CN105219669B - With oxidation resistance, the bacillus amyloliquefaciens TL for promoting growth of meat chicken to develop - Google Patents

Abstract

The invention belongs to the technical field of preparation of veterinary microbial additives, and in particular relates to bacillus amyloliquefaciens TL which has antioxidant capacity and promotes the growth and development of broilers. The present invention is characterized in that the Bacillus amyloliquefaciens TL is isolated from pig intestinal contents and has been sent to the Chinese Type Culture Collection Center in Wuhan University, Wuhan City, Hubei Province on June 4, 2015 (CCTCC) deposit, and its deposit number is CCTCC NO: M2015359. Through systematic experiments, it is verified that the strain has the characteristics of anti-oxidation ability, fast growth speed, strong stress resistance, safety, disease resistance and improvement of growth performance of livestock and poultry, especially broiler chickens, and can be used as a microbial additive for livestock and poultry feed.

Description

Bacillus amyloliquefaciens TL with antioxidant capacity and promoting growth and development of broiler chickens

technical field

The invention belongs to the technical field of veterinary microbial additives, and in particular relates to the screening and application of a strain of Bacillus amyloliquefaciens (Bacillus Amyloliquefaciens) TL which has strong antioxidant capacity and promotes the growth and development of broilers. The invention relates to the application field of antibiotic additives, and relates to the isolation and identification, safety evaluation, stress resistance performance and probiotic performance identification of a pig-derived probiotic Bacillus amyloliquefaciens strain and its application as a feed additive for livestock and poultry.

Background technique

In animal husbandry production, antibiotics are widely used due to their advantages in preventing and controlling animal diseases and improving the production performance of livestock and poultry. Therefore, to a certain extent, the breeding industry has shifted from free-range farming to intensive and large-scale production. , resulting in greater economic benefits. However, in recent years, due to the extensive and improper use of antibiotics, problems have followed one after another, such as the emergence of a large number of drug-resistant strains, the imbalance of normal flora in animals, and drug residues in animal products, which have seriously restricted the development of antibiotics. The development of the aquaculture industry also affects human health, which has attracted great attention from people from all walks of life. At present, all countries in the world have strict restrictions on the use of antibiotics for livestock and poultry, and it will be an inevitable trend to ban antibiotic additives in the future. In the current breeding environment, how to get out of the dilemma brought to the breeding industry due to the restriction and prohibition of antibiotics is the key to finding a feed additive that is non-toxic, residue-free, and drug-resistant, and can prevent disease and promote growth. , and probiotics have the above advantages one by one, so they are considered to be one of the most effective substitutes for antibiotics.

Bacillus amyloliquefaciens belongs to the genus Bacillus and is a bacterium that is highly related to Bacillus subtilis. During its growth, it can produce a series of metabolites that can inhibit the activity of fungi and bacteria. It is a beneficial bacterium that exists in the intestinal tract of humans and animals, can form spores, can withstand high temperature, acid, bile, and various antibiotics, does not inactivate when passing through the digestive tract, and can be stored stably in vitro. Bacillus amyloliquefaciens can enhance the body's antioxidant capacity, enhance the body's immune function, and metabolize antibacterial substances, including polypeptides, lipopeptides, and antibacterial proteins. Therefore, as a feed additive, it has unique advantages compared with other microbial feed additives.

At present, domestic and foreign researchers have reported many related reports on the isolation and identification of different Bacillus amyloliquefaciens strains and their metabolites in the environment. The results show that Bacillus amyloliquefaciens has a good inhibitory effect on a variety of pathogenic fungi and bacteria. There are also Bacillus amyloliquefaciens isolated from animal feces and intestinal contents, and it is added to the diet as a probiotic additive for animal production, which has a better effect of inhibiting pathogenic bacteria and can significantly improve animal production. performance. Bacillus amyloliquefaciens has broad application prospects as an additive, and is expected to be widely used in animal husbandry as a probiotic preparation. However, there are not many reports about its growth promotion. The present invention will carry out a series of studies on the promotion of broiler growth and development by Bacillus amyloliquefaciens TL.

Contents of the invention

The purpose of the present invention is to provide a strain of bacillus amyloliquefaciens that is safe, strong in oxidation resistance, strong in disease resistance, especially capable of significantly promoting the growth and development of livestock and poultry (for example, promoting the growth and development of broilers) and can be used as a microbial feed additive.

The present invention is realized through the following technical solutions:

According to the screening target and the physiological, biochemical and genetic characteristics of probiotics, the present invention uses classical physiological and biochemical means and modern molecular biological methods to select a large number of rectal contents from healthy pigs from a certain place in Wuhan City, Hubei Province. A strain of Bacillus amyloliquefaciens, which has strong antioxidant capacity and can promote the growth and development of broilers, was isolated and screened from the intestinal contents of healthy pigs raised on pig farms. The applicant named the strain Bacillus amyloliquefaciens TL, Bacillus Amyloliquefaciens TL was sent to China. Wuhan. Wuhan University China Type Culture Collection Center (CCTCC) for preservation on June 4, 2015, and its preservation number is CCTCC NO: M2015359.

The bacteriological characteristic of Bacillus amyloliquefaciens TL of the present invention:

Bacillus amyloliquefaciens TL is a facultative anaerobic Gram-positive bacillus. When it grows on LB plates, it forms a milky white round or nearly round colony, which is opaque, with irregular edges, a raised center, and wrinkled surfaces. The bacterium is rod-shaped, and the spores are approximately oval, located in the center of the bacterium or slightly biased. The optimum growth temperature range is 28℃～30℃; the optimum growth pH range is pH 6.5～7.0. The physiological and biochemical characteristics of the strain conform to the description of Bacillus amyloliquefaciens in "Bergey's Handbook of Bacteria". The 16S rRNA gene was identified by PCR and sequenced, and the results were compared with Blastn at NCBI, and it was found that its 16S rRNA was homologous to the 16S rRNA sequences of Bacillus amyloliquefaciens, methylotrophic Bacillus and Bacillus subtilis. At the same time, its gyrB gene was identified by PCR and sequenced, and compared with Blastn, it was found that the strain had the highest homology with Bacillus amyloliquefaciens, and it was determined to be Bacillus amyloliquefaciens (Bacillus Amyloliquefaciens).

