CN118360183A - Bacillus subtilis for high yield of nattokinase and acetoin and application thereof - Google Patents
Bacillus subtilis for high yield of nattokinase and acetoin and application thereof Download PDFInfo
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- CN118360183A CN118360183A CN202410093664.0A CN202410093664A CN118360183A CN 118360183 A CN118360183 A CN 118360183A CN 202410093664 A CN202410093664 A CN 202410093664A CN 118360183 A CN118360183 A CN 118360183A
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- ROWKJAVDOGWPAT-UHFFFAOYSA-N Acetoin Chemical compound CC(O)C(C)=O ROWKJAVDOGWPAT-UHFFFAOYSA-N 0.000 title claims abstract description 56
- 244000063299 Bacillus subtilis Species 0.000 title claims abstract description 38
- 235000014469 Bacillus subtilis Nutrition 0.000 title claims abstract description 38
- GFAZHVHNLUBROE-UHFFFAOYSA-N hydroxymethyl propionaldehyde Natural products CCC(=O)CO GFAZHVHNLUBROE-UHFFFAOYSA-N 0.000 title claims abstract description 28
- 108010073682 nattokinase Proteins 0.000 title claims abstract description 18
- 229940086319 nattokinase Drugs 0.000 title claims abstract description 16
- 238000000855 fermentation Methods 0.000 claims abstract description 44
- 230000004151 fermentation Effects 0.000 claims abstract description 44
- 238000004519 manufacturing process Methods 0.000 claims abstract description 9
- 238000004321 preservation Methods 0.000 claims abstract description 8
- 244000005700 microbiome Species 0.000 claims abstract description 7
- 239000001963 growth medium Substances 0.000 claims description 48
- 238000009630 liquid culture Methods 0.000 claims description 45
- 239000007788 liquid Substances 0.000 claims description 39
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 21
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 claims description 14
- 239000008367 deionised water Substances 0.000 claims description 12
- 229910021641 deionized water Inorganic materials 0.000 claims description 12
- 239000002054 inoculum Substances 0.000 claims description 11
- 238000012258 culturing Methods 0.000 claims description 10
- 239000002609 medium Substances 0.000 claims description 10
- 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 8
- 239000008103 glucose Substances 0.000 claims description 8
- 239000007787 solid Substances 0.000 claims description 8
- 239000006228 supernatant Substances 0.000 claims description 8
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 7
- 244000068988 Glycine max Species 0.000 claims description 7
- 235000010469 Glycine max Nutrition 0.000 claims description 7
- 239000001110 calcium chloride Substances 0.000 claims description 7
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 7
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims description 7
- 238000011081 inoculation Methods 0.000 claims description 7
- 229910001629 magnesium chloride Inorganic materials 0.000 claims description 7
- 229910000403 monosodium phosphate Inorganic materials 0.000 claims description 7
- 235000019799 monosodium phosphate Nutrition 0.000 claims description 7
- 235000013557 nattō Nutrition 0.000 claims description 7
- 238000002360 preparation method Methods 0.000 claims description 7
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 claims description 7
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 4
- 235000019764 Soybean Meal Nutrition 0.000 claims description 4
- 235000002639 sodium chloride Nutrition 0.000 claims description 4
- 238000010563 solid-state fermentation Methods 0.000 claims description 4
- 239000004455 soybean meal Substances 0.000 claims description 4
- 239000001888 Peptone Substances 0.000 claims description 3
- 108010080698 Peptones Proteins 0.000 claims description 3
- 229930006000 Sucrose Natural products 0.000 claims description 3
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 3
- 238000001816 cooling Methods 0.000 claims description 3
- 235000019319 peptone Nutrition 0.000 claims description 3
- 239000002994 raw material Substances 0.000 claims description 3
- 238000002791 soaking Methods 0.000 claims description 3
- 239000011780 sodium chloride Substances 0.000 claims description 3
- 238000010025 steaming Methods 0.000 claims description 3
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- 238000002798 spectrophotometry method Methods 0.000 description 6
- 239000005018 casein Substances 0.000 description 5
- 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 5
- 235000021240 caseins Nutrition 0.000 description 5
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- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
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- 229910052751 metal Inorganic materials 0.000 description 3
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- KJCVRFUGPWSIIH-UHFFFAOYSA-N 1-naphthol Chemical compound C1=CC=C2C(O)=CC=CC2=C1 KJCVRFUGPWSIIH-UHFFFAOYSA-N 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 2
- 229920000936 Agarose Polymers 0.000 description 2
- 108010049003 Fibrinogen Proteins 0.000 description 2
- 102000008946 Fibrinogen Human genes 0.000 description 2
- 239000012880 LB liquid culture medium Substances 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- 108090000190 Thrombin Proteins 0.000 description 2
