CN113717895B - Antibacterial bacillus and liquid fermentation step and antibacterial test method thereof - Google Patents
Antibacterial bacillus and liquid fermentation step and antibacterial test method thereof Download PDFInfo
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- 241000193830 Bacillus <bacterium> Species 0.000 title claims abstract description 47
- 239000007788 liquid Substances 0.000 title claims abstract description 47
- 238000000855 fermentation Methods 0.000 title claims abstract description 45
- 230000004151 fermentation Effects 0.000 title claims abstract description 45
- 230000000844 anti-bacterial effect Effects 0.000 title claims abstract description 25
- 238000010998 test method Methods 0.000 title claims abstract description 8
- 230000003385 bacteriostatic effect Effects 0.000 claims abstract description 38
- 238000004321 preservation Methods 0.000 claims abstract description 24
- 241000607142 Salmonella Species 0.000 claims abstract description 7
- 238000009629 microbiological culture Methods 0.000 claims abstract description 7
- 238000001694 spray drying Methods 0.000 claims description 20
- 241000894006 Bacteria Species 0.000 claims description 19
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 18
- 238000012258 culturing Methods 0.000 claims description 18
- 239000001963 growth medium Substances 0.000 claims description 18
- 239000000523 sample Substances 0.000 claims description 18
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 18
- 230000001580 bacterial effect Effects 0.000 claims description 15
- 239000002609 medium Substances 0.000 claims description 15
- 229920001817 Agar Polymers 0.000 claims description 14
- 239000008272 agar Substances 0.000 claims description 14
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 claims description 12
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 12
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 12
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 12
- 239000008223 sterile water Substances 0.000 claims description 12
- 238000003756 stirring Methods 0.000 claims description 12
- 238000000034 method Methods 0.000 claims description 10
- 239000007787 solid Substances 0.000 claims description 9
- 239000000243 solution Substances 0.000 claims description 9
- 238000002360 preparation method Methods 0.000 claims description 8
- 229920002261 Corn starch Polymers 0.000 claims description 6
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 6
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 6
- 235000019764 Soybean Meal Nutrition 0.000 claims description 6
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 6
- 229910000019 calcium carbonate Inorganic materials 0.000 claims description 6
- 230000001276 controlling effect Effects 0.000 claims description 6
- 238000001816 cooling Methods 0.000 claims description 6
- 239000008120 corn starch Substances 0.000 claims description 6
- 239000012470 diluted sample Substances 0.000 claims description 6
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 6
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 6
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 6
- 229940099596 manganese sulfate Drugs 0.000 claims description 6
- 239000011702 manganese sulphate Substances 0.000 claims description 6
- 235000007079 manganese sulphate Nutrition 0.000 claims description 6
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 claims description 6
- 239000001301 oxygen Substances 0.000 claims description 6
- 229910052760 oxygen Inorganic materials 0.000 claims description 6
- 239000000843 powder Substances 0.000 claims description 6
- 239000002994 raw material Substances 0.000 claims description 6
- 239000011780 sodium chloride Substances 0.000 claims description 6
- 239000004455 soybean meal Substances 0.000 claims description 6
- 230000001954 sterilising effect Effects 0.000 claims description 6
- 201000010099 disease Diseases 0.000 claims description 5
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 5
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 claims description 3
- 239000012880 LB liquid culture medium Substances 0.000 claims description 3
- 241001052560 Thallis Species 0.000 claims description 3
- 230000003213 activating effect Effects 0.000 claims description 3
- 230000004913 activation Effects 0.000 claims description 3
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 3
- 235000011114 ammonium hydroxide Nutrition 0.000 claims description 3
- 235000010216 calcium carbonate Nutrition 0.000 claims description 3
- 239000011248 coating agent Substances 0.000 claims description 3
- 238000000576 coating method Methods 0.000 claims description 3
- 239000000498 cooling water Substances 0.000 claims description 3
- 238000007865 diluting Methods 0.000 claims description 3
- 238000010790 dilution Methods 0.000 claims description 3
- 239000012895 dilution Substances 0.000 claims description 3
- 230000000694 effects Effects 0.000 claims description 3
- 238000005187 foaming Methods 0.000 claims description 3
- 238000011068 loading method Methods 0.000 claims description 3
- 238000002156 mixing Methods 0.000 claims description 3
- 230000001105 regulatory effect Effects 0.000 claims description 3
