CN117384742A - Simple Aspergillus niger culture device for rapidly and efficiently expressing glucose oxidase gene - Google Patents
Simple Aspergillus niger culture device for rapidly and efficiently expressing glucose oxidase gene Download PDFInfo
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- CN117384742A CN117384742A CN202311687942.7A CN202311687942A CN117384742A CN 117384742 A CN117384742 A CN 117384742A CN 202311687942 A CN202311687942 A CN 202311687942A CN 117384742 A CN117384742 A CN 117384742A
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- 241000228245 Aspergillus niger Species 0.000 title claims abstract description 88
- 108010015776 Glucose oxidase Proteins 0.000 title claims abstract description 46
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims abstract description 105
- 229910052760 oxygen Inorganic materials 0.000 claims abstract description 105
- 239000001301 oxygen Substances 0.000 claims abstract description 105
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 64
- 238000007789 sealing Methods 0.000 claims abstract description 24
- 239000002245 particle Substances 0.000 claims abstract description 22
- 238000004519 manufacturing process Methods 0.000 claims abstract description 10
- 239000004816 latex Substances 0.000 claims abstract description 4
- 229920000126 latex Polymers 0.000 claims abstract description 4
- 239000001963 growth medium Substances 0.000 claims description 48
- 239000007787 solid Substances 0.000 claims description 41
- 239000010935 stainless steel Substances 0.000 claims description 27
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 claims description 25
- 239000010949 copper Substances 0.000 claims description 25
- 229910000838 Al alloy Inorganic materials 0.000 claims description 24
- 239000004033 plastic Substances 0.000 claims description 18
- 229920003023 plastic Polymers 0.000 claims description 18
- 229910001256 stainless steel alloy Inorganic materials 0.000 claims description 18
- 229910000881 Cu alloy Inorganic materials 0.000 claims description 17
- 230000002093 peripheral effect Effects 0.000 claims description 17
- 240000008042 Zea mays Species 0.000 claims description 12
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 12
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 12
- 235000005822 corn Nutrition 0.000 claims description 12
- 239000012153 distilled water Substances 0.000 claims description 12
- 239000011521 glass Substances 0.000 claims description 12
- -1 polyethylene Polymers 0.000 claims description 12
- 238000012258 culturing Methods 0.000 claims description 10
- 238000000855 fermentation Methods 0.000 claims description 10
- 230000004151 fermentation Effects 0.000 claims description 10
- 229910000617 Mangalloy Inorganic materials 0.000 claims description 9
- 239000004698 Polyethylene Substances 0.000 claims description 9
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 9
- 229920000573 polyethylene Polymers 0.000 claims description 9
- 229910001220 stainless steel Inorganic materials 0.000 claims description 9
- 238000003756 stirring Methods 0.000 claims description 9
- 229910052802 copper Inorganic materials 0.000 claims description 8
- 239000007788 liquid Substances 0.000 claims description 8
- 239000000843 powder Substances 0.000 claims description 8
- 239000000853 adhesive Substances 0.000 claims description 7
- 230000001070 adhesive effect Effects 0.000 claims description 7
- 238000009630 liquid culture Methods 0.000 claims description 7
- 229920001817 Agar Polymers 0.000 claims description 6
- 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 6
- 239000008272 agar Substances 0.000 claims description 6
- 239000008103 glucose Substances 0.000 claims description 6
- 229910052751 metal Inorganic materials 0.000 claims description 6
- 239000002184 metal Substances 0.000 claims description 6
- 238000002360 preparation method Methods 0.000 claims description 6
- 230000001954 sterilising effect Effects 0.000 claims description 6
- 238000003466 welding Methods 0.000 claims description 6
- 238000010438 heat treatment Methods 0.000 claims description 4
- 238000012546 transfer Methods 0.000 claims description 4
- 244000046052 Phaseolus vulgaris Species 0.000 claims description 3
- 235000010627 Phaseolus vulgaris Nutrition 0.000 claims description 3
- 229910000639 Spring steel Inorganic materials 0.000 claims description 3
- 230000001154 acute effect Effects 0.000 claims description 3
- 239000011324 bead Substances 0.000 claims description 3
- 239000003795 chemical substances by application Substances 0.000 claims description 3
- 239000008367 deionised water Substances 0.000 claims description 3
- 229910021641 deionized water Inorganic materials 0.000 claims description 3
- VTIIJXUACCWYHX-UHFFFAOYSA-L disodium;carboxylatooxy carbonate Chemical compound [Na+].[Na+].[O-]C(=O)OOC([O-])=O VTIIJXUACCWYHX-UHFFFAOYSA-L 0.000 claims description 3
- 229920001971 elastomer Polymers 0.000 claims description 3
- 238000011081 inoculation Methods 0.000 claims description 3
- 238000002156 mixing Methods 0.000 claims description 3
- 238000005096 rolling process Methods 0.000 claims description 3
- 229940045872 sodium percarbonate Drugs 0.000 claims description 3
- 239000003381 stabilizer Substances 0.000 claims description 3
- 238000005303 weighing Methods 0.000 claims description 3
- 239000002023 wood Substances 0.000 claims description 3
- 238000011049 filling Methods 0.000 claims description 2
- 125000006850 spacer group Chemical group 0.000 abstract description 2
- 244000005700 microbiome Species 0.000 description 14
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 12
- 235000021186 dishes Nutrition 0.000 description 6
- 238000000034 method Methods 0.000 description 6
- 238000010586 diagram Methods 0.000 description 5
- RGHNJXZEOKUKBD-SQOUGZDYSA-N D-gluconic acid Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C(O)=O RGHNJXZEOKUKBD-SQOUGZDYSA-N 0.000 description 4
- 235000013339 cereals Nutrition 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 230000000813 microbial effect Effects 0.000 description 3
- 238000009629 microbiological culture Methods 0.000 description 3
- 108010065511 Amylases Proteins 0.000 description 2
- 102000013142 Amylases Human genes 0.000 description 2
- RGHNJXZEOKUKBD-UHFFFAOYSA-N D-gluconic acid Natural products OCC(O)C(O)C(O)C(O)C(O)=O RGHNJXZEOKUKBD-UHFFFAOYSA-N 0.000 description 2
- 241000233866 Fungi Species 0.000 description 2
- ORNBQBCIOKFOEO-YQUGOWONSA-N Pregnenolone Natural products O=C(C)[C@@H]1[C@@]2(C)[C@H]([C@H]3[C@@H]([C@]4(C)C(=CC3)C[C@@H](O)CC4)CC2)CC1 ORNBQBCIOKFOEO-YQUGOWONSA-N 0.000 description 2
- 235000019418 amylase Nutrition 0.000 description 2
- NXQOQNROJJFYCJ-FZFXZXLVSA-N androst-16-ene Chemical compound C1CCC[C@]2(C)[C@H]3CC[C@](C)(C=CC4)[C@@H]4[C@@H]3CCC21 NXQOQNROJJFYCJ-FZFXZXLVSA-N 0.000 description 2
- 238000005452 bending Methods 0.000 description 2
- 235000013351 cheese Nutrition 0.000 description 2
- 239000003086 colorant Substances 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- LNTHITQWFMADLM-UHFFFAOYSA-N gallic acid Chemical compound OC(=O)C1=CC(O)=C(O)C(O)=C1 LNTHITQWFMADLM-UHFFFAOYSA-N 0.000 description 2
- 239000000174 gluconic acid Substances 0.000 description 2
- 235000012208 gluconic acid Nutrition 0.000 description 2
- 230000005484 gravity Effects 0.000 description 2
- 229960000249 pregnenolone Drugs 0.000 description 2
- 235000015067 sauces Nutrition 0.000 description 2
- 239000002689 soil Substances 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 108091005508 Acid proteases Proteins 0.000 description 1
- 244000291564 Allium cepa Species 0.000 description 1
- 235000002732 Allium cepa var. cepa Nutrition 0.000 description 1
- 239000004382 Amylase Substances 0.000 description 1
- 241000228212 Aspergillus Species 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 240000004160 Capsicum annuum Species 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 108010059892 Cellulase Proteins 0.000 description 1
- 235000007516 Chrysanthemum Nutrition 0.000 description 1
- 244000189548 Chrysanthemum x morifolium Species 0.000 description 1
- 229920000742 Cotton Polymers 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 239000004366 Glucose oxidase Substances 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- ZOKXTWBITQBERF-UHFFFAOYSA-N Molybdenum Chemical compound [Mo] ZOKXTWBITQBERF-UHFFFAOYSA-N 0.000 description 1
- CBENFWSGALASAD-UHFFFAOYSA-N Ozone Chemical compound [O-][O+]=O CBENFWSGALASAD-UHFFFAOYSA-N 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 108010059820 Polygalacturonase Proteins 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 235000021307 Triticum Nutrition 0.000 description 1
- 244000098338 Triticum aestivum Species 0.000 description 1
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 description 1
- 229940025131 amylases Drugs 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 230000000840 anti-viral effect Effects 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000003124 biologic agent Substances 0.000 description 1
- 235000021329 brown rice Nutrition 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 229940106157 cellulase Drugs 0.000 description 1
- 229960004106 citric acid Drugs 0.000 description 1
- 235000015165 citric acid Nutrition 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 238000005260 corrosion Methods 0.000 description 1
- 230000007797 corrosion Effects 0.000 description 1
- 239000006071 cream Substances 0.000 description 1
- 238000012364 cultivation method Methods 0.000 description 1
- 102000038379 digestive enzymes Human genes 0.000 description 1
- 108091007734 digestive enzymes Proteins 0.000 description 1
- 229940079919 digestives enzyme preparation Drugs 0.000 description 1
- 238000002845 discoloration Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- 108010093305 exopolygalacturonase Proteins 0.000 description 1
- 239000003337 fertilizer Substances 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 235000019634 flavors Nutrition 0.000 description 1
- 235000012055 fruits and vegetables Nutrition 0.000 description 1
- 229940074391 gallic acid Drugs 0.000 description 1
- 235000004515 gallic acid Nutrition 0.000 description 1
- 229950006191 gluconic acid Drugs 0.000 description 1
- 229940116332 glucose oxidase Drugs 0.000 description 1
- 235000019420 glucose oxidase Nutrition 0.000 description 1
- WPBNNNQJVZRUHP-UHFFFAOYSA-L manganese(2+);methyl n-[[2-(methoxycarbonylcarbamothioylamino)phenyl]carbamothioyl]carbamate;n-[2-(sulfidocarbothioylamino)ethyl]carbamodithioate Chemical compound [Mn+2].[S-]C(=S)NCCNC([S-])=S.COC(=O)NC(=S)NC1=CC=CC=C1NC(=S)NC(=O)OC WPBNNNQJVZRUHP-UHFFFAOYSA-L 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 229910052750 molybdenum Inorganic materials 0.000 description 1
- 239000011733 molybdenum Substances 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 235000005985 organic acids Nutrition 0.000 description 1
- 230000001706 oxygenating effect Effects 0.000 description 1
- 210000000557 podocyte Anatomy 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 235000020418 red date juice Nutrition 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 238000003900 soil pollution Methods 0.000 description 1
- 239000008400 supply water Substances 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 235000021419 vinegar Nutrition 0.000 description 1
- 239000000052 vinegar Substances 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
- 239000011701 zinc Substances 0.000 description 1
- 229910052725 zinc Inorganic materials 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/02—Form or structure of the vessel
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/20—Material Coatings
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/22—Transparent or translucent parts
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/38—Caps; Covers; Plugs; Pouring means
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/48—Holding appliances; Racks; Supports
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12M27/00—Means for mixing, agitating or circulating fluids in the vessel
- C12M27/16—Vibrating; Shaking; Tilting
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
- C12M41/12—Means for regulation, monitoring, measurement or control, e.g. flow regulation of temperature
- C12M41/18—Heat exchange systems, e.g. heat jackets or outer envelopes
- C12M41/22—Heat exchange systems, e.g. heat jackets or outer envelopes in contact with the bioreactor walls
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- C12M43/00—Combinations of bioreactors or fermenters with other apparatus
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- 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
- C12N1/14—Fungi; Culture media therefor
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- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
- C12R2001/66—Aspergillus
- C12R2001/685—Aspergillus niger
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Abstract
Simple and easy high-efficient expression glucose oxidase gene's Aspergillus niger culture apparatus belongs to microbiology technical field, comprises flourishing ware bucket (1), support (2), its characterized in that: the device is characterized in that pendulum shafts (33) at two ends of a transverse frame rod (32) at the upper end of a dish barrel (1) are placed in shaft placing grooves (6) of a transverse support (3), a dish barrel wall (15) is of an inner layer and an outer layer of double-layer structure, a dish barrel wall cavity (16) is used for containing hot water (37), dish barrel hangers (26) and dish barrel swinging arms (29) at two sides are located in the same plane, a dish spacer block (7) and a perforated culture dish (10) are sleeved on a dish barrel center Kong Tongzhu (21), an oxygen production dish (34) is used for containing oxygen production particles (35) and water (36), and oxygen produced after reaction of the oxygen production dish is conveyed into a device in a dish barrel cavity (19) from an oxygen production cavity (24) through an oxygen production dish center hole cylinder (25), and a sealing cushion layer (27) is a latex film.
