CN106754836A - A kind of method of utilization bacillus HS17 fermenting and producing Collagenases and its application - Google Patents
A kind of method of utilization bacillus HS17 fermenting and producing Collagenases and its application Download PDFInfo
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- 102000029816 Collagenase Human genes 0.000 title claims abstract description 62
- 108060005980 Collagenase Proteins 0.000 title claims abstract description 62
- 241000193830 Bacillus <bacterium> Species 0.000 title claims abstract description 17
- 238000000034 method Methods 0.000 title claims abstract description 15
- 102000004190 Enzymes Human genes 0.000 claims abstract description 41
- 108090000790 Enzymes Proteins 0.000 claims abstract description 41
- 238000000855 fermentation Methods 0.000 claims abstract description 39
- 230000004151 fermentation Effects 0.000 claims abstract description 39
- 229910000357 manganese(II) sulfate Inorganic materials 0.000 claims abstract description 27
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 claims abstract description 26
- 108010010803 Gelatin Proteins 0.000 claims abstract description 25
- 239000008273 gelatin Substances 0.000 claims abstract description 25
- 229920000159 gelatin Polymers 0.000 claims abstract description 25
- 235000019322 gelatine Nutrition 0.000 claims abstract description 25
- 235000011852 gelatine desserts Nutrition 0.000 claims abstract description 25
- 229910000396 dipotassium phosphate Inorganic materials 0.000 claims abstract description 24
- 229930091371 Fructose Natural products 0.000 claims abstract description 20
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 claims abstract description 20
- 239000005715 Fructose Substances 0.000 claims abstract description 20
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims abstract description 19
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims abstract description 19
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims abstract description 14
- 229910000397 disodium phosphate Inorganic materials 0.000 claims abstract description 14
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 claims abstract description 12
- 229910000368 zinc sulfate Inorganic materials 0.000 claims abstract description 12
- XLYOFNOQVPJJNP-ZSJDYOACSA-N heavy water Substances [2H]O[2H] XLYOFNOQVPJJNP-ZSJDYOACSA-N 0.000 claims abstract description 11
- 239000012138 yeast extract Substances 0.000 claims abstract description 11
- 239000011686 zinc sulphate Substances 0.000 claims abstract description 11
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims abstract description 10
- 230000004913 activation Effects 0.000 claims description 33
- 239000007788 liquid Substances 0.000 claims description 32
- 239000002609 medium Substances 0.000 claims description 28
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 20
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 20
- 238000012360 testing method Methods 0.000 claims description 17
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 claims description 16
- 239000001963 growth medium Substances 0.000 claims description 12
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 11
- 238000001914 filtration Methods 0.000 claims description 11
- 239000008103 glucose Substances 0.000 claims description 11
- 239000002054 inoculum Substances 0.000 claims description 11
- 230000001954 sterilising effect Effects 0.000 claims description 11
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 10
- 238000011218 seed culture Methods 0.000 claims description 10
- 239000011780 sodium chloride Substances 0.000 claims description 10
- 238000012546 transfer Methods 0.000 claims description 10
- 239000000284 extract Substances 0.000 claims description 6
- 239000012530 fluid Substances 0.000 claims description 6
- 239000006228 supernatant Substances 0.000 claims description 6
- 241000283725 Bos Species 0.000 claims description 5
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 5
- 235000013372 meat Nutrition 0.000 claims description 5
- 229910052760 oxygen Inorganic materials 0.000 claims description 5
- 239000001301 oxygen Substances 0.000 claims description 5
- 230000001681 protective effect Effects 0.000 claims description 5
- 238000004659 sterilization and disinfection Methods 0.000 claims description 5
- 229960002424 collagenase Drugs 0.000 abstract description 54
- 102000008186 Collagen Human genes 0.000 abstract description 49
- 108010035532 Collagen Proteins 0.000 abstract description 49
- 229920001436 collagen Polymers 0.000 abstract description 47
- 229940088598 enzyme Drugs 0.000 abstract description 40
- 108090000765 processed proteins & peptides Proteins 0.000 abstract description 14
- 229920001184 polypeptide Polymers 0.000 abstract description 13
- 102000004196 processed proteins & peptides Human genes 0.000 abstract description 13
- 230000007062 hydrolysis Effects 0.000 abstract description 8
- 238000006460 hydrolysis reaction Methods 0.000 abstract description 8
- 230000000694 effects Effects 0.000 description 30
- 238000005457 optimization Methods 0.000 description 17
- 230000001580 bacterial effect Effects 0.000 description 15
- 238000013461 design Methods 0.000 description 15
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 14
- 238000004321 preservation Methods 0.000 description 11
- 238000006243 chemical reaction Methods 0.000 description 7
- 229910052757 nitrogen Inorganic materials 0.000 description 7
- 102000035195 Peptidases Human genes 0.000 description 6
- 108091005804 Peptidases Proteins 0.000 description 6
- 241000193744 Bacillus amyloliquefaciens Species 0.000 description 5
- 241000894006 Bacteria Species 0.000 description 5
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 5
- 239000004365 Protease Substances 0.000 description 5
- 235000015278 beef Nutrition 0.000 description 5
- 229910052799 carbon Inorganic materials 0.000 description 5
- 239000003292 glue Substances 0.000 description 5
- 239000000843 powder Substances 0.000 description 5
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 4
- 239000002253 acid Substances 0.000 description 4
- 229940041514 candida albicans extract Drugs 0.000 description 4
- 239000006071 cream Substances 0.000 description 4
- 238000004519 manufacturing process Methods 0.000 description 4
- 230000002572 peristaltic effect Effects 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 230000004044 response Effects 0.000 description 4
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 3