- 238000004659 sterilization and disinfection Methods 0.000 claims description 3
- 239000011550 stock solution Substances 0.000 claims description 3
- 238000009423 ventilation Methods 0.000 claims description 3
- 238000005303 weighing Methods 0.000 claims description 3
- 238000003745 diagnosis Methods 0.000 claims 4
- 230000002401 inhibitory effect Effects 0.000 claims 4
- 241000193403 Clostridium Species 0.000 claims 1
- 241000588722 Escherichia Species 0.000 claims 1
- 241000191940 Staphylococcus Species 0.000 claims 1
- 241000588724 Escherichia coli Species 0.000 abstract description 6
- 244000052616 bacterial pathogen Species 0.000 abstract description 6
- 230000009286 beneficial effect Effects 0.000 abstract description 2
- 239000003795 chemical substances by application Substances 0.000 abstract 2
- 239000002253 acid Substances 0.000 abstract 1
- 239000003833 bile salt Substances 0.000 abstract 1
- 230000007774 longterm Effects 0.000 abstract 1
- 230000000813 microbial effect Effects 0.000 abstract 1
- 239000003242 anti bacterial agent Substances 0.000 description 12
- 229940088710 antibiotic agent Drugs 0.000 description 12
- 244000144972 livestock Species 0.000 description 4
- 241000193468 Clostridium perfringens Species 0.000 description 3
- 206010059866 Drug resistance Diseases 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 3
- 241000191967 Staphylococcus aureus Species 0.000 description 3
- 241000282414 Homo sapiens Species 0.000 description 2
- 239000012620 biological material Substances 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 239000007952 growth promoter Substances 0.000 description 2
- 238000009776 industrial production Methods 0.000 description 2
- 239000007921 spray Substances 0.000 description 2
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
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- 230000007547 defect Effects 0.000 description 1
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- 230000029087 digestion Effects 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 230000007413 intestinal health Effects 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 244000144977 poultry Species 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 230000003595 spectral effect Effects 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
Classifications
-
- 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
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/18—Testing for antimicrobial activity of a material
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention relates to a bacteriostatic bacillus and a liquid fermentation step and a bacteriostatic test method thereof; the antibacterial bacillus deposit place is China general microbiological culture Collection center (China Committee); the preservation time is 2021, 4 and 6; the preservation registration number is CGMCC No.22124. The invention has the advantages that: the microbial agent still has remarkable antibacterial performance on pathogenic bacteria such as escherichia coli, salmonella and the like after liquid fermentation, has high long-term storage stability, is resistant to acid and bile salt, can be produced in industrial scale, and is more beneficial to the replacement of microecological agent products.
Description
Technical Field
The invention belongs to the technical field of newly preserved strain types; in particular to a liquid fermentation step and a bacteriostasis test method of the strain.
Background
Antibiotics are one of the biggest findings in the 20 th century, and penicillin alone has saved about 2 hundred million lives in the last century. Antibiotics bring great economic benefits to the breeding industry while bringing benefit to human beings. 1951. Over the years, over the counter antibiotics were added to feed with the first approval by the U.S. Food and Drug Administration (FDA). Thus, low doses of antibiotics are widely used in the livestock industry as growth promoters (Antibiotics as Growth Promoter, AGP) to be added to conventional feeds worldwide. It also witnessed the rapid development of the international animal husbandry. Over 60 antibiotics have been used in the livestock industry in succession for nearly 70 years. However, due to the wide use of antibiotics in livestock and poultry feeds, particularly the overproof use, bacteria are provided with enough opportunities to evolve the drug resistance against various drugs, thereby leading to the occurrence of drug resistance of various pathogenic bacteria, which directly threatens the livestock and human health. Along with 1986, the addition of antibiotics in the feed is completely prohibited in Sweden, countries such as abroad begin to continuously prohibit the antibiotics, along with the promulgation of No. 194 of the agricultural rural department in 2019, china begins to completely prohibit the antibiotics in No. 1 of 7 months in 2020, and the microecological preparation promotes the intestinal health of animals by changing the balance of intestinal flora, improves the immunity and the digestion and absorption rate of the animals, can generate bacteriostatic metabolites, has strong bacteriostatic effect on various pathogenic bacteria, does not generate drug resistance, and is one of the primary means for replacing antibiotics.
