CN117106630A - Bacillus megaterium LY114 and application thereof in cheese making - Google Patents
Bacillus megaterium LY114 and application thereof in cheese making Download PDFInfo
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- CN117106630A CN117106630A CN202310856005.3A CN202310856005A CN117106630A CN 117106630 A CN117106630 A CN 117106630A CN 202310856005 A CN202310856005 A CN 202310856005A CN 117106630 A CN117106630 A CN 117106630A
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- 241000194107 Bacillus megaterium Species 0.000 title claims abstract description 32
- 235000013351 cheese Nutrition 0.000 title abstract description 18
- 108090000746 Chymosin Proteins 0.000 claims abstract description 83
- 229940080701 chymosin Drugs 0.000 claims abstract description 82
- GNOLWGAJQVLBSM-UHFFFAOYSA-N n,n,5,7-tetramethyl-1,2,3,4-tetrahydronaphthalen-1-amine Chemical compound C1=C(C)C=C2C(N(C)C)CCCC2=C1C GNOLWGAJQVLBSM-UHFFFAOYSA-N 0.000 claims abstract description 82
- 238000000855 fermentation Methods 0.000 claims abstract description 26
- 230000004151 fermentation Effects 0.000 claims abstract description 26
- 102000004190 Enzymes Human genes 0.000 claims description 16
- 108090000790 Enzymes Proteins 0.000 claims description 16
- 229940088598 enzyme Drugs 0.000 claims description 16
- 238000000034 method Methods 0.000 claims description 15
- 239000007788 liquid Substances 0.000 claims description 14
- 238000004108 freeze drying Methods 0.000 claims description 13
- 239000006228 supernatant Substances 0.000 claims description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 12
- 238000012258 culturing Methods 0.000 claims description 10
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 9
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 8
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 8
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 8
- 239000008367 deionised water Substances 0.000 claims description 8
- 229910021641 deionized water Inorganic materials 0.000 claims description 8
- 239000000047 product Substances 0.000 claims description 8
- 238000003756 stirring Methods 0.000 claims description 8
- 239000001963 growth medium Substances 0.000 claims description 7
- 229920002271 DEAE-Sepharose Polymers 0.000 claims description 6
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 claims description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 6
- 239000007853 buffer solution Substances 0.000 claims description 6
- 239000002609 medium Substances 0.000 claims description 6
- 238000002156 mixing Methods 0.000 claims description 6
- 239000002244 precipitate Substances 0.000 claims description 6
- 230000001954 sterilising effect Effects 0.000 claims description 6
- 238000002360 preparation method Methods 0.000 claims description 5
- 238000004321 preservation Methods 0.000 claims description 5
- 239000000843 powder Substances 0.000 claims description 4
- 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 3
- LEVWYRKDKASIDU-IMJSIDKUSA-N L-cystine Chemical compound [O-]C(=O)[C@@H]([NH3+])CSSC[C@H]([NH3+])C([O-])=O LEVWYRKDKASIDU-IMJSIDKUSA-N 0.000 claims description 3
- 239000004158 L-cystine Substances 0.000 claims description 3
- 235000019393 L-cystine Nutrition 0.000 claims description 3
- 241000209140 Triticum Species 0.000 claims description 3
- 235000021307 Triticum Nutrition 0.000 claims description 3
- 238000005349 anion exchange Methods 0.000 claims description 3
- 238000005571 anion exchange chromatography Methods 0.000 claims description 3
- 239000000872 buffer Substances 0.000 claims description 3
- 229940041514 candida albicans extract Drugs 0.000 claims description 3
- 239000011248 coating agent Substances 0.000 claims description 3
- 238000000576 coating method Methods 0.000 claims description 3
- 229960003067 cystine Drugs 0.000 claims description 3
- 238000000502 dialysis Methods 0.000 claims description 3
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims description 3
- 238000010828 elution Methods 0.000 claims description 3
- 238000007710 freezing Methods 0.000 claims description 3
- 230000008014 freezing Effects 0.000 claims description 3
- 239000008103 glucose Substances 0.000 claims description 3
- 239000002054 inoculum Substances 0.000 claims description 3
- 239000012528 membrane Substances 0.000 claims description 3
- 230000001376 precipitating effect Effects 0.000 claims description 3
- 108010009004 proteose-peptone Proteins 0.000 claims description 3
- 235000017557 sodium bicarbonate Nutrition 0.000 claims description 3
- 229910000030 sodium bicarbonate Inorganic materials 0.000 claims description 3