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- 239000003153 chemical reaction reagent Substances 0.000 description 2
- CVSVTCORWBXHQV-UHFFFAOYSA-N creatine Chemical compound NC(=[NH2+])N(C)CC([O-])=O CVSVTCORWBXHQV-UHFFFAOYSA-N 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 238000003028 enzyme activity measurement method Methods 0.000 description 2
- 229940012952 fibrinogen Drugs 0.000 description 2
- 239000003527 fibrinolytic agent Substances 0.000 description 2
- 239000000796 flavoring agent Substances 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
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- 239000000203 mixture Substances 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 229960004072 thrombin Drugs 0.000 description 2
- 230000002537 thrombolytic effect Effects 0.000 description 2
- 238000010200 validation analysis Methods 0.000 description 2
- 201000001320 Atherosclerosis Diseases 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 101100010253 Bacillus subtilis (strain 168) dnaN gene Proteins 0.000 description 1
- 241000186016 Bifidobacterium bifidum Species 0.000 description 1
- 101100039285 Clostridium perfringens (strain 13 / Type A) rpsM gene Proteins 0.000 description 1
- 108700039887 Essential Genes Proteins 0.000 description 1
- 101100419195 Leptospira borgpetersenii serovar Hardjo-bovis (strain L550) rpsC2 gene Proteins 0.000 description 1
- 101100363550 Leptospira borgpetersenii serovar Hardjo-bovis (strain L550) rpsE2 gene Proteins 0.000 description 1
- 101100529965 Leptospira borgpetersenii serovar Hardjo-bovis (strain L550) rpsK2 gene Proteins 0.000 description 1
- 101100088535 Leptospira interrogans serogroup Icterohaemorrhagiae serovar Lai (strain 56601) rplP gene Proteins 0.000 description 1
- 101100037096 Methanococcus maripaludis (strain S2 / LL) rpl6 gene Proteins 0.000 description 1
- 101100038261 Methanococcus vannielii (strain ATCC 35089 / DSM 1224 / JCM 13029 / OCM 148 / SB) rpo2C gene Proteins 0.000 description 1
- 101100200099 Methanopyrus kandleri (strain AV19 / DSM 6324 / JCM 9639 / NBRC 100938) rps13 gene Proteins 0.000 description 1
- 101100254826 Methanopyrus kandleri (strain AV19 / DSM 6324 / JCM 9639 / NBRC 100938) rps5 gene Proteins 0.000 description 1
- 241001195348 Nusa Species 0.000 description 1
- 101150078442 RPL5 gene Proteins 0.000 description 1
- 101150102982 RpS10 gene Proteins 0.000 description 1
- 108010022999 Serine Proteases Proteins 0.000 description 1
- 102000012479 Serine Proteases Human genes 0.000 description 1
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- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 1
- 101150003155 dnaG gene Proteins 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
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- 238000003209 gene knockout Methods 0.000 description 1
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- 101150077178 infC gene Proteins 0.000 description 1
- 238000002386 leaching Methods 0.000 description 1
- 230000031700 light absorption Effects 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- 101150073438 nusA gene Proteins 0.000 description 1
- 101150047627 pgk gene Proteins 0.000 description 1
- 101150079312 pgk1 gene Proteins 0.000 description 1
- 101150095149 pgkA gene Proteins 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
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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
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L11/00—Pulses, i.e. fruits of leguminous plants, for production of food; Products from legumes; Preparation or treatment thereof
- A23L11/50—Fermented pulses or legumes; Fermentation of pulses or legumes based on the addition of microorganisms
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
- C12N9/54—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea bacteria being Bacillus
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- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/24—Preparation of oxygen-containing organic compounds containing a carbonyl group
- C12P7/26—Ketones
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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)
- C12Y304/21—Serine endopeptidases (3.4.21)
- C12Y304/21062—Subtilisin (3.4.21.62)
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- 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
- C12R2001/125—Bacillus subtilis ; Hay bacillus; Grass bacillus
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Abstract
The invention discloses bacillus subtilis for high yield of nattokinase and acetoin and application thereof, and belongs to the technical field of microorganisms. The bacillus subtilis is preserved in the collection center of Chinese classical collection microorganisms in 2023, 9 and 11 days, the preservation address is the university of Wuhan, jiu, hubei province, and the preservation number is CCTCC NO: m20231585. The strain has the characteristics of high yield of nattokinase and acetoin, the enzyme production level is improved by 167.3% compared with the wild strain, and the acetoin production level is improved by 86.1%. Meanwhile, the strain has simple fermentation nutrition conditions, is easy to culture, can be stably passaged, and has great commercial application potential.
