CN113234641A - Bacillus S77 capable of resisting low temperature and producing protease and application thereof - Google Patents
Bacillus S77 capable of resisting low temperature and producing protease and application thereof Download PDFInfo
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- 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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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
- C12N9/54—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea bacteria being Bacillus
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Abstract
The invention relates to the technical field of microorganisms, in particular to a low-temperature-resistant protease-producing bacillus S77 and application thereof. The bacillus of the invention S77, Latin name: bacillus S77, the name of the depository is: china center for type culture Collection, addresses: wuhan university, the preservation date is: 2021, 5 month and 13 days, with a deposit number: CCTCC NO: m2021528. The strain has good low temperature resistance, acid resistance, choline resistance, gastric juice resistance and intestinal juice resistance; has good adhesive capacity; the antibiotic is sensitive, and has no drug resistance, so that the safety of the antibiotic is ensured; also has the capability of producing protease and low temperature resistance, and is expected to be applied to the production of biological fermentation feed as probiotics.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to a low-temperature-resistant protease-producing bacillus S77 and application thereof.
Background
Probiotics, also known as prebiotics, are microbial agents that produce beneficial effects on animals. The probiotics is used as an additive of animal feed, and has the functions of promoting growth, resisting and treating diseases, improving feed digestibility and the like. Ideally the probiotic should have the following effects: (1) has resistance to acid and bile salt, and can maintain its activity when passing through stomach and small intestine, i.e. has acid resistance, bile salt resistance, intestinal juice resistance, and gastric juice resistance. (2) Can be attached to the mucous epithelial cells of the intestinal tract, and the generated mucus is beneficial to the colonization of the intestinal tract. I.e. has a certain adhesive capacity. (3) The antibiotic-resistant enteric coated film is sensitive to antibiotics and has no drug resistance, and the drug resistance gene level carried by the antibiotic-resistant enteric coated film cannot be transferred to pathogenic bacteria in intestinal tracts, so that the pathogenic bacteria in the intestinal tracts can obtain the drug resistance, and the safety of the pathogenic bacteria is ensured. However, the prior art does not find such a strain which has the above effects at the same time.
Disclosure of Invention
The invention aims to provide a strain which has the functions of acid resistance, cholate resistance, intestinal juice resistance and gastric juice resistance, and has the adhesive capacity and the drug resistance to meet the defects of the prior art. In particular to a bacillus S77 which is low temperature resistant and can produce protease and application thereof.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a bacillus strain S77, which has the Latin name: bacillus S77, the name of the depository is: china center for type culture Collection, addresses: wuhan university, the preservation date is: 2021, 5 month and 13 days, with a deposit number: CCTCC NO: m2021528.
Preferably, the sequence of the 16S rDNA of the bacillus S77 is shown in SEQ ID NO. 1.
The invention also provides application of the bacillus S77 in preparing low temperature resistant products
The invention also provides application of the bacillus S77 in preparation of acid-resistant products.
The invention also provides application of the bacillus S77 in preparation of choline-resistant products.
The invention also provides application of the bacillus S77 in preparing gastric juice resistant products.
The invention also provides application of the bacillus S77 in preparation of an intestinal juice resistant product.
The invention also provides application of the bacillus S77 in preparing products with adhesive properties.
The invention also provides application of the bacillus S77 in preparation of drug-resistant products.
The invention also provides application of the bacillus S77 in preparation of protease products.
The invention provides a bacillus S77 strain which is low temperature resistant and produces protease and application thereof, and the bacillus S77 of the invention has the following advantages:
(1) the bacillus S77 has good low temperature resistance, acid resistance, choline resistance, gastric juice resistance and intestinal juice resistance, and can keep the activity of the bacillus S77 when passing through the stomach and the small intestine;
(2) the bacillus S77 also has good adhesion capability, can be attached to mucosal epithelial cells of the intestinal tract, and produces mucus which is beneficial to the permanent planting of the bacillus S77 in the gastrointestinal tract;
(3) the bacillus S77 also has the characteristic of sensitivity to antibiotics and no drug resistance, and cannot transmit the level of a drug resistance gene carried by the bacillus S77 to pathogenic bacteria in intestinal tracts, so that the pathogenic bacteria in the intestinal tracts obtain the drug resistance, and the safety of the pathogenic bacteria is ensured;
(4) bacillus S77 of the present application also has the ability to produce protease;
(5) the bacillus S77 of the present application also has the ability to withstand low temperatures;
(6) the bacillus S77 is preferably used as a probiotic bacterium in the production of biological fermentation feed.