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- 238000000205 computational method Methods 0.000 description 3
- 238000010790 dilution Methods 0.000 description 3
- 239000012895 dilution Substances 0.000 description 3
- 230000003301 hydrolyzing effect Effects 0.000 description 3
- 239000008363 phosphate buffer Substances 0.000 description 3
- 238000012545 processing Methods 0.000 description 3
- 238000000611 regression analysis Methods 0.000 description 3
- 241000894007 species Species 0.000 description 3
- 239000000758 substrate Substances 0.000 description 3
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 241000726221 Gemma Species 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- 241000193159 Hathewaya histolytica Species 0.000 description 2
- 229910052770 Uranium Inorganic materials 0.000 description 2
- 235000011130 ammonium sulphate Nutrition 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 230000009194 climbing Effects 0.000 description 2
- 230000037319 collagen production Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 230000002255 enzymatic effect Effects 0.000 description 2
- 238000007689 inspection Methods 0.000 description 2
- 230000000813 microbial effect Effects 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 125000001477 organic nitrogen group Chemical group 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 239000003531 protein hydrolysate Substances 0.000 description 2
- 241000251468 Actinopterygii Species 0.000 description 1
- 239000004375 Dextrin Substances 0.000 description 1
- 229920001353 Dextrin Polymers 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 208000001953 Hypotension Diseases 0.000 description 1
- IOVCWXUNBOPUCH-UHFFFAOYSA-M Nitrite anion Chemical compound [O-]N=O IOVCWXUNBOPUCH-UHFFFAOYSA-M 0.000 description 1
- 208000001132 Osteoporosis Diseases 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- 108010009736 Protein Hydrolysates Proteins 0.000 description 1
- 206010052428 Wound Diseases 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 230000009102 absorption Effects 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000008351 acetate buffer Substances 0.000 description 1
- 235000019270 ammonium chloride Nutrition 0.000 description 1
- 230000003712 anti-aging effect Effects 0.000 description 1
- 230000003266 anti-allergic effect Effects 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 230000000767 anti-ulcer Effects 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 235000006708 antioxidants Nutrition 0.000 description 1
- 238000009360 aquaculture Methods 0.000 description 1
- 244000144974 aquaculture Species 0.000 description 1
- 206010003246 arthritis Diseases 0.000 description 1
- 244000052616 bacterial pathogen Species 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 235000019425 dextrin Nutrition 0.000 description 1
- 238000013401 experimental design Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 239000008236 heating water Substances 0.000 description 1
- 210000001320 hippocampus Anatomy 0.000 description 1
- 208000021822 hypotensive Diseases 0.000 description 1
- 230000001077 hypotensive effect Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- WBJZTOZJJYAKHQ-UHFFFAOYSA-K iron(3+) phosphate Chemical compound [Fe+3].[O-]P([O-])([O-])=O WBJZTOZJJYAKHQ-UHFFFAOYSA-K 0.000 description 1
- 229910000399 iron(III) phosphate Inorganic materials 0.000 description 1
- 238000002386 leaching Methods 0.000 description 1
- 239000012263 liquid product Substances 0.000 description 1
- 229940099596 manganese sulfate Drugs 0.000 description 1
- 239000011702 manganese sulphate Substances 0.000 description 1
- 235000007079 manganese sulphate Nutrition 0.000 description 1
- 238000005374 membrane filtration Methods 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 229910021645 metal ion Inorganic materials 0.000 description 1
- 238000009629 microbiological culture Methods 0.000 description 1
- FEMOMIGRRWSMCU-UHFFFAOYSA-N ninhydrin Chemical compound C1=CC=C2C(=O)C(O)(O)C(=O)C2=C1 FEMOMIGRRWSMCU-UHFFFAOYSA-N 0.000 description 1
- 229940005654 nitrite ion Drugs 0.000 description 1
- 238000013433 optimization analysis Methods 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 239000006041 probiotic Substances 0.000 description 1
- 235000018291 probiotics Nutrition 0.000 description 1
- 238000004451 qualitative analysis Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000005211 surface analysis Methods 0.000 description 1
- 238000004809 thin layer chromatography Methods 0.000 description 1
- 239000003053 toxin Substances 0.000 description 1
- 231100000765 toxin Toxicity 0.000 description 1
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 description 1
- 239000012137 tryptone Substances 0.000 description 1
- 238000002255 vaccination Methods 0.000 description 1
- 230000029663 wound healing Effects 0.000 description 1
- 229960001763 zinc sulfate Drugs 0.000 description 1
Classifications
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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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
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- Chemical & Material Sciences (AREA)
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- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Molecular Biology (AREA)
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- General Health & Medical Sciences (AREA)
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- Microbiology (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biomedical Technology (AREA)
- General Chemical & Material Sciences (AREA)
- Toxicology (AREA)
- Gastroenterology & Hepatology (AREA)
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- Proteomics, Peptides & Aminoacids (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Fertilizers (AREA)
Abstract
Method and its application the invention discloses a kind of utilization bacillus HS17 fermenting and producing Collagenases, belong to biological technical field.The present invention utilizes following fermentation medium：2~4 g/L gelatin, 40~50 g/L fructose, 3~6 g/L yeast extracts, 3~5 g/L ZnSO4, 3~4 g/L MnSO4, 2~4 g/L FePO4.2H2O, 5~6 g/L (NH4)2SO4, 3~5 g/L Na2HPO4, 4~6 g/L K2HPO4.Cultural method of the invention produces Collagenase efficiently, and vigor is high, and institute's producing enzyme is high to the percent hydrolysis of collagen, has application prospect in the producer face of collagen polypeptide.
