Bacillus cereus 13 and its application

Abstract

The present invention relates to a strain of Bacillus cereus 13 and its application, belonging to the technical field of microorganisms. Bacillus cereus ) 13 is deposited in the General Microbiology Center of the China Microorganism Culture Collection Administration Committee, with a deposit number of CGMCC No.29171 and a deposit date of November 29, 2023. The address of the depository institution is: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. The present invention also provides the use of Bacillus cereus 13 as feed. A strain of Bacillus cereus 13 of the present invention can promote the growth of broilers, improve feed conversion rate, enhance the antioxidant capacity of broilers, promote the expression of intestinal mucosal tight junction proteins, and increase immunoglobulin levels. At the same time, it can improve the distribution of intestinal flora and promote the secretion of short-chain fatty acids. Enhance the immune regulation ability of broiler intestines against microbial infections in many aspects.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and in particular to a strain of Bacillus cereus 13 and application thereof. Background Art

[0002] With the improvement of living standards, people have shifted from pursuing fullness to eating well, and food safety issues have attracted everyone's attention. Food safety from farm to table is a complex project. Since antibiotics play an important role in the aquaculture industry, their current application status has attracted much attention. In the aquaculture industry, antibiotics are widely used in animal husbandry to promote growth, prevent and treat diseases. Although antibiotics are very effective in promoting growth and treating bacterial infections, the abuse of antibiotics has led to increasingly prominent problems of antibiotic residues and bacterial resistance. Since January 1, 2020, all growth-promoting drug feed additives except traditional Chinese medicine have been withdrawn. Therefore, there is an urgent need to find more effective alternatives to solve this problem.

[0003] Probiotics have become an effective alternative to antibiotics due to their non-toxic, non-residue and non-resistance characteristics. Bacillus cereus is a type of Gram-positive bacteria that is widely present in nature. As a probiotic, it is widely used in many fields such as medical care, animal husbandry and agricultural production. Its main mechanism of action is reflected in regulating intestinal flora, activating immune cells, and regulating gastrointestinal function. In 1989, the U.S. Food and Drug Administration (FDA) and the American Feed Industry Association (AFIA) allowed Bacillus cereus to be used as a feed additive in animal husbandry production. Screening Bacillus cereus that can prevent animal diseases, promote animal growth, increase feed remuneration, and improve the quality of livestock and poultry products has become one of the main goals of probiotics.

[0004] The existing Bacillus cereus can be used as broiler feed to reduce the feed-to-meat ratio, and has achieved good results. However, in terms of anti-oxidation and improving immunoglobulin levels, the effect is not ideal. Therefore, it is necessary to find a strain of Bacillus cereus to solve these problems. Summary of the invention

[0005] In view of the problem that the existing Bacillus cereus has unsatisfactory effects in anti-oxidation and improving immunoglobulin levels, the present invention provides a strain of Bacillus cereus 13 and its application to solve the above problems. The Bacillus cereus 13 in the present invention has the function of anti-oxidation and improving immunoglobulin levels, and the improvement effect is excellent, which is of great significance to improve the matrix strength of broilers when used as broiler feed.

[0006] In a first aspect, the present invention provides a strain of Bacillus cereus 13, wherein the Bacillus cereus ( Bacillus cereus)13 is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number CGMCCNo.29171. The deposit date is November 29, 2023. The address of the deposit institution is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing.

[0007] In a second aspect, the present invention provides an application of Bacillus cereus 13 in preparing a feed for improving the antioxidant capacity of broiler chickens.

[0008] In a third aspect, the present invention provides an application of Bacillus cereus 13 in the preparation of feed for improving immunoglobulin in broiler chickens.

[0009] Furthermore, the immunoglobulins include IgG, IgA and IgM.

[0010] In a fourth aspect, the present invention provides a use of Bacillus cereus 13 in preparing feed for reducing feed-to-meat ratio.

[0011] In a fifth aspect, the present invention provides a use of Bacillus cereus 13 in the preparation of a product for inhibiting harmful intestinal bacteria.