Drawings
FIG. 1 is a colony morphology map.
FIG. 2 is a graph showing the gram staining results of the strains.
FIG. 3 is a diagram showing the amplification of the 16S rDNA gene of the strain.
FIG. 4 is a phylogenetic tree of the strains.
FIG. 5 is a graph showing the results of the strain resistant to low temperature culture at 10 ℃ for 24 hours.
FIG. 6 is a histogram of strain acid tolerance.
FIG. 7 is a histogram of bacterial strains with bile salt tolerance.
FIG. 8 is a bar graph of the strain's gastric juice tolerance.
FIG. 9 is a histogram of intestinal juice resistance of the strain.
FIG. 10 is a graph showing the results of the sensitivity of the strains to antibiotics.
Deposit description
Bacillus S77, latin name: bacillus S77, the name of the depository is: china center for type culture Collection, addresses: wuhan university, the preservation date is: 2021, 5 month and 13 days, with a deposit number: CCTCC NO: m2021528.
Detailed Description
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1 isolation and identification of the Strain
Weighing 5g tomato stems from Manchu Mongolian autonomous county in the fence of Chengde City of Hebei province, adding into a test tube containing 50ml sterile water, and diluting to 10%-1The sample diluent is diluted to 10-2、10-3、10-4、10-5、10-6Next, 100. mu.l of each dilution was applied to LB solid medium and incubated at 37 ℃ for 48 hours. Selecting colony, continuously streaking, separating and purifying, and selecting single colony with colony morphologyAs shown in fig. 1. After the microscopic examination confirms that the seeds are pure, the seeds are inoculated into a slant culture medium to be cultured for 24 hours and then are stored in a refrigerator at 4 ℃ for later use.
Gram staining and physiological and biochemical identification are carried out on the strains identified as the pure strains, the gram staining result is shown in figure 2, and the physiological and biochemical result is shown in table 1.
TABLE 1S 77 results of physiological and biochemical identification
The DNA of the strain was extracted, 16S rDNA amplification was performed, and the amplified product was subjected to agarose gel electrophoresis, the results of which are shown in FIG. 3. And (3) sending the 16S rDNA obtained by amplification to Dalian Meilun biotechnology limited for sequencing, wherein the sequencing result is shown as SEQ ID NO. 1.
And then comparing the sequencing results, constructing a phylogenetic tree, and identifying the species relationship of the strains. The phylogenetic tree results are shown in FIG. 4.
The LB solid medium comprises the following components in concentration: peptone 1g/L, yeast powder 0.5g/L, sodium chloride 0.5g/L, agar 15 g/L.
The pH value of the LB solid medium is 7.3.
The colonies shown in FIG. 1 were milky white, 5mm in diameter, smooth in surface, irregular in edge, and slightly protruded in center.
The microscopic examination is in the shape of short rod, (1-1.3) mum multiplied by (1-1.6) mum, and the single, paired and short-chain arrangement is used for naming the pure strains as S77.
FIG. 2 shows that gram staining is positive and aerobic.
FIG. 4 shows that the S77 strain has 99.43% homology with Bacillus (Bacillus).
The strain isolated in this application was finally identified as bacillus sp.s 77.
EXAMPLE 2 Low temperature resistance test of Strain
Activating the strain to obtain a strain with a thallus concentration of 107cfu/ml bacterial suspension is respectively inoculated in 10ml LB liquid culture medium according to the inoculation amount of 1 percent, and the temperature is 10 ℃, the temperature is 15℃,After culturing at 25 deg.C, 30 deg.C, 37 deg.C and 44 deg.C for 24h, the absorbance values are measured at 600nm wavelength, and 3 parallel averages are made. As a result, the growth of the cells was shown in FIG. 5 after culturing at 10 ℃ for 24 hours, as shown in Table 2.
TABLE 2 Low temperature resistance test results of Bacillus
The results show that: the strain can grow at a low temperature of 10 ℃, the thallus grows well at a temperature of 15 ℃, the optimal temperature is 25 ℃, and the strain S77 is a low-temperature bacillus strain and can better play a role in a low-temperature environment.
EXAMPLE 3 Strain acid resistance experiment
Activating the strain to obtain a strain with a thallus concentration of 107The method comprises the steps of inoculating 4% of bacterial suspension into LB liquid culture media with pH values of 1, 2 and 3 respectively, carrying out static culture at 37 ℃, sucking the bacterial suspension from the LB liquid culture media for 0, 1, 2 and 3 hours respectively, carrying out gradient dilution, coating the bacterial suspension onto corresponding solid culture media, carrying out inverted culture in a constant-temperature incubator at 37 ℃ for 48 hours, selecting plates with the colony number of 30-300 for counting, and carrying out 3 parallel averaging. The acid tolerance of the resulting strain is shown in FIG. 6.