Description
Technical field
The present invention relates to a kind of method of utilization bacillus HS17 fermenting and producing Collagenases, and its in collagen
Application in polypeptide preparation, belongs to biological technical field.
Background technology
Collagen has unique superhelix（3 right-handed helixs of α peptide chains）, stable in properties, general processing
Temperature and short time heating all can not decompose it, be difficult the utilization that is absorbed by the body.And be collagen polypeptide by collagen hydrolysate,
With easy absorption, antiulcer, antiallergy, hypotensive, antibacterial, anti-oxidant, norcholesterol, anti-aging, promotion wound healing, prevention
Various physiologically actives such as osteoporosis and arthritis, promotion corneal epithelial wound reparation so that collagen polypeptide turns into aquatic products processing
The study hotspot of accessory substance higher value application.
Collagenase is a kind of proteolytic enzyme for hydrolyzing natural collagen, can be special under physiological pH and temperature conditionss
Property ground hydrolysis natural collagen protein three-dimensional spiral structure, without damage other oroteins and tissue.The degraded of Collagenase
The factors such as vigor, stability turn into efficiently crucial using collagen.Microbial source Collagenase is certain micro-organisms in spy
Determine the protease with degraded collagen ability secreted under environment, microbe-derived Collagenase is mainly bacterium collagen
Enzyme, the microorganism Collagenase source of current most study is clostridium histolyticum（Clostridium histolyticum）, but it is pathogen, is produced with toxin, and nonideal clostridiopetidase A is originated.
The present situation of a large amount of fish-skin leftover bits and pieces is produced based on current aquatic products processing enterprise, the present invention is to obtain one plant of promotion sea
The bacillus of horse healthy aquaculture（The A of patent of invention CN 104232541）On the basis of, the bacterial strain is probiotics, special through culture
Journal of Sex Research finds the bacterial strain also high yield Collagenase, determines its cultural method for producing Collagenase, and with this conversion of substrate
Collagen produces collagen polypeptide, realizes the higher value application of the leftover bits and pieces such as fish-skin.
The content of the invention
The deficiency that the present invention exists for above-mentioned technology, there is provided one kind utilizes bacillus HS17 fermenting and producing high activity glue
The cultural method of former protease and its application, with bacillus amyloliquefaciens HS17 as starting strain, give birth to through the method for liquid fermentation
Collagenase is produced, the enzyme can produce collagen polypeptide by effectively hydrolyzing collagen.
Bacterial strain used by the present invention is bacillus amyloliquefaciens HS17, its can be produced on the culture plate of addition gelatin compared with
Big degraded circle（Accompanying drawing 1）, with high yield Collagenase, its preservation is entitled：HS17, depositary institution：China Microbiological
Culture presevation administration committee common micro-organisms center, preservation date：On August 20th, 2014, preserving number is CGMCC
No.9532, Classification And Nomenclature：Bacillus amyloliquefaciens（Bacillus amyloliquefaciens）.
The present invention is achieved by the following technical solution：
1. actication of culture
Take in the strain transfer activation medium of test tube slant preservation, mixed in 18~24h of activation under 37 DEG C, 200 rpm to thalline
It is turbid, activated seed liquid is obtained；Activation medium used：Seed culture medium：5 g/L glucose, 4 g/L beef extracts, 1 g/L is bright
Glue, 3 g/L NaCl, 1 g/L K2HPO4, 0.4 g/L MgSO4, 0.2 g/L MnSO4, pH7.2.
2. Shaking culture fermenting and producing Collagenase
Using preferred fermentation medium：2~4 g/L gelatin, 40~50 g/L fructose, 3~6 g/L yeast extracts,3~5
g/L ZnSO4, 3~4 g/L MnSO4, 2~4 g/L FePO4.2H2O, 5~6 g/L (NH4)2SO4, 3~5 g/L
Na2HPO4, 4~6 g/L K2HPO4, initial pH 6~7,50~100 mL fermented and cultureds of packing in every 500 mL conical flasks
Base, 115 DEG C of 20 min of sterilizing.