[0012] Furthermore, the harmful intestinal bacteria include Escherichia coli, Staphylococcus aureus and Salmonella enteritidis.

[0013] In a sixth aspect, the present invention provides an application of Bacillus cereus 13 in preparing feed for improving immunity of broiler chickens.

[0014] The beneficial effects of the present invention are:

[0015] The Bacillus cereus 13 provided by the present invention has good high temperature resistance, acid resistance and bile salt resistance, and can produce amylase, protease and cellulase at the same time, and has a certain inhibitory ability against common intestinal pathogens Escherichia coli, Staphylococcus aureus and Salmonella enteritidis. It can promote the growth of broilers, improve feed conversion rate, enhance the antioxidant capacity of broilers, promote the expression of intestinal mucosal tight junction proteins, and improve the level of immunoglobulins. At the same time, it can improve the distribution of intestinal flora and promote the secretion of short-chain fatty acids. The immune regulation ability of broiler intestinal tract against microbial infection is enhanced from many aspects. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0017] Figure 1 This is a plate image of Bacillus cereus 13.

[0018] Figure 2 This is the Gram stain of Bacillus cereus 13.

[0019] Figure 3 It is a line graph of the survival rate of Bacillus cereus 13 at different temperatures.

[0020] Figure 4 It is a line graph of the survival rate of Bacillus cereus 13 at different pH values.

[0021] Figure 5 It is a line graph of the survival rate of Bacillus cereus 13 at different bile salt concentrations.

[0022] Figure 6 This is a graph showing the ability of Bacillus cereus 13 to hydrolyze starch.

[0023] Figure 7 This is a graph showing the ability of Bacillus cereus 13 to decompose proteins.

[0024] Figure 8 This is a graph showing the ability of Bacillus cereus 13 to degrade cellulose. In the figure, A is a graph showing the state of cellulose after being degraded by Bacillus cereus 13; and B is a graph showing the state after being stained with Congo red.

[0025] Fig. 9 This is the antibacterial experiment diagram of Bacillus cereus 13.

[0026] Fig.10 This is a comparison chart of liver antioxidant capacity test in Example 5. In the figure, A is the T-AOC content in the liver of AA broiler chickens; B is the MDA content in the liver of AA broiler chickens; C is the SOD content in the liver of AA broiler chickens; and D is the GSH-Px content in the liver of AA broiler chickens.

[0027] Fig.11 : is a comparison chart of serum antioxidant capacity test in Example 5. In the figure, A is the T-AOC content in AA broiler serum; B is the MDA content in AA broiler serum; C is the SOD content in AA broiler serum; and D is the GSH-Px content in AA broiler serum.

[0028] Fig.12 is the mRNA content of jejunal tight protein ZO-1.

[0029] Fig.13 It is the mRNA content of jejunal tight protein Claudin.

[0030] Fig.14 It is the mRNA content of jejunal tight protein Occludin.

[0031] Fig.15: is a comparison chart of the immunoglobulin test results in the jejunal mucosa in Example 7. In the figure, A is the content of IgA in the intestinal tissue of AA broilers; B is the content of IgM in the intestinal tissue of AA broilers; C is the content of IgG in the intestinal tissue of AA broilers. DETAILED DESCRIPTION

[0032] In order to enable those skilled in the art to better understand the technical solutions in the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings 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 should fall within the scope of protection of the present invention.

[0033] Example 1

[0034] Isolation, Screening and Identification of Bacillus cereus 13

[0035] (1) Sampling: On December 10, 2021, soil was collected from the broiler farm in Linyi City, Shandong Province and placed in self-sealing bags for future use.

[0036] (2) Separation: Take 5 g of the soil collected in step (1) and add it to a conical flask containing 45 mL of sterile pure water. Incubate it in a water bath at 100°C for 10 minutes. After standing, take the upper solution and dilute it into samples of different concentration gradients. Use a spreader to spread it on LB solid culture medium and incubate it in an incubator at 37°C overnight.

