CN113621539B - Screening and application of enzyme-producing antibacterial bacillus subtilis with vomitoxin detoxification function - Google Patents
Screening and application of enzyme-producing antibacterial bacillus subtilis with vomitoxin detoxification function Download PDFInfo
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- A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
- A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
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Abstract
The invention belongs to the technical field of agricultural microorganism application, and particularly relates to screening and application of enzyme-producing antibacterial bacillus subtilis with vomitoxin (DON) detoxification function. Vomitoxin (DON) is one of main mycotoxins polluting feed and raw materials, and the bacillus subtilis GRH-Bs007 which can efficiently remove vomitoxin and efficiently inhibit bacteria and produce enzymes is obtained by screening, and the preservation number of the bacillus subtilis GRH-Bs007 in China center for type culture collection is CCTCC NO: m20211008, and the strain has the characteristics of high growth speed and strong stress resistance, and can be used as a microbial additive for feeding. The strain has good detoxication effect on vomitoxin, and can relieve the influence of vomitoxin (DON) on animal production performance.
Description
Technical Field
The invention belongs to the technical field of microbial additives for animals, relates to the field of antibiotic additives, and in particular relates to screening and application of an enzyme-producing antibacterial bacillus subtilis with a vomitoxin detoxification function.
Background
Vomitoxin (DON for short) is produced mainly by a variety of Fusarium species, and is commonly found in feeds, severely affecting animal productivity and receiving general attention from all countries of the world (Cui et al 2013). Therefore, the mycotoxin pollution in the feed is effectively controlled and solved, and the method has important significance for improving animal production performance. And along with the long-term and large-scale use of antibiotics in animal production, the defects of the antibiotics are increasingly prominent, so that the problems of low animal immune function, unbalanced intestinal flora, increased drug-resistant strains, drug residues and the like are caused, the healthy development of the breeding industry is seriously jeopardized, and the safety of human foods is influenced. It has therefore been urgent to develop new antibiotic alternatives.
The probiotics have the advantages of naturalness, safety, no residue, no pollution and the like, have the effects of preventing animal diseases, enhancing animal immunity and improving feed utilization rate and animal production performance, and become one of the best substitutes for antibiotics to be widely researched and developed. Meanwhile, researches show that the probiotics and the metabolites thereof have good detoxification effects, have the characteristics of rapid growth, rapid propagation and easy culture, are very easy to mass production and popularization and use, and become mycotoxin detoxification agents with great development potential.
The invention develops the enzyme-producing antibacterial feed microbial additive with the vomitoxin detoxification function by screening the probiotics which have the functions of efficiently removing vomitoxin, efficiently inhibiting bacteria and producing enzyme, identifying the probiotics, analyzing biological characteristics and verifying detoxification effects.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention provides an enzyme-producing antibacterial bacillus subtilis (Bacillus subtilis) with a vomitoxin detoxification function and application of the strain in vomitoxin detoxification.
In order to achieve the above purpose, the present invention is realized by the following technical scheme:
in the invention, in the existing 212 probiotic bacteria libraries separated, purified and stored in animal intestinal tracts in a laboratory, DON analogue ethylene oxide is selected as a unique carbon source for preliminary screening culture, and 8 dominant strains are obtained through screening. And then carrying out detoxification and rescreening test on 8 strains obtained by primary screening by taking DON pure products as a unique carbon source, and screening out probiotic strains GRH-Bs007 which have the functions of efficiently removing vomit toxin and efficiently inhibiting bacteria and producing enzymes by combining comprehensive evaluation of an antibacterial test and an enzyme production test. The strain GRH-Bs007 is determined to be bacillus subtilis (Bacillus subtilis) by morphological characteristics, physiological and biochemical indexes and amplification sequencing comparison of 16S rRNA gene, the nucleotide sequence of the 16S rRNA gene is shown as SEQ ID No.1, the bacillus subtilis is preserved in China center for type culture collection (CCTCC for short, address: in the university of Wuhan in Wuhan, hubei province) on the 8 th month 10 th year of 2021, and the proposed classification is named bacillus subtilis (Bacillus subtilis) GRH-Bs007, the preservation number is CCTCC NO: m20211008.