Disclosure of Invention
The invention aims to solve the defects, and provides bacteriostatic bacillus and a liquid fermentation step and a bacteriostatic test method thereof. The bacillus strain is obtained through screening, and the liquid fermentation shows that the bacillus strain has a spectral antibacterial effect on common pathogenic bacteria such as escherichia coli and salmonella, has a remarkable antibacterial effect, and provides a new possibility for replacing antibiotics with microecological preparations.
The invention is realized by adopting the following technical scheme.
The bacteriostatic bacillus is bacillus licheniformisBacillus licheniformisThe method comprises the steps of carrying out a first treatment on the surface of the The preservation place is China general microbiological culture Collection center; the preservation time is 2021, 4 and 6; the preservation registration number is CGMCC No.22124.
The antibacterial bacillus reference biological material is OKX101.
The liquid fermentation step of the bacteriostatic bacillus provided by the invention comprises the following steps:
step 1) activating strains; step 2) preparing first-stage seeds; step 3) preparing a secondary seed tank; step 4) preparing a liquid fermentation medium; step 5) liquid fermentation; step 6) spray drying.
The liquid fermentation step of the invention comprises the following steps,
step 1) activation of the seed
Inoculating glycerol tube of bacteriostatic bacillus with preservation number of CGMCC No.22124 in glycerol tube onto LB solid plate by streaking, culturing at 37deg.C for 18-24 hr, inoculating single colony into LB liquid shake flask, shake culturing at 37deg.C for about 200r/min for 12-16 hr, diluting and coating on LB plate, and culturing at 37deg.C for 12-18 hr to obtain seed plate;
step 2) preparation of first seed
Picking single bacterial colony from the seed flat plate by using an inoculating loop, inoculating the single bacterial colony into a triangular shake flask filled with LB culture medium, and standing and culturing for 12-18h at 30-40 ℃ to serve as a primary seed shake flask;
step 3) preparation of a secondary seed tank
Inoculating 1-10% of the strain into a seed tank filled with LB liquid culture medium, wherein the liquid loading amount is 20-40L, the fermentation temperature is 30-40 ℃, the stirring rotation speed is 150-250 rpm, and culturing for 12-18h to obtain a secondary seed tank;
step 4) liquid fermentation Medium configuration
The culture medium comprises the following components: corn starch, yeast powder, soybean meal, sodium chloride, dipotassium hydrogen phosphate, magnesium sulfate, manganese sulfate and calcium carbonate, wherein the raw materials are 800-1000 parts of water in L by kg; stirring to dissolve the culture medium completely, adjusting pH to 6.5-7.5 with NaOH or ammonia water, sterilizing at 105-121deg.C for 15-30min, and cooling with cooling water to 30-40deg.C to obtain fermentation culture medium;
step 5) liquid fermentation
Inoculating seeds of a seed tank into a sterilized fermentation medium according to the proportion of 1-10%, uniformly stirring, controlling the fermentation temperature to be 30-40 ℃, culturing for 12-24h, controlling the dissolved oxygen to be 10-50% by stirring and ventilation, regulating the pH to be 6.0-7.5 by NaOH or phosphoric acid, stopping fermentation when the dissolved oxygen is continuously risen for 2h, and cooling to wait for spray drying;
step 6) spray drying
The fermentation liquor of OKX101 is spray dried in a spray drying tower, the air inlet temperature of the spray drying tower is 130-170 ℃, the air outlet temperature of the spray drying tower is 60-90 ℃, and the liquid inlet speed of the fermentation liquor is 30-60L/h. Under the spray drying condition, OKX101 can be obtained, the number of living bacteria of the product can be kept, the antibacterial performance of the product can also be kept, and the product obtained by the method has good stability and is suitable for large-scale industrial production.
The culture medium comprises the following components in parts by weight: 10-40 parts of corn starch, 2-5 parts of yeast powder, 10-50 parts of soybean meal, 1-5 parts of sodium chloride, 0-5 parts of dipotassium hydrogen phosphate, 1-5 parts of magnesium sulfate, 0-0.5 part of manganese sulfate, 10-20 parts of calcium carbonate and 800-1000 parts of water in L when the raw materials are calculated in kg.
