CN113774002B - Bacillus amyloliquefaciens culture medium and application thereof - Google Patents
Bacillus amyloliquefaciens culture medium and application thereof Download PDFInfo
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- 241000193744 Bacillus amyloliquefaciens Species 0.000 title claims abstract description 36
- 239000001963 growth medium Substances 0.000 title claims abstract description 30
- 238000000855 fermentation Methods 0.000 claims abstract description 80
- 230000004151 fermentation Effects 0.000 claims abstract description 80
- 239000007788 liquid Substances 0.000 claims abstract description 42
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 claims abstract description 31
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 claims abstract description 29
- 239000000600 sorbitol Substances 0.000 claims abstract description 29
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 claims abstract description 23
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims abstract description 21
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims abstract description 21
- 235000011130 ammonium sulphate Nutrition 0.000 claims abstract description 21
- 239000008101 lactose Substances 0.000 claims abstract description 21
- 239000004202 carbamide Substances 0.000 claims abstract description 18
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims abstract description 11
- 239000001301 oxygen Substances 0.000 claims abstract description 11
- 229910052760 oxygen Inorganic materials 0.000 claims abstract description 11
- VWDWKYIASSYTQR-UHFFFAOYSA-N sodium nitrate Chemical compound [Na+].[O-][N+]([O-])=O VWDWKYIASSYTQR-UHFFFAOYSA-N 0.000 claims description 30
- 239000002609 medium Substances 0.000 claims description 22
- 238000000034 method Methods 0.000 claims description 20
- 239000004317 sodium nitrate Substances 0.000 claims description 15
- 235000010344 sodium nitrate Nutrition 0.000 claims description 15
- 230000008569 process Effects 0.000 claims description 12
- 238000003756 stirring Methods 0.000 claims description 10
- 238000012258 culturing Methods 0.000 claims description 8
- 238000004321 preservation Methods 0.000 claims description 6
- 230000001580 bacterial effect Effects 0.000 claims description 5
- 239000012880 LB liquid culture medium Substances 0.000 claims description 3
- 238000009629 microbiological culture Methods 0.000 claims description 3
- 239000007787 solid Substances 0.000 claims description 3
- 230000003213 activating effect Effects 0.000 claims description 2
- 239000000463 material Substances 0.000 claims description 2
- 238000012136 culture method Methods 0.000 claims 2
- -1 ammonium ions Chemical class 0.000 claims 1
- 230000001502 supplementing effect Effects 0.000 claims 1
- LXBIFEVIBLOUGU-JGWLITMVSA-N duvoglustat Chemical compound OC[C@H]1NC[C@H](O)[C@@H](O)[C@@H]1O LXBIFEVIBLOUGU-JGWLITMVSA-N 0.000 abstract description 74
- LXBIFEVIBLOUGU-UHFFFAOYSA-N Deoxymannojirimycin Natural products OCC1NCC(O)C(O)C1O LXBIFEVIBLOUGU-UHFFFAOYSA-N 0.000 abstract description 37
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 abstract description 12
- 238000004519 manufacturing process Methods 0.000 abstract description 9
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 abstract description 8
- 229910052799 carbon Inorganic materials 0.000 abstract description 8
- 229910052757 nitrogen Inorganic materials 0.000 abstract description 6
- FBPFZTCFMRRESA-JGWLITMVSA-N D-glucitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-JGWLITMVSA-N 0.000 abstract description 4
- 230000000813 microbial effect Effects 0.000 abstract description 4
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 abstract description 2
- 230000009286 beneficial effect Effects 0.000 abstract 1
- 239000006052 feed supplement Substances 0.000 abstract 1
- 239000002207 metabolite Substances 0.000 abstract 1
- 238000004659 sterilization and disinfection Methods 0.000 description 32
- 230000001954 sterilising effect Effects 0.000 description 30
- 238000002360 preparation method Methods 0.000 description 12
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 5
- 235000013305 food Nutrition 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 244000063299 Bacillus subtilis Species 0.000 description 4
- 235000014469 Bacillus subtilis Nutrition 0.000 description 4
- 240000000249 Morus alba Species 0.000 description 4
- 235000008708 Morus alba Nutrition 0.000 description 4
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 description 3
- 241000193830 Bacillus <bacterium> Species 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 3
- 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
- 230000001276 controlling effect Effects 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 208000002249 Diabetes Complications Diseases 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- 229930006000 Sucrose Natural products 0.000 description 2
- 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 description 2
- 229960000583 acetic acid Drugs 0.000 description 2