According to the measurement, the enzyme-producing antibacterial bacillus subtilis GRH-Bs007 with the vomit toxin detoxification function has good vomit toxin detoxification capability, and 84.45% of vomit toxin can be removed after 24 hours of detoxification; has good antibacterial activity on Escherichia coli K88, O157, O139, O157 and salmonella and staphylococcus aureus; has good amylase production capacity (enzyme activity is 64.6U/mL), stable growth speed, about 2 hours into logarithmic phase, about 4 hours into logarithmic phase, and high tolerance of spore bodies to temperature, gastric acid and bile salts.
According to the invention, the animal model sensitive to DON is used for verifying that bacillus subtilis GRH-Bs007 has good detoxification effect on BALB/c mice infused with stomach DON.
The invention respectively verifies the excellent detoxification effect of the bacillus subtilis GRH-Bs007 on DON through in vivo and in vitro tests, and the bacillus subtilis has good antibacterial effect, amylase activity and stress resistance, and can be used as a feed additive.
The invention has the beneficial effects that:
(1) The bacillus subtilis GRH-Bs007 has strong detoxication capability on vomitoxin;
(2) The bacillus subtilis GRH-Bs007 disclosed by the invention has a good antibacterial effect on common enteropathogenic bacteria (escherichia coli, salmonella and staphylococcus aureus);
(3) The bacillus subtilis GRH-Bs007 has good amylase production capacity;
(4) The bacillus subtilis GRH-Bs007 has high growth speed and high tolerance to temperature, gastric acid and bile salt.
(5) The bacillus subtilis GRH-Bs007 has better detoxification effect on vomitoxin in animals and good beneficial effects.
The more detailed technical scheme is seen in the detailed description.
Drawings
FIG. 1 shows a graph of the growth of the strain of the invention.
Detailed Description
Example 1: screening of strains
(1) Detoxification preliminary screening
The probiotic bacterial strain is primarily screened by taking phenyl ethylene oxide as the only carbon element source. The isolated and stored probiotic bacterial strain is streaked and inoculated into a solid primary screening culture medium mixed with phenyl ethylene oxide, the phenyl ethylene oxide is ensured to be the only carbon element source, and the probiotic bacterial strain is placed in a 37 ℃ incubator for culturing for 1 week, and the growth condition of the bacterial strain is observed. In order to avoid the interference of carbon sources of bacteria in the original environment, single colonies which grow out are selected, pure culture is carried out for 3 times continuously, and finally, strains which can grow on a culture medium are preserved, and the growth conditions of the strains are recorded and analyzed. 8 dominant strains were obtained by screening. The strain numbers are GRH-Bs003, GRH-Bs007, GRH-Bs035, GRH-Bs041, GRH-Bs077, GRH-Bs134, GRH-Bs145, GRH-Bs202, respectively.
(2) Detoxication re-screening device
Re-screening the strain detoxification capacity by taking DON pure products as substrates, respectively picking the primary screened strains into 50mL of LB liquid medium, culturing for 24 hours at 37 ℃ and 200r/min, and taking the primary screened strains as seed liquid; inoculating the seed solution into 100mL of LB liquid medium with an inoculum size of 1%, and culturing at 37 ℃ under 200r/min for 24 hours to obtain fermentation liquor; taking 990 μl of fermentation broth of the primary strain, adding 10 μl of DON standard working solution (initial concentration 100 mg/L), reacting at 37deg.C under 200r/min for 24 hr, adding equivalent LB liquid culture medium and equivalent DON standard working solution as control group, and repeating for 3 times for each sample. And uniformly mixing the liquid to be detected, centrifuging for 5min under the condition of 10000r/min, taking the supernatant after centrifugation, filtering by a 0.22 mu m sterile filter, and then measuring the DON content. And (3) measuring the DON residual content in the liquid to be detected by adopting a vomitoxin (DON) enzyme-linked immunosorbent assay kit, and making a compound hole during detection. The results are shown in Table 1.