Description
Technical Field
The invention belongs to the field of microorganisms, and particularly relates to bacillus subtilis for high yield of nattokinase and acetoin and application thereof.
Background
Nattokinase (Nattokinase, NK), also known as subtilisin NAT (subtilisin NAT), is a serine protease produced by Bacillus subtilis during fermentation, and NK has been widely studied and used for its unique thrombolytic function since it was discovered in 1980. Compared with the traditional thrombolytic drugs, NK not only can hydrolyze thrombus into amino acid and small molecular peptide, but also can inhibit thrombus formation through an indirect thrombolytic mechanism, and simultaneously has the characteristics of high safety, various pharmacological actions such as improving blood circulation, reducing blood pressure, preventing atherosclerosis and the like. In a word, NK is a novel thrombolytic agent with great application potential, and has been widely used in functional foods, health products and common foods based on the advantages of safety, effectiveness, oral administration, mass production and the like.
Acetoin is a widely used flavour compound and is also a metabolic substance that can be produced by a variety of microorganisms. Because of the special cream flavor, the acetoin has important application in the industries of foods, spices, cosmetics and the like, and can be used as an intermediate for synthesizing medicaments, thereby having important application in the synthesis of medicaments.
At present, the yield and enzyme activity of NK are difficult to meet the production requirement, and screening of microorganism strains with high NK yield is an important research direction of nattokinase production enterprises. Meanwhile, bacillus has an ability to produce acetoin, but acetoin is generally used as a fermentation byproduct and has a low yield.
Disclosure of Invention
This section is intended to outline some aspects of embodiments of the application and to briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section as well as in the description of the application and in the title of the application, which may not be used to limit the scope of the application.
The present invention has been made in view of the above and/or problems occurring in the prior art.
Therefore, the invention aims to overcome the defects in the prior art and provide the bacillus subtilis (Bacilussubtilis) JNC002.001 which is preserved in the China collection microbiological culture Collection center (China center for type culture collection) for 9 and 4 days in 2023, wherein the preservation number is CCTCC NO: m20231585, the preservation address is China university of Wuhan.
The invention also aims to overcome the defects in the prior art and provide the application of the bacillus subtilis JNC002.001 in preparation of acetoin.
As a preferred embodiment of the application according to the invention, wherein: inoculating bacillus subtilis JNC002.001 into a primary seed liquid for culture to obtain a primary seed liquid culture;
Transferring the first-level seed liquid culture to the second-level seed liquid culture for expansion culture to obtain a second-level seed liquid culture;
Inoculating the secondary seed liquid culture into a high-sugar culture medium for fermentation, and centrifuging to obtain a supernatant containing acetoin.
As a preferred embodiment of the application according to the invention, wherein: inoculating the bacillus subtilis JNC002.001 into a primary seed liquid for culture, wherein the primary seed liquid is an LB culture medium, and the culture parameters are as follows: shaking culture at 37 deg.c and 200rpm for 8-10 hr; the first-stage seed liquid culture is transferred to the second-stage seed liquid culture for expansion culture, wherein the second-stage seed liquid is LB culture medium, and the culture parameters are as follows: shaking culture is carried out for 2.5 to 3.5 hours at 37 ℃ and 200rpm, and the inoculation amount is 2 weight percent.
As a preferred embodiment of the application according to the invention, wherein: the high-sugar culture medium comprises the following components in percentage by weight: 10% of glucose, 2% of soybean peptone, 0.2% of disodium hydrogen phosphate, 0.1% of sodium dihydrogen phosphate, 0.05% of magnesium chloride, 0.02% of calcium chloride and the balance of deionized water, wherein the pH value is 7-7.5.
As a preferred embodiment of the application according to the invention, wherein: the secondary seed liquid culture is inoculated into a high-sugar culture medium for fermentation, wherein the fermentation condition is 200-250 rpm, the culture time is 36h, and the inoculation amount of the secondary seed liquid culture is 2-4wt%.
It is a further object of the present invention to overcome the deficiencies in the prior art and to provide the use of bacillus subtilis in the production of nattokinase, including,
Inoculating bacillus subtilis JNC002.001 into the primary seed liquid for culture to obtain a primary seed liquid culture;
Transferring the first-level seed liquid culture to the second-level seed liquid culture for expansion culture to obtain a second-level seed liquid culture;
Inoculating the secondary seed liquid culture into a fermentation medium for fermentation, centrifuging and taking supernatant to obtain nattokinase liquid.