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- 239000005720 sucrose Substances 0.000 description 2
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- 238000012360 testing method Methods 0.000 description 2
- SXAMGRAIZSSWIH-UHFFFAOYSA-N 2-[3-[2-(2,3-dihydro-1H-inden-2-ylamino)pyrimidin-5-yl]-1,2,4-oxadiazol-5-yl]-1-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)ethanone Chemical compound C1C(CC2=CC=CC=C12)NC1=NC=C(C=N1)C1=NOC(=N1)CC(=O)N1CC2=C(CC1)NN=N2 SXAMGRAIZSSWIH-UHFFFAOYSA-N 0.000 description 1
- 241001669263 Bacillus amyloliquefaciens DSM 7 Species 0.000 description 1
- 241000194110 Bacillus sp. (in: Bacteria) Species 0.000 description 1
- 241000255789 Bombyx mori Species 0.000 description 1
- BTBUEUYNUDRHOZ-UHFFFAOYSA-N Borate Chemical compound [O-]B([O-])[O-] BTBUEUYNUDRHOZ-UHFFFAOYSA-N 0.000 description 1
- 206010012655 Diabetic complications Diseases 0.000 description 1
- 229930091371 Fructose Natural products 0.000 description 1
- 239000005715 Fructose Substances 0.000 description 1
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 description 1
- 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 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 229940122069 Glycosidase inhibitor Drugs 0.000 description 1
- 102000005744 Glycoside Hydrolases Human genes 0.000 description 1
- 108010031186 Glycoside Hydrolases Proteins 0.000 description 1
- 102100024295 Maltase-glucoamylase Human genes 0.000 description 1
- BGMYHTUCJVZIRP-UHFFFAOYSA-N Nojirimycin Natural products OCC1NC(O)C(O)C(O)C1O BGMYHTUCJVZIRP-UHFFFAOYSA-N 0.000 description 1
- 208000008589 Obesity Diseases 0.000 description 1
- 241000187747 Streptomyces Species 0.000 description 1
- 241001052560 Thallis Species 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 102000004139 alpha-Amylases Human genes 0.000 description 1
- 108090000637 alpha-Amylases Proteins 0.000 description 1
- 108010028144 alpha-Glucosidases Proteins 0.000 description 1
- 229940024171 alpha-amylase Drugs 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
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- 238000007796 conventional method Methods 0.000 description 1
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- 238000002474 experimental method Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- IRXSLJNXXZKURP-UHFFFAOYSA-N fluorenylmethyloxycarbonyl chloride Chemical compound C1=CC=C2C(COC(=O)Cl)C3=CC=CC=C3C2=C1 IRXSLJNXXZKURP-UHFFFAOYSA-N 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 239000012362 glacial acetic acid Substances 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 239000003316 glycosidase inhibitor Substances 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 230000002218 hypoglycaemic effect Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 235000012054 meals Nutrition 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 229930014626 natural product Natural products 0.000 description 1
- BGMYHTUCJVZIRP-GASJEMHNSA-N nojirimycin Chemical compound OC[C@H]1NC(O)[C@H](O)[C@@H](O)[C@@H]1O BGMYHTUCJVZIRP-GASJEMHNSA-N 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 235000020824 obesity Nutrition 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 229930000732 piperidine alkaloid Natural products 0.000 description 1
- 150000003053 piperidines Chemical class 0.000 description 1
- 230000000291 postprandial effect Effects 0.000 description 1
- 230000003389 potentiating effect Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000007790 scraping Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 210000000813 small intestine Anatomy 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
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- 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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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/10—Nitrogen as only ring hetero atom
- C12P17/12—Nitrogen as only ring hetero atom containing a six-membered hetero ring
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
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Abstract
The invention discloses a bacillus amyloliquefaciens (Bacillus amyloliquefaciens) culture medium and application thereof, and belongs to the field of microbial fermentation. The culture medium of the invention uses lactose and sorbitol as carbon sources, urea and ammonium sulfate as nitrogen sources, and the liquid fermentation of bacillus amyloliquefaciens is carried out on a large scale, and the metabolite 1-deoxynojirimycin can reach more than 6 g/L; after the dissolved oxygen is controlled by the intervention of the feed supplement, the 1-deoxynojirimycin can reach more than 10 g/L. The culture medium is used for fermenting bacillus amyloliquefaciens, has high yield of 1-deoxynojirimycin, is simple in formula, is cheap and easy to obtain, is safe and environment-friendly, has simple and controllable fermentation process and short fermentation period, and is beneficial to industrial expansion production.