TABLE 1 DON pure detoxification and rescreening results
| Strain | GRH-Bs003 | GRH-Bs007 | GRH-Bs035 | GRH-Bs041 | GRH-Bs077 | GRH-Bs134 | GRH-Bs145 | GRH-Bs202 |
| Detoxification rate | 27.31％ | 84.45％ | 45.38％ | 55.72％ | 88.68％ | 33.32％ | 76.78％ | 44.61％ |
(3) Enzyme production test screening
Inoculating the primary screening strain into 50ml of LB liquid medium, culturing at 37 ℃ for 24 hours at 200r/min, and taking the strain as seed liquid. Centrifuging 5.00ml seed solution 12000r/min for 2min, discarding supernatant, and adjusting OD value of the precipitate to about 0.5 (about 5×10) with sterile water 7 cfu/ml), inoculating to enzyme-producing culture medium (tryptone 10g/L, yeast extract 5g/L, soluble starch 5g/L, KH) 2 PO 4 2g/L，MgSO 4 ·7H 2 O 0.5g/L，CaCl 2 ·2H 2 O0.2 g/L, water), culturing at 37deg.C for 24 hr/min, centrifuging to remove thallus to obtain supernatant, measuring amylase activity by Yoo improvement method, collecting 5mL of 0.5% soluble starch solution, preheating in water bath at 37deg.C for 10min, adding 0.5mL of properly diluted supernatant, shaking in water bath at 37deg.C, reacting for 5min, adding 5mL of 0.lmol/L H 2 S04, terminating the reaction. 0.5mL of the reaction solution was developed with 5mL of a dilute iodine solution, and the optical density value was measured at 620 nm. The enzyme activity was calculated using 0.5mL of water instead of 0.5mL of the reaction solution as a blank control and using no fermentation broth (the same volume of buffer was added) as a control tube, and the results are shown in Table 2.
The enzyme activity was calculated according to the following formula: enzyme activity (U) = (R) 0 -R)×50×D/R 0
Wherein: r is R 0 R represents the optical density value of the control and the reaction solution, and D represents the dilution factor of the enzyme. Adjust D to make (R) 0 -R)/R 0 0.2-0.7. The enzyme activity is defined as: the amount of enzyme hydrolyzing 1mg of starch at 37℃and pH6.0 in 5min was 1 activity unit.
TABLE 2 enzyme Activity measurement results
| Strain | GRH-Bs003 | GRH-Bs007 | GRH-Bs035 | GRH-Bs041 | GRH-Bs077 | GRH-Bs134 | GRH-Bs145 | GRH-Bs202 |
| Enzyme activity | 8.3U/mL | 64.6U/mL | 22.3U/mL | 18.7U/mL | 54.2U/mL | 15.9U/mL | 45.4U/mL | 23.7U/mL |
(4) Antibacterial test screening
Inoculating indicator bacteria (Salmonella, staphylococcus aureus, escherichia coli K88, K99, O139) into 5ml LB medium, shake culturing at 37deg.C for 12 hr, and diluting to 10 by turbidimetry 6 CFU/mL was ready for use. Primary screening of strainsAfter the activation culture, the bacterial solutions are respectively inoculated into 50mL EP pipes filled with LB according to the inoculum size of 2 percent, and are subjected to shaking culture at 37 ℃, are respectively taken out after being cultured for 24 hours and 48 hours, are centrifuged at 4 ℃ for 15 minutes at 8000r/min, and the bacterial bodies are removed to retain fermentation supernatant. The whole plate is uniformly coated with the indicator bacteria dipped with a cotton swab at least four times. 3 oxford cups were placed on each plate, slightly pressed, 200. Mu.l of the fermentation supernatant of the primary strain was added to the oxford cups, the mixture was placed at 4℃for 8 hours, the antibacterial effect was observed after 10 hours in a 37℃incubator, and the diameter of the antibacterial zone was measured with a ruler, and the results are shown in Table 3.
TABLE 3 in vitro bacteriostasis test of fermentation supernatant of Primary screening Strain
Conclusion: through comprehensive analysis of the screening results of detoxification preliminary screening, detoxification complex enzyme production test screening and bacteriostasis test screening, the strain GRH-Bs007 has the characteristics of high-efficiency removal of vomit toxin, high-efficiency bacteriostasis and enzyme production.