- 239000011780 sodium chloride Substances 0.000 claims description 3
- 239000012138 yeast extract Substances 0.000 claims description 3
- 229920002472 Starch Polymers 0.000 claims description 2
- 240000008042 Zea mays Species 0.000 claims description 2
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 2
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 2
- 235000005822 corn Nutrition 0.000 claims description 2
- 239000000203 mixture Substances 0.000 claims description 2
- 239000008107 starch Substances 0.000 claims description 2
- 235000019698 starch Nutrition 0.000 claims description 2
- 239000001166 ammonium sulphate Substances 0.000 claims 2
- 238000004659 sterilization and disinfection Methods 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 32
- 230000001580 bacterial effect Effects 0.000 abstract description 14
- 238000004519 manufacturing process Methods 0.000 abstract description 13
- 238000000746 purification Methods 0.000 abstract description 13
- 238000011031 large-scale manufacturing process Methods 0.000 abstract description 3
- 238000009776 industrial production Methods 0.000 abstract description 2
- 238000011160 research Methods 0.000 description 9
- 235000020183 skimmed milk Nutrition 0.000 description 9
- 235000013336 milk Nutrition 0.000 description 7
- 239000008267 milk Substances 0.000 description 7
- 210000004080 milk Anatomy 0.000 description 7
- 230000002797 proteolythic effect Effects 0.000 description 6
- 239000000243 solution Substances 0.000 description 6
- 235000013365 dairy product Nutrition 0.000 description 5
- 244000309466 calf Species 0.000 description 4
- 238000011161 development Methods 0.000 description 4
- 239000000758 substrate Substances 0.000 description 4
- 210000003165 abomasum Anatomy 0.000 description 3
- 229940042399 direct acting antivirals protease inhibitors Drugs 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 3
- 238000012545 processing Methods 0.000 description 3
- 238000012216 screening Methods 0.000 description 3
- 108020004465 16S ribosomal RNA Proteins 0.000 description 2
- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 description 2
- 108010076119 Caseins Proteins 0.000 description 2
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 102000005741 Metalloproteases Human genes 0.000 description 2
- 108010006035 Metalloproteases Proteins 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 108091005804 Peptidases Proteins 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- 239000004365 Protease Substances 0.000 description 2
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 2
- 239000008186 active pharmaceutical agent Substances 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 229910001424 calcium ion Inorganic materials 0.000 description 2
- 239000005018 casein Substances 0.000 description 2
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 description 2
- 235000021240 caseins Nutrition 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- NKLPQNGYXWVELD-UHFFFAOYSA-M coomassie brilliant blue Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=C1 NKLPQNGYXWVELD-UHFFFAOYSA-M 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
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- 230000007062 hydrolysis Effects 0.000 description 2
- 238000006460 hydrolysis reaction Methods 0.000 description 2
- 238000009629 microbiological culture Methods 0.000 description 2
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 239000012224 working solution Substances 0.000 description 2
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 241000193744 Bacillus amyloliquefaciens Species 0.000 description 1
- 244000063299 Bacillus subtilis Species 0.000 description 1
- 235000014469 Bacillus subtilis Nutrition 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 241000282832 Camelidae Species 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 1
- 241000283903 Ovis aries Species 0.000 description 1
- 102000012479 Serine Proteases Human genes 0.000 description 1
- 108010022999 Serine Proteases Proteins 0.000 description 1
- 239000005862 Whey Substances 0.000 description 1
- 108010046377 Whey Proteins Proteins 0.000 description 1
- 102000007544 Whey Proteins Human genes 0.000 description 1
- YVNQAIFQFWTPLQ-UHFFFAOYSA-O [4-[[4-(4-ethoxyanilino)phenyl]-[4-[ethyl-[(3-sulfophenyl)methyl]amino]-2-methylphenyl]methylidene]-3-methylcyclohexa-2,5-dien-1-ylidene]-ethyl-[(3-sulfophenyl)methyl]azanium Chemical compound C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C(=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S(O)(=O)=O)C)C=2C(=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S(O)(=O)=O)C)C=C1 YVNQAIFQFWTPLQ-UHFFFAOYSA-O 0.000 description 1