According to 3~6%（v/v）Inoculum concentration access the seed activation liquid of activation, after to enter train in the rotary shaker of 200 rpm
Support, control temperature for 30~33 DEG C of temperature, cultivated after 36~48 h of fermentation and finished, determine collagen enzyme activity.Zymotic fluid is passed through
8000r/min is centrifuged 6 min, is collected after removal thalline and retains supernatant, is then removed through the micro porous filtration membrane filtration of 0.22 um again
Bacterium, i.e., use as liquid enzymes.
3. fermentation tank culture fermenting and producing Collagenase
Fermented and cultured component is loaded with 70% ratio, through 118 DEG C of steam sterilizing 20min after, under flame ring protective condition according to 3~
6%（v/v）Ratio access activation strain, by adjust soda acid peristaltic pump control fermentation process in pH maintain pH all the time
6~7, using the heater regulation cultivation temperature of fermentation tank at 30~33 DEG C, dissolved oxygen parameter is by adjusting into the aseptic of tank
The size of air capacity and speed of agitator makes it maintain more than 6% all the time.Fermented 30~42 h after fermentation is finished, and determines collagen
Prolease activity.Zymotic fluid is centrifuged 6 min through 8000 r/min, is collected after removal thalline and retains supernatant, then passes through again
The micropore filtering film filtration sterilization of 0.22um, i.e., use as liquid enzymes.Optimal Medium used：2~4 g/L gelatin, 40
~50 g/L fructose, 3~6 g/L yeast extracts,3~5 g/L ZnSO4, 3~4 g/L MnSO4, 2~4 g/L
FePO4.2H2O, 5~6 g/L (NH4)2SO4, 3~5 g/L Na2HPO4, 4~6 g/L K2HPO4。
4. the preparation of collagen polypeptide
A certain amount of cod collagen powder, according to 1：5 ratio adds phosphate buffer（pH 7.2）In, it is subsequently adding glue
Former liquid of protease（Substrate is 2 with enzyme amount ratio：1）, the terminating reaction after 10~20 h of slow shaking reaction under the conditions of 35 DEG C.Collagen
Protein hydrolyzate heats the min of enzyme 10 that goes out through 90 DEG C, and 8 min removal albumen precipitations are centrifuged through 10000 r/min, cold at -75 DEG C
Lyophilized dry acquisition collagen polypeptide powder.
Collagenase vigour-testing method used：The fish scale collagen of 1mg is dissolved with 0.5 mL phosphate buffers
（pH7.4）, add 0.1ml dilution enzyme liquids, 37 DEG C of reaction 40min.Plus 0.5 mL trichloroacetic acid（10%,m/v）Terminating reaction, then
Add 0.9mL acetate buffers（pH5.4）Mixed with 1mL ninhydrins nitrite ion.Heating water bath 10min, uses after cooling at 100 DEG C
The 60%% ethanol dilution of 3 mL, the colorimetric estimation under 570 nm.
The control group treatment of enzyme activity determination used：Disturbed to eliminate, the dilution enzyme liquid of 0.1 mL is taken first at 100 DEG C
Lower water-bath is boiled 10min and is inactivated enzyme, the same enzyme activity determination of remaining step.
Enzyme activity computational methods used：Generate 1ug's per min with every mL enzyme liquids Hydrolyzed Collagen under the conditions of 37 DEG C
The amount of glycine is an enzyme activity unit（U）.
Enzyme activity computational methods used：Maximum sample enzyme activity in sample enzyme activity in every group/every group ×
100%。
The beneficial effects of the present invention are：
（1）Bacillus HS17 used by the present invention is the interior raw bacillus amyloliquefaciens from hippocampus enteron aisle, and non-pathogenic bacteria is raw
The Collagenase of product is safe.
（2）Cultural method gears to actual circumstances, and fermentation condition is easy to operate, and yield of enzyme is high
Using cultural method of the invention, the Collagenase energy value of fermenting and producing compares, its enzyme with the cultural method at initial stage
Output increased more than 20 times, the yield of cultural method is protruded.
（3）Collagenase is produced using the bacterial strain high to the hydrolysis efficiency of collagen, for collagen polypeptide
Production will have broad application prospects.
Brief description of the drawings
Fig. 1 is the gelatin degradation circle of bacillus HS17 flat board cultures；
Fig. 2 cultivates curve for bacillus HS17 in fermentation tank；
Fig. 3 is the thin-layer chromatography qualitative analysis of enzymolysis liquid, and wherein label 1 is collagen, and label 2 is enzymolysis liquid product.
Specific embodiment
The present invention is described in further detail below in conjunction with specific embodiment, these embodiments are used to understand rather than limit
Protection scope of the present invention processed.
Embodiment 1：The single factor test optimization of the culture medium of fermenting and producing Collagenase
（1）Actication of culture：Take test tube slant preservation strain transfer activation medium in, under 37 DEG C, 200 rpm activation 18~
24h is muddy to thalline, and activated seed liquid is obtained；Activation medium used：Seed culture medium：5 g/L glucose, 4 g/L oxen
Meat extract, 1 g/L gelatin, 3 g/L NaCl, 1 g/L K2HPO4, 0.4 g/L MgSO4, 0.2 g/L MnSO4, pH7.2.