Description
Technical Field
The invention relates to the technical field of microbial fermentation, in particular to a bacillus amyloliquefaciens culture medium and application thereof.
Background
1-deoxynojirimycin (1-DNJ) is a naturally occurring polyhydroxy piperidine alkaloid, and is known as3, 4, 5-trihydroxy-2-hydroxymethyl tetrahydropyridine, which naturally occurs in the branches, leaves and roots of mulberry, and in the larvae and pupae of silkworm, etc. The 1-DNJ has strong inhibition effect on alpha-glucosidase and alpha-amylase, can delay the absorption of sugar by the small intestine after meal, and achieves the aim of inhibiting postprandial blood sugar rise of diabetics. Based on its potent glycosidase inhibitory activity, nojirimycin can be used for treating diabetes and diabetic complications, obesity and related disorders, etc., and 1-DNJ can be used for food
In the field, foods using mulberry leaves as raw materials are allowed to be marketed as functional hypoglycemic products in east Asian countries such as Japan, and the like, and show a broad prospect in the field of health care foods of 1-DNJ.
The most common source of 1-DNJ is purification from mulberry leaves, however, the content of 1-DNJ in mulberry leaves is as low as 0.1%, the separation and purification of 1-DNJ are complex, and the cost of large-scale preparation is high due to the loss in the extraction process. The microbial fermentation has the characteristics of rapid growth, simple nutrition requirement, easy culture and the like, and the preparation of the natural compound with biological activity from a microbial source has great advantages, and the current literature reports that microorganisms such as Bacillus sp and Streptomyces sp can produce 1-DNJ. DC Stein et al describe a strain of Bacillus subtilis DSM704 producing 1-DNJ and its characteristics (Characterization of Bacillus subtilis DSM704 and its production of 1-deoxynojirimycin [ J ]. Appl Environ Microbiol,1984,48 (2): 280-284), which has a priority of cultivation carbon source of fructose, glucose, sorbitol, sucrose, etc., respectively, and the highest fermentation yield can reach 1mg/ml.
Screening 750 microorganisms by Shinji on et al determined other Bacillus strains producing 1-DNJ in large amounts, such AS Bacillus amyloliquefaciens AS385 and Bacillus subtilis B4 (Production of the. Alpha. -glycosidase inhibitor 1-deoxynojirimycin from Bacillus species [ J ]. Food Chem,2013,138 (1): 516-523), wherein the extracellular 1-DNJ concentration was up to 460mg/L when Bacillus amyloliquefaciens AS385 was cultured in sorbitol-supplemented medium. Kenji Yamagishi et al found that Bacillus amyloliquefaciens DSM7 had an optimal culture carbon to nitrogen ratio of 6.25:1, lactose is the most preferred carbon source, and the culture level can reach 1140mg/L (Lactose Increases the Production of 1-deoxynojirimycin in Bacillus amyloliquefaciens [ J ]. Food Science and Technology Research,2017,23 (2): 349-353) in 5 days. The Chinese patent CN20181012651. X provides a bacillus amyloliquefaciens engineering bacterium for high yield of 1-deoxynojirimycin, wherein main carbon and nitrogen sources in the fermentation of the bacillus amyloliquefaciens engineering bacterium are lactose and malt extract, and the fermentation level is 297.38mg/L. Chinese patent 202011249484.5 provides a strain of naturally bred bacillus amyloliquefaciens with high yield of 1-deoxynojirimycin, wherein a culture medium for fermentation culture takes sucrose as a carbon source and ammonium sulfate as a nitrogen source, and the fermentation level can reach 0.9g/L.
Bacillus amyloliquefaciens (Bacillus amyloliquefaciens, BA) belongs to gram-positive bacillus, has very high affinity with bacillus subtilis, and can fully improve the 1-DNJ production capacity of the bacillus amyloliquefaciens, improve the production efficiency and reduce the production cost by optimizing a fermentation medium and a process.