Description
Technical Field
The invention relates to an Aspergillus niger culture device for simply, quickly and efficiently expressing glucose oxidase genes, belonging to the technical field of microbiology.
Background
Aspergillus niger is a common species of fungi of the genus Aspergillus of the family Courosporaceae, the order Cellularomycetes. Is widely distributed in grains, plant products and soil around the world. The method is mainly used for producing amylase, acid protease, cellulase, pectase, glucose oxidase, citric acid, gluconic acid, gallic acid and the like in the fermentation industry. Some strains also convert hydroxy pregnenolone to androstene. The growth temperature is 37 ℃, the minimum relative humidity is 88 percent, and the mildew of grains and other industrial equipment with higher moisture can be caused. Under the high-temperature and high-humidity environment, aspergillus niger is easy to grow and propagate in a large amount to generate enzyme heat, flowers fall or rotting bell are caused when the aspergillus niger infects cotton, or the aspergillus niger is grown on succulent fruits to cause rotting or soft rot, and a large amount of black powder is generated when the aspergillus niger infects the surfaces of onion scales; the plum rain season is also liable to cause mildew of clothes and the like. Aspergillus niger hypha 15-20pm in diameter and about 1-3mm long, and smooth in wall thickness; the top forms a spherical top bag, which is covered with a layer of stalk base and a layer of small stalk, and the small stalk is grown with a string of brown black spheres with diameter of 2.5-4.0 μm. The conidiophore is spherical, has a diameter of 700-800 μm, and is brown and black. Aspergillus niger spreads rapidly, initially white, and turns into bright yellow until it is black, thick velvet-like, colorless on the back or slightly yellow brown in the center. Some Aspergillus niger strains can also convert hydroxy pregnenolone to androstene. Aspergillus niger hyphae are black brown, big spherical in top sac and double-layered in small peduncles, and conidia are spherical, black brown, smooth or rough. Aspergillus niger has strong resistance to ultraviolet rays and ozone. Aspergillus niger has developed hyphae, multiple branches and polynuclear multicellular fungi. Conidiophores of Aspergillus niger grow vertically from specialized thick-walled and enlarged hyphal cells (podocytes); conidiophores like "chrysanthemum". Hyphae and spores of aspergillus niger usually have various colors, such as black, brown, green, yellow, orange, brown and the like, and different strains and colors. Aspergillus niger can cause mold spoilage of foods, grains and fruits and vegetables. Aspergillus niger is the main strain for making sauce, brewing wine and vinegar. Aspergillus niger is a species that produces enzyme preparations (proteases, amylases, pectinases) and can be used to produce organic acids (e.g., citric acid, gluconic acid, etc.). Agricultural aspergillus niger can be used as a strain for producing saccharified feed. Aspergillus niger can be used for measuring microelements such as manganese, copper, molybdenum, zinc and the like and is used as a mould corrosion test bacterium. The contamination of the cheese during the maturation of Aspergillus niger can cause the surface of the cheese to become black and bad, and can cause discoloration of the cream. Aspergillus niger is often used as a microorganism to be cultivated in a laboratory or industry, is widely applied to production and scientific research practice, and can be particularly used for expressing glucose oxidase genes. In the experimental process, aspergillus niger capable of expressing glucose oxidase gene with high efficiency is often required to be cultivated, but is limited by instruments and equipment in many cases, and the instruments and equipment are expensive for many researchers, so that many experiments are difficult to develop. At present, the patent related to aspergillus niger mostly relates to a fermentation tank for culturing aspergillus niger, such as a device for producing citric acid by using wheat deep processing residual waste liquid, with application number 201620101721.6; an Aspergillus niger cultivation method and application thereof in catalyzing and synthesizing white spirit flavor ester, application number 201910935019.8; application of water-soluble carbon cage in preparing citric acid by Aspergillus niger fermentation and application method thereof are disclosed, with application number 202110616494.6. Still other patents describe how to use Aspergillus niger, such as a microbial fertilizer, application No. 201711313024.2; preparation method of germinated brown rice jujube juice ferment powder/tablet rich in natural digestive enzyme, application number 20201048661. X; a biological agent capable of replacing feed antibiotics, application number 202110164774.8; a preparation method of high-lysine biological feed, application number 202110585963.2. Still other subjects of some patent protection are microbial culture devices, such as a microbial culture device, application number 201811174573.0; an antiviral fermented Chinese medicine for fowl and its preparation method, application number 201911413831.0; a microbial cultivation device for food safety detection, application No. 201920971955.X; a microorganism culture device for detecting chilli sauce products, which is applied for the application number 201922146208.5; a microorganism culture apparatus, application number 202020327023.4; a microorganism culture device for soil pollution treatment, application number 202020474744.8; a microorganism culture apparatus, application number 202021034989.5; a microbial culture apparatus for biomedical analysis, application number 202022040672.9; a microorganism culture device convenient for temperature detection, application number 202022355908.8; a microorganism culture device for environmental engineering, application number 202110021159.1; a soil microorganism culture device and a method, application number 202111289670.6; a microorganism culture apparatus, application number 202121090056.2; a microorganism culture apparatus, application number 202121531499.0; a microorganism culture apparatus, application No. 202122028524.X; a microorganism culture device convenient for sampling and detection, application number 202123166219.3; microorganism culture apparatus, application number 202223389482.3; a microbial cultivation device with a layered structure, application number 202320906653.0. All of these patents refer to the culture of Aspergillus niger at first glance, but either the structure is too complex to be expensive or only reference is made to how Aspergillus niger is used, and none relate to the purpose of the present invention. Therefore, how to design a device for cultivating aspergillus niger, which can cultivate aspergillus niger conveniently and rapidly and is cheap and good, is a great problem to be solved urgently, so that the aspergillus niger strain capable of expressing glucose oxidase genes efficiently is inoculated on a cultivating plate by utilizing the principle of clock swinging, so that the cultivating plate can swing for a long time under the slight pushing of manpower, the surface of the cultivating plate is fully contacted with oxygen, and meanwhile, on an ultra-clean workbench, all the cultivating plates are sealed in a sealed space, and oxygen supply water is generated by utilizing hot water heating and oxygen production particles to react with water in the sealed space, so that the aspergillus niger is in a suitable wet and hot growth environment, and the aspergillus niger cultivating device capable of expressing glucose oxidase genes simply, rapidly and efficiently is necessary.
Disclosure of Invention
In order to overcome the difficulty of how to design a device for culturing the aspergillus niger, which can conveniently and rapidly culture the aspergillus niger and is cheap and good, the invention provides a simple and rapid aspergillus niger culturing device for expressing the glucose oxidase gene in high efficiency.
