultralow temperature freezer.

[0035] Through the above method, a colony was isolated from the donkey intestinal contents, which was flat, dry, and mat on the surface, opaque, and white; the colony had an irregular edge, belonged to Gram-positive cocci, and grew under aerobic conditions at an optimum growth temperature of 37°C. The colony was preliminarily identified as B. subtilis, as shown in FIGS. 1 and 2.

[0036] Example 2 Sequencing identification of B. subtilis

[0037] Forward primer P1 (5'-AGAGTTTGATCCTGGCTCAG- 3’, SEQ ID NO: 1) and reverse primer P2 (S'-GGTTACCTTGTTACGACTT-3', SEQ ID NO: 2) were used. The primers were synthesized by Shanghai Lifei Biotechnology Co., Ltd. (Beijing) and subjected to 16S rDNA amplification. The PCR system included: 30 uL of 2x PCR Mix, 2.5 pL each of primers, 2 uL of 4 bacterial suspension, and 23 uL of ddH,O. The PCR program was as follows: 95°C for 5 min; 30 903129 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 90 s; finally extension at 72°C for 10 min.

The PCR product was run on a 1.0% agarose gel (containing a 1/10,000 concentration of SYBR®

Green I) for 30 min and observed under an ultraviolet lamp; after determining the success of the

PCR product and meet the requirement, the PCR product was submitted to Shanghai Lifei

Biotechnology Co., Ltd. (Beijing) for sequencing. The sequencing result was subjected to the homology analysis on NCBI. Through Genbank sequence alignment, the result showed a 100% similarity to the 16S rDNA sequence of B. subtilis, as shown in FIG. 3. Through bacterial morphology, physiological and biochemical characteristics, suitable growth conditions, and sequencing identification results, the strain DK-03 was determined as B. subtilis.

[0038] Example 3 Safety analysis of B. subtilis

[0039] A single colony of B. subtilis DK-03 was seeded on a blood agar plate and cultured in a 37°C incubator for 24-48 h to observe whether there was a zone of hemolysis and what type of the zone of hemolysis was.

[0040] There are the following three types of hemolysis for the colony:

[0041] (1) a-hemolysis: as known as green hemolysis, a narrow (1-2 mm), grass green zone of hemolysis formed around the colony, which is caused by hemoglobin;

[0042] (2) B-hemolysis: as known as complete hemolysis, a completely clear and transparent zone of hemolysis formed around the colony, which is caused by complete erythrocytolysis due to hemolysin produced by bacteria; and

[0043] (3) y-hemolysis: no hemolysis, without change in the culture medium around the colony and without erythrocytolysis or destruction of red blood cells.

[0044] After 48 h, the isolated B. subtilis DK-03 showed y-hemolysis, namely no hemolysis, on the hemolytic plate, with excellent safety, as shown in FIG. 4.

[0045] Example 4 Enzyme-producing capability tests for B. subtilis

[0046] (1) Amylase production test

[0047] Starch and agar were put in the LB broth to prepare a starch hydrolysate medium. The test strain was inoculated on the culture medium under sterile conditions, and cultured at 37°C overnight; iodine solution was added to the Gram stain reagent to prevent photolysis, but observation was conducted within 5 min. The iodine solution was dropped to the culture medium.

The result is shown in FIG. 5. A transparent zone appeared near the colony, demonstrating that 5. subtilis DK-03 can produce amylase.

[0048] (2) Cellulase production test

[0049] After the culture medium was prepared, at natural pH, the test strain was inoculated on a plate and cultured at a constant temperature for 12 h. A certain amount of 0.3% Congo Red solution 903129 was poured to completely immerse the colony; after shaking at 60 rpm for 50 min, Congo Red was discarded; the culture medium was replaced with NaCl for 20-30 min, and the colony was rinsed with NaCl twice. Later, NaCl was discarded to observe whether a transparent zone was formed.

After rinsing with NaCl twice, NaCl was discarded to observe under light. The result is shown in

FIG. 6. There was a transparent zone around the colony, demonstrating that B. subtilis DK-03 can produce cellulase.

[0050] (3) Lipase production test

[0051] Tween-80 medium was prepared with peptone, yeast extract, NaCl, CaCl, Tween-80 and agar. The test strain was inoculated on the culture medium in a clean bench, and cultured at a constant temperature for 24 h and at room temperature for 1-2 days. An apparent white pellet zone was observed, as shown in FIG. 7, demonstrating that B. subtilis DK-03 can produce lipase.

[0052] Example 5 Analysis of antibacterial activity of B. subtilis

[0053] Escherichia coli and Staphylococcus aureus were individually inoculated in a common broth and cultured at 37°C for 24 h. The bacterial suspensions were used as indicators for the pathogen. 100 uL each of the bacterial suspension was uniformly spread on a nutrient agar plate, and Oxford cups were placed horizontally; 100 uL of B. subtilis DK-03 suspension cultured overnight was added to the Oxford cups, respectively, and three duplicates were cultured at 37°C for 24 h. The diameter of inhibition zone was measured. Test results are shown in Table 1.