Disclosure of Invention
The invention aims to provide a bacillus amyloliquefaciens culture medium and application thereof in preparation of 1-deoxynojirimycin, so as to make up for the defects of the prior art.
The invention provides a bacillus amyloliquefaciens culture medium which is characterized in that a carbon source is lactose and sorbitol, and a nitrogen source is urea and ammonium sulfate. Further preferably, the formula of the culture medium for liquid fermentation culture is as follows: 15-50 g/L lactose, 10-50 g/L sorbitol, 1-10 g/L ammonium sulfate, 2-10 g/L urea, 2-10 g/L sodium nitrate and pH 6.5-8.5. More preferably, the liquid fermentation medium has the formula: lactose 25g/L, sorbitol 25g/L, ammonium sulfate 2g/L, urea 4g/L, sodium nitrate 3g/L, pH8.0.
The invention further provides a method for preparing 1-deoxynojirimycin by using the bacillus amyloliquefaciens culture medium for fermentation, which is characterized in that the bacillus amyloliquefaciens is bacillus amyloliquefaciens HDCC00252. In a preferred embodiment, the medium formulation for the fermentative preparation of 1-deoxynojirimycin is: the carbon source is lactose and sorbitol, and the nitrogen source is urea and ammonium sulfate. Further preferably, the formula of the culture medium for fermentation culture is: 15-50 g/L lactose, 10-50 g/L sorbitol, 1-10 g/L ammonium sulfate, 2-10 g/L urea, 2-10 g/L sodium nitrate and pH 6.5-8.5. More preferably, the formula of the culture medium for fermentation culture is: lactose 25g/L, sorbitol 25g/L, ammonium sulfate 2g/L, urea 4g/L, sodium nitrate 3g/L, pH8.0.
During the whole fermentation period, the sorbitol concentration of the fermentation liquid is controlled to be not lower than 15g/L, preferably 15-25 g/L; the feeding control means preferably feeds a feeding liquid. The ammonium ion concentration of the fermentation broth is controlled to be not less than 500ppm, preferably 500 to 1000ppm, by feeding. During the whole fermentation period, dissolved oxygen is controlled to be more than 30% alternately through air flow and stirring, and the culture is carried out for 4-5 days at 35-37 ℃. Further preferably, the bacillus amyloliquefaciens seed liquid is prepared by a method comprising the following steps: streaking the cryopreserved bacillus amyloliquefaciens on an LB solid medium plate for culture; and (3) picking single bacterial colony, inoculating the single bacterial colony into an LB liquid culture medium, and activating twice to obtain seed liquid.
The invention provides a bacillus amyloliquefaciens culture medium and a method for producing 1-deoxynojirimycin by utilizing the culture medium through fermentation, and the method has the following main advantages: (1) The titer of the 1-deoxynojirimycin produced by fermentation can reach more than 10g/L, which is greatly improved compared with the prior art; (2) The bacillus amyloliquefaciens culture medium has a simple formula, and all raw materials are cheap and easy to obtain, safe and environment-friendly; (3) The fermentation process is simple and controllable, the fermentation period is short, and the industrial expansion production is facilitated.
Drawings
FIG. 1 HPLC profile of strain fermentation broth;
FIG. 2 shows the yield of 1-deoxynojirimycin and the amount of Bacillus amyloliquefaciens (OD 600 nm) during fermentation.
Detailed Description
The invention utilizes the 1-DNJ high-yield strain obtained by the company: bacillus amyloliquefaciens (Bacillus amyloliquefaciens) HDCC00252 is preserved in North Chen Silu No. 1 and No. 3 of the Korean region of Beijing of China general microbiological culture collection center (CGMCC) with the preservation number of CGMCC No.22781 and the preservation date of 2021, 06 and 25. The experimental methods used in the following examples are conventional methods unless otherwise specified. Materials, reagents, and the like used in the following examples are commercially available products unless otherwise specified.
The invention will be further described by way of the following examples, which are not intended to limit the scope of the invention in any way. It will be appreciated by those skilled in the art that equivalent substitutions and modifications may be made to the teachings of the present invention, and that such modifications may still fall within the scope of the present invention.
Example 150L tank fermentation test
1. Strain activation
Thawing a working strain (CGMCC No. 22781) glycerol pipe at room temperature, sucking 0.1ml of bacterial suspension, inoculating to LB solid plate, coating uniformly, and culturing in a 37 deg.C incubator for 24 hr to obtain activated and revived lawn.