Example 2: identification of strains
(1) Morphological identification and physicochemical characteristic identification
The cell morphology and physicochemical characteristics of the GRH-Bs007 strain are shown in Table 4.
TABLE 4 cell morphology and physicochemical Properties of GRH-Bs007 Strain
| Identification index | Results | Identification index | Results |
| Colony color | White or yellowish | Acid production by glucose | + |
| Gram staining | Positive and negative | Xylose acid production | + |
| Cell morphology | Rod-shaped | Acid production by arabinose | + |
| Spore of spore | + | Mannitol acid production | + |
| Contact enzyme | + | By means of propionate | - |
| Hydrolyzed gelatin | + | Nitrate reduction | + |
| Indole formation | - | Hydrolyzed starch | + |
| Anaerobic growth | - | Growth of 7% sodium chloride | + |
Note that: "-" indicates negative; "+" indicates positive.
(2) Molecular characterization
The strain GRH-Bs007 was species identified by the strain 16SrRNA sequence. Primers used for PCR amplification of the bacillus 16SrRNA genome are F1 and R1 (sequences are shown below). The PCR amplification reaction procedure was: pre-denaturation at 95 ℃ for 10min,1 cycle; denaturation at 95℃for 1min, annealing at 56℃for 30s, extension at 72℃for 1.5min for 30 cycles; finally, the extension is carried out for 10min at 72 ℃. After amplification, the product was sent to Shanghai Biotechnology Co.Ltd for sequencing, and the 16S rRNA sequencing result of strain GRH-Bs007 was shown as SEQ ID No. 1.
F1：5’-AGAGTTTGATCCTGGCTCAG-3’
R1：5’-AAGGAGGTGATCCACCC-3’
As a result of Blastn comparison of the 16SrRNA sequence of GRH-Bs007 at NCBI, the search revealed that GRH-Bs007 had a high homology with the sequence of Bacillus subtilis in Genebank, and the similarity was 100% (https:// www.ncbi.nlm.nih.gov /), which proves that the strain GRH-Bs007 was Bacillus subtilis, and was formally designated as Bacillus subtilis (Bacillus subtilis) GRH-Bs007.
The bacillus subtilis 2021 is delivered to China Center for Type Culture Collection (CCTCC) in the university of Wuhan in Wuhan, hubei province for 8 months and has the preservation number of: cctccc NO: m20211008.
Example 3: determination of the biological Properties of Bacillus subtilis GRH-Bs007
(1) Growth curve determination
The bacillus subtilis GRH-Bs007 is selected and cultured in 50mL of LB liquid medium at 37 ℃ under the condition of 200r/min for 24 hours to be used as seed liquid, and the seed liquid is inoculated into 100mL of LB liquid medium with the inoculum size of 1 percent to be used as fermentation broth after being cultured at 37 ℃ under the condition of 200r/min for 24 hours. The fermentation broth is inoculated into 5mL of LB liquid culture medium with an inoculum size of 1%, the culture broth is taken out when 0h, 2h, 4h, 6h, 8h, 10h, 12h, 14h, 16h, 18h, 20h and 22h are carried out respectively, the culture broth is diluted by 10 times in gradient, the growth curve of each strain is measured by a pouring method, and three experimental groups are parallel. The growth curve of the strain was plotted with the incubation time on the abscissa and the log (CFU/mL) value on the ordinate. The growth curve results are shown in FIG. 1, GRH-Bs007 enter the logarithmic phase at about 2h, the logarithmic phase at about 4h, and then enter the stationary phase and the recession phase.
(2) High temperature tolerance test
GRH-Bs007 spore suspension was injected into a centrifuge tube, diluted 10-fold in gradient, and the residual viable count was measured by decanting. And then placing the centrifuge tubes filled with the residual spore suspension into water baths with the temperature of 75 ℃, the temperature of 80 ℃, the temperature of 85 ℃ and the temperature of 90 ℃ for heat treatment for 5min, taking the heat-treated bacterial liquid to perform stepwise 10-time gradient dilution, and measuring the residual viable count by a pouring method. Three replicates were run with equal amounts of spore suspension plus equal amounts of physiological saline as controls and GRH-Bs007 spore high temperature tolerance results are shown in Table 6.