The strain has a strong ability to survive in an acidic and high bile salt environment. When incubated at pH 3.0 for 3 hours, the number of viable bacteria is still in the same order of magnitude, both at 10 ⁸ CFU/mL, the survival rate reaches 67.19%, and it can tolerate 0.5 % bile salts 12h. It is speculated that the strain can resist the adverse effects of gastric acid and high concentration of bile salts in the small intestine, adhere to the intestinal tract and survive to play a probiotic effect, which has also been confirmed in animal experiments.

The strain can produce spores and has relatively strong tolerance to high temperature. At 80°C and 90°C, the processing time was set to 10min, 20min, and 30min, respectively. In 80°C water bath, with the prolongation of time, the number of viable bacteria decreased slowly, but still at the same order of magnitude, reaching 10 ⁸ , and the minimum survival rate still reached 59.25%; in 90°C water bath, with the extension of water bath time, the number of viable bacteria The number decreased, but remained at the same order of magnitude, reaching 10 ⁸ , and its minimum survival rate reached at 50.00%. It shows that the strain can tolerate the high temperature of feed maturation and granulation, which lays a good foundation for its practical production as a feed additive.

The results of the acute toxicity test on the strain showed that the signs of the mice in the experimental group were normal, and no poisoning or death occurred. This is consistent with the results of subsequent animal experiments, ensuring its safety as a feed additive.

The strain has obvious antibacterial effect on common intestinal pathogenic bacteria (Staphylococcus aureus, Escherichia coli, Salmonella, Listeria monocytogenes). This is consistent with the results of subsequent animal experiments, indicating that the strain has the effect of enhancing resistance.

The strain is sensitive to common veterinary drugs such as penicillin, chloramphenicol, erythromycin, tetracycline, gentamicin, spectinomycin, vancomycin, ciprofloxacin, norfloxacin, ampicillin and nitrofurantoin, which is The use of this strain in combination with antibiotics provides a basis.

The applicant made the Bacillus amyloliquefaciens TL of the present invention by conventional centrifugal spray-drying through liquid fermentation to make microecological preparations (or bacterial agents), which were applied to feed additives, and broiler feeding experiments were carried out to study its probiotic properties. The results showed that , reduce feed-to-meat ratio to broiler chicken, the effect of promoting growth, and test has reached expected effect, thereby completed task of the present invention.

Bacillus amyloliquefaciens TL of the present invention has the following advantages:

(1) Bacillus amyloliquefaciens TL is derived from the intestinal contents of healthy pigs. It is isolated and screened from a large number of candidate strains. It is effective against common intestinal pathogens such as Staphylococcus aureus, E. It has obvious antibacterial effect. It is completely resistant to gastric acid and high osmotic environment of intestinal bile salts, so it can exert a prebiotic effect in the intestinal tract. At the same time, the strain can produce spores and has strong stress resistance.

(2) Animal experiments have proved that Bacillus amyloliquefaciens TL can be used as a feed additive for livestock and poultry, and has an inhibitory effect on intestinal pathogenic bacteria of livestock and poultry, can promote the development of the body's immune system, improve the body's antioxidant capacity, and achieve a significant increase The effect of livestock and poultry production performance, and provide a reference for its addition in feed.

For a more detailed technical solution, see the content of the "Specific Implementation Plan".

Description of drawings

Sequence Listing SEQ ID NO: 1 is the partial nucleotide sequence of the 16S rDNA gene of the isolated strain Bacillus amyloliquefaciens TL of the present invention.

Sequence Listing SEQ ID NO: 2 is the nucleotide sequence of the gyrB gene of the isolated strain Bacillus amyloliquefaciens TL of the present invention.

Fig. 1: It is the growth state of Bacillus amyloliquefaciens TL isolated and screened by the present invention on the LB medium plate.

Fig. 2: It is the microscopic examination picture of the Gram staining reaction of Bacillus amyloliquefaciens TL isolated and screened in the present invention.

Figure 3: Picture of antibiotic susceptibility test of Bacillus amyloliquefaciens TL. Explanation of reference numerals: Fig. 3A is a diagram of chloramphenicol sensitivity test; Fig. 3B is a diagram of sensitivity test of ampicillin; Fig. 3C is a diagram of sensitivity test of nitrofurantoin; Fig. 3D is a diagram of sensitivity test of erythromycin; Fig. 3E is a diagram of sensitivity test of tetracycline; Fig. 3F is a diagram of gentamicin sensitivity test; Fig. 3G is a diagram of norfloxacin sensitivity test; Fig. 3H is a diagram of ciprofloxacin sensitivity test; Fig. 3I is a diagram of spectinomycin sensitivity test; Fig. 3J is a diagram of vancomycin sensitivity Test chart.

specific implementation plan

Example 1: Isolation and Identification of Bacillus Strains

1. Isolation of strains

(1) The biological material of the present invention collects the rectal content of a healthy pig from a pig farm in Wuhan, Hubei, dilutes the rectal content with phosphate buffered saline (PBS), and uses a cotton swab on (LB) solid medium (purchased from BD Company of the United States). ) and cultured by streaking, placed in a 37°C incubator for static culture for 18-24 hours.

(2) Pick the colonies that meet the characteristics of Bacillus colonies on the plate and inoculate them on a plate containing LB medium, place them in an incubator, and culture them statically at 37°C for 24 hours, and finally carry out staining microscope inspection.

The growth characteristics of the isolate (bacillus) of the present invention in LB medium are shown in Figure 1, and its Gram staining characteristics are shown in Figure 2.

2. Physiological and biochemical identification of isolates

Physiological and biochemical identification was carried out on the screened isolates (identification items are shown in Table 1), and the species was further identified. The identification results (see Table 2) were compared with the description on the identification of Bacillus species in the "Bergey's Bacteria Identification Manual". The isolate was initially determined to be Bacillus.

Table 1 Biochemical identification items of the isolate (Bacillus)

Table 2 Biochemical identification results of isolates (Bacillus)

3. Confirmation of isolate species

On the basis of the above-mentioned identification, further carry out 16S rRNA gene sequence detection and gyrB gene identification to the above-mentioned isolate (Bacillus isolate), and confirm and identify the species to which the strain belongs (see the sequence described in the sequence table SEQ ID NO: 1 ).

(1) Extraction steps of isolate genome DNA:

Genomic DNA of isolates was extracted using Tiangen Biochemical Technology (Beijing) Co., Ltd. Bacterial Genomic DNA Extraction Kit (operated according to the instructions of the kit).