The antibacterial test method of the antibacterial bacillus disclosed by the invention comprises the following steps:
step 1) sample pretreatment: firstly preparing a product stock solution, weighing 5g of sample, fixing the sample to 50ml of sterile water to prepare a 10-fold diluted sample solution, vibrating on a shaking table for 15 minutes, uniformly mixing, standing for a plurality of minutes, and then adopting sterile water for gradient dilution to prepare 100-fold, 200-fold, 500-fold, 1000-fold, 2000-fold, 4000-fold and 8000-fold diluted sample solutions for later use;
step 2) pouring 10 mL autoclaved plain agar (1.5% agar) into each sterilization culture dish in an ultra-clean workbench, paving the bottom, enabling the thickness of each culture dish to be uniform, and placing the sterilized oxford cup on the agar after the plain agar is cooled and solidified; 3-5 oxford cups are placed on each plate equidistantly;
step 3) absorbing 10 mu L of indicator bacteria liquid, adding the indicator bacteria liquid into 100 mL LB solid medium with constant temperature of 50+/-5 ℃, gently shaking the indicator bacteria liquid uniformly to avoid foaming, wherein the content of bacteria in the medium is 10 5 -10 6 CFU/ml；
Step 4) pouring the LB solid culture medium added with the bacterial liquid in the step 3 into a plain agar culture dish provided with oxford cups, pouring 15-20mL of each dish, and uniformly paving the layer until the layer is cooled and solidified;
step 5) taking out the oxford cup by using tweezers, sucking 150-200 mu L of sample to be detected, injecting the sample into a corresponding oxford cup hole, setting sterile water as a blank control, marking a culture dish, and placing the culture dish in a refrigerator at 2-8 ℃ for 2-4 hours to diffuse the sample;
step 6) the petri dishes are moved into a 37 ℃ incubator for cultivation. In the following 6-12 h, the growth condition and the antibacterial effect of the thalli are observed, and the antibacterial zone can be observed generally by 8-12 h, but different indicator bacteria and different plate paving operations at each time have differences and are observed in time;
and 7) observing the diameter of a bacteriostasis ring in the flat plate, and simultaneously judging the bacteriostasis effect by using sterile water as a contrast.
The bacteriostatic bacillus is used for bacteriostasis of escherichia coli.
The bacteriostatic bacillus disclosed by the invention is used for bacteriostasis of salmonella.
The bacteriostatic bacillus provided by the invention is used for bacteriostasis of staphylococcus aureus.
The bacteriostatic bacillus is used for bacteriostasis of clostridium perfringens.
The invention has the beneficial effects that the bacterial strain obtained by the invention has obvious antibacterial effect on common pathogenic bacteria such as escherichia coli, salmonella and the like through antibacterial evaluation, so that the bacterial strain can be used for treating various diseases caused by pathogenic bacteria.
The invention is further explained below with reference to the drawings and the detailed description.
Drawings
FIG. 1 is a graph showing the antibacterial effect of the present invention on Escherichia coli.
FIG. 2 is a graph showing the bacteriostatic effect of the invention on Salmonella salmeteriana.
FIG. 3 is a graph showing the bacteriostatic effect of the invention on Staphylococcus aureus.
Figure 4 is a graph showing the bacteriostatic effect of the invention on clostridium perfringens.
Detailed Description
The bacteriostatic bacillus is bacillus licheniformisBacillus licheniformisThe method comprises the steps of carrying out a first treatment on the surface of the The preservation place is China general microbiological culture Collection center; the preservation time is 2021, 4 and 6; the preservation registration number is CGMCC No.22124.
The antibacterial bacillus reference biological material is OKX101.
The liquid fermentation step of the bacteriostatic bacillus provided by the invention comprises the following steps:
step 1) activating strains; step 2) preparing first-stage seeds; step 3) preparing a secondary seed tank; step 4) preparing a liquid fermentation medium; step 5) liquid fermentation; step 6) spray drying.