As a preferred embodiment of the application according to the invention, wherein: inoculating the bacillus subtilis JNC002.001 into a primary seed liquid for culture, wherein the primary seed liquid is 4mL of LB culture medium, and the culture parameters are as follows: shaking culture is carried out for 8-10 h at 37 ℃ and 200 rpm.
As a preferred embodiment of the application according to the invention, wherein: the primary seed liquid culture is transferred to the secondary seed liquid culture for expansion culture, wherein the secondary seed liquid is 50mL of LB culture medium, and the culture parameters are as follows: shaking culture is carried out at 37 ℃ and 200rpm for 2.5-3.5 hours, and the inoculation amount is 2wt%.
As a preferred embodiment of the application according to the invention, wherein: the fermentation medium comprises the following components in percentage by weight: 2.5% of soybean meal, 2% of glucose, 0.2% of disodium hydrogen phosphate, 0.1% of sodium dihydrogen phosphate, 0.05% of magnesium chloride, 0.02% of calcium chloride and the balance of deionized water, wherein the pH value is 7-7.5.
As a preferred embodiment of the application according to the invention, wherein: the secondary seed liquid culture is inoculated into a fermentation culture medium for fermentation, wherein the fermentation condition is 200-250 rpm, the temperature is 37 ℃, and the culture time is 36-60 h.
As a preferred embodiment of the application according to the invention, wherein: the inoculation amount of the secondary seed liquid culture is 2-4wt%.
It is still another object of the present invention to overcome the disadvantages of the prior art, and to provide an application of the bacillus subtilis JNC002.001 in the preparation of natto, including,
Seed liquid preparation: inoculating bacillus subtilis JNC002.001 lawn into a liquid seed culture medium, and culturing at 37 ℃ and 200rpm for 8 hours to obtain seed liquid;
Fermentation of a solid culture medium: taking soybean raw materials, washing with deionized water, adding water for soaking for 14-18 h, adding 3% of table salt and 3% of sucrose after draining, steaming in a high-pressure steam sterilization pot for 20-40 min, taking out, and then putting into a sterile operation table for cooling to obtain solid fermentation culture;
Inoculating the seed liquid to the sterilized solid state fermentation culture according to the inoculum size of 2-4%, culturing for 48-60 h at 37 ℃, and stirring every 12 h.
The invention has the beneficial effects that:
(1) The bacillus subtilis Bacillusubtilis JNC002.001 is obtained by screening from commercial natto and performing physical mutagenesis, the maximum enzyme activity of nattokinase produced by fermentation in a shake flask can reach 294FU/mL, the acetoin content can reach 41.5g/L, and compared with the original wild strain, the bacillus subtilis Bacillusubtilis JNC002.001 is respectively improved by 167.3 percent and 86.1 percent, and the enzyme production capacity and the acetoin production level are greatly improved.
(2) The Bacillus subtilis JNC002.001 has good passage stability and great application potential after passage stability verification.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the description of the embodiments will be briefly described below, it being obvious that the drawings in the following description are only some embodiments of the present invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art. Wherein:
FIG. 1 is a photograph showing a method for screening a high-yield nattokinase strain using a casein medium and a fibrin plate, wherein the casein medium is on the left and the fibrin plate is on the right, in the embodiment of the present invention.
FIG. 2 shows the enzyme activity and OD changes of Bacillus subtilis (Bacilussubtilis) JNC002.001 at different fermentation times.
FIG. 3 is a passaging stability validation of Bacillus subtilis (Bacilussubtilis) JNC 002.001.
FIG. 4 is a phylogenetic tree constructed by MEGA 6.0 software selection NJ (Neighbor-Joining) method.
Detailed Description
In order that the above-recited objects, features and advantages of the present invention will become more apparent, a more particular description of the invention will be rendered by reference to specific embodiments thereof which are illustrated in the appended drawings.
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced in other ways other than those described herein, and persons skilled in the art will readily appreciate that the present invention is not limited to the specific embodiments disclosed below.
Further, reference herein to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic can be included in at least one implementation of the invention. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments.
The bacillus subtilis (Bacilussubtilis) JNC002.001 is preserved in the collection center of China classical collection microorganisms (CCTCC NO) at the 9 th month 4 th year 2023: m20231585, the preservation address is China university of Wuhan.
The raw materials in the invention are all common commercial products.