[0037] (3) Purification: Select single colonies with different morphologies, and purify the strains of each single colony by streaking on LB solid culture medium three times. Screen the strains according to the different colony morphologies and determine their numbers. Store them in test tube slants and store them in a 4°C refrigerator as test bacteria for later tests. At the same time, store them in 50% sterile glycerol tubes at -20°C for future use.

[0038] (4) Morphological identification

[0039] Strain 13 was inoculated into LB medium and cultured at 35°C and 180 rpm for 24 h. -7 After the gradient, take 100 μL and spread it on LB solid medium, culture at 37℃ for 48h, and continue to observe the color, shape and size of single colonies. Figure 1 As shown, strain 13 is a monoclonal colony.

[0040] (5) Stain with Gram stain. First, apply and fix the strain, and add crystal violet stain to the fixed culture. After 1 minute, pour out the crystal violet stain and rinse with water. Then, use iodine solution for 30 seconds. Next, pour out the iodine solution and rinse with water. Add a few drops of 95% alcohol and shake gently to decolorize. After 20 seconds, rinse with water and absorb the water. After staining with diluted safranin solution for 1 minute, rinse with distilled water. Finally, dry and examine under a microscope. The results are detailed in Figure 2 , the staining results showed Gram-positive.

[0041] (6) Growth characteristics

[0042] A single colony of strain 13 was selected and cultured in LB medium at 37°C with shaking, and then a fresh bacterial suspension was inoculated into LB liquid medium. The number of bacteria was measured every 30 minutes at OD 600 nm using a microplate reader (Tecan, Infinite 200 Pro Microplate Reader, Switzerland).

[0043] (7) 16S rRNA gene sequence analysis

[0044] DNA extraction kit (A29790, Invitrogen TM , USA) to extract the genomic DNA of the isolated strain, and sent the DNA to Sangon Biotech Co., Ltd. for sequencing. The sequencing results were entered into the NCBI nucleic acid database for sequence comparison. After BLAST gene comparison, the homology with Bacillus cereus in Genebank reached 100%, and the strain was preliminarily determined to be Bacillus cereus. Bacillus cereus Bacillus cereus 13 is deposited in the General Microbiological Center of China Microorganism Culture Collection Administration, with the deposit number CGMCC No.29171 and the deposit date November 29, 2023. The address of the depository institution is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. The 16S rDNA sequence is shown in SEQ ID NO.1.

[0045] Example 2

[0046] Detection of probiotic properties of Bacillus cereus 13

[0047] 1. High temperature resistance

[0048] After quantifying Bacillus cereus 13 using a turbidimeter, the fresh suspension of Bacillus cereus 13 was placed in a water bath at different temperatures (37°C, 70°C, 80°C, 90°C and 100°C) for 0 min, 3 min, 5 min, 10 min and 15 min. The plate count method was used to calculate the number of viable bacteria. The test results are detailed in Figure 3The results showed that Bacillus cereus 13 had a certain ability to resist high temperatures.

[0049] 2. Acid resistance

[0050] The bacteria were cultured in LB medium with different pH values ​​(2.0, 3.0, 4.0, 5.0) and then cultured at 37°C for 1 hour, 2 hours, 3 hours and 4 hours. The number of viable bacteria was calculated by plate count method. Figure 4 Bacillus cereus 13 has a certain acid resistance, and its growth is almost unaffected at pH = 5 and pH = 4. At pH = 3, the survival rate is about 70% after 2 hours of treatment, and at pH = 2, the survival rate is about 45% after 2 hours of treatment.

[0051] 3. Bile salt tolerance

[0052] Bacillus cereus 13 was incubated with LB medium containing 0.0%, 0.1%, 0.2%, 0.3% and 0.4% swine bile salt concentrations, and then incubated at 37°C for 1 hour, 2 hours, 3 hours and 4 hours. The number of viable bacteria was calculated by the plate count method. The test results are detailed in Figure 5 , indicating that Bacillus cereus 13 can tolerate bile salt treatment.