- 230000002053 acidogenic effect Effects 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 235000003704 aspartic acid Nutrition 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 235000019634 flavors Nutrition 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 230000005484 gravity Effects 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- FAXGPCHRFPCXOO-LXTPJMTPSA-N pepstatin A Chemical compound OC(=O)C[C@H](O)[C@H](CC(C)C)NC(=O)[C@H](C)NC(=O)C[C@H](O)[C@H](CC(C)C)NC(=O)[C@H](C(C)C)NC(=O)[C@H](C(C)C)NC(=O)CC(C)C FAXGPCHRFPCXOO-LXTPJMTPSA-N 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 239000012898 sample dilution Substances 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 238000010563 solid-state fermentation Methods 0.000 description 1
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- 230000009897 systematic effect Effects 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 230000035899 viability Effects 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
- 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
- C12N1/205—Bacterial isolates
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
- A23C19/00—Cheese; Cheese preparations; Making thereof
- A23C19/02—Making cheese curd
- A23C19/032—Making cheese curd characterised by the use of specific microorganisms, or enzymes of microbial origin
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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
- 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
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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)
- C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
- C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
- C12N9/6478—Aspartic endopeptidases (3.4.23)
- C12N9/6483—Chymosin (3.4.23.4), i.e. rennin
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
- C12Y304/23—Aspartic endopeptidases (3.4.23)
- C12Y304/23004—Chymosin (3.4.23.4), i.e. rennin
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
- C12R2001/11—Bacillus megaterium
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- General Engineering & Computer Science (AREA)
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- Virology (AREA)
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Abstract
Bacillus megaterium LY114 and application thereof belong to the technical field of biology. The invention provides a novel bacillus megatherium LY114 on one hand and the application of the bacillus megatherium LY114 on the other hand. The chymosin produced by the screened bacillus megatherium has higher activity, higher specific activity, higher yield and higher purification ratio after purification, can improve the purification efficiency of chymosin, greatly shortens the fermentation period, quickens the production speed, has low cost, can better meet the requirement of large-scale production of later cheese due to the characteristics, and provides important basis for further realizing the industrial production of bacterial chymosin.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to bacillus megatherium LY114 and application thereof in cheese making.
Background
Chymosin is a key enzyme preparation in cheese making and dairy processing. Chymosin can coagulate milk and affect the yield, texture, flavor, etc. of cheese. Traditional chymosin is mainly derived from animals such as calves, lambs, camels and the like. The most commonly used traditional chymosin is calf abomasum, which can be obtained from the fourth abomasum of a mammalian calf. However, the traditional chymosin has the disadvantages of long growth period, high production cost and the like, and great interest is brought to searching for a good-quality substitute of the traditional chymosin. At present, the research on chymosin in China is relatively lagged, the chymosin is mainly supplied by foreign production, and only some scientific research institutions in China perform systematic research on chymosin. The prior cheese production industry in China does not form large-scale production, and occupies less specific gravity in domestic dairy product consumption, so that the research on the production and processing of the prior cheese is relatively less. With the continuous expansion of the domestic cheese market and the development of the domestic food processing industry, the worldwide demand for chymosin will further increase. Therefore, the yield of chymosin is improved, the product quality is improved, and the chymosin is an important factor for promoting the development of the milk industry in China.