(2) Primers for amplifying the 16S rRNA gene:

16S rRNA gene amplification primers are universal primers (primers were synthesized by Beijing Qingke Xinye Biotechnology Co., Ltd.), and the primer sequences are as follows:

27F 5'-GAGTTTGATCCTGGCTCAG-3'

1492R 5'-ACGGCTACCTTGTTACGACTT-3'

(3) PCR amplification of 16S rRNA gene

The 16S rRNA genes of the genomes of the above isolates were respectively amplified with the above primer pairs. The reaction system is shown in Table 3. The PCR program was: 94°C for 5min, 94°C for 1min, 61.5°C for 1min, 72°C for 1.5min, and after 30 cycles, extend at 72°C for 10min. Get the PCR product and carry out electrophoresis detection on 0.8% agarose gel (containing ethidium bromide), and the target fragment size that amplifies obtains is about the specific band of 1542bp (see sequence table SEQ ID NO: 1 described sequence). The target fragment was recovered with a DNA purification kit (purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.), and sent to Beijing Qingke Xinye Biotechnology Co., Ltd. for sequencing. The sequencing results were compared by Blast in the NCBI database. Retrieval finds that the 16S rRNA sequence identity of the 16S rRNA of the isolated bacillus strain of the present invention and reported bacillus bacillus such as amyloliquefaciens, methylotrophic bacillus and bacillus subtilis all reaches 99% and above (see Table 4).

Table 3 PCR system for 16S rRNA amplification of isolate TL (Bacillus)

Table 4 BLAST results of TL 16S rRNA gene sequencing of isolates

(4) PCR amplification of gyrB gene

The reference primer sequence is as follows, and the gyrB gene of the isolate Bacillus is amplified by using the genome of the above isolate Bacillus as a template.

The DNA sequences of the primer pairs are as follows:

BS-F(5'-GAAGGCGGNACNCAYGAAG-3')

BS-R(5'-CTTCRTGNGTNCCGCCTTC-3')

PCR program: 94°C for 5min, 94°C for 1min, 61.5°C for 1min, 72°C for 1.5min, and after 30 cycles, extend at 72°C for 10min. The PCR reaction system is shown in Table 3. The PCR product was taken and detected by electrophoresis on 0.8% agarose gel (containing ethidium bromide), and the amplified target fragment was about 1146bp in size (see the sequence described in SEQ ID NO: 2 in the sequence table). The target fragment was recovered with a DNA purification kit (purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.), and sent to Beijing Qingke Xinye Biotechnology Co., Ltd. for sequencing. The sequencing results were compared by Blast in the NCBI database, and the results are shown in Table 5. The results show that the gyrB gene of the isolated Bacillus amyloliquefaciens TL of the present invention has the highest nucleotide sequence homology with the gyrB gene of the reported Bacillus amyloliquefaciens TL, and the similarity is 99% (the nucleotide sequence is referring to SEQ ID NO : 2; namely the gyrB gene nucleotide sequence of the reported bacillus). This shows that the Bacillus isolated from the present invention is Bacillus amyloliquefaciens.

Table 5 BLAST alignment results of gyrB gene sequences of Bacillus

The present invention obtained a strain of Bacillus amyloliquefaciens through final screening, and the applicant named the strain Bacillus amyloliquefaciens TL, Bacillus Amyloliquefaciens TL, and sent it to China. Wuhan. Wuhan University, China for the preservation of typical cultures on June 4, 2015 Center (CCTCC) deposit, its deposit number is CCTCC NO: M2015359.

Embodiment 2: The inhibitory action test of Bacillus amyloliquefaciens TL isolated by the present invention on enteropathogenic bacteria of livestock and poultry

1. Screening of control vector strains and preparation of fermentation broth

The vector strains of the control were Salmonella choleraesuis C78-1 (purchased from China Veterinary Drug Administration), porcine pathogenic E. O139 (gifted by Professor Wu Bin, State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University), Escherichia coli O138:K81:K88ac (purchased from China Veterinary Drug Control Institute), Escherichia coli K12:K99 (purchased from China Veterinary Drug Control Institute), Listeria monozen Bacteria LM201 (Huazhong Agricultural University Veterinary Microbiology and Immunology Laboratory preservation) and Staphylococcus aureus (ATCC25923, purchased from the Clinical Inspection Center of the Ministry of Health) are pathogenic bacteria indicator bacteria, and Bacillus amyloliquefaciens TL of the present invention is effective on livestock and poultry intestines. A systematic comparative study was carried out on the inhibitory effect on pathogenic bacteria.

Specific steps are as follows:

(1) Activation of Bacillus amyloliquefaciens TL: draw the freeze-dried bacterial powder of Bacillus amyloliquefaciens on the LB agar plate, and place it in a 37°C incubator for 24 hours (repeat the above steps twice to obtain pure strains) , pick a typical single colony and inoculate it in LB liquid medium, cultivate it in a constant temperature shaker at 37°C at 200r/min for 24h, then place it in a water bath at 80°C for 10min, then transfer it to LB liquid medium at 2% (v/v), and Place them in a constant temperature shaker at 37°C at 200r/min for cultivation, and keep them in the logarithmic growth phase for later use.

(2) Activation of control pathogenic bacteria: the above-mentioned Staphylococcus aureus, Salmonella choleraesuis C78-1, porcine pathogenic E. coli O157:H7, porcine E. coli O139, Escherichia coli O138:K81:K88ac, Escherichia coli K12:K99 and Listeria monocytogenes LM201 were transferred to inclined test tubes at the same time, and cultured at 37°C for 12 hours for later use.

(3) Preparation and processing of fermented liquid: the fresh bacterial liquid obtained in steps (1) and (2) is inoculated in LB broth medium at 2% (v/v), placed in a constant temperature shaker at 37°C at 200r/m Min cultured for 24 hours, centrifuged to take the supernatant, 10000r/min, 10min centrifuged, aspirated the supernatant, and measured the pH of each fermentation broth; set aside at 4°C.

2. In vitro anti-bacteria (inhibition) test of Bacillus amyloliquefaciens TL

(1) Operation steps:

1) Preparation of LB plates: pour about 25 mL of LB agar medium (thickness is about 4 mm) into each plate, and dry the plates before the test (after the indicator bacteria are evenly coated on the plates, the plate shall be subject to no visible water droplets).