[0053] 4. Ability to hydrolyze starch

[0054] Soluble starch agar (CN230550, Chinook, China) was used. A drop of Bacillus cereus 13 solution was dropped on the agar plate and incubated at 37°C for 24 hours. Then a few drops of Lugol's iodine solution (CN230370, Chinook, China) were dropped on the colony. After 3 minutes, the size of the transparent circle was observed. The results are detailed in Figure 6 , indicating that Bacillus cereus 13 has good ability to hydrolyze starch.

[0055] 5. Ability to degrade proteins

[0056] Casein agar (CN230555, Chinook, China) was used. After incubating a drop of Bacillus cereus 13 solution at 30°C for 24 hours, the size of the transparent circle was observed. Figure 7 , indicating that Bacillus cereus 13 has good protein hydrolysis ability.

[0057] 6. Detection and quantification of cellulase

[0058] Congo red sodium carboxymethylcellulose screening medium (CN230510, Chinook, China) was used. After culturing a drop of Bacillus cereus 13 solution at 30°C for 24 hours, it was stained with 1 mg / mL Congo red for 30 minutes, the waste liquid was discarded, and the formation of a transparent circle was observed. Based on the clarity of the transparent circle and the ratio to the colony diameter, it was preliminarily determined that Bacillus cereus 13 had the ability to degrade cellulose. The results are detailed in Figure 8 .

[0059] Example 3

[0060] Antibacterial experiment

[0061] Resuscitate three pathogenic indicator bacteria, Escherichia coli, Salmonella, Staphylococcus aureus and Bacillus cereus 13. After the bacteria are revived, pick a single colony with an inoculation loop and add it to LB liquid medium for bacterial growth. Adjust the indicator bacteria concentration to 10 8 CFU / mL, after mixing evenly, dilute 100 times, take 100μL, and spread it on LB agar medium; and take 5μL of Bacillus cereus 13 bacterial droplets to the center of the agar plate, wait for the bacterial liquid to stand and dry naturally, and then invert the culture dish in a 37℃ incubator for 24h. Fig. 9 This indicates that Bacillus cereus 13 can effectively inhibit the growth of common intestinal pathogenic microorganisms such as Escherichia coli, Staphylococcus aureus and Salmonella.

[0062] Example 4

[0063] Effects of Bacillus cereus 13 on the growth performance of broiler chickens

[0064] 1. Animal experiments and sample collection

[0065] A total of 240 one-day-old AA broilers were randomly divided into two treatment groups: a control group (Con, basal diet) and a Bacillus cereus 13 treatment group (BC13, basal diet + 0.5 mL bacterial solution, 1 × 10 9 / mL). Each treatment had 6 replicates, with 20 chickens in each replicate. The body weight and feed intake of AA broilers were recorded weekly, and the average daily weight gain, average daily feed intake and feed weight ratio were calculated based on the daily weight gain changes and feed intake. The results are shown in Table 1. It can be seen from Table 1 that the average body weight, average daily weight gain and feed conversion rate of the Bacillus cereus 13 treatment group were significantly different from those of the control group.

[0066] Table 1 - Effects of Bacillus cereus 13 on the growth of AA broilers

[0067]

[0068] Mean values ​​with different letters in the same original sample are significantly different (P<0.05).

[0069] Example 5

[0070] Effect of Bacillus cereus 13 on the antioxidant capacity of AA broilers: Seven broilers of 21 days and 42 days were randomly selected from the Bacillus cereus 13 treatment group of Example 4, and seven broilers of 21 days and 42 days were randomly selected from the control group of Example 4. Samples such as liver and serum were taken and stored in a -80°C refrigerator for analysis.

[0071] Liver and serum antioxidant capacity: The total antioxidant capacity (T-AOC), malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) concentrations of serum and liver were measured using commercially available kits (Nanjing Jiancheng). The test results are shown in Table 2 and Figures 10-11 shown.