The liquid fermentation step of the invention comprises the following steps,
step 1) activation of the seed
Inoculating bacteriostatic bacillus with the preservation number of GCMCC No.22124 in an glycerol pipe onto a flat plate of LB solid by streaking, inversely culturing for 18-24 hours at 37 ℃, then picking single bacterial colony, inoculating into a LB liquid shake flask, shake culturing for about 12-16 hours at 37 ℃ and 200r/min, diluting and coating the bacterial colony on the LB flat plate, inversely culturing for 12-18 hours at 37 ℃ to obtain a seed flat plate;
step 2) preparation of first seed
Picking single bacterial colony from the seed flat plate by using an inoculating loop, inoculating the single bacterial colony into a triangular shake flask filled with LB culture medium, and standing and culturing for 12-18h at 30-40 ℃ to serve as a primary seed shake flask;
step 3) preparation of a secondary seed tank
Inoculating 1-10% of the strain into a seed tank filled with LB liquid culture medium, wherein the liquid loading amount is 20-40L, the fermentation temperature is 30-40 ℃, the stirring rotation speed is 150-250 rpm, and culturing for 12-18h to obtain a secondary seed tank;
step 4) liquid fermentation Medium configuration
The culture medium comprises the following components: corn starch, yeast powder, soybean meal, sodium chloride, dipotassium hydrogen phosphate, magnesium sulfate, manganese sulfate and calcium carbonate, wherein the raw materials are 800-1000 parts of water in L by kg; stirring to dissolve the culture medium completely, adjusting pH to 6.5-7.5 with NaOH or ammonia water, sterilizing at 105-121deg.C for 15-30min, and cooling with cooling water to 30-40deg.C to obtain fermentation culture medium;
step 5) liquid fermentation
Inoculating seeds of a seed tank into a sterilized fermentation medium according to the proportion of 1-10%, uniformly stirring, controlling the fermentation temperature to be 30-40 ℃, culturing for 12-24h, controlling the dissolved oxygen to be 10-50% by stirring and ventilation, regulating the pH to be 6.0-7.5 by NaOH or phosphoric acid, stopping fermentation when the dissolved oxygen is continuously risen for 2h, and cooling to wait for spray drying;
step 6) spray drying
The fermentation liquor of OKX101 is spray dried in a spray drying tower, the air inlet temperature of the spray drying tower is 130-170 ℃, the air outlet temperature of the spray drying tower is 60-90 ℃, and the liquid inlet speed of the fermentation liquor is 30-60L/h. Under the spray drying condition, OKX101 can be obtained, the number of living bacteria of the product can be kept, the antibacterial performance of the product can also be kept, and the product obtained by the method has good stability and is suitable for large-scale industrial production.
The culture medium comprises the following components in parts by weight: 10-40 parts of corn starch, 2-5 parts of yeast powder, 10-50 parts of soybean meal, 1-5 parts of sodium chloride, 0-5 parts of dipotassium hydrogen phosphate, 1-5 parts of magnesium sulfate, 0-0.5 part of manganese sulfate, 10-20 parts of calcium carbonate and 800-1000 parts of water in L when the raw materials are calculated in kg.
The antibacterial test method of the antibacterial bacillus disclosed by the invention comprises the following steps:
step 1) sample pretreatment: firstly preparing a product stock solution, weighing 5g of sample, fixing the sample to 50ml of sterile water to prepare a 10-fold diluted sample solution, vibrating on a shaking table for 15 minutes, uniformly mixing, standing for a plurality of minutes, and then adopting sterile water for gradient dilution to prepare 100-fold, 200-fold, 500-fold, 1000-fold, 2000-fold, 4000-fold and 8000-fold diluted sample solutions for later use;
step 2) pouring 10 mL autoclaved plain agar (1.5% agar) into each sterilization culture dish in an ultra-clean workbench, paving the bottom, enabling the thickness of each culture dish to be uniform, and placing the sterilized oxford cup on the agar after the plain agar is cooled and solidified; 3-5 oxford cups are placed on each plate equidistantly;
step 3) absorbing 10 mu L of indicator bacteria liquid, adding the indicator bacteria liquid into 100 mL LB solid medium with constant temperature of 50+/-5 ℃, gently shaking the indicator bacteria liquid uniformly to avoid foaming, wherein the content of bacteria in the medium is 10 5 -10 6 CFU/ml；
Step 4) pouring the LB solid culture medium added with the bacterial liquid in the step 3 into a plain agar culture dish provided with oxford cups, pouring 15-20mL of each dish, and uniformly paving the layer until the layer is cooled and solidified;
step 5) taking out the oxford cup by using tweezers, sucking 150-200 mu L of sample to be detected, injecting the sample into a corresponding oxford cup hole, setting sterile water as a blank control, marking a culture dish, and placing the culture dish in a refrigerator at 2-8 ℃ for 2-4 hours to diffuse the sample;
step 6) the petri dishes are moved into a 37 ℃ incubator for cultivation. In the following 6-12 h, the growth condition and the antibacterial effect of the thalli are observed, and the antibacterial zone can be observed generally by 8-12 h, but different indicator bacteria and different plate paving operations at each time have differences and are observed in time;
and 7) observing the diameter of a bacteriostasis ring in the flat plate, and simultaneously judging the bacteriostasis effect by using sterile water as a contrast.