2) Preparation of indicator bacteria liquid: Preserved Staphylococcus aureus, Salmonella choleraesuis C78-1 and porcine pathogenic E. K99 and Listeria monocytogenes LM201 were streak cultured to obtain a single colony; a single colony was picked and placed in LB liquid medium, and shaken at 37°C for 12 hours for this test.

3) Dilution of the bacterial solution: use a McFarland turbidimetric tube to adjust the concentration of the bacterial solution of the indicator bacteria to No. 0.5 McFarland standard; then dilute the bacterial solution by an appropriate multiple. Spread the diluted bacterial solution evenly on the plate with a sterilized cotton swab.

4) After the water on the surface of the agar is dry, gently put the sterile Oxford cup into the petri dish with tweezers, and draw 0.25mL of the fermentation supernatant into the Oxford cup (be careful not to overflow the cup).

5) Place the plate in a refrigerator at 4°C for several hours, and then transfer it to an incubator at 37°C (be careful not to collapse the Oxford cup while moving). Observe and measure the size of the inhibition zone after 10-12 hours.

(2) Test results: See Table 6 for the results of the in vitro inhibition (anti)bacteria test of Bacillus amyloliquefaciens TL. According to the test result, it can be known that the Bacillus amyloliquefaciens TL isolated in the present invention is to Salmonella choleraesuis C78-1, porcine pathogenic E. K99, Staphylococcus aureus and Listeria monocytogenes LM201 all had certain antibacterial effects, especially for Staphylococcus aureus.

Table 6 In vitro antibacterial test of Bacillus amyloliquefaciens TL

Example 3: Safety Evaluation of Bacillus amyloliquefaciens TL

1. Acute toxicity test

The acute toxicity test was carried out with reference to the standard method stipulated in the national standard GB15193.3-2003 of the People's Republic of China for the test of the maximum tolerated dose.

Specific steps:

Bacillus amyloliquefaciens TL was inoculated in LB liquid medium at an inoculum amount of 1%, cultured at 37°C until the stationary phase, and the concentration of the bacterial solution was adjusted to 10 ⁹ CFU/mL.

40 SPF-grade Kunming mice (18g-22g) were randomly divided into a test group and a control group after 3 days of acclimatization, with 20 males and 20 males. The mice in the test group were given 0.2 mL of 109 CFU/mL bacterial solution, and the mice in the control group were given 0.2 mL of normal saline. The mice in each group were given food and water ad libitum once a day. The mental state, physical signs, poisoning performance, death status, body weight change and organ index of mice in each group were recorded during the experiment.

Calculate according to the following formula: organ index (g) = organ weight/body weight × 100

During the test period, the signs of the mice in each group were normal, no poisoning and death occurred, and the mental state, diet, drinking water, and excreta were normal; the weight of the mice in the test group continued to increase, and there was no significant difference compared with the control group (P>0.05). Table 7; After dissection, there was no significant difference between the test group and the control group, and the results of the influence on the mouse organ coefficient showed that Bacillus amyloliquefaciens TL had no significant effect on the mouse liver and spleen compared with the control group Sex difference (P>0.05) (see Table 8).

Table 7 Changes in body weight of mice in acute toxicity test

Note: P>0.05, no significant difference compared with the control group

Table 8 Effect of mouse organ coefficient in acute toxicity test

Note: P>0.05, no significant difference compared with the control group

2. Antibiotic sensitivity test

(1) Using the K-B disk diffusion method, selected penicillin, ampicillin, vancomycin, erythromycin, tetracycline, ciprofloxacin, norfloxacin, nitrofurantoin, chloramphenicol, gentamicin, spectinomycin A total of 11 commonly used antimicrobials were tested for susceptibility. Using M-H medium, the standard sensitive strain Staphylococcus aureus ATCC25923 was used as the quality control bacteria, and the test judgment standard was carried out according to the latest version of NCCLS standard provided by WHO (version 2009). The test steps are as follows:

1) Pick a colony of Bacillus amyloliquefaciens on an LB agar plate with an inoculating loop, inoculate it in 3-5 mL of LB broth medium, and place it in a constant temperature shaker at 37°C at 200r/min for cultivation.

2) Cultivate for 18-24 hours, and adjust the concentration of the bacterial solution to No. 0.5 McFarland standard with the white paper with black characters as the background. If the concentration of the bacterial solution is too thick, it can be diluted with broth or saline (the corrected bacterial solution should be used within 15 minutes).

3) Dip the bacterial solution with a sterile cotton swab, squeeze off the excess bacterial solution on the tube wall, and spread it on the M-H agar plate, rotate the plate 60 degrees each time, and finally wrap it around the periphery twice, repeat several times, Ensure uniform coating.

4) After the water on the plate is completely absorbed by the agar, use sterile tweezers to take the drug-sensitive paper and paste it on the surface of the plate (once the paper is pasted, it cannot be picked up again). Each plate is pasted with 3 drug-sensitive paper sheets, the distance between each drug-sensitive paper sheet is not less than 24mm, and the distance between the center of the drug-sensitive paper sheet and the edge of the plate is not less than 15mm.

5) Within 15 minutes after affixing the drug-sensitive paper, place the plate upside down in a 37°C oven; incubate for 24 hours to observe the results.

(2) The results of antibiotic susceptibility test of Bacillus amyloliquefaciens TL are shown in Table 9. The results showed that Bacillus amyloliquefaciens TL was sensitive to penicillin, chloramphenicol, erythromycin, tetracycline, gentamicin, spectinomycin, vancomycin, ciprofloxacin, norfloxacin, ampicillin and nitrofurantoin , consistent with relevant reports, thus indicating that the isolated Bacillus amyloliquefaciens TL of the present invention should pay attention to the compatibility with the above-mentioned antibiotics in use, but from the perspective of safety, the above-mentioned tests show that Bacillus amyloliquefaciens TL is used in livestock and poultry is relatively safe.

Table 9 Results of antibiotic susceptibility test of Bacillus amyloliquefaciens TL

The comprehensive test results of acute toxicity test and antibiotic susceptibility test show that Bacillus amyloliquefaciens TL is safe.