（2）Fermentation optimization：Activated seed liquid is according to 5%（v/v）After inoculum concentration accesses different fermentation mediums, 30 DEG C,
Fermented in the shaking table of 200 rpm and collagen enzyme activity is determined after 48 h.
Carbon source is shown in Table 1 to the influence that bacterial strain HS17 produces Collagenase, it can be seen that fructose produces glue as carbon source through fermentation
The effect of former protease is best, next to that glucose and dextrin.Fermentation medium used（g/L）：Carbon source 20（Its species is shown in Table
1）, peptone 8, gelatin 5, Na2HPO42, K2HPO43, pH 7.5.
The carbon source of table 1 produces the influence of Collagenase to bacterial strain
Nitrogen source is shown in Table 2, fermentation medium used to the influence that bacterial strain produces Collagenase（g/L）：：Fructose 20, nitrogen source 8（Its
Species is shown in Table 2）, gelatin 5, Na2HPO42, K2HPO43, pH 7.5.In the organic nitrogen source selected, fermented with yeast extract and given birth to
It is optimal to produce Collagenase, is secondly tryptone；In inorganic nitrogen-sourced, fermenting and producing collagen during with ammonium sulfate as nitrogen source
The effect of enzyme is preferably, secondly ammonium chloride.In Spawn incubation early stage quickly using inorganic nitrogen-sourced so that thalline is largely bred, and bacterium
After bulk concentration reaches to a certain degree, the metabolism enzyme system in microbial body is initially formed, and can slowly utilize organic nitrogen source, and
This stage being capable of a large amount of productive target product.The result of Collagenase influence is produced on bacillus HS17 according to nitrogen source, is chosen
Dusty yeast and ammonium sulfate are used as preferred nitrogen source.
The nitrogen source of table 2 produces the influence of Collagenase to bacterial strain
Slaine is shown in Table 3, fermentation medium used to the influence that bacterial strain produces Collagenase（g/L）：Fructose 20, dusty yeast 8,
Gelatin 5, Na2HPO42, K2HPO43, slaine 3（Its species is shown in Table 3）, pH 7.5.Compared with blank, to Collagenase
What fermenting and producing had active influence is manganese sulfate, zinc sulfate and ferric orthophosphate.
The slaine of table 3 produces the influence of Collagenase to bacterial strain
Embodiment 2：The multifactor optimization of the culture medium of fermenting and producing Collagenase
（1）Actication of culture：Take test tube slant preservation strain transfer activation medium in, under 37 DEG C, 200 rpm activation 18~
24h is muddy to thalline, and activated seed liquid is obtained；Activation medium used：Seed culture medium：5 g/L glucose, 4 g/L oxen
Meat extract, 1 g/L gelatin, 3 g/L NaCl, 1 g/L K2HPO4, 0.4 g/L MgSO4, 0.2 g/L MnSO4, pH7.2.
（2）Fermentation optimization：Activated seed liquid is according to 5%（v/v）After inoculum concentration accesses different fermentation mediums, 30 DEG C,
Fermented in the shaking table of 200 rpm and collagen enzyme activity is determined after 48 h.According to carbon source, nitrogen source and metal ion to gemma bar
Bacterium HS17 produces the optimum results of Collagenase, chooses gelatin, fructose, yeast extract, ZnSO4、MnSO4、FePO4.2H2O、
(NH4)2SO4、Na2HPO4And K2HPO4Carry out the Plackett-Burman design optimizations of the level of 9 factor 2, its factor and level
4 are shown in Table, 5 are the results are shown in Table, maximum collagenase activity is obtained at the 3rd group, be 105.81 U/mL.
The factor level of the Plackett-Burman design optimizations of table 4
The result of the Plackett-Burman design optimizations of table 5
Analysis is optimized to Plackett-Burman designs using Design Expert softwares, based on single order side after amendment
Journey and Regression Analysis Result, determine fructose, MnSO4(NH4)2SO4It is most notable factor, it means that improve fructose, MnSO4With
(NH4)2SO4Concentration is conducive to improving collagen production of enzyme.Using each conditions after Plackett-Burman design optimizations,
Climbing experiment is carried out, it the results are shown in Table 6.As can be seen from Table 6, maximum collagen enzyme activity is obtained at the 3rd group, is
125.42 U/mL, therefore with 45 g/L fructose, 3.5 g/L MnSO4With 3.5 g/L (NH4)2SO4Centered on point, carry out center
Combination Design and response surface analysis optimize.
Table 6 climbing experimental design and result
Using Design-Expert softwares respectively to fructose, MnSO4(NH4)2SO4This 3 factors carry out center combination and set
Meter, factor level coding is shown in Table 7, and its center combination design and enzymatic production the results are shown in Table 8.
The level and coding of the center combination design of table 7
Regression analysis, encoding Factor are carried out to the data obtained using Design Expert softwaresA（Fructose）、B（MnSO4）WithC
（(NH4)2SO4）Secondary multinomial regression equation：Collagenase activity（Y, U/mL）= 126.36 + 10.12A -12.88B +
8.09C - 19.36AB + 17.81AC – 1.87BC – 12.18A 2 – 5.32B 2 – 8.43C 2；The F=of the regression model
137.13,p<0.0001, show model significantly, only 0.01% optimization error；Lose and intend item F=2.10,p=0.2177>0.05,
I.e. equation model loses and intends not significantly, illustrating that the equation can preferably describe the relation of each fermentation condition and collagenase activity.