TABLE 5 high temperature tolerance of Bacillus subtilis GRH-Bs007
| Treatment temperature | 75℃ | 80℃ | 85℃ | 90℃ |
| Survival rate | 100％ | 96.2±2.7％ | 92.6±3.2％ | 61.5±2.8％ |
(3) Test of bile salt tolerance
0.5mL of GRH-Bs007 spore suspension was added to 4.5mL of 0.3% simulated bile salt (pH 8.0) and mixed rapidly on a shaker and then placed in an incubator at 37℃for stationary culture. And taking out the culture solution at 0h and 24h respectively, immediately performing stepwise 10-time gradient dilution, and measuring the residual viable count by a pouring method. The same amount of spore suspension and the same amount of physiological saline are used as a control, and each group of experiments is repeated in three times. GRH-Bs007 spores had a survival rate of 91.6.+ -. 6.3% after 24 hours in mock bile salts.
(4) Gastric juice tolerance test
0.5mL of GRH-Bs007 spore suspension was added to 4.5mL of simulated gastric fluid (pH 2.0) and mixed rapidly on a shaker and then placed in an incubator at 37℃for stationary culture. And taking out the culture solution at 0h, 2h and 4h respectively, immediately performing stepwise 10-time gradient dilution, and measuring the residual viable count by a pouring method. The results of the high temperature tolerance of Bacillus subtilis GRH-Bs007 spores were shown in Table 7 in triplicate with an equal amount of spore suspension plus an equal amount of physiological saline as a control.
TABLE 6 Bacillus subtilis GRH-Bs007 spores to mimic gastric juice resistance
| Processing time | For 2 hours | 4 hours |
| Survival rate | 97.1±1.3％ | 93.2±4.6％ |
Conclusion: experiments show that the strain GRH-Bs007 has the advantages of high propagation speed and strong tolerance.
Example 4: application of bacillus subtilis GRH-Bs007 in animal raising
50 3-week-old BALB/c male mice were pre-fed for 3d and randomly divided into 5 groups of 2 replicates each, 5 replicates each per cage, and the grouping and testing protocol is shown in Table 7, with the feeding ambient temperature being 23.+ -. 2 ℃, 12h of illumination per day, free drinking and feeding. Each group of BALB/c mice was analyzed for productivity at test 14d and 28d, respectively, and the effect of GRH-Bs007 on detoxification and its probiotic effect in mice was analyzed.
TABLE 7 animal test grouping
| Group of | 1-14d | 15-28d |
| Control group | PBS | PBS |
| Probiotics group | GRH-Bs007 | GRH-Bs007 |
| Toxin eliminating group | DON | DON |
| Preventive group | GRH-Bs007 | GRH-Bs007+DON |
| Treatment group | DON | GRH-Bs007 |
Note that: N/A represents no treatment; all test groups were fed standard mouse feed with DON at a dose of 2 mg/kg.BW, GRH-Bs007 as a live bacterial suspension (10 8 CFU/mL, resuspended after PBS wash); the administration mode is oral gastric lavage (0.2 mL/dose); test animals were fasted overnight (free drinking water) following day 14d and 28d, respectively, after the start of challenge) and test samples were collected the next day.
The results of the growth performance of the BALB/c mice in each treatment group are shown in Table 8. On average daily gain, the probiotic group (fed with bacillus subtilis GRH-Bs 007) was significantly higher than the other groups (P < 0.05), while the challenge group was significantly lower than the other groups (P < 0.05); on average daily feeding, mice in each treatment group had different tendencies, but the differences were not significant (P > 0.05), indicating that Bacillus subtilis GRH-Bs007 had the effect of promoting growth of mice, and was able to alleviate the effect of DON poisoning on average daily gain in BALB/c mice.