Compared with chymosin of plant source and animal source, the chymosin of microorganism source has low production cost, easy extraction, high economic benefit and wider biochemical diversity, and can well solve the problem of insufficient calf abomasum enzyme supply. At present, there are few researches on bacterial chymosin, and the related researches take bacillus subtilis and bacillus amyloliquefaciens as research objects, and the problems of low chymosin storage activity, high proteolytic activity and the like become bottlenecks affecting research, development and utilization of the bacterial chymosin. Compared with the solid state fermentation of fungi, the immersed fermentation of bacteria has obvious advantages in the aspects of controlling the fermentation degree and the material utilization rate, and simultaneously, the problem of lower MCA/PA ratio of the bacterial chymosin can be effectively solved by screening new strains and optimizing the fermentation process. Accordingly, there is also increasing research on bacterial chymosin.
The bacterial strain of high-yield chymosin is screened from the high-quality raw materials, an optimal fermentation process is determined, and the chymosin characteristics of the bacterial chymosin are analyzed to determine whether the chymosin characteristics meet the requirements of the later cheese production, so that the method has important significance for enriching chymosin-producing bacterial strain libraries and expanding development and production of chymosin-producing bacterial strain libraries. The chymosin separated from natural raw materials has low activity and long fermentation period, and has great loss of enzyme activity in the purification process, and the characteristics of the chymosin can not well meet the requirements of cheese production.
Disclosure of Invention
Aiming at the problems existing in the prior art, the invention aims to design and provide bacillus megatherium LY114 and a technical scheme applied to cheese making. The bacillus megatherium LY114 and the application thereof in cheese making have the characteristics of high chymosin activity, short fermentation time, high purification ratio and the like, and can be used for preparing chymosin with excellent properties to meet the cheese production requirement.
The invention is realized by the following technical scheme:
in a first aspect, the invention provides a strain of Bacillus megaterium LY114, classified under the name Bacillus megaterium. The strain is preserved in China general microbiological culture Collection center (CGMCC) in 03 and 03 days 2023, and has the preservation number of China center for China including national institute of microbiology (CGMCC) No. 3 of North West Lu No. 1, the Korean area of Beijing, and the preservation number of China center: CGMCC No.26730.
The strain is separated and screened from soil in milking area of dairy farm in Laiyang city in Shandong province.
The strain screening method comprises the following steps: the strain with chymosin producing capacity is determined according to the conditions of precipitation rings and hydrolysis rings formed by each strain on a casein plate in more than 90 strains screened in Ningxia, yunnan, heilongjiang, shandong and other dairy farms. The bacterial strain with chymosin producing capacity is further analyzed, and bacterial strain with relatively large ratio is selected. Fermenting and culturing the selected strains, measuring and analyzing the proteolytic activity of the protease at different fermentation times, and comprehensively comparing the chymosin activity, the proteolytic activity and the pH value of skim milk after chymosin.
In a second aspect, the invention provides the use of Bacillus megaterium LY114 in the preparation of chymosin.