The technical scheme adopted for solving the technical problems is as follows:
the invention relates to a simple and rapid Aspergillus niger culture device for expressing glucose oxidase genes with high efficiency, which consists of a dish containing barrel 1 and a bracket 2, and is characterized in that: the whole dish barrel 1 is cylindrical, the outer diameter of the dish barrel 1 is 15-20 cm, and the height is 15-25 cm; the swinging shafts 33 at two ends of the transverse frame rod 32 at the upper end of the dish barrel 1 are placed in the shaft placing grooves 6 of the transverse frame 3, the wall 15 of the dish barrel is the outer wall of the barrel body of the dish barrel 1, the inner layer is of a double-layer structure with an inner layer and an outer layer, the inner layer is of aluminum alloy, copper or stainless steel, the cross section is in a ring shape, the wall thickness of the inner layer is 0.3-0.5 mm, and the center of the inner surface of the bottom wall of the inner layer of the wall 15 of the dish barrel is provided with 1 round hole; the outer layer of the container barrel wall 15 is plastic, the cross section of the container barrel wall 15 is also circular, the thickness of the outer layer is 1-5 mm, the space between the inner layer structure and the outer layer structure of the container barrel wall 15 is the container barrel wall cavity 16, the width of the container barrel wall cavity 16, namely the distance between the inner layer structure and the outer layer structure is 0.5-1 cm, the container barrel wall cavity 16 is used for containing hot water 37, the added amount of hot water 37 is that the distance between the upper surface of the hot water 37 and the top end of the inner layer of the container barrel wall 15 is 1 cm; the ring fixing concave 17 is a groove arranged at the upper part of the outer surface of the outer layer of the vessel barrel wall 15, the cross section of the ring fixing concave is semicircular, the diameter of the circle where the semicircle is positioned is 0.5-1 cm, and the distance between the upper edge of the ring fixing concave 17 and the top edge of the outer layer of the vessel barrel wall 15 is 3-5 cm; the solid ring 18 is in a circular shape, the diameter of the cross section circle of the ring body is 0.45-0.9 cm, and the ring body of the solid ring 18 is copper, stainless steel or aluminum alloy; the dish barrel hanging frame 26 is made of copper, stainless steel or aluminum alloy, the cross section of the dish barrel hanging frame 26 is round, the diameter of the cross section of the dish barrel hanging frame 26 is 0.45-0.9 cm, the lower end of the dish barrel hanging frame 26 is provided with two free ends, the two free ends are welded on the outer surface of the fixed ring 18, and the two welding points are positioned at two ends of one diameter of the circle where the fixed ring 18 is positioned; the lower ends of the dish containing barrel racks 26 are firstly horizontally stretched to two sides for 1-2 cm, then vertically bent upwards, when reaching a plane 3-5 cm away from the upper surface of the dish containing barrel cover 23, the two metal strips are bent towards the middle, the acute angle formed after bending towards the middle is not more than 45 degrees, then the dish containing barrel racks 26 on the two sides are welded together at the contact position, and simultaneously the upper part of the welding position is welded with the lower end of the dish containing barrel swing arm 29, so that the dish containing barrel swing arm 29 is positioned in the same plane; the dish barrel swing arm 29 is made of copper, stainless steel or aluminum alloy, the cross section of the dish barrel swing arm 29 is round, the diameter of the cross section of the dish barrel swing arm 29 is 0.45-0.9 cm, the length of the dish barrel swing arm 29 is 10-20 cm, and the upper end of the dish barrel swing arm 29 is welded on the middle lower surface of the transverse frame rod 32; the cross frame rod 32 is a cross rod which supports and fixes the telescopic spring 30, the swing shaft sheath 31 and the swing shaft 33 and is connected with the dish containing barrel swing arm 29, the cross frame rod 32 is made of copper, stainless steel or aluminum alloy, the cross section is round, the diameter of the cross section of the cross frame rod 32 is 0.5-1.5 cm, the length is 3-5 cm, the centers of two ends of the cross frame rod 32 are respectively provided with a cylindrical groove, and the diameter of the cylindrical groove is 0.3-0.5 cm, and the depth is 1-1.5 cm; the balance staff sheath 31 is a structure for fixing the balance staff 33 and is made of manganese steel, a plurality of cylinders are sleeved together one by one, two adjacent cylinders are connected in a seamless mode, the cylinder inside is just inserted into the cylinder outside, the diameter of the cylinder inside is the smallest, the diameter of the cylinder inside is 0.3-0.5 mm, the telescopic spring 30 and the balance staff 33 are plugged in the cylinder cavity of the cylinder, the diameter of the cylinder outside is the largest, the wall thickness of each cylinder is the same and is 0.3-0.5 mm, the lengths of the cylinders are different, the lengths of the two adjacent cylinders are different by 1-2 mm, the cylinders are flush after being sleeved together, and the front ends of the cylinders form a cone; the telescopic spring 30 is made of spring steel and has the length of 3-5 mm; the pendulum shaft 33 is made of manganese steel and is cylindrical, the diameter of the cross section of the pendulum shaft 33 is 0.3-0.5 mm, and the length of the pendulum shaft is 2-3 cm; the fixed end of the pendulum shaft 33 is inserted into the cylinder cavity of the innermost cylinder of the pendulum shaft jacket 31, and the free end of the pendulum shaft 33 is 0.3-0.5 mm away from the outermost side of the shaft groove 6; the dish containing barrel cavity 19 is a space for containing the culture dish 10 with holes to cultivate the aspergillus niger, is cylindrical and is formed by the inner wall of the dish containing barrel wall 15 and the dish containing barrel cover 23; the water-through connection support ring 20 is a support ring between the lower surface of the bottom wall of the inner wall of the vessel barrel wall 15 and the upper surface of the bottom wall of the outer wall of the vessel barrel wall 15, and is made of copper, stainless steel or aluminum alloy, the cross section of the ring body of the water-through connection support ring 20 is rectangular, the length of the rectangle is 1-2 cm, and the width of the rectangle is 0.5-1 cm; the water-through connection support ring 20 is provided with a plurality of water-through holes at the center in the horizontal direction of the ring body so that the hot water 37 can pass through and enter the dish containing barrel center Kong Tongzhu; the dish containing barrel center Kong Tongzhu is a cylinder positioned in the center of the dish containing barrel cavity 19 and is made of aluminum alloy, copper or stainless steel, the cross section of the dish containing barrel center Kong Tongzhu is circular, the diameter of an inner ring is 0.5-1 cm, the diameter of an outer ring is 1.1-2 cm, the wall thickness of the outer ring is 0.3-0.5 mm, the lower end of the dish containing barrel center Kong Tongzhu is welded and fixed on the periphery of a central round hole in the inner surface of the bottom wall of the inner layer of the dish containing barrel wall 15, a cavity in the dish containing barrel center Kong Tongzhu is communicated with the lower dish containing barrel wall cavity 16, the upper end of the dish containing barrel center Kong Tongzhu is free, a cavity in the dish containing barrel center Kong Tongzhu is communicated with the upper dish containing barrel cavity 19, and the dish containing cushion 7 and the culture dish 10 with holes are sleeved on the dish containing barrel center Kong Tongzhu; the inter-dish cushion block 7 is made of rubber or plastic and is cylindrical, the solid structure of the periphery of the inter-dish cushion block 7 is an inter-dish cushion block body 8, and a cavity in the center of the inter-dish cushion block 7 is an inter-dish cushion block cavity 9; the thickness of the pad body 8 between dishes is 1-2 cm, and the height in the up-down direction is 1-2 cm; the cushion block cavity 9 between dishes is cylindrical and has a diameter of 1.2-2.1 cm; the culture dish 10 with holes is made of glass or polyethylene plastic, transparent, in a plane disk shape and without a cover, the culture dish 10 with holes consists of a central hole cylinder 11 with holes, a wall 12 with holes, a cavity 13 with holes and a solid culture medium 14, the central hole cylinder 11 with holes is positioned at the center of the bottom wall of the culture dish 10 with holes, is made of glass or polyethylene plastic, transparent, has a circular cross section, has an inner ring diameter of 1.2-2.1 cm and an outer ring diameter of 1.4-3.5 cm, has a wall thickness of 0.1-0.5 cm and a height of 1 cm, the lower end of the central hole cylinder 11 with holes is fixed on the periphery of the central round hole of the inner surface of the bottom wall of the culture dish 10 with holes, the cavity in the central hole cylinder 11 with holes is communicated with the outside, and the upper end of the central hole cylinder 11 with holes is free; the culture dish wall 12 with holes is the bottom wall and the peripheral wall of the culture dish 10 with holes, is vitreous or polyethylene plastic, transparent, has the thickness of 0.5-1 mm, and the periphery of the bottom wall of the culture dish 10 with holes is circular, and has the diameter of 10-12 cm; the peripheral wall of the culture dish 10 with holes is cylindrical, the height is 1 cm, the diameter of the inner ring of the circular ring with the cross section of the peripheral wall is 9.9-11.8 cm, the diameter of the outer ring is 10-12 cm, and the wall thickness is 0.5-1 mm; the culture dish cavity 13 with holes is a space surrounded by the culture dish wall 12 with holes and the culture dish central hole cylinder 11 with holes; the solid culture medium 14 is a culture medium for culturing aspergillus niger which efficiently expresses glucose oxidase genes; the container cover 23 is a cover which is buckled on the upper part of the container barrel 1 to seal the upper opening and provide oxygen for the container barrel cavity 19, is transparent glass, the lower part of the container cover 23 is cylindrical, the upper part of the container cover 23 is spherical, the diameter of a sphere where the upper part of the container cover 23 is positioned is 30-40 cm, the center of the outer surface of the upper part of the container cover 23 is provided with 1 container cover handle 28, the inner diameter of a cylinder at the lower part of the container cover 23 is 15-20 cm, and the height of the cylinder is 1-3 cm; the lower cylinder of the vessel cover 23 is provided with an upper bottom but not a lower bottom, the thickness of the upper bottom and the peripheral wall is 1.5-2 cm, the central part of the upper bottom is missing, the inner side wall surface of the formed cylindrical groove is provided with a spiral wire which is meshed with the spiral wire on the outer surface of the peripheral wall of the oxygen vessel 34 to form a fixed spiral wire 22, the vessel cover 23 consists of the fixed spiral wire 22, an oxygen producing cavity 24, a sealing cushion layer 27, a vessel cover handle 28, an oxygen producing vessel 34, oxygen producing particles 35 and water 36, the fixed spiral wire 22 is the spiral wire between the oxygen vessel 34 and the lower groove of the vessel cover 23, the width is 1-2 mm, the pitch between two adjacent spirals is 0.5-1 mm, when in screwing, a finger props against the sealing cushion layer 27 at the bottom of the oxygen vessel 34, the oxygen vessel 34 and the lower groove of the vessel cover 23 can be screwed together, and the oxygen vessel 34 can be unscrewed from the lower groove of the vessel cover 23 by anticlockwise rotation; the barrel cover handle 28 is made of vitreous or stainless steel and is arc-shaped and long, two ends of the barrel cover handle are fixed on the outer surface of the upper part of the dish barrel cover 23, and a distance between the lower part of the barrel cover handle 28 and the outer surface of the upper part of the dish barrel cover 23 is 2-3 cm, so that a finger can extend into the barrel to lift the whole dish barrel cover 23; the oxygen producing vessel 34 is a device for containing oxygen producing particles 35 and water 36, and conveying oxygen produced after the reaction of the oxygen producing particles 35 and the water 36 into the vessel barrel cavity 19 from the oxygen producing chamber 24 through the oxygen producing vessel central hole barrel 25, the oxygen producing vessel 34 is fixed at the center of the inner surface of the upper part of the vessel barrel cover 23 through a fixed rotary wire 22, a round hole with the diameter of 0.5-1 cm is formed in the center of the bottom wall of the oxygen producing vessel 34, the upper surface of the periphery of the round hole is connected with the lower end of the oxygen producing vessel central hole barrel 25, the space surrounded by the bottom wall of the oxygen producing vessel 34, the periphery wall of the oxygen producing vessel 34, the upper part of the vessel barrel cover 23 and the oxygen producing vessel central hole barrel 25 is the oxygen producing chamber 24, the oxygen producing particles 35 and the water 36 are placed in the oxygen producing vessel 34, and the oxygen producing particles 35 are commercially available white granular oxygenating agents made of sodium percarbonate, stabilizer and synergist and are cylindrical; the water 36 is autoclaved deionized water prepared in a laboratory, and the filling amount of the water 36 is half of the volume in the oxygen-generating vessel 34; the sealing cushion layer 27 is a latex film with the thickness of 1-2 mm, the lower surface of the bottom wall of the oxygen generating vessel 34 is adhered to the upper surface of the sealing cushion layer 27 by using an adhesive, a round hole is arranged in the center of the sealing cushion layer 27, the round hole faces the lower end of the oxygen generating vessel central hole barrel 25, and the diameter of the sealing cushion layer 27 is 15-20 cm.