FIG. 6 shows that the acid resistance of the strain is good, the growth of the strain occurs under the condition of pH3.0, and the survival rate of the strain decreases with the increase of the culture time at pH2.0 and pH 1.0.
EXAMPLE 4 bile salt resistance test of the Strain
Activating the strain to obtain a strain with a thallus concentration of 108The bacterial suspension of cfu/ml is respectively inoculated into LB liquid culture medium with bile salt concentration of 0.3, 1.5 and 3g/L according to the inoculum size of 4 percent, is statically cultured at 37 ℃, bacterial suspension is respectively absorbed from the LB liquid culture medium for 0 and 3 hours, is subjected to gradient dilution, is coated on a corresponding solid culture medium, is inversely cultured for 48 hours in a constant-temperature incubator at 37 ℃, plates with the colony number of 30-300 are selected for counting, and 3 parallel averages are calculated. The tolerance ability of the strain to bile salts was calculated, and the tolerance results are shown in FIG. 7.
Said strainThe tolerance to bile salts is calculated by the formula: bile salt tolerance (%) ═ Nt/N0×100％。
In the formula, Nt: 3h live bacteria count (cfu/ml) of the culture medium containing the bile salt;
N0: 0h viable count (cfu/ml) of the medium containing bile salts.
FIG. 7 shows: the strain S77 has the thallus growth condition in low-concentration bile salt, the tolerance capability of the strain can reach 138.75%, the tolerance capability of the strain is slightly reduced along with the increase of the concentration of the bile salt, the tolerance capability of the strain is still 89.11% after 3 hours, and the strain has better bile salt tolerance capability.
EXAMPLE 5 Strain resistance to Artificial gastric juice
The activated strain is prepared into a strain with the concentration of 107The bacterial suspension of cfu/ml is inoculated into the artificial gastric juice according to the inoculation amount of 4 percent, the bacterial suspension is respectively absorbed from the artificial gastric juice in 0 hour, 2 hours and 4 hours, the bacterial suspension is diluted by 10 times of gradient and is coated on an LB solid culture medium, the inverted culture is carried out at 37 ℃, plates with the colony number of 30-300 are selected for counting, and 3 parallel averages are calculated. And calculating the tolerance of the strain to artificial gastric juice. The tolerability results are shown in FIG. 8.
The strain tolerance to artificial gastric juice is calculated by the following formula: artificial gastric juice tolerance (%) ═ Nt/N0×100％。
In the formula, Nt: viable count of culture medium (cfu/ml) at each time period;
N0: viable count (cfu/ml) of 0h medium.
The preparation method of the artificial gastric juice comprises the following steps: 16.4mL of 20% hydrochloric acid was added and the pH was adjusted to 3.0. Adding pepsin (granules) to make its concentration be 0.01g/mL, fully dissolving them, filtering and sterilizing by using microporous membrane with 0.22 micrometer specification so as to obtain the invented product.
FIG. 8 shows: the survival rate of the strain is reduced to a certain extent along with the increase of the culture time, and is 71.16% at 4h, which shows that the strain has good gastric juice resistance.
EXAMPLE 6 Strain Artificial intestinal juice resistance test
The activated strain is prepared into a strain with the concentration of 107cfu/ml bacterial suspension, 4% inoculation amountInoculating the strain into artificial intestinal juice, respectively sucking bacterial suspensions from the artificial intestinal juice for 0 hour, 4 hours and 8 hours, performing gradient dilution by 10 times, coating the diluted bacterial suspensions on an LB solid culture medium, performing inverted culture at 37 ℃, selecting a flat plate with the colony number of 30-300 for counting, performing 3 parallel averaging, and calculating the tolerance of the strain to the artificial intestinal juice. The tolerability results are shown in FIG. 9.
The tolerance of the strain to the artificial intestinal juice is calculated by the following formula: artificial intestinal juice tolerance (%) ═ Nt/N0×100％。
In the formula, Nt: viable count of culture medium (cfu/ml) at each time period;
N0: viable count (cfu/ml) of 0h medium.
The preparation method of the artificial intestinal juice comprises the following steps: adding trypsin (granule) to concentration of 0.01g/mL, and filtering with 0.22 μm microporous membrane for sterilization after trypsin is dissolved sufficiently.