2. Primary seed preparation
Taking activated and revived lawn, scraping a loop of colony with an inoculating loop, inoculating into 500ml triangular flask containing 100ml LB liquid medium, wrapping, shake culturing at 37deg.C on a shaking table at 220rpm for 24 hr, and controlling OD of seed liquid 600nm The value is more than or equal to 5.0.
The pH value of the LB liquid medium is regulated to 7.0 before sterilization, and the sterilization condition is 121-123 ℃ for 30min.
3. Preparation of secondary seeds
Inoculating qualified first-stage seed at 1% (V/V) ratio into a seed tank filled with 10L LB liquid culture medium, setting temperature at 37deg.C, controlling dissolved oxygen to be more than 20%, air flow rate at 1vvm, initial stirring at 200rpm, culturing for 12 hr, and controlling OD of seed liquid 600nm ≥10。
The pH value of the LB liquid medium is regulated to 7.0 before sterilization, and the sterilization condition is 121-123 ℃ for 30min.
4. Fermentation culture
The secondary seeds which are qualified in culture are inoculated into a 50L tank filled with 27L of liquid fermentation culture medium according to the proportion of 10% (V/V), the culture temperature is 37 ℃, the dissolved oxygen is controlled to be more than 30%, the air flow is 1.0VVM, and the initial stirring rotating speed is 200rpm, and the culture is carried out for 5 days. The pH value of the fermentation medium is adjusted to 8.0 before sterilization, and the sterilization condition is 121-123 ℃ for 30min.
The formula of the liquid fermentation medium comprises the following components: lactose 25g/L, sorbitol 25g/L, ammonium sulfate 2g/L, urea 4g/L, sodium nitrate 3g/L, and pH is adjusted to 8.0 before sterilization, and sterilization conditions are 121-123 ℃ for 30min.
5. Fermentation tank control process
In the culture process, the concentration range of sorbitol is controlled to be 20+/-5 g/L by adding 500g/L sorbitol, and the concentration of dissolved ammonium is controlled to be 500-1000 ppm by feeding 20% ammonium sulfate solution.
6. Sample processing
After fermentation, 2ml of fermentation liquid is taken, high-speed centrifugation is carried out at 10000rpm for 10min to remove thalli, then supernatant is taken, and after equal volume of ethanol is added for uniform mixing, high-speed centrifugation at 10000rpm for 10min is carried out. 20. Mu.L of the supernatant was placed in a 1.5mL centrifuge tube, 40. Mu.L of a solution of FMOC-Cl in acetonitrile was added with 0.4mol/L borate buffer (pH=8.5) and 2mmol/L of the solution, and the mixture was stirred and mixed well, reacted in a water bath at 25℃for 20 minutes, and then 20. Mu.L of glycine was added with 1mol/L, and left standing for 5 minutes to complete the reaction of the redundant derivatizing reagent. Finally, 1.5mL of 0.1% (V/V) aqueous acetic acid solution was added, and the mixture was filtered through a 0.45 μm microporous filter membrane and subjected to liquid chromatography.
7. Liquid phase analysis method:
chromatographic column: kromasil C18 analytical column (4.6 mm. Times.250 mm,5 μm)
Mobile phase: acetonitrile-0.1% glacial acetic acid (50:50, V/V)
Flow rate: 1.0mL/min
Column temperature: 30 DEG C
Detection conditions: fluorescence detector, excitation wavelength 254nm, emission wavelength 322nm
Sample injection volume: 10 mu L
8. Experimental results:
the sample concentration was calculated from the standard concentration, the peak area and the sample peak area by using the sample derived by the same method as the standard for the known concentration 1-DNJ control, and it was confirmed that the 1-DNJ content in the fermentation broth obtained in this example was 10.6. 10.6 g/L. During fermentation, the cell quantity of the bacillus amyloliquefaciens is amplified with the increase of the yield of the 1-deoxynojirimycin, and the fermentation is carried out for 20 to 90 hours to form a high-speed prime period and also form a logarithmic growth phase of bacteria.