The solid culture medium 14 is a culture medium for culturing aspergillus niger which can efficiently express glucose oxidase genes, and the preparation method is as follows: firstly, absorbing 2 mL corn steep liquor in 50 mL distilled water by a liquid transfer device, stirring and dissolving, fixing the volume to 70 mL by distilled water, and adjusting the pH to 5.5-6.0 by sodium hydroxide to prepare corn steep liquor; next, weigh 2 g bean cake powder in 250 mL triangular flask, add 70 mL corn steep liquor; adding 1.5-2 g of agar powder into corn steep liquor, stirring while heating to fully dissolve agar and ensure no paste bottom, and sterilizing the triangular flask at 121deg.C and 0.1 MPa for 30 min to obtain fermentation culture solution; weighing 10 g glucose in 20 mL distilled water, stirring to dissolve, metering volume to 30 mL with distilled water, 121 deg.C, 0.1 MPa, and sterilizing for 30 min; finally, pouring 30 mL glucose solution into 70 mL fermentation culture liquid in an ultra-clean workbench, and fully mixing to prepare a solid culture medium 14; when the liquid culture medium is cooled to about 55 ℃, namely, when a triangular flask is held and slightly scalded, but not scalded, the culture medium can be rapidly poured into a perforated culture dish 10, when the poured liquid culture medium flows to the 3/4 area of the bottom wall of the perforated culture dish 10, the pouring is stopped, the whole bottom wall can be covered after the culture medium flows, the thickness of the culture medium is about 1-2 mm, then the perforated culture dish 10 is horizontally placed on the table top of an ultra-clean workbench, and the culture plate for inoculating Aspergillus niger capable of efficiently expressing glucose oxidase genes can be prepared after the culture medium is horizontally placed on the table top of the ultra-clean workbench for 1-2 hours; during inoculation, firstly, a pipetting gun is used for sucking 5-10 micro-elevating Aspergillus niger strains for efficiently expressing glucose oxidase genes, then 3-5 sterilized clean glass beads are used for rolling back and forth on the surface of the solid culture medium 14, so that the Aspergillus niger strains for efficiently expressing the glucose oxidase genes are uniformly distributed on the surface of the solid culture medium 14, a culture dish 10 with holes to be used can be prepared, and then the culture dish 10 is placed into a containing barrel 1, so that the Aspergillus niger for efficiently expressing the glucose oxidase genes can be cultured.
The bracket 2 is made of wood, plastic, stainless steel, copper or aluminum alloy and consists of a transverse bracket 3 and bracket legs 4, wherein the transverse bracket 3 is a bracket arranged in the horizontal direction, the length is 1-3 meters, each interval of the transverse bracket 3 is 30 cm, a swinging groove 5 is arranged on each interval of the transverse bracket 3, the cross section of the transverse bracket 3 is rectangular, and the length of the cross section of the transverse bracket 3 is 10-20 cm and the width is 2-5 cm; the support legs 4 are positioned below the two end parts of the transverse support 3 and are used for supporting the support of the transverse support 3, the height is 30-50 cm, the cross section is rectangular, the longitudinal section is trapezoidal, the width of the cross section of the support legs 4 is 2-5 cm, the length of the cross section of the upper part of the support legs 4 is 10-20 cm, and the length of the cross section of the upper part is 20-40 cm; the swinging groove 5 is a groove formed at one side of the transverse bracket 3, and the depth is 5-10 cm; the shaft placing groove 6 is made of manganese steel and is positioned above the grooves specially reserved on the two side walls of the swinging groove 5 of the transverse bracket 3, the shaft placing groove 6 is in a cuboid shape as a whole, a plurality of grooves with gradually enlarged diameters and semicircular cross sections are arranged in the center of the upper part of the shaft placing groove 6, the uppermost surface is also the groove farthest from the two side walls of the swinging groove 5 and is used for placing the swinging shaft 33, and the diameter of the groove is 0.4-0.6 mm; the outer groove is a space for containing the swing shaft sheath 31 and the cross bar 32, and the groove with the largest diameter is opened on the two side walls of the swing groove 5.
The Aspergillus niger culture device for simply and rapidly expressing the glucose oxidase gene has the beneficial effects that the Aspergillus niger strain capable of efficiently expressing the glucose oxidase gene is inoculated on the culture plate by utilizing the principle of clock swinging, so that the culture plate can swing for a long time under the slight pushing of manpower, the surface of the culture plate is fully contacted with oxygen, and meanwhile, all the culture plates are sealed in a sealed space on an ultra-clean workbench, and the sealed space utilizes hot water to supply heat and oxygen-producing particles to react with water to supply oxygen, so that the Aspergillus niger is in a suitable growth environment of damp and hot, thereby achieving the purpose of simply and rapidly culturing the Aspergillus niger for efficiently expressing the glucose oxidase gene. The invention has simple manufacture, can be used very conveniently.
Drawings
The invention is further described below with reference to the accompanying drawings.
FIG. 1 is a schematic diagram showing the overall structure of an Aspergillus niger culture device for simple, rapid and efficient expression of glucose oxidase genes.
FIG. 2 is a schematic diagram showing the structure of a vertical section of the joint between a vessel and a bracket of an Aspergillus niger culture device for simply, rapidly and efficiently expressing glucose oxidase genes.
FIG. 3 is a schematic diagram of the whole structure of a culture dish with holes of an Aspergillus niger culture device for simply, rapidly and efficiently expressing glucose oxidase genes.
FIG. 4 is a schematic diagram showing the overall structure of the spacer block between dishes of an Aspergillus niger culture device for simple, rapid and efficient expression of glucose oxidase gene.
FIG. 5 is a diagram showing colonies cultured in an Aspergillus niger culture device for simple, rapid and efficient expression of glucose oxidase gene.
In the figure, 1, a vessel bucket, 2, a bracket, 3, a cross bracket, 4, a bracket leg, 5, a swinging groove, 6, a shaft placing groove, 7, an inter-vessel cushion block, 8, an inter-vessel cushion block body, 9, an inter-vessel cushion block cavity, 10, a hole culture vessel, 11, a hole culture vessel center hole barrel, 12, a hole culture vessel wall, 13, a hole culture vessel cavity, 14, a solid culture medium, 15, a vessel bucket wall, 16, a vessel bucket wall cavity, 17, a fixed ring concave, 18, a fixed ring, 19, a vessel bucket cavity, 20, a water connection support ring, 21, a vessel bucket center Kong Tongzhu, 22, a fixed rotary wire, 23, a vessel bucket cover, 24, an oxygen generating cavity, 25, an oxygen generating vessel center hole barrel, 26, a vessel bucket handle, 27, a sealing cushion layer, 28, 29, a vessel bucket swing arm, 30, a spring, 31, a swing shaft jacket, 32, a cross bar, 33, 34, an oxygen generating vessel bucket cover, 35, oxygen generating particles, and 37.