FIG. 9 shows: the strain has a thallus growth phenomenon in the artificial intestinal juice, the survival rate is reduced and then increased along with the prolonging of the culture time, but the survival rate is kept over 90 percent all the time, and at 8h, the survival rate can reach 93.34 percent, which indicates that the strain has excellent intestinal juice resistance.
Example 7 Strain adhesion Performance test
The hydrocarbon Method (MATH) is used. Respectively taking 2mL of activated thallus with the concentration of 107centrifuging cfu/ml bacterial suspension at 8000r/min for 10min, discarding supernatant, washing thallus with PBS buffer solution for 3 times, re-suspending the thallus, and adjusting OD600 of the bacterial suspension to 0.8. Adding 4mL of the bacterial suspension and 1mL of n-hexane into a test tube, adding no n-hexane into a control group, sealing, violently oscillating for 60s by using a vortex oscillator, and standing for 15min until liquid is layered. Absorbing 3.0mL of lower phase aqueous solution, using PBS buffer solution to perform zero adjustment correction on a spectrophotometer, measuring the light absorption value at the wavelength of 600nm, and calculating the surface hydrophobicity (CSH) of the bacterial cell according to the formula:
in the formula: a. the0The OD value of the control group is obtained; a. theSThe OD value of the experimental group is shown.
The results are shown in Table 3. Table 3 shows that S77 has good surface aggregation property and can better adhere to epithelial cells of intestinal tract and the surface of mucous membrane, thereby exerting the probiotic effect.
TABLE 3 surface hydrophobicity and surface agglutination of Bacillus
| Strain (strain) | Hydrophobic (hydrophobic)% | Aggregation (clustering)% |
| S77 | 60.89±1.01 | 72.27±0.52 |
EXAMPLE 9 antibiotic resistance test of strains
The antibiotic resistance of the strains was evaluated by a drug sensitive test using K-B (drug sensitive paper agar diffusion). Taking the thallus concentration as 108A100. mu.L cfu/ml seed solution of Bacillus sp 77 was inoculated into LB solid medium at 40 ℃ and mixed well and poured into a dish. After the culture medium is cooled and solidified, the drug sensitive paper sheets are evenly and flatly pasted on the surface of the LB solid culture medium. The flat plates with the drug sensitive paper sheets stuck thereon are respectively placed in an incubator at 37 ℃ for culturing for 48h, and the diameter (D) of the inhibition zone of each drug sensitive paper sheet is measured by a vernier caliper. The results of drug resistance are shown in FIG. 10 and Table 4.
The content of each antibiotic in the drug sensitive paper sheet is as follows: amikacin (30 μ g), CTR (30 μ g), TE tetracycline (30 μ g), CFP cefoperazone (75 μ g), DX doxycycline (30 μ g), N neomycin (30 μ g), P penicillin (10 μ g), GM gentamicin (10 μ g), CZ cefotaxime (30 μ g), milbemycin (30 μ g), ofloxacin (5 μ g), E erythromycin (15 μ g).
TABLE 4 Bacillus resistance results
In the table, S represents sensitivity, I represents mediator, and R represents drug resistance.
The results show that: the strain S77 shows drug resistance to common antibiotic penicillin, relative drug resistance to gentamicin, and sensitivity to other antibiotics, wherein the sensitivity rate, the drug resistance rate and the intermediate rate are respectively 83.33%, 8.33% and 8.33%. Therefore, the strain is safe to be added into animal feed as probiotics, the strain S77 has no drug resistance gene, and the level of the drug resistance gene carried by the strain S77 cannot be transferred to pathogenic bacteria in intestinal tracts, so that the pathogenic bacteria acquire drug resistance.
EXAMPLE 10 protease Activity assay in Strain
The strain is selected from S77 strain slant and inoculated in 10ml LB broth culture medium for 24h as strain liquid. The strain liquid is inoculated on a casein selective medium and cultured for 72h at 37 ℃, and finally the diameter D (mm) of a hydrolysis ring on a flat plate is measured, and each group is processed for 3 times. The bacterial fluid enzyme activity was determined with reference to SB/T10317-1999 "protease activity assay". The results are shown in Table 5.