According to the analytical data, B. subtilis DK-03 can significantly inhibit the growth of Æ. coli and §. aureus and have excellent antibacterial activity.

[0054] Table 1 The antibacterial activity of B. subtilis DK-03

[0055] Herein, “+++” indicates significant antibacterial activity, with an inhibition zone of >12 mm.

[0056] Example 7 Effect of B. subtilis on growth performance of donkey foals

[0057] In view of excellent enzyme-producing and antibacterial properties of B. subtilis DK-03, this example used B. subtilis DK-03 in a donkey foal feed.

[0058] 1. Preparation of bacterial powder

[0059] After the B. subtilis DK-03 was activated, the strain was propagated in a fermentor, fermented, centrifuged, concentrated, and spray-dried at a low temperature to prepare a bacterial powder with a viable count of approximately 1 x 10° CFU/g. 6

[0060] 2. Test scheme and results 1000163

[0061] Test location: À large-scale donkey farm in Yucheng, Shandong Province. Grouping: A hundred healthy Dezhou donkey foals aged around 6 months with a medium body condition and close weights were divided into a control group and a probiotics group. The control group was fed with conventional feeds, and the probiotics group was fed with feeds supplemented with 1%o B. subtilis DK-03 powder. The experimental period was three months.

[0062] After culture experiment, test donkey foals were weighed and their average daily gain was calculated. It was found that the average daily gain of donkey foals in the probiotics group was 540 g/day, and that of test donkey foals was 462 g/day, which was increased by approximately 80 g/day, with a significant effect, as shown in FIG. 8. Meanwhile, donkey foals with diarrhea were counted. The statistical result was shown in Table 2. Herein, 22% of the donkey foals had diarrhea in the control group, and 10% of the donkey foals had diarrhea in the probiotics group, demonstrating that feeding B. subtilis DK-03 significantly reduces the incidence of diarrhea in donkey foals.

[0063] Table 2 The effect of B. subtilis DK-03 on the diarrhea rate of donkey foals

[0064] In conclusion, the donkey-derived B. subtilis DK-03 screened by the present disclosure has excellent enzyme-producing and antibacterial properties and high safety, which can be used as a feed additive to improve the balance of intestinal flora in donkey foals, increase the growth performance of donkey foals, reduce the probability of disease occurrence in donkey foals, and help improve economic benefits of donkey farms.

[0065] The above descriptions are only preferred examples of the present disclosure and are not intended to limit the present disclosure. Any modifications, equivalent substitutions, and improvements made within the spirit and principle of the present disclosure shall be included in the protection scope of the present disclosure. 7

Claims

CLAIMS: LU503123

1. Bacillus subtilis DK-03, the strain deposited at the China Center for Type Culture Collection (CCTCC) on 20 January 2021 under accession number M2021018.

2. A microbial inoculum comprising Bacillus subtilis DK-03 according to claim 1.

3. Use of Bacillus subtilis DK-03 according to claim 1 or of the microbial inoculum according to claim 2 in the field of feed additives for donkey foals.

4. A feed additive for donkey foals comprising at least one of the Bacillus subtilis DK-03 according to claim 1 and the microbial inoculum according to claim 2.

5. Use of the Bacillus subtilis DK-03 according to claim 1, the microbial inoculum according to claim 2 or the feed additive for donkey foals according to claim 4 in the field of donkey foal feed.

6. A donkey foal feed comprising at least one of the Bacillus subtilis DK-03 according to claim 1, the microbial inoculum according to claim 2 and the feed additive for donkey foals according to claim 4 in the field of donkey foal feed.

7. Use of the Bacillus subtilis DK-03 according to claim 1, the microbial inoculum according to claim 2, the feed additive for donkey foals according to claim 4 or the donkey foal feed according to claim 6 for the production of a product for balancing the intestinal flora of a donkey foal. 1

8. Use of the Bacillus subtilis DK-03 according to claim 1, LU503123 the microbial inoculum according to claim 2, the feed additive for donkey foals according to claim 4 or the donkey foal feed according to claim 6 for the preparation of a product for improving an immune function of a donkey foal.

9. Use of the Bacillus subtilis DK-03 according to claim 1, the microbial inoculum according to claim 2, the feed additive for donkey foals according to claim 4 or the donkey foal feed according to claim 6 for the manufacture of a product for reducing disease cases in donkey foals.

10. Use of the Bacillus subtilis DK-03 according to claim 1, the microbial inoculum according to claim 2, the feed additive for donkey foals according to claim 4 or the donkey foal feed according to claim 6 for the preparation of a product for increasing the efficiency of digestion and absorption of nutrients in the digestive tract of a donkey foal.

11. Use of the Bacillus subtilis DK-03 according to claim 1, the microbial inoculum according to claim 2, the feed additive for donkey foals according to claim 4 or the donkey foal feed according to claim 6 for the manufacture of a product for improving a growth performance of a donkey foal. 2