As shown in FIG. 1, the bacteriostatic bacillus is used for bacteriostasis of escherichia coli.
As shown in figure 2, the bacteriostatic bacillus is used for bacteriostasis of salmonella.
As shown in figure 3, the bacteriostatic bacillus is used for bacteriostasis of staphylococcus aureus.
As shown in FIG. 4, the bacteriostatic bacillus is used for bacteriostasis of clostridium perfringens.
The foregoing is only a portion of specific embodiments of the present invention (since the technical solutions of the present invention relate to numerical ranges, the embodiments are not exhaustive, and the numerical ranges and other technical gist ranges of the present invention are covered by the protection ranges disclosed herein), and the details or common knowledge in the solutions are not described herein. It should be noted that the above embodiments do not limit the present invention in any way, and it is within the scope of the present invention for those skilled in the art to obtain the technical solution by equivalent substitution or equivalent transformation. The protection scope of the present application shall be subject to the content of the claims, and the description of the specific embodiments and the like in the specification can be used for explaining the content of the claims.
Claims (7)
1. The application of the bacteriostatic bacillus is characterized in that the bacteriostatic bacillus is used for inhibiting escherichia coliEscherichia coli) The application isNot related to diagnosis and treatment of disease; the bacteriostatic bacillus is bacillus licheniformisBacillus licheniformis) The method comprises the steps of carrying out a first treatment on the surface of the The preservation place is China general microbiological culture Collection center; the preservation time is 2021, 4 and 6; the preservation registration number is CGMCC No.22124.
2. The application of the bacteriostatic bacillus is characterized in that the bacteriostatic bacillus is used for inhibiting salmonella @Salmonella spp.), the use does not involve diagnosis and treatment of disease; the bacteriostatic bacillus is bacillus licheniformisBacillus licheniformis) The method comprises the steps of carrying out a first treatment on the surface of the The preservation place is China general microbiological culture Collection center; the preservation time is 2021, 4 and 6; the preservation registration number is CGMCC No.22124.
3. The application of the bacteriostatic bacillus is characterized in that the bacteriostatic bacillus is used for inhibiting staphylococcus aureusStaphylococcus aureus) The application does not involve diagnosis and treatment of disease; the bacteriostatic bacillus is bacillus licheniformisBacillus licheniformis) The method comprises the steps of carrying out a first treatment on the surface of the The preservation place is China general microbiological culture Collection center; the preservation time is 2021, 4 and 6; the preservation registration number is CGMCC No.22124.
4. The application of the bacteriostatic bacillus is characterized in that the bacteriostatic bacillus is used for inhibiting clostridium perfringensClostridium perfringens) The application does not involve diagnosis and treatment of disease; the bacteriostatic bacillus is bacillus licheniformisBacillus licheniformis) The method comprises the steps of carrying out a first treatment on the surface of the The preservation place is China general microbiological culture Collection center; the preservation time is 2021, 4 and 6; the preservation registration number is CGMCC No.22124.