In a third aspect, the present invention provides a method for preparing chymosin using Bacillus megaterium LY114, comprising the steps of:
1) Coating and streaking Bacillus megaterium LY114, culturing for 12h, and then inoculating in a modified TYC culture medium, and shake culturing at 37 ℃ and 180r/min for 10-12 h to obtain seed liquid;
2) Inoculating the seed liquid into a bran culture medium according to the inoculum size of 5% by volume, shake-culturing at 37 ℃ and 180r/min for 36-48 hours to obtain a fermentation liquid, and centrifuging the obtained fermentation liquid at a low temperature to obtain a supernatant;
3) Mixing ammonium sulfate with the supernatant obtained in the step 2), precipitating for 2 hours at 4 ℃, centrifuging at a low temperature and high speed, discarding the supernatant, collecting the precipitate, redissolving the precipitate in 20mmol/L Tris-HCl buffer solution with pH of 7.0, stirring and dialyzing for 12 hours at 4 ℃, and freeze-drying to obtain crude chymosin powder;
4) Dissolving crude enzyme in 20mmol/L Tris-HCl buffer solution with pH of 7.0, passing through 0.22 μm filter membrane, separating and purifying by DEAE-Sepharose Fast Flow anion exchange chromatography, dialyzing, and lyophilizing to obtain chymosin product.
Further, the modified TYC medium in step 1) is: glucose 50g, casein peptone 15g, sodium chloride 1g, sodium bicarbonate 2g, L-cystine 0.2g, disodium hydrogen phosphate 2g, yeast extract 5g, deionized water to 1000mL, sterilizing at 115 ℃ for 20min; the bran culture medium in the step 2) is as follows: 1000mL of deionized water, 20-60 g of cooked wheat fine bran and sterilizing for 20min at 115 ℃.
Further, the saturation of ammonium sulfate in the mixed solution formed by mixing the ammonium sulfate in the step 3) with the supernatant is 60%.
Further, the freeze-drying parameters in step 3) and the freeze-drying parameters in step 4) are: pre-freezing at-80deg.C for 300min; freeze-drying at-80deg.C for 800min.
Further, the DEAE-Sepharose Fast Flow anion exchange column in step 4) was 5mL in volume, and the initial elution condition was 20mmol/L Tris-HCl buffer pH 8.0 at a flow rate of 0.5mL/min.
Further, the dialysis conditions in step 4) are: and (3) stirring and dialyzing at 4 ℃ for 12 hours.
In a fourth aspect the invention provides chymosin produced by any of the methods described above.
Chymosin activity according to the present invention means: the amount of enzyme in the aggregated l mL 10% skim milk powder for 40min was defined as one activity unit SU (Soxhlet unit).
Curd activity (SU) = (number of milk tested/chymosin amount) ×2400/t×n, where: 2400 min converted to seconds; n is dilution multiple; t is the curd time, s.
On the basis of conforming to the common knowledge in the field, the above preferred conditions can be arbitrarily combined to obtain the preferred examples of the invention.
The reagents and materials used in the present invention are commercially available.
The invention has the beneficial effects that: the Bacillus megaterium LY114 screened by the invention can reach the maximum curd activity by fermenting for 48 hours after 10-12 hours shake culture, can reach more than 440SU/mL, has the specific enzyme activity of 7532SU/mg after purification, has the yield of 34.17 percent and the purification ratio of 4.83. In addition, the curd activity is low at a lower pH of the substrate concentration, so that the chymosin is easier to discharge in the post-production period of cheese.
Therefore, the chymosin produced by the bacillus megatherium LY114 screened by the invention has higher activity, higher specific activity, higher yield and higher purification rate after purification, can improve the purification efficiency of chymosin, greatly shortens the fermentation period, accelerates the production speed, has low cost, and has the characteristics of better meeting the requirement of large-scale production of later cheese, thereby providing an important basis for further realizing the industrial production of bacterial chymosin.
Drawings
FIG. 1 B.megaterium LY114 16S rDNA phylogenetic tree;
FIG. 2 is a graph showing the pH stability of LY114 chymosin;
FIG. 3 is a graph showing the thermostability of LY114 chymosin;
FIG. 4 is a graph showing the effect of substrate calcium ion concentration on LY114 chymosin activity;
FIG. 5 is a curd form of LY114 chymosin;
FIG. 6 shows a cheese made with LY114 chymosin.