Embodiment 4: Stress resistance characteristics of Bacillus amyloliquefaciens TL

(1) Bile salt tolerance test

After activating the Bacillus amyloliquefaciens TL strain for 2 generations, take 1 mL and inoculate it in 9 mL LB broth containing 0%, 0.15%, 0.3%, 0.5% bile salts, culture it on a constant temperature shaker at 37°C at 200r/min for 12 hours, and dilute to 10 ⁶ CFU/mL, take 0.1mL and spread it on LB plates, observe the growth and count. The results showed (see Table 10) that with the increase of bile salt concentration, the number of viable bacteria decreased slowly, but still reached 10 ⁷ , while the mass fraction of bile salt in pig small intestine fluctuated in the range of 0.03% to 0.3%. It shows that Bacillus amyloliquefaciens TL can tolerate the intestinal bile salt environment.

Table 10 Tolerance of Bacillus amyloliquefaciens TL to bile salts

(2) Acid resistance test

The activated Bacillus amyloliquefaciens TL was respectively inoculated into the LB liquid medium with pH 3.0 at an inoculum amount of 2%, and cultured on a constant temperature shaker at 37° C. at 200 r/min. Take the culture solution inoculated for 0h, 1h, 2h, and 3h, dilute it to 10 ⁶ CFU/mL, take 0.1mL and spread it on the LB plate, observe the growth and count. The results are shown in Table 11, which indicated that after the Bacillus amyloliquefaciens TL tolerated pH 3.0 and 3 hours, the number of viable bacteria was still at the same order of magnitude, both at 10 ⁸ CFU/mL, and the survival rate reached 67.19%. This shows that Bacillus amyloliquefaciens TL can still grow and reproduce in a low pH (strongly acidic) environment, and has good acid resistance, indicating that it can survive in the gastrointestinal tract of animals.

Table 11 Tolerance of Bacillus amyloliquefaciens TL to different pH values

The pH value in the stomach of piglets at birth is generally 5-6, and then the pH value gradually drops to about 4 due to the colonization of lactic acid bacteria, and the pH value remains at about 3 before 2 months of age. Usually the pH value of gastric acid is around 3.0, and the liquid food stays in the stomach for 1 to 2 hours. In a short time after eating, the pH value in the stomach can rise to about 6.0. When the probiotics pass through the gastrointestinal tract, chyme acts as a protective agent to reduce the degree of damage to the probiotics. Once they survive through the stomach and duodenum, the bacteria that enter the ileum and cecum along with the chyme will rapidly Increase. It can be seen from the test results that the number of live bacteria of Bacillus amyloliquefaciens TL isolated in the present invention is still at the same order of magnitude after several hours in a low pH environment (Table 11). It can be known that the Bacillus amyloliquefaciens TL of the present invention has a high probability of survival in a low-pH gastric acid environment, and enough bacteria survive to reach the intestinal tract smoothly.

(3) High temperature tolerance test of Bacillus amyloliquefaciens TL

Inoculate 2% of the activated Bacillus amyloliquefaciens TL strain in LB liquid medium, and culture in a constant temperature shaker at 37° C. at 200 r/min for 24 hours. The bacterial liquid was heat-treated at 80°C and 90°C, and 37°C was used as the control. The bacterial liquid was taken for 10 min, 20 min and 30 min, and then diluted to 10 ⁶ CFU/mL, and the growth was observed and counted with an LB plate.

The results showed that compared with the control group, in the 80°C water bath, with the prolongation of the water bath time, the number of viable bacteria of Bacillus amyloliquefaciens TL decreased slowly, but it was still at the same order of magnitude, reaching 10 ⁸ , and its minimum survival rate was still Reached 59.25%. In the 90°C water bath, the number of viable bacteria of Bacillus amyloliquefaciens TL decreased with the prolongation of the water bath time, but the number of viable bacteria was still in the same order of magnitude, reaching 10 ⁸ , and the minimum survival rate reached 50.00%. This shows that Bacillus amyloliquefaciens TL has good high temperature resistance, can withstand the high temperature of feed ripening and pelleting, and facilitates its application as a feed additive (see Table 12).

Table 12 Tolerance of Bacillus amyloliquefaciens TL to high temperature

In summary, resistance to low pH gastric acid environment and tolerance to bile are the primary indicators for screening probiotics. The Bacillus amyloliquefaciens TL isolated in the present invention has a high survival rate in a very low pH gastric acid environment, and has Sufficient bacteria survived to reach the intestinal tract, and the tolerance of Bacillus amyloliquefaciens TL to intestinal bile salts allowed it to successfully colonize the host intestinal tract and exert a beneficial effect.

In addition, probiotics must withstand high-temperature heat treatment during the concentration of the bacterial solution or during the preparation process. Therefore, the tolerance to high temperature becomes another key indicator for the selection of probiotic strains. The Bacillus amyloliquefaciens TL of the present invention can form spores to resist adverse environments, and the survival rate of the spores at 80°C for 10 minutes is 83.33%, which provides favorable conditions for the formulation production and development of Bacillus amyloliquefaciens TL and exerts its probiotic effect .

Embodiment 5: as the application embodiment of microbial additive (broiler chicken feeding test)

Add the Bacillus amyloliquefaciens TL preparation of the present invention (manufactured by Wuhan Huada Ruier Technology Co., Ltd., the number of effective viable bacteria ≥ 2×10 ¹⁰ CFU/g) to broiler feed, the purpose is to study the effect of the strain of the present invention on broiler chickens The probiotic properties and the antioxidant capacity of the strain of the present invention, the impact on the disease resistance of broiler chickens.

1. Test materials and groups

Feed animals are white-feathered broiler chickens, totally 9000, which are randomly divided into 3 groups, which are respectively set as control group (basal diet without adding any medicine and probiotics), antibiotic group (adding 50g of probiotics on the basis of basic diet) Aureomycin/t) and probiotic group (200g/t of Bacillus amyloliquefaciens TL preparation added on the basis of basal diet, that is, 4×10 ¹² CFU/t), each group had 6 replicates, each replicate 500 chickens. The test period is 42 days. See Table 13 for the grouping treatments of the feeding test.

The basic diet in this example is a commercially available full-price feed (purchased from the feed factory of Wuhan Dangyang Zhengyang Breeding Cooperative). The mixing, processing and granulation of probiotic preparations, antibiotics and basic diets are uniformly produced by the feed factory of Wuhan Dangyang Zhengyang Breeding Cooperative.