TABLE 8 influence of Bacillus subtilis GRH-Bs007 on growth performance in mice
| Group of | Average daily gain (g) | Average daily feed (g) |
| Control group | 0.302±0.04 b | 3.31±0.02 |
| Probiotics group | 0.332±0.05 c | 3.55±0.02 |
| Toxin eliminating group | 0.197±0.03 a | 3.02±0.04 |
| Preventive group | 0.283±0.04 b | 3.21±0.03 |
| Treatment group | 0.276±0.03 b | 3.27±0.04 |
Note that: data in the tables are expressed as M+ -SD; between the same column of data, the non-shoulder-labeled person or shoulder-labeled person has the same letter indicating that the difference is not significant (P > 0.05), and the shoulder-labeled person has the same letter indicating that the difference is significant (P < 0.05).
Sequence listing
<110> screening and application of enzyme-producing antibacterial bacillus subtilis with vomitoxin detoxification function
Wuhan Guanya Biotechnology Co.Ltd
<120> screening and application of enzyme-producing antibacterial bacillus subtilis with vomitoxin detoxification function
<130> 2021-08-08
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1415
<212> DNA
<213> Bacillus subtilis (Bacillus subtilis)
<400> 1
tgcagtcgag cggacagatg ggagcttgct ccctgatgtt agcggcggac gggtgagtaa 60
cacgtgggta acctgcctgt aagactggga taactccggg aaaccggggc taataccgga 120
tggttgtttg aaccgcatgg ttcaaacata aaaggtggct tcggctacca cttacagatg 180
gacccgcggc gcattagcta gttggtgagg taacggctca ccaaggcaac gatgcgtagc 240
cgacctgaga gggtgatcgg ccacactggg actgagacac ggcccagact cctacgggag 300
gcagcagtag ggaatcttcc gcaatggacg aaagtctgac ggagcaacgc cgcgtgagtg 360
atgaaggttt tcggatcgta aagctctgtt gttagggaag aacaagtacc gttcgaatag 420
ggcggtacct tgacggtacc taaccagaaa gccacggcta actacgtgcc agcagccgcg 480
gtaatacgta ggtggcaagc gttgtccgga attattgggc gtaaagggct cgcaggcggt 540
ttcttaagtc tgatgtgaaa gcccccggct caaccgggga gggtcattgg aaactgggga 600
acttgagtgc agaagaggag agtggaattc cacgtgtagc ggtgaaatgc gtagagatgt 660
ggaggaacac cagtggcgaa ggcgactctc tggtctgtaa ctgacgctga ggagcgaaag 720
cgtggggagc gaacaggatt agataccctg gtagtccacg ccgtaaacga tgagtgctaa 780
gtgttagggg gtttccgccc cttagtgctg cagctaacgc attaagcact ccgcctgggg 840
agtacggtcg caagactgaa actcaaagga attgacgggg gcccgcacaa gcggtggagc 900
atgtggttta attcgaagca acgcgaagaa ccttaccagg tcttgacatc ctctgacaat 960
cctagagata ggacgtcccc ttcgggggca gagtgacagg tggtgcatgg ttgtcgtcag 1020
ctcgtgtcgt gagatgttgg gttaagtccc gcaacgagcg caacccttga tcttagttgc 1080
cagcattcag ttgggcactc taaggtgact gccggtgaca aaccggagga aggtggggat 1140
gacgtcaaat catcatgccc cttatgacct gggctacaca cgtgctacaa tggacagaac 1200
aaagggcagc gaaaccgcga ggttaagcca atcccacaaa tctgttctca gttcggatcg 1260
cagtctgcaa ctcgactgcg tgaagctgga atcgctagta atcgcggatc agcatgccgc 1320
ggtgaatacg ttcccgggcc ttgtacacac cgcccgtcac accacgagag tttgtaacac 1380
ccgaagtcgg tgaggtaacc ttttaggagc cagcc 1415
Claims (2)
1. Enzyme-producing antibacterial bacillus subtilis with vomitoxin detoxification functionBacillus subtilis) GRH-Bs007, characterized in that: the bacillus subtilis GRH-Bs007 is preserved in China center for type culture Collection (China) on 8 th and 10 th of 2021, and the proposed classification is named as bacillus subtilis GRH-Bs007, and the preservation number is CCTCC NO: m20211008.
2. Use of bacillus subtilis GRH-Bs007 of claim 1 for the removal of vomitoxin.
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