Mathematical Modeling according to being set up enters the response surface optimality analysis of line parameter, 50 g/L fructose, 3 g/L MnSO4
With 6 g/L (NH4)2SO4When, theoretical Collagenase most highly active value is 170.58 U/mL.It is the reliability of the method for inspection,
Using optimization fermented and cultured component（g/L）：Gelatin 3, fructose 50, yeast extract 4,ZnSO4 4, MnSO4 3,
FePO4.2H2O 3, (NH4)2SO4 6, Na2HPO4 4, K2HPO45, fermenting and producing Collagenase is carried out with this understanding
3 parallel tests, actually obtain collagenase activity average value for 171.85U/mL, the error between actual value and predicted value
About 0.74%.Therefore using the condition reliability of response surface optimization bacterial strain HS17 fermenting and producing Collagenases, with practical valency
Value.
The center combination design of table 8 and result
Embodiment 3：The single factor test optimization of the condition of culture of fermenting and producing Collagenase
（1）Actication of culture：Take test tube slant preservation strain transfer activation medium in, under 37 DEG C, 200 rpm activation 18~
24h is muddy to thalline, and activated seed liquid is obtained；Activation medium used：Seed culture medium：5 g/L glucose, 4 g/L oxen
Meat extract, 1 g/L gelatin, 3 g/L NaCl, 1 g/L K2HPO4, 0.4 g/L MgSO4, 0.2 g/L MnSO4, pH7.2.
（2）Fermentation optimization：Under the conditions of different vaccination amount, initial pH, cultivation temperature and liquid amount, determined after 48 h of fermentation
Collagen enzyme activity.Fermentation medium used（g/L）:Gelatin 3, fructose 50, yeast extract 4,ZnSO4 4, MnSO4
3, FePO4.2H2O 3, (NH4)2SO4 6, Na2HPO4 4, K2HPO4 5。
Inoculum concentration is shown in Table 9 to the influence that bacillus HS17 produces Collagenase, and most suitable initial pH is as can be seen from Table 9
4%（v/v）.The initial pH that ferments is shown in Table 10 to the influence that bacillus HS17 produces Collagenase, and maximum producing is obtained in pH 6.5
Collagenase ability.Cultivation temperature is shown in Table 11 to the influence that bacillus HS17 produces Collagenase, most preferably produces collagen
The cultivation temperature of enzyme is 32 DEG C.Liquid amount is shown in Table 12 to the influence that bacillus HS17 produces Collagenase, and optimal liquid amount is
100 mL/500 mL conical flasks.
The inoculum concentration of table 9 produces the influence of Collagenase to bacterial strain HS17
The initial pH of table 10 produces the influence of Collagenase to bacterial strain HS17
The cultivation temperature of table 11 produces the influence of Collagenase to bacterial strain HS17
The liquid amount of table 12 produces the influence of Collagenase to bacterial strain HS17
Embodiment 4：The multifactor optimization of the condition of culture of fermenting and producing Collagenase
（1）Actication of culture：Take test tube slant preservation strain transfer activation medium in, under 37 DEG C, 200 rpm activation 18~
24h is muddy to thalline, and activated seed liquid is obtained；Activation medium used：Seed culture medium：5 g/L glucose, 4 g/L oxen
Meat extract, 1 g/L gelatin, 3 g/L NaCl, 1 g/L K2HPO4, 0.4 g/L MgSO4, 0.2 g/L MnSO4, pH7.2.
（2）Fermentation optimization：According to the result of the test that single factor test optimizes, inoculum concentration, initial pH and cultivation temperature are to gemma bar
Bacterium HS17 produces Collagenase large effect.Using Design-Expert softwares to inoculum concentration, initial pH and culture temperature
Spending this 3 factors carries out center combination design, and factor level coding is shown in Table 13, and it the results are shown in Table 14.
The level and coding of the center combination design of table 13
Regression analysis, encoding Factor are carried out to the data obtained using Design Expert softwaresA（Inoculum concentration）、B（Culture temperature
Degree）WithC（(initial pH）Secondary multinomial regression equation：Collagenase activity（Y, U/mL）= 263.39 + 6.98A -
11.99B - 8.95C – 25.16AB + 10.01AC – 25.03BC – 45.55A 2 – 7.52B 2 + 3.29C 2；Return
The F=185.90 of model,p<0.0001, show model significantly, only 0.01% optimization error；Lose and intend item F=2.91,p=
0.1331>0.05, i.e. equation model lose to be intended not significantly, illustrating that the equation can preferably describe each fermentation condition and Collagenase
The relation of activity.