Detailed Description
Example 1
As shown in the figure, the simple Aspergillus niger culture device for expressing glucose oxidase genes simply, quickly and efficiently comprises a dish barrel 1, a support 2, a cross support 3, support legs 4, a swinging groove 5, an axle placing groove 6, an inter-dish cushion block 7, an inter-dish cushion block body 8, an inter-dish cushion block cavity 9, a culture dish 10 with holes, a central hole barrel 11 with holes, a culture dish wall 12 with holes, a culture dish cavity 13 with holes, a solid culture medium 14, a dish barrel wall 15, a dish barrel wall cavity 16, a solid ring concave 17, a solid ring 18, a dish barrel cavity 19, a water connection supporting ring 20, a dish barrel center Kong Tongzhu, a fixed spin wire 22, a dish barrel cover 23, an oxygen producing cavity 24, an oxygen producing dish central hole barrel 25, a dish barrel hanging frame 26, a sealing cushion layer 27, a dish barrel cover handle 28, a dish barrel swing arm 29, a telescopic spring 30, a swinging axle sheath 31, a cross support rod 32, a swinging axle 33, oxygen producing dish 34, oxygen producing particles 35, water 36 and hot water 37. The dish containing barrel 1 is of a barrel-shaped structure capable of swinging for Aspergillus niger to grow, the whole dish containing barrel 1 is cylindrical, the outer diameter of the dish containing barrel 1 is 15-20 cm, and the height of the dish containing barrel is 15-25 cm; the swinging shafts 33 at the two ends of the transverse frame rod 32 at the upper end of the dish containing barrel 1 are placed in the shaft placing grooves 6 of the transverse frame 3, when the external force vertical to the direction of the frame 2 is applied to push, the swinging shafts 33 are extremely hard and thin, and the contact surface between the swinging shafts 33 and the shaft placing grooves 6 is extremely small, so that the whole dish containing barrel 1 can swing for a long time under the inertia effect, and the swinging of the dish containing barrel 1 can enable oxygen to fully contact the surface of the solid culture medium 14, so that the aspergillus niger grows well, and the growth of the aspergillus niger is accelerated; the container barrel wall 15 is the outer wall of the container barrel 1 barrel body and is of a double-layer structure with an inner layer and an outer layer, the inner layer is made of aluminum alloy, copper or stainless steel, the cross section of the inner layer is in a ring shape, the wall thickness of the inner layer is 0.3-0.5 mm, and the center of the inner surface of the bottom wall of the inner layer of the container barrel wall 15 is provided with 1 round hole; the outer layer of the vessel wall 15 is plastic, the cross section is also circular, the thickness of the outer layer is 1-5 mm, the space between the inner layer and the outer layer of the vessel wall 15 is the vessel wall cavity 16, the width of the vessel wall cavity 16, namely the distance between the inner layer and the outer layer is 0.5-1 cm, the vessel wall cavity 16 is used for containing hot water 37, the added amount of the hot water 37 is 1 cm from the upper surface of the hot water 37 to the top end of the inner layer of the vessel wall 15, when the vessel cover 23 is covered, the upper opening of the vessel wall cavity 16 is blocked, the hot water 37 cannot overflow in the swinging process, and the hot water 37 transfers heat into the vessel wall cavity 19 through the heat conduction of the inner layer of the vessel wall 15 so as to keep the temperature in the vessel wall cavity 19 and be used for the growth of aspergillus niger inoculated on the surface of the solid culture medium 14 in the vessel 10; the ring fixing concave 17 is a groove for arranging the ring fixing 18 to firmly fix the ring fixing 18 on the upper part of the outer surface of the outer layer of the vessel wall 15, the cross section is semicircular, the diameter of the circle where the semicircle is positioned is 0.5-1 cm, and the distance between the upper edge of the ring fixing concave 17 and the top edge of the outer layer of the vessel wall 15 is 3-5 cm; the solid ring 18 is in a circular shape, the diameter of the cross section circle of the ring body is 0.45-0.9 cm, the ring body of the solid ring 18 is made of copper, stainless steel or aluminum alloy, when the solid ring is arranged, a metal strip with preset thickness is firstly used for encircling the solid ring concave 17, redundant parts are sheared off after encircling, and the metal strips at two ends are welded together to form the solid ring 18; the dish barrel hanging frame 26 is made of copper, stainless steel or aluminum alloy, the cross section of the dish barrel hanging frame 26 is round, the diameter of the cross section of the dish barrel hanging frame 26 is 0.45-0.9 cm, the lower end of the dish barrel hanging frame 26 is provided with two free ends, the two free ends are welded on the outer surface of the fixed ring 18, the two welding points are positioned at two ends of one diameter of the circle where the fixed ring 18 is positioned, namely, the two free ends of the lower end of the dish barrel hanging frame 26 are welded on two side surfaces of the dish barrel wall 15; the lower ends of the dish containing barrel racks 26 are firstly horizontally stretched to two sides for 1-2 cm, then vertically bent upwards, when reaching a plane 3-5 cm away from the upper surface of the dish containing barrel cover 23, the two metal strips are bent towards the middle, the acute angle formed after bending towards the middle is not more than 45 degrees, then the dish containing barrel racks 26 on the two sides are welded together at the contact position, and simultaneously the upper part of the welding position is welded with the lower end of the dish containing barrel swing arm 29, so that the dish containing barrel swing arm 29 is positioned in the same plane; the dish barrel swing arm 29 is made of copper, stainless steel or aluminum alloy, the cross section of the dish barrel swing arm 29 is round, the diameter of the cross section of the dish barrel swing arm 29 is 0.45-0.9 cm, the length of the dish barrel swing arm 29 is 10-20 cm, and the upper end of the dish barrel swing arm 29 is welded on the middle lower surface of the transverse frame rod 32; the transverse frame rod 32 is a transverse rod which supports and fixes the telescopic spring 30, the swing shaft sheath 31 and the swing shaft 33 and is connected with the dish containing barrel swing arm 29, the transverse frame rod 32 is made of copper, stainless steel or aluminum alloy, the cross section of the transverse frame rod 32 is round, the diameter of the transverse frame rod 32 is 0.5-1.5 cm, the length of the transverse frame rod is 3-5 cm, the centers of two ends of the transverse frame rod 32 are respectively provided with a cylindrical groove, the diameter of each cylindrical groove is 0.3-0.5 cm, the depth of each cylindrical groove is 1-1.5 cm, the cylindrical grooves are used for containing the swing shaft sheath 31, when the swing shaft sheath 31 is arranged, one end of the swing shaft sheath 31, which is not provided with the swing shaft 33, is coated with a layer of adhesive, then the adhesive is inserted into the grooves at two ends of the transverse frame rod 32, and the adhesive is commercially available glass adhesive; the balance staff sheath 31 is a structure for fixing the balance staff 33 and is made of manganese steel, a plurality of cylinders are sleeved together one by one, two adjacent cylinders are connected in a seamless mode, the cylinder inside is just inserted into the cylinder outside, the diameter of the cylinder inside is the smallest, the diameter of the cylinder inside is 0.3-0.5 mm, the telescopic spring 30 and the balance staff 33 are plugged in the cylinder cavity of the cylinder, the diameter of the cylinder outside is the largest, the wall thickness of each cylinder is the same and is 0.3-0.5 mm, the lengths of the cylinders are different, the lengths of the two adjacent cylinders are different by 1-2 mm, the rear ends of the cylinders are flush after being sleeved together, the front ends of the cylinders form a cone, and the tip of the cone is the balance staff 33; the telescopic spring 30 is made of spring steel and has the length of 3-5 mm, so that the pendulum shaft 33 is prevented from being inserted too tightly and not easy to replace after the friction time is too long; the pendulum shaft 33 is made of manganese steel and is cylindrical, the diameter of the cross section of the pendulum shaft 33 is 0.3-0.5 mm, and the length of the pendulum shaft is 2-3 cm; the fixed end of the pendulum shaft 33 is inserted into the cylinder cavity of the innermost cylinder of the pendulum shaft jacket 31, when the pendulum shaft is arranged, the telescopic spring 30 is plugged into the cylinder cavity of the cylinder, then the pendulum shaft 33 is inserted, the part of the pendulum shaft 33 exposed out of the innermost cylinder of the shaft jacket 31 is provided with a length of 0.5-1 cm and is placed in the shaft placing groove 6, and the free end of the pendulum shaft 33 is 0.3-0.5 mm away from the outermost side of the shaft placing groove 6; the dish containing barrel cavity 19 is a space for containing the culture dish 10 with holes to cultivate the aspergillus niger, is cylindrical and is formed by the inner wall of the dish containing barrel wall 15 and the dish containing barrel cover 23; the water-through connection support ring 20 is a support ring between the lower surface of the bottom wall of the inner wall of the vessel barrel wall 15 and the upper surface of the bottom wall of the outer wall of the vessel barrel wall 15, and is made of copper, stainless steel or aluminum alloy, the cross section of the ring body of the water-through connection support ring 20 is rectangular, the length of the rectangle is 1-2 cm, and the width of the rectangle is 0.5-1 cm; a plurality of water permeable holes are arranged at the center of the water permeable connection support ring 20 in the horizontal direction so that the hot water 37 can pass through and enter the dish barrel center Kong Tongzhu; the dish containing barrel center Kong Tongzhu 21 is a cylinder positioned in the center of the dish containing barrel cavity 19 and is made of aluminum alloy, copper or stainless steel, the cross section of the dish containing barrel center Kong Tongzhu is circular, the diameter of an inner ring is 0.5-1 cm, the diameter of an outer ring is 1.1-2 cm, the wall thickness of the outer ring is 0.3-0.5 mm, the lower end of the dish containing barrel center Kong Tongzhu is welded and fixed on the periphery of a central round hole in the inner surface of the bottom wall of the inner layer of the dish containing barrel wall 15, a cavity in the dish containing barrel center Kong Tongzhu is communicated with the lower dish containing barrel wall cavity 16, the upper end of the dish containing barrel center Kong Tongzhu is free, a cavity in the dish containing barrel center Kong Tongzhu is communicated with the upper dish containing barrel cavity 19, so that the dish containing barrel cavity 19 keeps humidity stable, the requirements of Aspergillus niger on moisture in the culture process are ensured, the dish containing barrel center Kong Tongzhu is sleeved with the culture dish 10 with holes, namely, when in use, the culture dish 10 is sterilized, pouring a liquid solid culture medium 14 into a flat plate, inoculating Aspergillus niger strains to be cultured on the upper surface of the solid culture medium 14 after the solid culture medium 14 is solidified, sleeving an inter-dish cushion block 7 on a dish containing barrel center Kong Tongzhu, sliding the inter-dish cushion block 7 downwards to the lower end of the dish containing barrel center Kong Tongzhu by gravity, enabling the lower surface to be in contact with the bottom wall of the inner layer of the dish containing barrel wall 15, sleeving an inoculated culture dish 10 with holes on the dish containing barrel center Kong Tongzhu, and sliding the culture dish 10 with holes downwards to the upper surface of the inter-dish cushion block 7 on the lower part of the dish containing barrel center Kong Tongzhu by gravity, so that the inter-dish cushion block 7 and the culture dish 10 with holes are sequentially arranged, and finishing the arrangement of the inter-dish cushion block 7 and the culture dish 10 with holes when the upper surface of the last culture dish 10 with holes is 1 cm from the plane where the upper edge of the dish containing barrel center Kong Tongzhu is positioned; the inter-dish cushion block 7 is a cushion block for supporting and spacing the culture dishes 10 with holes, is made of rubber or plastic, is cylindrical, the solid structure of the periphery of the inter-dish cushion block 7 is an inter-dish cushion block body 8, a cavity in the center of the inter-dish cushion block 7 is an inter-dish cushion block cavity 9, that is to say, the inter-dish cushion block body 8 is the outer wall of the inter-dish cushion block 7, and the inter-dish cushion block cavity 9 is the inner cavity of the inter-dish cushion block 7; the thickness of the pad body 8 between dishes is 1-2 cm, and the height in the up-down direction is 1-2 cm; the cushion block cavity 9 between dishes is cylindrical and has a diameter of 1.2-2.1 cm; the culture dish 10 is a culture dish for culturing aspergillus niger, is vitreous or polyethylene plastic, is transparent, takes the shape of a plane disk without a cover, the culture dish 10 is composed of a central hole cylinder 11 of the culture dish, a wall 12 of the culture dish with holes, a culture dish cavity 13 with holes and a solid culture medium 14, the central hole cylinder 11 of the culture