Example 1
Screening of nattokinase-producing strains:
(1) 10g of natto is weighed and placed in 90mL of sterile physiological saline to prepare bacterial suspension, after the bacterial suspension is diluted to 10 -8 in a gradient way, 100 mu L of bacterial suspension with three dilutions of 10 -6、10-7 and 10 -8 are dripped on casein culture medium and uniformly coated (see figure 1);
Placing the culture medium in an incubator at 37 ℃ for inverted culture for 24 hours, selecting a plurality of single bacterial colonies with larger proteolytic transparent rings as primary screening strains, carrying out flat streaking on the primary screening strains to realize separation and purification, and preserving in a slant culture medium;
Wherein, the formula of the casein culture medium is as follows: 0.2% of casein, 0.1% of glucose, 0.1% of yeast extract powder, 0.1% of dipotassium hydrogen phosphate, 0.05% of potassium dihydrogen phosphate, 0.01% of magnesium sulfate and 2% of agar.
(2) Selecting a primary screening strain, inoculating the primary screening strain into a primary seed liquid culture medium, performing shake culture at 37 ℃ and 200rpm for 8-10 hours, transferring the primary screening strain into a secondary seed liquid culture medium according to an inoculum size of 2 percent, performing shake culture at 200rpm for 2.5-3.5 hours, inoculating the primary screening strain into a fermentation culture medium according to an inoculum size of 2 percent, performing shake fermentation at 37 ℃ and 200rpm for 60 hours, and centrifuging the primary screening strain at 4 ℃ and 8000rpm for 3 minutes to obtain a supernatant which is crude enzyme liquid;
performing enzyme activity detection on the crude enzyme solution by using a fibrin plate method, placing the fibrin plate with the spotted sample in a constant temperature incubator, reacting for 10-18 hours at 37 ℃, measuring the diameter of a dissolved transparent ring, and selecting a strain with a larger transparent ring as a starting strain (see figure 1);
Wherein, the fermentation culture medium comprises the following components in percentage by weight: 2.5% of soybean meal, 2% of glucose, 0.2% of disodium hydrogen phosphate, 0.1% of sodium dihydrogen phosphate, 0.05% of magnesium chloride, 0.02% of calcium chloride and the balance of deionized water, wherein the pH value is 7-7.5;
The primary seed liquid and the secondary seed liquid are both LB liquid culture mediums, and the difference is that the primary seed liquid culture medium and the secondary seed liquid culture medium are respectively 4mL of culture medium are filled in a 15mL test tube, and the secondary seed liquid culture medium is 50mL of culture medium are filled in a 250mL conical flask.
The specific method for detecting the enzyme activity by the fibrin plate method is as follows:
preparing a fibrinogen solution with a concentration of 1.5mg/mL by using PBS buffer solution (0.01 mol/L, pH 6.8, the same applies below), dissolving in a water bath at 37 ℃, and filtering the solution with filter paper to obtain a solution which can be used for experiments;
preparing 1.5% agarose solution by using PBS buffer solution, heating and dissolving, and then preserving heat in a water bath at 50 ℃; 1-2U/mL thrombin solution was prepared with 0.9% sodium chloride solution. 10mL of fibrinogen solution is sucked into a 50mL beaker, heated in a water bath at 50 ℃ for about 30s and kept at a constant temperature, 10mL of 1.5% agarose solution is added into the 50mL beaker, 1mL of thrombin solution is added into the 10mL beaker after the mixture is uniformly mixed, the beaker is gently shaken for about 1min, the mixed solution is poured into a 90mm culture dish, and the mixture is kept stand for about 30min, so that the flat plate can be used after complete solidification.
A plurality of small holes (6-8) are punched on a fibrin flat plate by using a puncher with the diameter of 2mm, water in the small holes is sucked out by using a liquid-transferring gun, 5-10 mu L of enzyme liquid to be detected is added into the small holes, and the edges of the flat plate are wrapped by using a sealing film to prevent the flat plate from drying.
Placing the fibrin plate with the sample application in a constant temperature incubator, and reacting for 10-18h at 37 ℃.
(3) And (3) accurately measuring the enzyme activity of the crude enzyme solution of the original strain by an ultraviolet spectrophotometry, and selecting the strain with higher enzyme activity as the mutagenesis original strain.
The enzyme activity of the strain SD2 is 110FU/mL.
The ultraviolet spectrophotometry is an NK enzyme activity detection method identified by the Japanese natto Association, has standard operation specifications, and has accurate detection results, but complicated operation and higher cost; the screening efficiency can be improved by only using the enzyme to accurately measure the enzyme activity.