Example 2
1. Seed liquid preparation the same as in example 1
2. Fermentation culture
Fermentation culture: the qualified liquid seeds are inoculated into a 50L tank filled with 27L of liquid fermentation medium at a proportion of 10% (V/V), the culture temperature is 37 ℃, the dissolved oxygen is controlled to be more than 30%, the air flow is 1.0VVM, and the initial stirring rotating speed is 200rpm, and the culture is carried out for 5 days. The pH value of the fermentation tank culture medium is adjusted to 8.0 before sterilization, and the sterilization condition is 121-123 ℃ for 30min. The process is not fed, and the 1-DNJ content in the fermentation broth at the fermentation end point is 7.2g/L.
The formula of the liquid fermentation medium comprises the following components: lactose 25g/L, sorbitol 25g/L, ammonium sulfate 2g/L, urea 4g/L, sodium nitrate 3g/L, and pH is adjusted to 8.0 before sterilization, and sterilization conditions are 121-123 ℃ for 30min.
Example 3
1. Seed liquid preparation the same as in example 1
2. Fermentation culture: the qualified liquid seeds are inoculated into a 50L tank filled with 27L of optimized liquid fermentation medium at a proportion of 10% (V/V), the culture temperature is 37 ℃, the dissolved oxygen is controlled to be more than 30%, the air flow is 1.0VVM, and the initial stirring rotation speed is 200rpm, and the culture is carried out for 4.5 days. The pH value of the fermentation tank culture medium is adjusted to 8.0 before sterilization, and the sterilization condition is 121-123 ℃ for 30min. The process is not fed, and the 1-DNJ content in the fermentation broth at the fermentation end point is 5.4g/L.
The formula of the liquid fermentation medium comprises the following components: 15g/L lactose, 10g/L sorbitol, 1g/L ammonium sulfate, 2g/L urea and 2g/L sodium nitrate, and adjusting the pH to 6.5 before sterilization, wherein the sterilization condition is 121-123 ℃ for 30min.
Example 4
1. Seed liquid preparation the same as in example 1
2. Fermentation culture: inoculating qualified liquid seeds in a proportion of 10% (V/V) into a 50L tank filled with 27L of optimized liquid fermentation culture medium, wherein the culture temperature is 37 ℃, the dissolved oxygen is controlled to be more than 30%, the air flow is 1.0VVM, and the initial stirring rotation speed is 200rpm, and culturing is carried out for 4-5 days. The pH value of the fermentation tank culture medium is adjusted to 8.0 before sterilization, and the sterilization condition is 121-123 ℃ for 30min. The process is not fed, and the 1-DNJ content in the fermentation broth at the fermentation end point is 6.2g/L.
The formula of the liquid fermentation medium comprises the following components: : lactose 50g/L, sorbitol 50g/L, urea 10g/L, ammonium sulfate 10g/L, sodium nitrate 10g/L, pH value adjusted to 8.5 before disinfection, and disinfection conditions of 121-123 ℃ for 30min.
Example 5
1. Seed liquid preparation the same as in example 1
2. Fermentation culture: inoculating qualified liquid seeds in a proportion of 10% (V/V) into a 50L tank filled with 27L of optimized liquid fermentation culture medium, wherein the culture temperature is 37 ℃, the dissolved oxygen is controlled to be more than 30%, the air flow is 1.0VVM, and the initial stirring rotation speed is 200rpm, and culturing is carried out for 4-5 days. The pH value of the fermentation tank culture medium is adjusted to 8.0 before sterilization, and the sterilization condition is 121-123 ℃ for 30min.
The formula of the liquid fermentation medium comprises the following components: : lactose 25g/L, sorbitol 25g/L, ammonium sulfate 2g/L, urea 4g/L, sodium nitrate 3g/L, and pH is adjusted to 8.0 before sterilization, and sterilization conditions are 121-123 ℃ for 30min.
The fermentation tank control process comprises the following steps: the concentration range of sorbitol is controlled to 15+/-2 g/L by adding 500g/L sorbitol in the culture process.
Finally, under the scheme conditions, the 1-DNJ content in the fermentation broth is 7.6g/L.
Example 6
1. Seed liquid preparation the same as in example 1
2. Fermentation culture: the qualified liquid seeds are inoculated into a 50L tank filled with 27L of optimized liquid fermentation medium at a proportion of 10% (V/V), the culture temperature is 37 ℃, the dissolved oxygen is controlled to be more than 30%, the air flow is 1.0VVM, and the initial stirring rotation speed is 200rpm, and the culture is carried out for 5 days. The pH value of the fermentation tank culture medium is adjusted to 8.0 before sterilization, and the sterilization condition is 121-123 ℃ for 30min.