dish with holes is positioned at the center of the bottom wall of the culture dish 10, is vitreous or polyethylene plastic, is transparent, has a circular cross section, has an inner ring diameter of 1.2-2.1 cm, an outer ring diameter of 1.4-3.5 cm and a wall thickness of 0.1-0.5 cm, has a height of 1 cm, the lower end of the central hole cylinder 11 of the culture dish with holes is fixedly arranged at the periphery of a central round hole of the inner surface of the bottom wall 10 of the culture dish with holes, a cavity in the central hole cylinder 11 of the culture dish with holes is communicated with the outside, the upper end of the central hole cylinder 11 of the culture dish with holes is also communicated with the outside, so that the whole culture dish 10 with holes can be sleeved on the central cylinder Kong Tongzhu of the culture dish with the cushion block 577 to form a mutual supporting relation; the culture dish wall 12 with holes is the bottom wall and the peripheral wall of the culture dish 10 with holes, is vitreous or polyethylene plastic, transparent, has the thickness of 0.5-1 mm, and the periphery of the bottom wall of the culture dish 10 with holes is circular, and has the diameter of 10-12 cm; the peripheral wall of the culture dish 10 with holes is cylindrical, the height is 1 cm, the diameter of the inner ring of the circular ring with the cross section of the peripheral wall is 9.9-11.8 cm, the diameter of the outer ring is 10-12 cm, and the wall thickness is 0.5-1 mm; the culture dish cavity 13 with holes is a space surrounded by the culture dish wall 12 with holes and the culture dish central hole cylinder 11 with holes and is used for containing a solid culture medium 14; the solid culture medium 14 is a culture medium for culturing aspergillus niger which can efficiently express glucose oxidase genes, and the preparation method is as follows: firstly, absorbing 2 mL corn steep liquor in 50 mL distilled water by a liquid transfer device, stirring and dissolving, fixing the volume to 70 mL by distilled water, and adjusting the pH to 5.5-6.0 by sodium hydroxide to prepare corn steep liquor; next, weigh 2 g bean cake powder in 250 mL triangular flask, add 70 mL corn steep liquor; adding 1.5-2 g of agar powder into corn steep liquor, stirring while heating to fully dissolve agar and ensure no paste bottom, and sterilizing the triangular flask at 121deg.C and 0.1 MPa for 30 min to obtain fermentation culture solution; weighing 10 g glucose in 20 mL distilled water, stirring to dissolve, metering volume to 30 mL with distilled water, 121 deg.C, 0.1 MPa, and sterilizing for 30 min; finally, pouring 30 mL glucose solution into 70 mL fermentation culture liquid in an ultra-clean workbench, and fully mixing to prepare a solid culture medium 14; when the liquid culture medium is cooled to about 55 ℃, namely, when a triangular flask is held and slightly scalded, but not scalded, the culture medium can be rapidly poured into a perforated culture dish 10, when the poured liquid culture medium flows to the 3/4 area of the bottom wall of the perforated culture dish 10, the pouring is stopped, the whole bottom wall can be covered after the culture medium flows, the thickness of the culture medium is about 1-2 mm, then the perforated culture dish 10 is horizontally placed on the table top of an ultra-clean workbench, and the culture plate for inoculating Aspergillus niger capable of efficiently expressing glucose oxidase genes can be prepared after the culture medium is horizontally placed on the table top of the ultra-clean workbench for 1-2 hours; during inoculation, firstly sucking Aspergillus niger strains which are 5-10 micro-liters and efficiently express glucose oxidase genes by a pipetting gun, then rolling back and forth on the surface of a solid culture medium 14 by 3-5 sterilized clean glass beads, so that the Aspergillus niger strains which are efficiently express the glucose oxidase genes are uniformly distributed on the surface of the solid culture medium 14, namely a culture dish 10 with holes to be used can be prepared, and then the culture dish is placed in a containing barrel 1, so that Aspergillus niger which is efficiently express the glucose oxidase genes can be cultivated; as the Aspergillus niger strain used in the experiment can efficiently express the glucose oxidase gene, and the invention can provide proper conditions of temperature, humidity, oxygen and the like required by the growth and the propagation of the Aspergillus niger, a shaking table or an incubator which occupies a larger space and is more expensive is not needed, and the invention is simple and rapid, therefore, the invention is provided with a simple and rapid Aspergillus niger culture device for efficiently expressing the glucose oxidase gene. The container cover 23 is a cover which is buckled on the upper part of the container barrel 1 to seal the upper opening and provide oxygen for the container barrel cavity 19, is transparent glass and is used for observing the condition of the container barrel 1, the lower part of the container cover 23 is cylindrical, the upper part of the container cover 23 is spherical, the diameter of a sphere where the upper part of the container cover 23 is positioned is 30-40 cm, the center of the outer surface of the upper part of the container cover 23 is provided with 1 container cover handle 28 which is used for lifting or covering the whole container cover 23, and the inner diameter of the cylinder at the lower part of the container cover 23 is 15-20 cm and the height is 1-3 cm; the lower cylinder of the vessel cover 23 is provided with an upper bottom but not a lower bottom, the thickness of the upper bottom and the peripheral wall is 1.5-2 cm, the central part of the upper bottom is missing, the inner side wall surface of the formed cylindrical groove is provided with a spiral wire which is used for being meshed with the spiral wire on the outer surface of the peripheral wall of the oxygen producing vessel 34 to form a fixed spiral wire 22, so that the oxygen producing vessel 34 is fixed at the central part of the inner surface of the upper part of the vessel cover 23 through the fixed spiral wire 22, the vessel cover 23 consists of the fixed spiral wire 22, an oxygen producing cavity 24, a sealing cushion layer 27, a vessel cover handle 28, the oxygen producing vessel 34, oxygen producing particles 35 and water 36, the fixed spiral wire 22 is a spiral wire between the oxygen producing vessel 34 and the lower groove of the vessel cover 23, the width is 1-2 mm, the pitch between the adjacent two spirals is 0.5-1 mm, when the spiral wires are screwed, the sealing cushion layer 27 at the bottom of the oxygen producing vessel 34 is propped against the oxygen producing vessel 34 by fingers, the sealing cushion layer 27 is screwed together by clockwise rotation, and the oxygen producing vessel 34 can be unscrewed from the lower groove of the vessel cover 23 by anticlockwise rotation; the barrel cover handle 28 is made of vitreous or stainless steel and is arc-shaped and long, two ends of the barrel cover handle are fixed on the outer surface of the upper part of the dish barrel cover 23, and a distance between the lower part of the barrel cover handle 28 and the outer surface of the upper part of the dish barrel cover 23 is 2-3 cm, so that a finger can extend into the barrel to lift the whole dish barrel cover 23; the oxygen-producing vessel 34 is a device for containing oxygen-producing particles 35 and water 36, and conveying oxygen produced after the reaction of the oxygen-producing particles 35 and the water 36 into the vessel barrel cavity 19 from the oxygen-producing vessel 24 through the oxygen-producing vessel central hole barrel 25, a round hole with the diameter of 0.5-1 cm is formed in the center of the bottom wall of the oxygen-producing vessel 34, the upper surface of the periphery of the round hole is connected with the lower end of the oxygen-producing vessel central hole barrel 25, so that the whole oxygen-producing vessel central hole barrel 25 is communicated up and down, the space surrounded by the bottom wall of the oxygen-producing vessel 34, the peripheral wall of the oxygen-producing vessel 34, the upper part of the vessel barrel cover 23 and the oxygen-producing vessel central hole barrel 25 is the oxygen-producing cavity 24, the oxygen-producing particles 35 and the water 36 are placed in the oxygen-producing vessel 34, and the oxygen-producing particles 35 are white granular agents which are commercially available and are made of sodium percarbonate, stabilizer and synergist, are cylindrical, and can be directly sunk into the bottom of the water 36 to react with the water 36, and a large amount of oxygen is produced, so as to provide oxygen-enriched aspergillus niger strains for the high-efficiency expression glucose oxidase genes on the solid medium 14 for growth; the water 36 is autoclaved deionized water prepared in a laboratory, the pouring amount of the water 36 is half of the volume in the oxygen-generating vessel 34, and because the vessel cover 23 is transparent, an experimenter can intuitively observe specific conditions of oxygen-generating particles 35 and water 36 placed in the oxygen-generating vessel 34, once no bubbles are generated any more, the vessel barrel 1 can be taken down, and after the surface of the vessel is disinfected by alcohol, the oxygen-generating particles 35 are replaced or the water 36 is added in an ultra-clean workbench; the sealing cushion layer 27 is a latex film with the thickness of 1-2 mm, the lower surface of the bottom wall of the oxygen producing vessel 34 is adhered to the upper surface of the sealing cushion layer 27 by an adhesive, a round hole is arranged in the center of the sealing cushion layer 27 and faces the lower end of the oxygen producing vessel central hole barrel 25 so that oxygen can pass through, the diameter of the sealing cushion layer 27 is 15-20 cm, the sealing cushion layer is the same as the inner diameter of the lower part of the vessel barrel cover 23, and after the vessel barrel cover 23 is buckled on the vessel barrel wall 15, a gap between the sealing cushion layer and the vessel barrel cover can be blocked so as to prevent microorganisms in the outside air from entering the polluted solid culture medium 14. The bracket 2 is a bracket for supporting the dish containing barrel 1 to enable the dish containing barrel 1 to swing in the swinging groove 5, is made of wood, plastic, stainless steel, copper or aluminum alloy, and consists of a transverse bracket 3 and bracket legs 4, wherein the transverse bracket 3 is a bracket arranged in the horizontal direction, the length is 1-3 m, the swinging groove 5 is arranged every 30 cm of the transverse bracket 3, the cross section of the transverse bracket 3 is rectangular, and the length of the cross section of the transverse bracket 3 is 10-20 cm and the width is 2-5 cm; the support legs 4 are positioned below the two end parts of the transverse support 3 and used for supporting the support of the transverse support 3, the height is 30-50 cm, the cross section is rectangular, the longitudinal section is trapezoidal and used for keeping stability, the width of the cross section of the support legs 4 is 2-5 cm, the length of the cross section of the upper part of the support legs 4 is 10-20 cm, and the length of the cross section of the upper part is 20-40 cm; the swinging groove 5 is a groove formed at one side of the transverse bracket 3, has a depth of 5-10 cm and is used for placing the upper end part of the dish containing barrel 1, so that the dish containing barrel 1 swings back and forth below the transverse bracket 3; the shaft placing groove 6 is made of manganese steel and is positioned above the grooves specially reserved on the two side walls of the swinging groove 5 of the transverse bracket 3, the shaft placing groove 6 is integrally cuboid, a plurality of grooves with gradually enlarged diameters and semicircular cross sections are formed in the center of the upper part of the shaft placing groove 6, the uppermost groove is the groove farthest from the two side walls of the swinging groove 5 and is used for placing the swinging shaft 33, the swinging shaft 33 is in contact with the shaft placing groove 6, the diameter of the groove is 0.4-0.6 mm, and as a small part of the lower surface of the swinging shaft 33 is in contact with the inner surface of the shaft placing groove 6, the friction force is extremely small, so that the whole dish containing barrel 1 can swing for a long time at the swinging groove 5 after being pushed by artificial external force; the outer grooves are spaces for accommodating the balance staff sheath 31 and the cross frame rod 32, and the grooves with the largest diameters are opened on the two side walls of the swinging groove 5, but the inner surfaces of the grooves are not contacted with the balance staff sheath 31 and the cross frame rod 32, namely the balance staff sheath 31 and the cross frame rod 32 are suspended in the air, so that the whole dish containing barrel 1 can swing back and forth around the balance staff 33. Under normal conditions, the Aspergillus niger capable of efficiently expressing the glucose oxidase gene can be cultivated in a fermentation tank or a shaking table in 11 days, and experiments prove that the task can be finished in advance by using a simple and rapid Aspergillus niger cultivation device capable of efficiently expressing the glucose oxidase gene to cultivate the Aspergillus niger capable of efficiently expressing the glucose oxidase gene, as shown in FIG. 5, the time cost is greatly saved, and meanwhile, the structure is simple, the price is low, and more cost can be saved in a cultivation facility.