Table 1 comparison of enzyme activities of selected strains
| Strain name | Fermentation broth enzyme activity (FU/mL) |
| DS1 | 105.53 |
| DS4 | 83.75 |
| DS5 | 107.06 |
| CSDC4 | 90.45 |
| YJ1 | 102.18 |
| YJ3 | 93.76 |
| ZW5 | 90 |
| SD2 | 111.67 |
| SD3 | 100.38 |
Example 2
ARTP mutagenesis breeding of nattokinase-producing strains:
(1) The starting strain SD2 is inoculated into LB culture medium to be cultivated to a logarithmic phase, bacterial liquid is collected and centrifuged (8000 rpm,5 min), bacterial cells are washed 2 times by physiological saline, and the bacterial cells are re-suspended by the physiological saline to ensure that the OD600 value is 0.6-0.8.
(2) Transferring 10 mu L of bacterial suspension, uniformly coating on the surface of a sterilized metal slide, placing the metal slide in an ARTP mutagenesis instrument, setting the air flow to be 10SLM, the power to be 120W and the treatment time to be 0-3min, and carrying out ARTP mutagenesis.
(3) After mutagenesis, the metal slide glass is placed in an EP tube containing 1mL of sterile physiological saline, the EP tube is subjected to shake elution, bacterial liquid in the EP tube is subjected to ten-time gradient dilution, 100 mu L of dilution liquid is coated on an LB solid plate, and the culture is carried out at 37 ℃ for 12-24 hours.
(4) After the culture is completed, single colony on the flat plate is selected, inoculated into a primary seed liquid culture medium, and subjected to shaking culture at 37 ℃ and 200rpm for 8-10 hours;
Transferring into secondary seed liquid culture medium according to 2% inoculum size, shake culturing at 37deg.C and 200rpm for 2.5-3.5 hr;
Inoculating into fermentation medium according to 2% inoculum size, fermenting at 37deg.C with 200rpm for 60 hr, centrifuging at 4deg.C and 8000rpm for 3min to obtain supernatant as crude enzyme solution.
The primary seed liquid and the secondary seed liquid are both LB liquid culture mediums, wherein the difference is that the primary seed liquid culture medium and the secondary seed liquid culture medium are respectively 4mL of culture medium which is filled in a 15mL test tube and 50mL of culture medium which is filled in a 250mL conical flask. The fermentation medium comprises the following components in percentage by weight: 2.5% of soybean meal, 2% of glucose, 0.2% of disodium hydrogen phosphate, 0.1% of sodium dihydrogen phosphate, 0.05% of magnesium chloride, 0.02% of calcium chloride and the balance of deionized water, wherein the pH value is 7-7.5.
Judging whether the mutant strain has forward mutation according to a fibrin plate method (a crude enzyme solution of an initial strain is added on a plate to be used as a contrast), accurately measuring the enzyme activity of the crude enzyme solution which is preliminarily judged to be the forward mutant strain by adopting an ultraviolet spectrophotometry, and preserving the microbial strain with the improved enzyme activity after the mutation.
The high-yield NK mutant strain is obtained through mutation screening, and the enzyme activity of fermentation liquor reaches 285.83FU/mL (see figure 2). The strain is sampled to Shanghai Meiji biological company for second generation genome sequencing, 19 strains closest to the species level are selected based on 31 housekeeping genes (dnaG,frr,infC,nusA,pgk,pyrG,rplA,rplB,rplC,rplD,rplE,rplF,rplK,rplL,rplM,rplN,rplP,rplS,rplT,rpmA,rpoB,rpsB,rpsC,rpsE,rpsI,rpsJ,rpsK,rpsM,rpsS,smpB,tsf) by comparison with a database, a phylogenetic tree is constructed by a MEGA 6.0 software selection NJ (Neighbor-Joining) method, and the strain JNC002.001 obtained by screening is shown to belong to Bacillus subtilis genus in FIG. 4; the bacillus subtilis is named as bacillus subtilis JNC002.001 and is preserved in China collection microbiological culture collection center (CCTCC) in the 9 th month 4 days of 2023, wherein the preservation number is CCTCC NO: m20231585, the preservation address is China university of Wuhan.
(5) The initial strain SD2 and Bacillus subtilis JNC002.001 were fermented and cultured, the fermentation broth was sampled every 12 hours, the enzyme activity was measured by ultraviolet spectrophotometry on the crude enzyme solution of the two strains, and the OD600 value change of the mutagenized strain Bacillus subtilis JNC002.001 was measured, and the measurement results are shown in FIG. 2.
Example 3
Bacillus subtilis jnc002.001 passage stability validation:
the obtained primary bacillus subtilis Bacillusubtilis JNC002.001 was subcultured to 10 generations on an agar slant medium, and 2-, 3-, 5-, and 10-generation strains were subjected to fermentation culture.