The formula of the liquid fermentation medium comprises the following components: : lactose 25g/L, sorbitol 25g/L, ammonium sulfate 2g/L, urea 4g/L, sodium nitrate 3g/L, and pH is adjusted to 8.0 before sterilization, and sterilization conditions are 121-123 ℃ for 30min.
The fermentation tank control process comprises the following steps: the concentration range of sorbitol is controlled to 25+/-5 g/L by adding 500g/L sorbitol in the culture process.
Finally, under the scheme conditions, the 1-DNJ content in the fermentation broth is 7.4g/L.
Example 7 1 ton (T) tank fermentation test
1. Seed liquid preparation the same as in example 1
2. Fermentation culture: inoculating qualified secondary seeds at a ratio of 10% (V/V) into a 1.5T tank filled with 1000L liquid fermentation culture medium, wherein the culture temperature is 37 ℃, the dissolved oxygen is controlled to be more than 30%, the air flow is 1.0VVM, the initial stirring rotating speed is 20Hz, and culturing is carried out for 4-5 days. The pH value of the fermentation medium is adjusted to 8.0 before sterilization, and the sterilization condition is 121-123 ℃ for 30min.
The formula of the liquid fermentation medium comprises the following components: lactose 25g/L, sorbitol 25g/L, ammonium sulfate 2g/L, urea 4g/L, sodium nitrate 3g/L, and pH is adjusted to 8.0 before sterilization, and sterilization conditions are 121-123 ℃ for 30min. In the culture process, the concentration range of sorbitol is controlled to be 20+/-5 g/L by adding 500g/L sorbitol, and the concentration of dissolved ammonium is controlled to be 500-1000 ppm by feeding 20% ammonium sulfate solution.
Finally, under the scheme conditions, the 1-DNJ content in the fermentation broth is 10.2g/L.
Claims (7)
1. An application of a culture medium in culturing bacillus amyloliquefaciens HDCC00252, which is characterized in that the culture medium consists of the following components: 15-50 g/L of lactose, 10-50 g/L of sorbitol, 1-10 g/L of ammonium sulfate, 2-10 g/L of urea, 2-10 g/L of sodium nitrate and pH of 6.5-8.5; the bacillus amyloliquefaciens HDCC00252 is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.22781 and the preservation date of 2021, 06 and 25 days.
2. Use according to claim 1, characterized in that the culture medium consists of the following components: lactose 25g/L, sorbitol 25g/L, ammonium sulfate 2g/L, urea 4g/L, sodium nitrate 3g/L, pH8.0.
3. A method for culturing bacillus amyloliquefaciens, comprising the steps of: inoculating bacillus amyloliquefaciens seed liquid into a culture medium for fermentation culture, wherein the culture medium consists of the following components: 15-50 g/L of lactose, 10-50 g/L of sorbitol, 1-10 g/L of ammonium sulfate, 2-10 g/L of urea, 2-10 g/L of sodium nitrate and pH of 6.5-8.5; the bacillus amyloliquefaciens HDCC00252 is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.22781 and the preservation date of 2021, 06 and 25 days.
4. A method according to claim 3, characterized in that the medium consists of the following components: lactose 25g/L, sorbitol 25g/L, ammonium sulfate 2g/L, urea 4g/L, sodium nitrate 3g/L, pH8.0.
5. The culture method according to claim 4, wherein: in the fermentation process, controlling the concentration of sorbitol in the fermentation liquid to be 15-25 g/L through material supplementing; and controlling the concentration of ammonium ions in the fermentation liquor to be 500-1000 ppm through feeding.
6. The culture method according to any one of claims 4 to 5, wherein: in the fermentation culture process, air flow and stirring are alternately controlled to be more than 30% of dissolved oxygen, and the culture is carried out for 4-5 days at 35-37 ℃.
7. The method of claim 6, wherein the bacillus amyloliquefaciens seed liquid is prepared by a method comprising the following steps: streaking the cryopreserved bacillus amyloliquefaciens on an LB solid medium plate for culture; and (3) picking single bacterial colony, inoculating the single bacterial colony into an LB liquid culture medium, and activating twice to obtain seed liquid.
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