The foregoing has shown and described the basic principles and main features of the present invention and the advantages of the present invention. It will be understood by those skilled in the art that the present invention is not limited to the foregoing embodiments, which have been described in the foregoing embodiments and description merely illustrates the principles of the invention, and that various changes and modifications may be effected therein without departing from the spirit and scope of the invention as defined in the appended claims and their equivalents.
Claims (3)
1. Simple and easy high-efficient Aspergillus niger culture apparatus who expresses glucose oxidase gene comprises flourishing ware bucket (1), support (2), its characterized in that: the whole dish barrel (1) is cylindrical, the outer diameter of the dish barrel (1) is 15-20 cm, and the height of the dish barrel is 15-25 cm; the swinging shafts (33) at two ends of the transverse frame rod (32) at the upper end of the dish containing barrel (1) are placed in the shaft placing grooves (6) of the transverse frame (3), the wall (15) of the dish containing barrel is the outer wall of the barrel body of the dish containing barrel (1), the inner layer is of a double-layer structure with an inner layer and an outer layer, the inner layer is of aluminum alloy, copper or stainless steel, the cross section is of a ring shape, the wall thickness of the inner layer is 0.3-0.5 mm, and the center of the inner surface of the bottom wall of the inner layer of the wall (15) of the dish containing barrel is provided with 1 round hole; the outer layer of the container barrel wall (15) is plastic, the cross section of the container barrel wall is also circular, the thickness of the outer layer is 1-5 mm, a space between the inner layer structure and the outer layer structure of the container barrel wall (15) is a container barrel wall cavity (16), the width of the container barrel wall cavity (16), namely the distance between the inner layer structure and the outer layer structure is 0.5-1 cm, the container barrel wall cavity (16) is used for containing hot water (37), the added amount of the hot water (37) is 1 cm from the upper surface of the hot water (37) to the top end of the inner layer of the container barrel wall (15); the ring fixing concave (17) is a groove arranged at the upper part of the outer surface of the outer layer of the vessel wall (15), the cross section is semicircular, the diameter of the circle where the semicircle is positioned is 0.5-1 cm, and the distance between the upper edge of the ring fixing concave (17) and the top edge of the outer layer of the vessel wall (15) is 3-5 cm; the solid ring (18) is in a circular ring shape, the diameter of the cross section circle of the ring body is 0.45-0.9 cm, and the ring body of the solid ring (18) is copper, stainless steel or aluminum alloy; the dish barrel hanging frame (26) is made of copper, stainless steel or aluminum alloy, the cross section of the dish barrel hanging frame is circular, the diameter of the cross section of the dish barrel hanging frame (26) is 0.45-0.9 cm, two free ends are shared at the lower end of the dish barrel hanging frame (26), the two free ends are welded on the outer surface of the fixed ring (18), and the two welding points are positioned at the two ends of one diameter of the circle where the fixed ring (18) is positioned; the lower ends of the dish containing barrel racks (26) extend to two sides in the horizontal direction, the extending distance is 1-2 cm, then the dish containing barrel racks are vertically bent upwards, when reaching a plane 3-5 cm away from the upper surface of the dish containing barrel cover (23), the two metal strips are bent towards the middle, an acute angle formed after the metal strips are bent towards the middle is not more than 45 degrees, then the dish containing barrel racks (26) on the two sides are welded together at the contact position, and the upper part of the welding position and the lower end of the dish containing barrel swing arm (29) are welded together, so that the dish containing barrel swing arm (29) is positioned in the same plane; the dish barrel swing arm (29) is made of copper, stainless steel or aluminum alloy, the cross section of the dish barrel swing arm (29) is round, the diameter of the cross section of the dish barrel swing arm (29) is 0.45-0.9 cm, the length of the dish barrel swing arm is 10-20 cm, and the upper end of the dish barrel swing arm (29) is welded on the middle lower surface of the transverse frame rod (32); the cross frame rod (32) is a cross rod which supports and fixes the telescopic spring (30), the swing shaft sheath (31) and the swing shaft (33) and is connected with the dish barrel swing arm (29), the cross frame rod (32) is made of copper, stainless steel or aluminum alloy, the cross section is round, the diameter of the cross section of the cross frame rod (32) is 0.5-1.5 cm, the length of the cross frame rod is 3-5 cm, a cylindrical groove is respectively arranged in the center of two ends of the cross frame rod (32), and the diameter of the cylindrical groove is 0.3-0.5 cm, and the depth of the cylindrical groove is 1-1.5 cm; the balance staff sheath (31) is of a structure for fixing a balance staff (33), is made of manganese steel, is formed by sleeving a plurality of cylinders one by one, is in seamless connection with two adjacent cylinders, wherein the cylinder inside is just inserted into the cylinder outside, the diameter of the cylinder inside is the smallest, the diameter of the cylinder inside is 0.3-0.5 mm, a telescopic spring (30) and the balance staff (33) are plugged in the cylinder cavity of the cylinder, the diameter of the cylinder outside is the largest, the wall thickness of each cylinder is the same, the wall thickness of each cylinder is 0.3-0.5 mm, the lengths of the cylinders are different, the lengths of the two adjacent cylinders are different by 1-2 mm, the rear ends of the cylinders are flush after being sleeved together, and the front ends of the cylinders form a cone; the telescopic spring (30) is made of spring steel and has the length of 3-5 mm; the pendulum shaft (33) is made of manganese steel and is cylindrical, the diameter of the cross section of the pendulum shaft (33) is 0.3-0.5 mm, and the length of the pendulum shaft is 2-3 cm; the fixed end of the pendulum shaft (33) is inserted into the cylinder cavity of the innermost cylinder of the pendulum shaft sheath (31), and the free end of the pendulum shaft (33) is 0.3-0.5 mm away from the outermost side of the shaft groove (6); the dish barrel cavity (19) is a space for containing a culture dish (10) with holes to cultivate Aspergillus niger, is cylindrical and is surrounded by the inner wall of the dish barrel wall (15) and the dish barrel cover (23); the water-through connection support ring (20) is a support ring between the lower surface of the bottom wall of the inner wall of the vessel barrel wall (15) and the upper surface of the bottom wall of the outer wall of the vessel barrel wall (15), and is made of copper, stainless steel or aluminum alloy, the cross section of the ring body of the water-through connection support ring (20) is rectangular, and the length of the rectangle is 1-2 cm and the width is 0.5-1 cm; the center of the water-through connection support ring (20) in the horizontal direction is provided with a plurality of water-through holes so that hot water (37) can pass through and enter the dish containing barrel center Kong Tongzhu (21); the dish containing barrel center Kong Tongzhu (21) is a cylinder positioned in the center of the dish containing barrel cavity (19) and is made of aluminum alloy, copper or stainless steel, the cross section of the dish containing barrel center Kong Tongzhu is circular, the diameter of an inner ring is 0.5-1 cm, the diameter of an outer ring is 1.1-2 cm, the wall thickness of the outer ring is 0.3-0.5 mm, the lower end of the dish containing barrel center Kong Tongzhu (21) is fixedly welded on the periphery of a central round hole in the inner surface of the bottom wall of the inner layer of the dish containing barrel wall (15), a cavity in the dish containing barrel center Kong Tongzhu (21) is communicated with a dish containing barrel wall cavity (16) below, the upper end of the dish containing barrel center Kong Tongzhu (21) is free, a cavity in the dish containing barrel center Kong Tongzhu (21) is communicated with the dish containing barrel cavity (19) above, and the dish-to-be-used cushion block (7) and the culture dish (10) with holes are sleeved on the dish containing barrel center Kong Tongzhu (21); the inter-dish cushion block (7) is made of rubber or plastic and is cylindrical, the solid structure of the periphery of the inter-dish cushion block (7) is an inter-dish cushion block body (8), and a cavity in the center of the inter-dish cushion block (7) is an inter-dish cushion block cavity (9); the thickness of the cushion block body (8) between dishes is 1-2 cm, and the height in the up-down direction is 1-2 cm; the cushion block cavity (9) between dishes is cylindrical, and the diameter is 1.2-2.1 cm; the culture dish with holes (10) is made of glass or polyethylene plastic, transparent, in a plane disk shape, without a cover, the culture dish with holes (10) consists of a central hole cylinder (11) of the culture dish with holes, a wall (12) of the culture dish with holes, a cavity (13) of the culture dish with holes and a solid culture medium (14), the central hole cylinder (11) of the culture dish with holes is positioned at the center of the bottom wall of the culture dish with holes (10) and is made of glass or polyethylene plastic, the culture dish with holes is transparent, the cross section of the culture dish with holes is in a circular ring shape, the diameter of an inner ring is 1.2-2.1 cm, the diameter of an outer ring is 1.4-3.5 cm, the wall thickness is 0.1-0.5 cm, the height is 1 cm, the lower end of the central hole cylinder (11) of the culture dish with holes is fixedly arranged at the periphery of the central round hole of the inner surface of the bottom wall of the culture dish with holes (10), and the cavity in the central hole cylinder (11) of the culture dish with holes is communicated with the outside, and the upper end of the central hole cylinder (11) of the culture dish with holes is free; the culture dish wall (12) with holes is a bottom wall and a peripheral wall of the culture dish (10) with holes, is vitreous or polyethylene plastic, transparent, has the thickness of 0.5-1 mm, and has the periphery of the bottom wall of the culture dish (10) with holes of a circular shape and the diameter of 10-12 cm; the peripheral wall of the culture dish (10) with holes is cylindrical, the height is 1 cm, the diameter of the inner ring of the circular ring with the cross section of the peripheral wall is 9.9-11.8 cm, the diameter of the outer ring is 10-12 cm, and the wall thickness is 0.5-1 mm; the culture dish cavity (13) with holes is a space surrounded by the culture dish wall (12) with holes and the culture dish central hole cylinder (11) with holes; the solid culture medium (14) is a culture medium for culturing Aspergillus niger which can efficiently express glucose oxidase genes; the container cover (23) is a cover which is buckled on the upper part of the container barrel (1) to seal the upper opening and provide oxygen for the container barrel cavity (19), the transparent glass is adopted, the lower part of the container cover (23) is cylindrical, the upper part of the container cover is spherical, the diameter of a sphere where the upper part of the container cover (23) is positioned is 30-40 cm, the center of the outer surface of the upper part of the container cover (23) is provided with 1 container cover handle (28), the inner diameter of a cylinder at the lower part of the container cover (23) is 15-20 cm, and the height of the cylinder is 1-3 cm; the lower cylinder of the vessel cover (23) is provided with an upper bottom but not a lower bottom, the thickness of the upper bottom and the peripheral wall is 1.5-2 cm, the central part of the upper bottom is missing, the inner side wall surface of the formed cylindrical groove is provided with spiral threads which are meshed with the spiral threads on the outer surface of the peripheral wall of the oxygen vessel (34) to form a fixed spiral thread (22), the vessel cover (23) consists of the fixed spiral thread (22), an oxygen production cavity (24), a sealing cushion layer (27), a vessel cover handle (28), the oxygen vessel (34), oxygen production particles (35) and water (36), the fixed spiral thread (22) is the spiral threads between the oxygen vessel (34) and the lower groove of the vessel cover (23), the width of the spiral threads between two adjacent spirals is 0.5-1 mm, when the spiral threads are screwed, the sealing cushion layer (27) at the bottom of the oxygen vessel (34) is propped against a finger, the oxygen vessel (34) can be screwed together with the lower groove of the vessel cover (23) by clockwise rotation, and the oxygen vessel cover (34) can be unscrewed from the lower groove of the vessel cover (23) by anticlockwise rotation; the barrel cover handle (28) is made of glass or stainless steel and is arc-shaped and long, two ends of the barrel cover handle are fixed on the outer surface of the upper part of the dish barrel cover (23), and the distance between the lower part of the barrel cover handle (28) and the outer surface of the upper part of the dish barrel cover (23) is 2-3 cm, so that a finger can extend into the barrel to lift the whole dish barrel cover (23); the oxygen generating vessel (34) is a device for containing oxygen generating particles (35) and water (36) and conveying oxygen generated after reaction of the oxygen generating particles and the water into the vessel barrel cavity (19) from the oxygen generating vessel central hole barrel (25), the oxygen generating particles (35) and the water (36) are fixedly arranged at the center of the inner surface of the upper part of the vessel barrel cover (23) through fixed rotary threads (22), a round hole with the diameter of 0.5-1 cm is formed in the center of the bottom wall of the oxygen generating vessel (34), the upper surface of the periphery of the round hole is connected with the lower end of the oxygen generating vessel central hole barrel (25), the space surrounded by the bottom wall of the oxygen generating vessel (34), the peripheral wall of the oxygen generating vessel (34), the upper part of the vessel barrel cover (23) and the oxygen generating vessel central hole barrel (25) is the oxygen generating cavity (24), the oxygen generating particles (35) and the water (36) are placed in the oxygen generating vessel (35) and are white granular oxygen-enriching agents made of sodium percarbonate, stabilizer and synergist sold in the market, and are cylindrical; the water (36) is autoclaved deionized water prepared in a laboratory, and the filling amount of the water (36) is half of the inner volume of the oxygen-generating vessel (34); the sealing cushion layer (27) is a latex film, the thickness is 1-2 mm, the lower surface of the bottom wall of the oxygen producing dish (34) is adhered to the upper surface of the sealing cushion layer (27) by using an adhesive, a round hole is formed in the center of the sealing cushion layer (27), the round hole faces the lower end of the central hole barrel (25) of the oxygen producing dish, and the diameter of the sealing cushion layer (27) is 15-20 cm.
2. The simple and rapid aspergillus niger cultivation device for expressing glucose oxidase gene with high efficiency according to claim 1, wherein the device is characterized in that: the solid culture medium (14) is a culture medium for culturing aspergillus niger capable of efficiently expressing glucose oxidase genes, and the preparation method is as follows: firstly, absorbing 2 mL corn steep liquor in 50 mL distilled water by a liquid transfer device, stirring and dissolving, fixing the volume to 70 mL by distilled water, and adjusting the pH to 5.5-6.0 by sodium hydroxide to prepare corn steep liquor; next, weigh 2 g bean cake powder in 250 mL triangular flask, add 70 mL corn steep liquor; adding 1.5-2 g of agar powder into corn steep liquor, stirring while heating to fully dissolve agar and ensure no paste bottom, and sterilizing the triangular flask at 121deg.C and 0.1 MPa for 30 min to obtain fermentation culture solution; weighing 10 g glucose in 20 mL distilled water, stirring to dissolve, metering volume to 30 mL with distilled water, 121 deg.C, 0.1 MPa, and sterilizing for 30 min; finally, pouring 30 mL glucose solution into 70 mL fermentation culture liquid in an ultra-clean workbench, and fully mixing to prepare a solid culture medium (14); when the liquid culture medium is cooled to about 55 ℃, namely, when a triangular flask is held and slightly scalded but not scalded, the liquid culture medium can be poured into a holed culture dish (10), the pouring is stopped when the liquid culture medium flows to 3/4 area of the bottom wall of the holed culture dish (10), the whole bottom wall can be covered after the culture medium flows, the thickness of the culture medium is about 1-2 mm, then the holed culture dish (10) is horizontally placed on the table surface of an ultra-clean workbench, and the culture plate can be prepared after standing for 1-2 hours, and the aspergillus niger capable of efficiently expressing glucose oxidase genes can be inoculated; during inoculation, firstly, a pipetting gun is used for sucking 5-10 micro-elevating Aspergillus niger strains for efficiently expressing glucose oxidase genes, then 3-5 sterilized clean glass beads are used for rolling back and forth on the surface of a solid culture medium (14), so that the Aspergillus niger strains for efficiently expressing the glucose oxidase genes are uniformly distributed on the surface of the solid culture medium (14), a culture dish (10) with holes to be used can be prepared, and then the culture dish is placed into a dish containing barrel (1), so that Aspergillus niger for efficiently expressing the glucose oxidase genes can be cultured.
3. The simple and rapid aspergillus niger cultivation device for expressing glucose oxidase gene with high efficiency according to claim 1, wherein the device is characterized in that: the support (2) is made of wood, plastic, stainless steel, copper or aluminum alloy and consists of a transverse support (3) and support legs (4), wherein the transverse support (3) is a support arranged in the horizontal direction, the length is 1-3 m, each interval of 30 cm of the transverse support (3) is provided with one swinging groove (5), the cross section of the transverse support (3) is rectangular, and the length of the cross section of the transverse support (3) is 10-20 cm and the width of the cross section of the transverse support is 2-5 cm; the support legs (4) are positioned below the two end parts of the transverse support (3) and are used for supporting the support of the transverse support (3), the height of the support is 30-50 cm, the cross section of the support is rectangular, the longitudinal section of the support is trapezoidal, the width of the cross section of the support legs (4) is 2-5 cm, the length of the cross section of the upper part of the support legs (4) is 10-20 cm, and the length of the cross section of the upper part of the support legs is 20-40 cm; the swinging groove (5) is a groove formed at one side of the transverse bracket (3) and has a depth of 5-10 cm; the shaft placing groove (6) is made of manganese steel and is positioned above a groove specially reserved on two side walls of the swinging groove (5) of the transverse bracket (3), the shaft placing groove (6) is integrally cuboid, a plurality of grooves with gradually enlarged diameters and semicircular cross sections are arranged in the center of the upper part of the shaft placing groove (6), the uppermost surface is a groove farthest from the two side walls of the swinging groove (5) and is used for placing a swinging shaft (33), and the diameter of the groove is 0.4-0.6 mm; the outside groove is a space for containing the swing shaft sheath (31) and the cross bar (32), and the groove with the largest diameter is opened on the two side walls of the swing groove (5).
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0731460A (en) * | 1993-07-16 | 1995-02-03 | Hiranuma Sangyo Kk | Apparatus for automatic preparation of nutrient medium |
| CN105695341A (en) * | 2016-03-16 | 2016-06-22 | 江苏国信协联能源有限公司 | Mouldy bran culture medium for enlarged culture of aspergillus niger and culture method |
| CN108441448A (en) * | 2018-04-02 | 2018-08-24 | 郭庆 | A kind of aerobic method for cultivation of bacteria |
| CN215828648U (en) * | 2021-08-16 | 2022-02-15 | 茂名沃尔田生物科技有限公司 | A high-efficient fermentation cylinder for bio-organic fertilizer |
| CN216688128U (en) * | 2021-11-23 | 2022-06-07 | 吉林省正恒检测有限公司 | A bacteria cultivation device for soil detection |
-
2023
- 2023-12-11 CN CN202311687942.7A patent/CN117384742B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0731460A (en) * | 1993-07-16 | 1995-02-03 | Hiranuma Sangyo Kk | Apparatus for automatic preparation of nutrient medium |
| CN105695341A (en) * | 2016-03-16 | 2016-06-22 | 江苏国信协联能源有限公司 | Mouldy bran culture medium for enlarged culture of aspergillus niger and culture method |
| CN108441448A (en) * | 2018-04-02 | 2018-08-24 | 郭庆 | A kind of aerobic method for cultivation of bacteria |
| CN215828648U (en) * | 2021-08-16 | 2022-02-15 | 茂名沃尔田生物科技有限公司 | A high-efficient fermentation cylinder for bio-organic fertilizer |
| CN216688128U (en) * | 2021-11-23 | 2022-06-07 | 吉林省正恒检测有限公司 | A bacteria cultivation device for soil detection |
Non-Patent Citations (2)
| Title |
|---|
| XIAORAN ZHANG等: "Development and temperature gradientonline monitoring of a vehicular rotarysolid-state bioreactor: a novel devicefor large-scale preparation ofAspergillus nigerspore inoculum", 《CHEM TECHNOL BIOTECHNOL》, vol. 94, 18 September 2019 (2019-09-18), pages 3883 * |
| 程伟等: "一种酿酒用工业微生物简易扩大培养装置的设计与应用", 《酿酒科技》, no. 11, 31 December 2016 (2016-12-31), pages 72 - 79 * |
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