Parameter optimization analysis, inoculum concentration 4.46% are carried out according to the Mathematical Modeling set up（v/v）, 30 DEG C of cultivation temperature
When with initial pH being 7, the theoretical Collagenase most highly active value of modeling is 268.68 U/mL.For the method for inspection can
By property, using the optimal fermented and cultured component and condition that obtain, fermenting and producing Collagenase is carried out, 3 are carried out with this understanding
Secondary parallel test, actually obtains collagenase activity average value for 267.15 U/mL, the error between actual value and predicted value
About 0.68%.Therefore using the condition reliability of response surface optimization bacillus HS17 fermenting and producing Collagenases, with reality
With value.
The center combination design of table 14 and result
Embodiment 5：Shake flask fermentation produces Collagenase
Actication of culture：Take test tube slant preservation strain transfer activation medium in, under 37 DEG C, 200 rpm activate 18~24h
It is muddy to thalline, activated seed liquid is obtained；Activation medium used：Seed culture medium：5 g/L glucose, 4 g/L beef
Cream, 1 g/L gelatin, 3 g/L NaCl, 1 g/L K2HPO4, 0.4 g/L MgSO4, 0.2 g/L MnSO4, pH7.2.
Fermenting and producing：Using the optimal fermented and cultured component and condition that obtain:3 g/L gelatin, 50 g/L fructose, 4
G/L yeast extracts,4 g/L ZnSO4, 3 g/L MnSO4, 3 g/L FePO4.2H2O, 6 g/L (NH4)2SO4, 4 g/L
Na2HPO4, 5 g/L K2HPO4, the mL/500 mL of liquid amount 100, inoculum concentration 4.46%（v/v）, 30 DEG C of cultivation temperature and initial
PH 7, carries out fermenting and producing Collagenase, the rpm of shaking speed 200, period sampling measuring enzymatic activity, when 42 h is fermented
Maximum enzyme activity is obtained, is 284.71 U/mL.
Embodiment 6：Ferment tank produces Collagenase
（1）Comparative example 1
Actication of culture：Take test tube slant preservation strain transfer activation medium in, under 37 DEG C, 200 rpm activate 18~24h
It is muddy to thalline, activated seed liquid is obtained；Activation medium used：Seed culture medium：5 g/L glucose, 4 g/L beef
Cream, 1 g/L gelatin, 3 g/L NaCl, 1 g/L K2HPO4, 0.4 g/L MgSO4, 0.2 g/L MnSO4, pH7.2.
Fermenting and producing：Using the optimal fermented and cultured component and condition that obtain:2 g/L gelatin, 40 g/L fructose, 3
G/L yeast extracts,3 g/L ZnSO4, 3 g/L MnSO4, 2 g/L FePO4.2H2O, 5 g/L (NH4)2SO4, 3 g/L
Na2HPO4, 4 g/L K2HPO4.Fermented and cultured component, after 118 DEG C of steam sterilizing 20min, flame ring are loaded with 70% ratio
According to 3% under protective condition（v/v）Ratio access activation strain, by adjust soda acid peristaltic pump control fermentation process in
PH maintains pH 6 all the time, using the heater regulation cultivation temperature of fermentation tank at 30 DEG C, dissolved oxygen parameter by adjust into
The filtrated air amount of tank and the size of speed of agitator make it maintain more than 6% all the time, results of regular determination collagen enzyme activity,
Ferment 36 h when to obtain maximum collagen enzyme activity be 365.32 U/mL..Zymotic fluid is centrifuged 6 min through 8000 r/min, goes
Retain supernatant except collecting after thalline, then used as liquid enzymes through the micropore filtering film filtration sterilization of 0.22um again.
（2）Comparative example 2
Actication of culture：Take test tube slant preservation strain transfer activation medium in, under 37 DEG C, 200 rpm activate 18~24h
It is muddy to thalline, activated seed liquid is obtained；Activation medium used：Seed culture medium：5 g/L glucose, 4 g/L beef
Cream, 1 g/L gelatin, 3 g/L NaCl, 1 g/L K2HPO4, 0.4 g/L MgSO4, 0.2 g/L MnSO4, pH7.2.
Fermenting and producing：Using the optimal fermented and cultured component and condition that obtain:4 g/L gelatin, 50 g/L fructose, 6
G/L yeast extracts,5 g/L ZnSO4, 4 g/L MnSO4, 4 g/L FePO4.2H2O, 6 g/L (NH4)2SO4, 5 g/L
Na2HPO4, 6 g/L K2HPO4.Fermented and cultured component, after 118 DEG C of steam sterilizing 20min, flame ring are loaded with 70% ratio
According to 6% under protective condition（v/v）Ratio access activation strain, by adjust soda acid peristaltic pump control fermentation process in
PH maintains pH 7 all the time, using the heater regulation cultivation temperature of fermentation tank at 33 DEG C, dissolved oxygen parameter by adjust into
The filtrated air amount of tank and the size of speed of agitator make it maintain more than 6% all the time, results of regular determination collagen enzyme activity,
Ferment 40 h when to obtain maximum collagen enzyme activity be 389.27 U/mL.Zymotic fluid is centrifuged 6 min through 8000 r/min, goes
Retain supernatant except collecting after thalline, then used as liquid enzymes through the micropore filtering film filtration sterilization of 0.22um again.