[0072] Table 2 - Effects of Bacillus cereus 13 on free radicals in serum and liver of AA broilers

[0073]

[0074] From Table 2 and Figures 10-11 The test results show that Bacillus cereus 13 can significantly reduce the level of oxidative free radical MDA in the liver and serum, and increase the levels of antioxidant indicators T-AOC, SOD and GSH-Px. In general, this strain has the ability to improve the antioxidant level of broilers.

[0075] Example 6

[0076] Effects of Bacillus cereus 13 on intestinal immunity of AA broilers

[0077] Tight junction protein RNA level detection

[0078] Total RNA was isolated from the jejunal mucosa of broilers in the Bacillus cereus 13-treated group and the control group selected in Example 5 using TRIzol. TM RNA was reverse transcribed into cDNA using the RT kit. All qRT-PCR reactions were performed and analyzed using the CFX96 Touch Deep Well Real-Time PCR Detection System and SYBR Green PCR Master Mix. Table 3 lists the qPCR primers for mRNA encoding tight junction proteins (ZO-1, Claudin, Occludin). ΔΔCT Relative mRNA levels were assessed by PCR. β-actin was used as an internal standard for data analysis and normalization.

[0079] Table 3 - qPCR primer sequences

[0080]

[0081] like Figures 12-14 As shown, compared with the control group, the mRNA levels of ZO-1, Claudin and Occludin were significantly increased, indicating that Bacillus cereus 13 can improve the tight junction of the intestine of AA broiler chickens, prevent the invasion and infection of harmful intestinal bacteria to a certain extent, and improve the immunity of the chickens.

[0082] Example 7

[0083] Immunoglobulin levels

[0084] The levels of jejunal mucosal immune-related indicators, i.e., immunoglobulin IgG, IgA, and IgM, were detected by ELISA kits (Jiancheng, China). The intestinal tissues of broilers in the Bacillus cereus 13 treatment group and the control group selected in Example 5 were ground, centrifuged, and the supernatant was taken. The levels of immunoglobulin IgG, IgA, and IgM were detected by ELISA kits, respectively. The specific experimental operations were carried out according to the experimental instructions. The results are shown in Table 4 and Fig.15 shown.

[0085] Table 4 - Effects of Bacillus cereus 13 on AA broiler immunoglobulins IgG, IgA and IgM

[0086]

[0087] From Table 4 and Fig.15 It can be seen that the levels of immunoglobulins IgG, IgA and IgM in the jejunal mucosa of Bacillus cereus 13 were significantly increased compared with the control group.

[0088] Although the present invention has been described in detail with reference to the accompanying drawings and in combination with the preferred embodiments, the present invention is not limited thereto. Without departing from the spirit and essence of the present invention, a person of ordinary skill in the art may make various equivalent modifications or substitutions to the embodiments of the present invention, and these modifications or substitutions shall be within the scope of the present invention. Any person of ordinary skill in the art may easily think of changes or substitutions within the technical scope disclosed by the present invention, and these shall be within the scope of protection of the present invention.

Claims

1. A strain of Bacillus cereus 13, characterized in that: Bacillus cereus ( Bacillus cereus )13 is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number CGMCC No.29171. The deposit date is November 29, 2023. The address of the deposit institution is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing.

2. Use of the Bacillus cereus 13 as claimed in claim 1 in preparing feed for improving the antioxidant capacity of broilers.

3. Use of the Bacillus cereus 13 as claimed in claim 1 in preparing feed for improving immunoglobulin in broiler chickens.

4. The use according to claim 3, characterized in that Immunoglobulins are IgG, IgA and IgM.

5. Use of the Bacillus cereus 13 as claimed in claim 1 in preparing feed for reducing the feed-to-meat ratio of broiler chickens.

6. A use of the Bacillus cereus 13 as claimed in claim 1 in the preparation of a product for inhibiting harmful intestinal bacteria; characterized in that: The harmful bacteria are Escherichia coli, Staphylococcus aureus and Salmonella enteritidis.

7. Use of the Bacillus cereus 13 as claimed in claim 1 in preparing feed for improving immunity of broiler chickens.

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