Table 13 Test grouping

2. Test process

The animal experiment was carried out in Zhengyang broiler farm in Yichang City, Hubei Province in May 2014. The chicken house, all test equipment and the surrounding environment of the farm were cleaned and fully disinfected one week before the official breeding. The test period is 42 days, which is divided into two stages, 1-21d is the first stage, the early stage, and 22-42d is the second stage, the late stage. The experiment was carried out in a closed chicken house, and the method of flat raising on the Internet was adopted. The whole period was free to eat and drink. In principle, the feed was fed 3 times a day, and the measurement was unlimited. The chicken house is heated by electric heating tubes. The temperature in the 1-7-day-old house is 33-35°C. After that, it will be reduced by 1-2°C every week. The light time for 1-3 days is 24 hours. 1 hour dark. Free ventilation with extractor fans (as applicable). During the test period, the chicken coop was cleaned and disinfected regularly, the condition of the chicken flock was observed, and the morbidity, death and diarrhea of the chickens were recorded. All chickens were vaccinated according to routine procedures.

3. Measuring indicators

1. Determination of production performance indicators

Weigh on an empty stomach in the morning on the 1st and 42nd day after the test, and accurately record the initial weight, final weight, feed consumption, number of dead chickens and dead weight, and finally calculate the average daily gain, average daily feed consumption, and feed-to-meat ratio and mortality.

Calculation formula:

Average body weight (g) = total weight of random weighing / number of weighing only;

Average daily weight gain (g) = (final weight - initial weight) / test days;

Ratio of feed to meat = total consumption (kg) / total weight of slaughtered chickens (kg;)

Determination and calculation of mortality: During the test period, the mortality of the flock was recorded every day, and finally the number of dead chickens was counted in groups.

Mortality rate (%)=(number of dead animals in a certain group during the test period/total number of animals in a certain group during the test period).

2. Determination of serum biochemical indicators

Serum biochemical indicators detected mainly include blood glucose (GLU), total protein (TP), albumin (ALB), serum urea nitrogen (BUN), cholesterol (CHOL), triglyceride (TG), alanine aminotransferase (ALT), Aspartate aminotransferase (AST).

On the 21d and 42d after the test, 2 broiler chickens were randomly selected for each repetition in each group, and blood was collected under the wing using a blood collection needle and a vacuum coagulation tube. About 5mL of blood can be collected from each feather, and the blood sample of the procoagulant tube is quickly placed in a 37°C constant temperature incubator for 1 hour, and then transferred to a 4°C refrigerator for about 2 hours (depending on the test situation). After the blood coagulation serum seeps out, pass through Centrifuge in a refrigerated centrifuge at 3000r/min for 15min, use a pipette to absorb the serum and aliquot it, and store it at -20°C for the determination of serum biochemical indicators. Serum biochemical indicators were detected by Hitachi's automatic biochemical analyzer 7100, and the detection index items and detection methods are shown in Table 14.

Table 14 Detection items and methods of serum biochemical indicators

3. Determination of antioxidant indicators

The antioxidant indicators detected by the kit method (purchased from Nanjing Jiancheng Bioengineering Institute) included superoxide dismutase (SOD), glutathione peroxidase (GSH-PX) and malondialdehyde (MDA), See its instruction manual for specific operation methods.

4. Determination of immune organ index

On the 21d and 42d after the experiment, 2 broiler chickens were randomly selected for each repetition in each group. After slaughtering, the thymus, spleen and bursa of Fabricius were quickly removed, and the excess water and blood on the surface were sucked off with filter paper, and the other broilers were peeled off. Excess tissues were weighed to calculate their immune organ index, and the calculation formula was as follows:

Immune organ index (g) = fresh weight of immune organs / live weight of chicken

5. Determination of Morphological and Structural Indexes of Intestinal Mucosa

On the 21st day and 42nd day after the test, 2 broiler chickens were randomly selected for each repetition in each group. After slaughter, tissue samples of about 1 ^cm2 in size were cut from the middle section of the ileum and cecum and placed in 4% formalin solution. After fixation, dehydration, wax infiltration, embedding, sectioning and other processing steps, and then hematoxylin-eosin (HE) staining, the Japanese Nikon 80i biological optical microscope combined with NIS-Elements high-definition color graphic analysis system was used for observation , take pictures and take pictures, select 10 typical visual fields (the villi are complete, and go straight) from the tissue section, measure the villi length (VH) and the crypt depth (CD), and calculate the ratio of the villi length to the crypt depth ( VH/CD).

3. Results

1. Production performance

It can be seen from Table 15 that there was no significant difference in the initial body weight of broilers in each group (P>0.05). In terms of final weight, the Bacillus amyloliquefaciens TL group was higher than the control group, the difference was significant (P<0.05), which was basically the same as that of the antibiotic group; in terms of average daily gain, the Bacillus amyloliquefaciens TL group was higher than the control group, the difference was not significant (P >0.05), which was basically the same as that of the antibiotic group; in terms of average daily feed consumption and feed-to-meat ratio, the Bacillus amyloliquefaciens TL group was lower than the control group and the antibiotic group, and the difference was not significant (P>0.05); in terms of mortality, each group There was no significant difference between them (P>0.05). This shows that adding Bacillus amyloliquefaciens TL to the broiler diet can improve the production performance of broilers to a certain extent, promote the growth and development of broilers, and increase the feed conversion rate at the same time.

Table 15 Effect of Bacillus amyloliquefaciens TL preparation on broiler performance (g)

Note: The same lowercase letters or no letters on the shoulders of the same peers indicate no significant difference (P>0.05), different lowercase letters indicate significant differences (P<0.05), completely different uppercase letters indicate extremely significant differences (P<0.01).