5. The liquid fermentation method of the bacteriostatic bacillus is characterized by comprising the following steps of:
step 1) activating strains; step 2) preparing first-stage seeds; step 3) preparing a secondary seed tank; step 4) preparing a liquid fermentation medium; step 5) liquid fermentation; step 6) spray drying;
specifically, the step 1) of strain activation
Inoculating bacteriostatic bacillus with the preservation number of CGMCC No.22124 in glycerol pipe onto a LB solid plate through streaking, inversely culturing for 18-24h at 37 ℃, then picking single bacterial colony, inoculating into a LB liquid shake flask, shake culturing for about 12-16h at 37 ℃ and 200r/min, diluting and coating the bacterial colony on the LB plate, inversely culturing for 12-18h at 37 ℃ to obtain a seed plate;
step 2) preparation of first seed
Picking single bacterial colony from the seed flat plate by using an inoculating loop, inoculating the single bacterial colony into a triangular shake flask filled with LB culture medium, and standing and culturing for 12-18h at 30-40 ℃ to serve as a primary seed shake flask;
step 3) preparation of a secondary seed tank
Inoculating 1-10% of the strain into a seed tank filled with LB liquid culture medium, wherein the liquid loading amount is 20-40L, the fermentation temperature is 30-40 ℃, the stirring rotation speed is 150-250 rpm, and culturing for 12-18h to obtain a secondary seed tank;
step 4) liquid fermentation Medium configuration
The culture medium comprises the following components: corn starch, yeast powder, soybean meal, sodium chloride, dipotassium hydrogen phosphate, magnesium sulfate, manganese sulfate and calcium carbonate, wherein the raw materials are 800-1000 parts of water in L by kg; stirring to dissolve the culture medium completely, adjusting pH to 6.5-7.5 with NaOH or ammonia water, sterilizing at 105-121deg.C for 15-30min, and cooling with cooling water to 30-40deg.C to obtain fermentation culture medium;
step 5) liquid fermentation
Inoculating seeds of a seed tank into a sterilized fermentation medium according to the proportion of 1-10%, uniformly stirring, controlling the fermentation temperature to be 30-40 ℃, culturing for 12-24h, controlling the dissolved oxygen to be 10-50% by stirring and ventilation, regulating the pH to be 6.0-7.5 by sodium hydroxide or phosphoric acid, stopping fermentation when the dissolved oxygen is continuously risen for 2 hours, and cooling to wait for spray drying;
step 6) spray drying
The spray drying is carried out in a spray drying tower, the air inlet temperature of the spray drying tower is 130-170 ℃, the air outlet temperature is 60-90 ℃, and the liquid inlet speed of the fermentation liquid is 30-60L/h.
6. The liquid fermentation method according to claim 5, wherein the culture medium comprises the following components in parts by weight: 10-40 parts of corn starch, 2-5 parts of yeast powder, 10-50 parts of soybean meal, 1-5 parts of sodium chloride, 0-5 parts of dipotassium hydrogen phosphate, 1-5 parts of magnesium sulfate, 0-0.5 part of manganese sulfate, 10-20 parts of calcium carbonate and 800-1000 parts of water in L when the raw materials are calculated in kg.
7. A bacteriostasis test method of bacillus bacteriostasis is characterized by comprising the following steps:
step 1) sample pretreatment: the sample is bacteriostatic bacillus with a preservation number of CGMCC No. 22124; firstly preparing a product stock solution, weighing 5g of sample, fixing the volume in 50ml of sterile water, preparing a 10-fold diluted sample solution, vibrating on a shaking table for 15 minutes, uniformly mixing, standing for several minutes, and then adopting sterile water for gradient dilution to prepare 100-fold, 200-fold, 500-fold, 1000-fold, 2000-fold, 4000-fold and 8000-fold diluted sample solutions for later use;
step 2) pouring 10 mL of autoclaved raw agar into each sterilization culture dish in an ultra-clean workbench for bottom laying, wherein each culture dish has uniform thickness, and placing the sterilized oxford cup on the agar after the raw agar is cooled and solidified; 3-5 oxford cups are placed on each plate equidistantly;
step 3) absorbing 10 mu L of indicator bacteria liquid, adding the indicator bacteria liquid into 100 mL LB solid medium with constant temperature of 50+/-5 ℃, gently shaking the indicator bacteria liquid uniformly to avoid foaming, wherein the content of bacteria in the medium is 10 5 -10 6 CFU/ml；
Step 4) pouring the LB solid culture medium added with the bacterial liquid in the step 3) into an oxford cup-placed plain agar culture dish, pouring 15-20mL of each dish, and uniformly paving the layer until the layer is cooled and solidified;
step 5) taking out the oxford cup by using tweezers, sucking 150-200 mu L of sample to be detected, injecting the sample into a corresponding oxford cup hole, setting sterile water as a blank control, marking a culture dish, and placing the culture dish in a refrigerator at 2-8 ℃ for 2-4 hours to diffuse the sample;
step 6), transferring the culture dish into a 37 ℃ incubator for culture; in the following 6-12 h, the growth condition and the antibacterial effect of the thalli are observed, and the antibacterial ring can be observed by 8-12 h, but different indicator bacteria and different plate paving operations can be different, and the bacteria are observed in time;
and 7) observing the diameter of a bacteriostasis ring in the flat plate, and simultaneously judging the bacteriostasis effect by using sterile water as a contrast.
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