Detailed Description
The invention is further illustrated by way of examples which follow, but are not thereby limited to the examples described. The experimental methods, in which specific conditions are not noted in the following examples, were selected according to conventional methods and conditions, or according to the commercial specifications.
The test methods used in the examples are regarded as conventional methods unless otherwise specified; the materials, reagents and the like used, unless otherwise specified, are all commercially available.
The procedure for Measuring Chymosin Activity (MCA) in the examples is as follows:
v taking s The undiluted or diluted Bacillus megaterium fermentation supernatant with a certain multiple of 0.05 mol/L, pH 7.0.0 PBS is incubated in a water bath at 35deg.C for 10 min, and added to V E Volume of 10 mM CaCl 2 In 10% (w/v) skim milk solution, samples were taken every 15. 15 s and observed for sample status at 45 ° angles, and if there was no change, rapidly returned to the water bath, the timing was stopped when discrete particles were formed, and recorded as time T.
MCA＝(2400×V S ×N)/(T×V E ). Wherein: MCA is chymosin activity (SU/mL); v (V) s For skimmed milk substrate volume (mL), V E Enzyme liquid volume (mL), T is curd time(s), N is MCE dilution factor.
In the examples, the protein content of the sample was determined by the coomassie brilliant blue method and a kjeldahl apparatus, wherein the coomassie brilliant blue method is as follows:
the preparation method of the Bradford working solution comprises the following steps: 100 mg Coomassie brilliant blue G250, 40 mL 95% ethanol, 100 mL 85% phosphoric acid, deionized water to a volume of 1L; drawing a protein sample standard curve; add 20. Mu.L of sample dilution and 200. Mu.L of Bradford working solution, mix rapidly, react at 25-30℃for 5min, and then measure A595 values for each well on a microplate reader.
Example 1: acquisition of Bacillus megaterium LY114
The strain with chymosin producing capacity is determined according to the conditions of precipitation rings and hydrolysis rings formed by each strain on a casein plate in 90 strains obtained by screening in Ningxia, yunnan, heilongjiang, shandong and other dairy farms, wherein the morphology and curd potential of part of preferred strains are shown in Table 1. The bacterial strain with chymosin producing capacity is further analyzed, and bacterial strain with relatively large ratio is selected. And (3) fermenting and culturing the selected strains, measuring and analyzing the proteolytic activity of the protease at different fermentation times, and selecting the strain with high chymosin activity and low proteolytic activity and non-acidogenic characteristic from the chymosin, the proteolytic activity and the pH value of the skim milk after chymosin. Through 16S rDNA sequencing, drawing homology phylogenetic tree (see figure 1), API 20E physicochemical experiment, API 50 CHB identification and enzyme production condition rescreening, a bacillus megatherium LY114 with high chymosin yield is obtained, a login number of ON714523 is obtained, and the bacillus megatherium LY114 is preserved in China general microbiological culture collection center with a preservation number of CGMCC No.26730.
Table 1 morphology and curd potential of part of the preferred strains
Note that: positive for "+"; "-" is negative.