Table 16 Effect of Bacillus amyloliquefaciens TL on serum biochemical indicators of 21d broiler chickens

Note: The same lowercase letters or no letters on the shoulders of the same peers indicate no significant difference (P>0.05), different lowercase letters indicate significant differences (P<0.05), completely different uppercase letters indicate extremely significant differences (P<0.01)

2. Effect of Bacillus amyloliquefaciens TL on serum biochemical indicators

The results in Table 16 and Table 17 show that at the age of 21 days, the content of total protein (TP) in the serum of the Bacillus amyloliquefaciens TL group and the antibiotic group increased by 9.79% and 8.14% respectively compared with the control group, and the difference was significant (P<0.05); The content of aspartate aminotransferase (AST) in the serum of Bacillus amyloliquefaciens TL group was 3.56% lower than that of the control group, the difference was significant (P<0.05), and it was 1.52% lower than that of the antibiotic group, the difference was not significant (P>0.05); Bacillus amyloliquefaciens The contents of blood glucose (GLU) and albumin (ALB) in serum of TL group increased by 4.42%, 10.02%, 2.78%, and 0.50% respectively compared with the control group and antibiotic group (P>0.05); the Bacillus amyloliquefaciens TL group Serum cholesterol (CHOL), blood urea nitrogen (BUN), triglyceride (TG) and alanine aminotransferase (ALT) decreased by 8.60%, 1.35%, 21.35% and 1.77% respectively compared with the control group, the difference was not significant (P>0.05) , respectively decreased by 4.01%, 8.75%, 0% and 3.82% compared with the antibiotic group, the difference was not significant (P>0.05). At the age of 42 days, the serum albumin (ALB) content of the Bacillus amyloliquefaciens TL group increased by 12.65% compared with the control group, the difference was significant (P<0.05); the serum aspartate aminotransferase (AST) content of the Bacillus amyloliquefaciens TL group It was 4.46% lower than the control group, the difference was significant (P<0.05), and it was 0.99% lower than the antibiotic group, the difference was not significant (P>0.05); the total protein (TP) content in the serum of Bacillus amyloliquefaciens TL group was higher than that of the control group 5.77%, the difference was not significant (P>0.05); the content of alanine aminotransferase (ALT) in the serum of the Bacillus amyloliquefaciens TL group was 9.37% and 3.24% lower than that of the control group and the antibiotic group, the difference was not significant (P>0.05); The contents of cholesterol (CHOL) and blood urea nitrogen (BUN) in the serum of the Bacillus amyloliquefaciens TL group decreased by 14.60%, 15.14%, 2.86%, and 9.33% respectively compared with the control group and the antibiotic group, and the difference was not significant (P>0.05) .

Table 17 Effect of Bacillus amyloliquefaciens TL on serum biochemical indicators of 42d broiler chickens

Note: The same lowercase letters or no letters on the shoulders of the same peers indicate no significant difference (P>0.05), different lowercase letters indicate significant differences (P<0.05), completely different uppercase letters indicate extremely significant differences (P<0.01)

As a result of the test, the contents of blood glucose, total protein, albumin, urea nitrogen, cholesterol, triglyceride, alanine aminotransferase and aspartate aminotransferase in the serum of the Bacillus amyloliquefaciens TL group were better than those of the control group and the antibiotic group in varying degrees, thus indicating that , Bacillus amyloliquefaciens is beneficial to improve some biochemical indicators of broiler serum to a certain extent, promote the digestibility of non-starch polysaccharides and starch polysaccharides in the feed, and protein synthesis, and has no damage to heart and liver functions. This is Bacillus amyloliquefaciens TL The application in formulation production provides a basis.

3. Effect of Bacillus amyloliquefaciens TL on antioxidant indexes

The results showed (see Table 18), at the age of 21 days, the content of malondialdehyde (MDA) in the serum of the Bacillus amyloliquefaciens TL group was 12.08% and 10.46% lower than that of the control group and the antibiotic group, and the difference was significant (P<0.05) ; The content of superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX) in the serum of Bacillus amyloliquefaciens TL group increased by 15.85% and 34.94% respectively compared with the control group, the difference was significant (P<0.05) , respectively increased by 3.71% and 16.12% compared with the antibiotic group, the difference was not significant (P>0.05). At the age of 42 days, the content of malondialdehyde (MDA) in the serum of the Bacillus amyloliquefaciens TL group was 9.43% lower than that of the control group, the difference was significant (P<0.05), and it was 8.12% lower than that of the antibiotic group, the difference was not significant (P>0.05 ); the content of glutathione peroxidase (GSH-PX) in the serum of Bacillus amyloliquefaciens TL group increased by 22.45% compared with the matched group, the difference was significant (P<0.05), and it was increased by 1.36% than the antibiotic group, the difference was not significant ( P>0.05); the content of superoxide dismutase (SOD) in the serum of the Bacillus amyloliquefaciens TL group increased by 11.30% and 6.31% respectively compared with the control group and the antibiotic group, the difference was not significant (P>0.05).

Table 18 Effect of Bacillus amyloliquefaciens TL on antioxidant indexes of broilers

Note: The same lowercase letters or no letters on the shoulders of the same peers indicate no significant difference (P>0.05), different lowercase letters indicate significant differences (P<0.05), and completely different uppercase letters indicate extremely significant differences (P<0.01).

The test results showed that at the age of 21 days, the contents of SOD and GSH-PX in the serum of the Bacillus amyloliquefaciens TL group were significantly higher than those in the control group and the antibiotic group (P<0.05), and the contents of MDA were significantly lower (P<0.05); At the age of 42 days, the GSH-PX in the serum of the Bacillus amyloliquefaciens TL group was significantly higher than that of the control group (P<0.05); the content of MDA was significantly lower than that of the control group and antibiotic group (P<0.05). This shows that Bacillus amyloliquefaciens TL can improve the antioxidant capacity of the body to a certain extent, and reduce or alleviate the oxidative damage of the body.

4. Effect of Bacillus amyloliquefaciens TL on immune organ index

The results showed (Table 19), at the age of 21 days, the thymus index and the bursa index of the Bacillus amyloliquefaciens TL group increased by 28.71% and 17.69% respectively compared with the control group, the difference was significant (P<0.05), and the spleen index was higher than that of the control group. Increased by 31.87%; the difference was not significant (P>0.05) The thymus index, spleen index and bursa index of the Bacillus amyloliquefaciens TL group increased by 18.18%, 22.45% and 13.07% respectively compared with the antibiotic group, the difference was not significant (P>0.05 ). At the age of 42 days, the thymus index of the Bacillus amyloliquefaciens TL group increased by 48.97% and 50.52% respectively compared with the control group and the antibiotic group, and the difference was significant (P<0.05); the spleen index of the Bacillus amyloliquefaciens TL group was higher than that of the control group and the antibiotic group. The Bacillus amyloliquefaciens TL group increased by 30.43% and 27.66% respectively, the difference was not significant (P>0.05); the bursa index of the Bacillus amyloliquefaciens TL group increased by 26.32% and 33.33% respectively compared with the control group and the antibiotic group, the difference was not significant (P>0.05 ).