Inoculating each passage strain into a primary seed liquid culture medium, performing shake culture at 37 ℃ and 200rpm for 8-10h, transferring into a secondary seed liquid culture medium according to 2% of the inoculum size, performing shake culture at 37 ℃ and 200rpm for about 2.5-3.5h, inoculating into a fermentation culture medium according to 2% of the inoculum size, performing shake fermentation at 37 ℃ and 200rpm for 60h, and centrifuging at 4 ℃ and 8000rpm for 3min to obtain a crude enzyme solution.
And (3) carrying out enzyme activity measurement on each passage strain by adopting an ultraviolet spectrophotometry, wherein the enzyme activity is shown as a figure 3, and the strain can still maintain the characteristic of high yield NK after being passed for ten times.
Example 4
The application of preparing fermented natto by using bacillus subtilis is carried out according to the following steps:
(1) Seed liquid preparation:
Inoculating the bacterial lawn of the bacillus bifidus JNC002.001 to a liquid LB culture medium, and culturing for 8 hours at 37 ℃ and 200rpm to obtain seed liquid.
(2) Fermentation of a solid culture medium: selecting full, mildew-free, deformity-free and yellowish commercial organic soybeans, washing with deionized water until no impurities exist, soaking the soybeans for 14-18 hours according to the mass ratio of 1:3, draining, adding 3% of salt and 3% of sucrose, subpackaging into triangular bottles, putting 25g of triangular bottles into a high-pressure steam sterilization pot, steaming for 20min, and taking out the triangular bottles and putting the triangular bottles into a sterile operation table for cooling.
Inoculating the seed solution to the sterilized solid state fermentation culture according to the inoculum size of 4%, culturing at 37 ℃ for 48 hours, and stirring every 12 hours. Weighing 5g of fermented natto, dissolving in 10mL of sterile deionized water, leaching for 24 hours at 4 ℃, mashing with a glass rod, centrifuging for 10min at 12000 Xg, adding 10mL of sterile deionized water into a centrifuge tube, mashing and centrifuging, and obtaining a supernatant as a solid fermentation crude enzyme solution.
And (3) performing enzyme activity measurement on the crude enzyme liquid by adopting an ultraviolet spectrophotometry to obtain 237.5FU/g of enzyme activity of the crude enzyme liquid in solid state fermentation.
Example 5
As a result of comparing the genome re-sequencing of the mutant strain JNC002.001 with that of the original strain SD2, it was found that partial base mutation and deletion occurred in the gene acoA related to acetoin metabolism in the mutant strain. In the past studies, the acetoin of acoA gene knockout strain was hardly degraded, and thus the accumulation of acetoin was increased.
Therefore, the invention explores the level change of the acetoin produced by fermenting the mutagenesis strain JNC002.001, and provides an application of preparing the acetoin by using bacillus subtilis, which is carried out according to the following steps:
Respectively streaking the strains SD2 and JNC002.001 stored in the glycerol pipe on LB solid medium, and culturing overnight in a 37 ℃ incubator;
Picking single colony on the plate, inoculating into a culture medium of 4mLLB, and shake culturing at 37 ℃ and 200rpm for 8-10h; transferring into 50mL high sugar culture medium according to 2% inoculum size, shake culturing at 37deg.C and 200rpm for 36 hr; centrifuging the fermentation liquor at 8000rpm for 3min to obtain supernatant, thus obtaining fermentation liquor containing acetoin;
The high-sugar culture medium comprises the following components in percentage by weight: 10% of glucose, 2% of soybean peptone, 0.2% of disodium hydrogen phosphate, 0.1% of sodium dihydrogen phosphate, 0.05% of magnesium chloride, 0.02% of calcium chloride and the balance of deionized water, wherein the pH value is 7-7.5.
The modified volt-wave test method is adopted for measuring the acetoin content:
A pure acetoin sample (Sigma) dissolved in water shows a maximum light absorption at 560nm by the Volts-wave test reaction. The test was performed as follows: to 200. Mu.l of acetoin standard or culture supernatant of appropriate dilution, 140. Mu.l of creatine (0.5% by mass/volume in water), 200. Mu.l of alpha-naphthol (5% by mass/volume in 95% ethanol) and 200. Mu.l of KOH (40% by mass/volume in water) were added sequentially. Vortex shaking is required for each reagent added to the sample. After all reagents were added, incubated at room temperature for 15min, vortexed again before measurement, and absorbance was measured at 560nm using a spectrophotometer.
According to the measurement, the acetoin content of the starting strain SD2 after 36 hours of fermentation is 22.3g/L, and the acetoin content of the strain JNC002.001 after 36 hours of fermentation is 41.5g/L.
Thus the acetoin yield of the mutant strain JNC002.001 was increased by 86.1% compared to the starting strain SD 2.