Example 2
(1) Coating and streaking Bacillus megaterium LY114 for 12h, inoculating to an improved TYC culture medium, and shake culturing at 37 ℃ for 10-12 h at 180r/min to obtain seed liquid, wherein the strain is Bacillus megaterium (Bacillus megaterium) LY114 with a preservation number of CGMCC No.26730; the modified TYC medium was: glucose 50g, casein peptone 15g, sodium chloride 1g, sodium bicarbonate 2g, L-cystine 0.2g, disodium hydrogen phosphate 2g, yeast extract 5g, deionized water to 1000mL, sterilizing at 115 ℃ for 20min;
(2) Inoculating the seed liquid into a fermentation culture medium with an inoculum size of 5%, shake culturing at 37 ℃ and 180r/min for 36-48 hours to obtain a fermentation liquid, centrifuging the obtained fermentation liquid at a low temperature, and taking supernatant, wherein the volume percentage of the fermentation liquid is as follows: 1000mL of deionized water, 60g of cooked wheat fine bran, 10g of soluble starch, 3g of corn steep liquor and sterilizing at 115 ℃ for 20min;
(3) Mixing ammonium sulfate with the supernatant obtained in the step (2), precipitating for 2 hours at 4 ℃ with the saturation degree of ammonium sulfate being 60%, centrifuging at low temperature and high speed, discarding the supernatant, collecting the precipitate, redissolving the precipitate in 20mmol/L Tris-HCl buffer solution with the pH of 7.0, stirring and dialyzing for 12 hours at 4 ℃, and obtaining crude chymosin powder after freeze drying under the following freeze drying conditions: pre-freezing at-80deg.C for 300min; freeze-drying at-80deg.C for 800min;
(4) Dissolving crude enzyme in Tris-HCl buffer solution with the pH of 20mmol/L and 7.0, passing through a 0.22 mu m filter membrane, separating and purifying by DEAE-Sepharose Fast Flow anion exchange chromatography, dialyzing, and lyophilizing to obtain chymosin product, wherein the lyophilization condition is-80 ℃ prefreezing for 300min; freeze-drying at-80 ℃ for 800min, DEAE-Sepharose Fast Flow anion exchange column volume of 5mL, initial elution condition of 20mmol/L Tris-HCl buffer pH 8.0, flow rate of 0.5mL/min, dialysis condition: stirring and dialyzing for 12h at 4 ℃;
(5) And (3) dissolving the chymosin product obtained in the step (4) in deionized water, and exploring the excellent properties of the bacillus megaterium chymosin in the later cheese making process.
On the basis of example 2, different fermentation times were used for comparison.
TABLE 2 variation of MCA of Bacillus megaterium LY114 MCE with fermentation time
The results in Table 2 demonstrate that the curd viability reached a maximum of 448SU/ml when LY114 was fermented for 48 hours with modified TYC as the medium. Meanwhile, the LY114 strain has higher natural fermentation chymosin activity than common bacillus, so the fermentation time selected by the invention is 48 hours.
Example 3
TABLE 3 purification of chymosin
As shown in Table 3, after the preparation method of the Bacillus megaterium chymosin of the embodiment is adopted on the basis of the fermentation medium of the embodiment 2, the specific enzyme activity is 7532SU/mg, the yield is 34.17%, the purification ratio reaches 4.83, and the specific enzyme activity, the yield and the purification ratio are all improved after chymosin is purified.
Example 4
TABLE 4 Effect of different protease inhibitors on LY114 MCE
As shown in table 3, LY114 chymosin can be significantly inhibited by EDTA (1-25 mM) without being affected by other protease inhibitors. Different protease inhibitors may determine the type of enzyme. Pepstatin a, EDTA, PMSF and DTT are aspartic acid, metalloprotease, serine protease and cysteine inhibitors, respectively. The results indicate that LY114 chymosin is a metalloprotease.
Example 5
And (3) taking chymosin finished products, respectively preparing skim milk solutions with pH values of 5.5, 6.0, 6.5, 7.0 and 7.5, and measuring chymosin activity under different pH conditions. As a result, as shown in FIG. 2, the chymosin enzyme activity gradually decreased as the pH increased, and was substantially deactivated when the pH reached 7.5, and the relative enzyme activity was maximized in the skim milk solution at pH5.5, and the curd morphology was shown in FIG. 5.
Example 6
Taking chymosin finished products, respectively incubating for 10, 20, 30, 40 and 50min in water baths at 35, 40, 45, 50, 55 and 60 ℃ respectively, and measuring chymosin activity by taking skim milk solution as a substrate. As a result, as shown in FIG. 3, when the temperature was increased to 45℃and the enzyme activity tended to be significantly decreased, the enzyme activity was almost completely disappeared in the water bath at 60℃for 30 minutes.