Table 19 Effect of Bacillus amyloliquefaciens TL on the immune organ index of broiler chickens

Note: The same lowercase letters or no letters on the shoulders of the same peers indicate no significant difference (P>0.05), different lowercase letters indicate significant differences (P<0.05), and completely different uppercase letters indicate extremely significant differences (P<0.01).

The test results showed that at the age of 21 days, the thymus index, spleen index, and bursa index of the Bacillus amyloliquefaciens TL group increased by 28.71% (P<0.05), 31.87% (P>0.05), and 17.69% (P>0.05) respectively compared with the control group. <0.05); 18.18% (P>0.05), 22.45% (P>0.05) and 13.07% (P>0.05) higher than the antibiotic group respectively. At the age of 42 days, the thymus index, spleen index, and bursa index of the Bacillus amyloliquefaciens TL group increased by 48.97 (P<0.05), 30.43% (P>0.05), and 26.32% (P>0.05) respectively compared with the control group; Antibiotic group increased by 50.52% (P<0.05), 27.66% (P>0.05), 33.33% (P>0.05). This shows that Bacillus amyloliquefaciens TL can promote the development of immune organs, thereby improving the immune level of animals.

5. Effect of Bacillus amyloliquefaciens TL on the morphology of intestinal mucosa

(1) Effects on the morphology of ileal mucosa

As can be seen from the results in Table 20, at the age of 21 days, the villi length (VH) of the Bacillus amyloliquefaciens TL group ileum increased by 18.23% and 13.49% compared with the control group and the antibiotic group, and the difference was extremely significant (P<0.01); The ileal crypt depth (CD) of the Bacillus amyloliquefaciens TL group was not significantly lower than that of the control group and the antibiotic group, and the difference was not significant (P>0.05); the ileal villi length/crypt depth (VH /CD) increased by 18.92% and 17.65% respectively compared with the control group and the antibiotic group, the difference was extremely significant (P<0.01). At the age of 42 days, the villi length (VH) of the ileum in the Bacillus amyloliquefaciens TL group increased by 10.89% compared with the control group, the difference was extremely significant (P<0.01), and it increased by 5.37% compared with the antibiotic group, the difference was significant (P<0.05); The ileal crypt depth (CD) of the Bacillus amyloliquefaciens TL group was not significantly lower than that of the control group and the antibiotic group, and the difference was not significant (P>0.05); the ileal villi length/crypt depth (VH /CD) increased by 18.24% compared with the control group, the difference was extremely significant (P<0.01), and it increased by 10.33% compared with antibiotics, the difference was significant (P<0.05).

(2) Effects on the morphology of the cecal mucosa

As can be seen from the results in Table 21, at the age of 21 days, the villi length (VH) of the cecum of the Bacillus amyloliquefaciens TL group increased by 12.47% and 11.19% respectively compared with the control group and the antibiotic group, and the difference was significant (P<0.05); The cecal crypt depth (CD) in the TL group was not significantly lower than that in the control group and the antibiotic group, and the differences were not significant (P>0.05); the cecal villi length/crypt depth (VH/CD) ) increased by 22.69% and 19.92% respectively compared with the control group and the antibiotic group, the difference was significant (P<0.05). At the age of 42 days, the length of cecal villi (VH) in the Bacillus amyloliquefaciens TL group was 10.65% higher than that in the control group, the difference was significant (P<0.05), and it was 7.23% higher than that in the antibiotic group, the difference was not significant (P>0.05); The cecal crypt depth (CD) of the Bacillus TL group was not significantly lower than that of the control group and the antibiotic group, and the difference was not significant (P>0.05); the cecal villi length/crypt depth (VH/ CD) increased by 21.72% and 19.92% respectively compared with the control group and the antibiotic group, the difference was significant (P<0.05).

Table 20 Effect of Bacillus amyloliquefaciens TL on morphology of broiler ileum mucosa

Note: The same lowercase letters or no letters on the shoulders of the same peers indicate no significant difference (P>0.05), different lowercase letters indicate significant differences (P<0.05), completely different uppercase letters indicate extremely significant differences (P<0.01)

Table 21 Effect of Bacillus amyloliquefaciens TL on morphology of cecal mucosa in broilers

Note: The same lowercase letters or no letters on the shoulders of the same peers indicate no significant difference (P>0.05), different lowercase letters indicate significant differences (P<0.05), completely different uppercase letters indicate extremely significant differences (P<0.01)

The test results showed that at the age of 21 and 42 days, the length of ileum and cecum villi and the ratio of villi length to crypt depth in the Bacillus amyloliquefaciens TL group were significantly or extremely significantly higher than those in the control group and the antibiotic group. This shows that the Bacillus amyloliquefaciens TL preparation is beneficial to maintain the integrity of the intestinal mucosal structure and function, and promote the body's digestion and absorption of nutrients.

In summary, the addition of Bacillus amyloliquefaciens TL prepared by the present invention is beneficial to promote the growth of broilers, reduce the average daily feed consumption, and improve the feed conversion rate; it can improve some serum biochemical indicators of broilers to a certain extent; enhance the antioxidant capacity; Improve the development of immune organs and the morphology of ileum and cecum mucosa in broilers. Therefore the Bacillus amyloliquefaciens TL preparation prepared by the present invention can be used as a kind of microbial additive for feeding in the poultry industry, and it is recommended that the addition of the Bacillus amyloliquefaciens TL bacterial preparation of the present invention be 200g/t (i.e. 4×10 ¹² CFU/t).

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Claims (3)

1. A strain of Bacillus amyloliquefaciens (Bacillus Amyloliquefaciens) TL isolated from pig intestines that has obvious antibacterial effects on pathogenic bacteria Staphylococcus aureus, Escherichia coli, Salmonella, and Listeria monocytogenes in livestock and poultry intestines, preserved In China Center for Type Culture Collection, its collection number is CCTCC NO: M2015359. 2. The microbial preparation prepared by the Bacillus amyloliquefaciens (Bacillus Amyloliquefaciens) TL described in claim 1. 3. the application of Bacillus amyloliquefaciens (Bacillus Amyloliquefaciens) TL described in claim 1 in the preparation of microbial additives for livestock and poultry.