TABLE 5 determination of protease Activity by Strain S77
| Strain name | Diameter of hydrolysis ring (mm) | Protease activity (u/g) |
| S77 | 24.2 | 767.27 |
The embodiment shows that the bacillus S77 with low temperature resistance and protease production and the application thereof are provided, and the bacillus S77 has the following advantages:
(1) the bacillus S77 has good low temperature resistance, acid resistance, choline resistance, gastric juice resistance and intestinal juice resistance;
(2) the bacillus S77 also has good adhesion capability, and facilitates the colonization of the bacillus S77 in the gastrointestinal tract;
(3) the bacillus S77 also has the characteristic of sensitivity to antibiotics, has no drug resistance, and ensures the safety of the bacillus S77;
(4) bacillus S77 of the present application also has the ability to produce protease;
(5) the bacillus S77 of the present application also has the ability to withstand low temperatures;
(6) the bacillus S77 is preferably used as a probiotic bacterium in the production of biological fermentation feed.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
<110> northwest national university
<120> bacillus S77 with low temperature resistance and protease production and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1400
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
ggattgagaa gcttgctctc aagaagttag cggcggacgg gtgagtaaca cgtgggtaac 60
ctgcccataa gactgggata actccgggaa accggggcta ataccggata acattttgaa 120
ctgcatggtt cgaaattgaa aggcggcttc ggctgtcact tatggatgga cccgcgtcgc 180
attagctagt tggtgaggta acggctcacc aaggcaacga tgcgtagccg acctgagagg 240
gtgatcggcc acactgggac tgagacacgg cccagactac tacgggaggc agcagtaggg 300
aatcttccgc aatggacgaa agtctgacgg agcaacgccg cgtgagtgat gaaggctttc 360
gggtcgtaaa actctgttgt tagggaagaa caagtgctag ttgaataagc tggcaccttg 420
acggtaccta accagaaagc cacggctaac tacgtgccag cagccgcggt aatacgtagg 480
tggcaagcgt tatccggaat tattgggcgt aaagcgcgcg caggtggttt cttaagtctg 540
atgtgaaagc ccacggctca accgtggagg gtcattggaa actgggagac ttgagtgcag 600
aagaggaaag tggaattcca tgtgtagcgg tgaaatgcgt agagatatgg aggaacacca 660
gtggcgaagg cgactttctg gtctgtaact gacactgagg cgcgaaagcg tggggagcaa 720
acaggattag ataccctggt agtccacgcc gtaaacgatg agtgctaagt gttagagggt 780
ttccgccctt tagtgctgaa gttaacgcat taagcactcc gcctggggag tacggccgca 840
aggctgaaac tcaaaggaat tgacgggggc ccgcacaagc ggtggagcat gtggtttaat 900
tcgaagcaac gcgaagaacc ttaccaggtc ttgacatcct ctgaaaaccc tagagatagg 960
gcttctcctt cgggagcaga gtgacaggtg gtgcatggtt gtcgtcagct cgtgtcgtga 1020
gatgttgggt taagtcccgc aacgagcgca acccttgatc ttagttgcca tcattaagtt 1080
gggcactcta aggtgactgc cggtgacaaa ccggaggaag gtggggatga cgtcaaatca 1140
tcatgcccct tatgacctgg gctacacacg tgctacaatg gacggtacaa agagctgcaa 1200
gaccgcgagg tggagctaat ctcataaaac cgttctcagt tcggattgta ggctgcaact 1260
cgcctacatg aagctggaat cgctagtaat cgcggatcag catgccgcgg tgaatacgtt 1320
cccgggcctt gtacacaccg cccgtcacac cacgaggaag tttgtaaaca cccggagagt 1380
cggtggggta aacctttatg 1400
Claims (10)
1. A bacillus strain S77, characterized in that the Latin name is: bacillus S77, the name of the depository is: china center for type culture Collection, addresses: wuhan university, the preservation date is: 2021, 5 month and 13 days, with a deposit number: CCTCC NO: m2021528.
2. The Bacillus S77, wherein the sequence of the 16S rDNA of Bacillus S77 is shown in SEQ ID NO. 1.
3. Use of the bacillus sp.s 77 of claim 1 or 2 for the preparation of a low temperature resistant product.
4. Use of the bacillus sp.s 77 of claim 1 or 2 for the preparation of acid resistant products.
5. Use of the bacillus sp.s 77 of claim 1 or 2 for the preparation of a choline-resistant product.
6. Use of bacillus sp.s 77 according to claim 1 or 2 for the preparation of a gastric juice resistant product.
7. Use of the bacillus sp 77 of claim 1 or 2 for the preparation of an intestinal juice resistant product.
8. Use of the bacillus sp 77 according to claim 1 or 2 for the preparation of a product with adhesive properties.
9. Use of the bacillus sp 77 of claim 1 or 2 for the preparation of a drug-free product.
10. Use of the bacillus sp 77 of claim 1 or 2 for the preparation of a protease product.
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