Example 7
Providing chymosin product, and preparing with 0, 10, 20, 30, 40, 50, 60, 70, 80, 90 and 100mM CaCl 2 And (3) measuring chymosin activity at different calcium ion concentrations. As shown in FIG. 4, the curd activity was improved in the presence of 30mM Ca 2+ Maximum is reached in the skim milk solution of (2) and is 1.8 times that of the control group.
Example 8
Pasteurizing milk, cooling, adding activated starter, fermenting and pre-acidifying. Adding 0.1g/L CaCl when the pH of milk is reduced to 6.3 2 And adding chymosin solution immediately after uniformly mixing, and waiting for curd. After the curd is finished, it is cut into uniform milk pieces and left to stand for 3-5min. Stirring and heating, cutting the aggregated milk into small pieces after the whey is discharged, adding salt, and stirring uniformly. Finally, it was molded and allowed to mature at 6℃for 14 days, and the results are shown in FIG. 6.
Claims (9)
1. Bacillus megaterium (Bacillus megaterium) LY114 with a preservation number of CGMCC No.26730.
2. Use of bacillus megatherium LY114 of claim 1 in the preparation of chymosin.
3. A method for preparing chymosin using bacillus megatherium LY114 of claim 1, comprising the steps of:
1) Coating and streaking Bacillus megaterium LY114, culturing for 12h, and then inoculating in a modified TYC culture medium, and shake culturing at 37 ℃ and 180r/min for 10-12 h to obtain seed liquid;
2) Inoculating the seed liquid into a fermentation culture medium according to the inoculum size of 5% by volume, shake-culturing at 37 ℃ and 180r/min for 36-48 hours to obtain a fermentation liquid, and centrifuging the obtained fermentation liquid at a low temperature to obtain a supernatant;
3) Mixing ammonium sulfate with the supernatant obtained in the step 2), precipitating for 2 hours at 4 ℃, centrifuging at a low temperature and high speed, discarding the supernatant, collecting the precipitate, redissolving the precipitate in 20mmol/L Tris-HCl buffer solution with pH of 7.0, stirring and dialyzing for 12 hours at 4 ℃, and freeze-drying to obtain crude chymosin powder;
4) Dissolving crude enzyme in 20mmol/L Tris-HCl buffer solution with pH of 7.0, passing through 0.22 μm filter membrane, separating and purifying by DEAE-Sepharose Fast Flow anion exchange chromatography, dialyzing, and lyophilizing to obtain chymosin product.
4. A method according to claim 3, wherein the modified TYC medium of step 1) is: glucose 50g, casein peptone 15g, sodium chloride 1g, sodium bicarbonate 2g, L-cystine 0.2g, disodium hydrogen phosphate 2g, yeast extract 5g, deionized water to 1000mL, sterilizing at 115 ℃ for 20min; the fermentation medium in the step 2) is as follows: 1000mL of deionized water, 20-60 g of cooked wheat fine bran, 10g of soluble starch, 3g of corn steep liquor and 20min of sterilization at 115 ℃.
5. A method according to claim 3, wherein the ammonium sulphate saturation in the mixture formed by mixing the ammonium sulphate with the supernatant in step 3) is 60%.
6. A method according to claim 3, wherein the lyophilization in step 3) and the lyophilization parameters in step 4) are: pre-freezing at-80deg.C for 300min; freeze-drying at-80deg.C for 800min.
7. The method according to claim 3, wherein the DEAE-Sepharose Fast Flow anion exchange column in step 4) has a volume of 5mL, and the initial elution condition is 20mmol/L Tris-HCl buffer pH 8.0 at a flow rate of 0.5mL/min.
8. A method according to claim 3, characterized in that the dialysis conditions in step 4) are: and (3) stirring and dialyzing at 4 ℃ for 12 hours.
9. Chymosin obtainable by a process according to any of claims 3 to 8.
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