（3）Comparative example 3
Actication of culture：Take test tube slant preservation strain transfer activation medium in, under 37 DEG C, 200 rpm activate 18~24h
It is muddy to thalline, activated seed liquid is obtained；Activation medium used：Seed culture medium：5 g/L glucose, 4 g/L beef
Cream, 1 g/L gelatin, 3 g/L NaCl, 1 g/L K2HPO4, 0.4 g/L MgSO4, 0.2 g/L MnSO4, pH7.2.
Fermenting and producing：The fermented and cultured component and condition of use:3 g/L gelatin, 50 g/L fructose, the leaching of 4 g/L yeast
Powder,4 g/L ZnSO4, 3 g/L MnSO4, 3 g/L FePO4.2H2O, 6 g/L (NH4)2SO4, 4 g/L Na2HPO4,
5 g/L K2HPO4.With 70% ratio load fermented and cultured component, through 118 DEG C of steam sterilizing 20min after, under flame ring protective condition
According to 4.46%（v/v）Ratio access activation strain, by adjust soda acid peristaltic pump control fermentation process in pH tie up all the time
Hold in pH 7, using the heater regulation cultivation temperature of fermentation tank at 30 DEG C, dissolved oxygen parameter is by adjusting into the aseptic of tank
The size of air capacity and speed of agitator makes it maintain more than 6% all the time.Zymotic fluid is centrifuged 6 min through 8000 r/min, goes degerming
Collected after body and retain supernatant, then used as liquid enzymes through the micropore filtering film filtration sterilization of 0.22um again.Fermentation 38
Maximum cell concentration 40.56 is obtained during h（OD600）, it is 415.39 U/mL that maximum collagen production of enzyme is obtained during 35 h of fermentation
（See accompanying drawing 2）.
Embodiment 7：Hydrolyzed Collagen prepares collagen polypeptide
10 g cod collagen powder are weighed, according to 1：5 ratio adds phosphate buffer（pH 7.2）In, it is subsequently adding glue
Former liquid of protease（Substrate is 2 with enzyme amount ratio：1）, the terminating reaction after 20 h of slow shaking reaction under the conditions of 35 DEG C.Collagen
Hydrolyzate heats the enzyme 10min that goes out through 90 DEG C, and 8 min removal albumen precipitations are centrifuged through 10000 r/min, is freezed at -75 DEG C dry
The dry g of acquisition collagen polypeptide powder 6.12.By accompanying drawing 3 as can be seen that producing more than 6 kinds of collagen egg after collagen protein enzymolysis
White polypeptide, and with the extension in reaction time, the yield of collagen polypeptide is more, therefore the clostridiopetidase A of production has preferable collagen
PD effect.
By protein hydrolyzate through 10000r/min refrigerated centrifuges（5℃）8min, hydrolysis egg is determined using ninhydrin
- NH in white liquor2Content, the percent hydrolysis that collagen is determined after calculating degree of hydrolysis is 68%.Degree of hydrolysis computational methods used are such as
Under：Degree of hydrolysis（DH）=h/hb× 100%, h is the peptide bond mM number that every gram of collagen is cleaved after hydrolyzing, h in formulabIt is every
The peptide bond mM number of gram collagen raw material（It is 8.41）.
Claims (2)
1. a kind of method of utilization bacillus HS17 fermenting and producing Collagenases and its application, it is characterised in that it is included such as
Lower step：
（1）Actication of culture：Test tube slant preserve strain transfer activation medium in, under 37 DEG C, 200 rpm activation 18~
24h is muddy to thalline, and activated seed liquid is obtained；Activation medium used：Seed culture medium：5 g/L glucose, 4 g/L oxen
Meat extract, 1 g/L gelatin, 3 g/L NaCl, 1 g/L K2HPO4, 0.4 g/L MgSO4, 0.2 g/L MnSO4, pH7.2；
（2）Fermenting and producing：According to 3~6%（v/v）Inoculum concentration by activated seed liquid access fermentation medium in, in 200 rpm
Rotary shaker in cultivate, control temperature is cultivated and finished at 30~33 DEG C after 36~48 h of fermentation；Or in fermentation tank with
70% ratio load fermented and cultured component, through 118 DEG C of min of steam sterilizing 20 after, according to 3~6% under flame ring protective condition
（v/v）Ratio access the strain of activation after carry out fermented and cultured, the pH in control fermentation process maintains pH 6~7, adjusts
At 30~33 DEG C, dissolved oxygen maintains more than 6% to cultivation temperature, and fermented 30~42 h after fermentation is finished；The hair fermented after finishing
Zymotic fluid is centrifuged 6 min through 8000 r/min, is collected after removal thalline and retains supernatant, then again through the micropore filtering film of 0.22um
Filtration sterilization, i.e., use as liquid enzymes.
2. a kind of method of utilization bacillus HS17 fermenting and producing Collagenases as claimed in claim 1 and its application,
It is characterized in that described fermentation medium is 2~4 g/L gelatin, 40~50 g/L fructose, 3~6 g/L yeast extracts, 3
~5 g/L ZnSO4, 3~4 g/L MnSO4, 2~4 g/L FePO4.2H2O, 5~6 g/L (NH4)2SO4, 3~5 g/
L Na2HPO4, 4~6 g/L K2HPO4。
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