It should be noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the same, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the present invention may be modified or substituted without departing from the spirit and scope of the technical solution of the present invention, and it should be covered in the scope of the present invention.
Claims (10)
1. Bacillus subtilis (Bacilussubtilis) JNC002.001 was deposited at the collection center of classical collection microorganisms in china at 9 and 4 of 2023 with the deposit number cctccc NO: m20231585, the preservation address is China university of Wuhan.
2. The use of bacillus subtilis JNC002.001 according to claim 1 for the preparation of acetoin.
3. The use according to claim 2, wherein: inoculating bacillus subtilis JNC002.001 into a primary seed liquid for culture to obtain a primary seed liquid culture;
Transferring the first-level seed liquid culture to the second-level seed liquid culture for expansion culture to obtain a second-level seed liquid culture;
Inoculating the secondary seed liquid culture into a high-sugar culture medium for fermentation, and centrifuging to obtain a supernatant containing acetoin.
4. A use according to claim 3, wherein: inoculating the bacillus subtilis JNC002.001 into a primary seed liquid for culture, wherein the primary seed liquid is an LB culture medium, and the culture parameters are as follows: shaking culture at 37 deg.c and 200rpm for 8-10 hr; the first-stage seed liquid culture is transferred to the second-stage seed liquid culture for expansion culture, wherein the second-stage seed liquid is LB culture medium, and the culture parameters are as follows: shaking culture is carried out for 2.5 to 3.5 hours at 37 ℃ and 200rpm, and the inoculation amount is 2 weight percent.
5. Use according to claim 3 or 4, characterized in that: the high-sugar culture medium comprises the following components in percentage by weight: 10% of glucose, 2% of soybean peptone, 0.2% of disodium hydrogen phosphate, 0.1% of sodium dihydrogen phosphate, 0.05% of magnesium chloride, 0.02% of calcium chloride and the balance of deionized water, wherein the pH value is 7-7.5.
6. A use according to claim 3, wherein: the secondary seed liquid culture is inoculated into a high-sugar culture medium for fermentation, wherein the fermentation condition is 200-250 rpm, the culture time is 36h, and the inoculation amount of the secondary seed liquid culture is 2-4wt%.
7. Use of bacillus subtilis according to claim 1 for the production of nattokinase, characterized in that: comprising the steps of (a) a step of,
Inoculating bacillus subtilis JNC002.001 into a primary seed liquid for culture to obtain a primary seed liquid culture, wherein the primary seed liquid is an LB culture medium, and the culture parameters are as follows: shaking culture at 37 deg.c and 200rpm for 8-10 hr;
Transferring the primary seed liquid culture to a secondary seed liquid culture for expansion culture to obtain a secondary seed liquid culture, wherein the secondary seed liquid is an LB culture medium, and the culture parameters are as follows: shaking culture is carried out for 2.5 to 3.5 hours at 37 ℃ and 200rpm, and the inoculum size is 2 weight percent;
Inoculating the secondary seed liquid culture into a fermentation medium for fermentation, centrifuging and taking supernatant to obtain nattokinase liquid.
8. The use according to claim 7, wherein: the fermentation medium comprises the following components in percentage by weight: 2.5% of soybean meal, 2% of glucose, 0.2% of disodium hydrogen phosphate, 0.1% of sodium dihydrogen phosphate, 0.05% of magnesium chloride, 0.02% of calcium chloride and the balance of deionized water, wherein the pH value is 7-7.5.
9. The use according to claim 7, wherein: inoculating the secondary seed liquid culture into a fermentation culture medium for fermentation, wherein the fermentation condition is 200-250 rpm, the temperature is 37 ℃, and the culture time is 36-60 h; the inoculation amount of the secondary seed liquid culture is 2-4wt%.
10. The use of bacillus subtilis JNC002.001 according to claim 1 for the preparation of natto, characterized in that: comprising the steps of (a) a step of,
Seed liquid preparation: inoculating bacillus subtilis JNC002.001 lawn into a liquid seed culture medium, and culturing at 37 ℃ and 200rpm for 8 hours to obtain seed liquid;
Fermentation of a solid culture medium: taking soybean raw materials, washing with deionized water, adding water for soaking for 14-18 h, adding 3% of table salt and 3% of sucrose after draining, steaming in a high-pressure steam sterilization pot for 20-40 min, taking out, and then putting into a sterile operation table for cooling to obtain solid fermentation culture;
Inoculating the seed liquid to the sterilized solid state fermentation culture according to the inoculum size of 2-4%, culturing for 48-60 h at 37 ℃, and stirring every 12 h.
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