CN110699338A - Application of aspergillus niger LCCC30112 in production of glucoamylase and fermentation of tobacco leaves - Google Patents
Application of aspergillus niger LCCC30112 in production of glucoamylase and fermentation of tobacco leaves Download PDFInfo
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- CN110699338A CN110699338A CN201910912478.4A CN201910912478A CN110699338A CN 110699338 A CN110699338 A CN 110699338A CN 201910912478 A CN201910912478 A CN 201910912478A CN 110699338 A CN110699338 A CN 110699338A
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- aspergillus niger
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- 241000228245 Aspergillus niger Species 0.000 title claims abstract description 57
- 238000000855 fermentation Methods 0.000 title claims abstract description 44
- 230000004151 fermentation Effects 0.000 title claims abstract description 44
- 241000208125 Nicotiana Species 0.000 title claims abstract description 42
- 235000002637 Nicotiana tabacum Nutrition 0.000 title claims abstract description 42
- 108010073178 Glucan 1,4-alpha-Glucosidase Proteins 0.000 title claims abstract description 30
- 102100022624 Glucoamylase Human genes 0.000 title claims abstract description 29
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 17
- 108090000790 Enzymes Proteins 0.000 claims abstract description 37
- 102000004190 Enzymes Human genes 0.000 claims abstract description 37
- 239000001963 growth medium Substances 0.000 claims description 19
- 238000000034 method Methods 0.000 claims description 19
- 239000000843 powder Substances 0.000 claims description 11
- 239000007788 liquid Substances 0.000 claims description 10
- 239000006228 supernatant Substances 0.000 claims description 10
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 9
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 9
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 9
- 239000008367 deionised water Substances 0.000 claims description 9
- 229910021641 deionized water Inorganic materials 0.000 claims description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 9
- VWDWKYIASSYTQR-UHFFFAOYSA-N sodium nitrate Chemical compound [Na+].[O-][N+]([O-])=O VWDWKYIASSYTQR-UHFFFAOYSA-N 0.000 claims description 8
- 238000009630 liquid culture Methods 0.000 claims description 7
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 6
- 239000007853 buffer solution Substances 0.000 claims description 6
- 238000004108 freeze drying Methods 0.000 claims description 6
- 238000011068 loading method Methods 0.000 claims description 6
- 238000005571 anion exchange chromatography Methods 0.000 claims description 5
- 238000001641 gel filtration chromatography Methods 0.000 claims description 5
- 239000002609 medium Substances 0.000 claims description 5
- 229920002261 Corn starch Polymers 0.000 claims description 4
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 4
- 229920005654 Sephadex Polymers 0.000 claims description 4
- 239000012507 Sephadex™ Substances 0.000 claims description 4
- 229920002684 Sepharose Polymers 0.000 claims description 4
- 235000019764 Soybean Meal Nutrition 0.000 claims description 4
- 229930006000 Sucrose Natural products 0.000 claims description 4
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 4
- 238000005273 aeration Methods 0.000 claims description 4
- 239000008120 corn starch Substances 0.000 claims description 4
- 239000008103 glucose Substances 0.000 claims description 4
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 4
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 4
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 claims description 4
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 claims description 4
- 235000010344 sodium nitrate Nutrition 0.000 claims description 4
- 239000004317 sodium nitrate Substances 0.000 claims description 4
- 239000004455 soybean meal Substances 0.000 claims description 4
- 229960004793 sucrose Drugs 0.000 claims description 4
- 238000009423 ventilation Methods 0.000 claims description 4
- 229920001817 Agar Polymers 0.000 claims description 3
- 244000061456 Solanum tuberosum Species 0.000 claims description 3
- 235000002595 Solanum tuberosum Nutrition 0.000 claims description 3
- 239000008272 agar Substances 0.000 claims description 3
- 238000005119 centrifugation Methods 0.000 claims description 3
- CBMPTFJVXNIWHP-UHFFFAOYSA-L disodium;hydrogen phosphate;2-hydroxypropane-1,2,3-tricarboxylic acid Chemical compound [Na+].[Na+].OP([O-])([O-])=O.OC(=O)CC(O)(C(O)=O)CC(O)=O CBMPTFJVXNIWHP-UHFFFAOYSA-L 0.000 claims description 3
- 239000002054 inoculum Substances 0.000 claims description 3
- 239000008055 phosphate buffer solution Substances 0.000 claims description 3
- 239000002244 precipitate Substances 0.000 claims description 3
- 238000000746 purification Methods 0.000 claims description 3
- 238000000502 dialysis Methods 0.000 claims description 2
- 238000004090 dissolution Methods 0.000 claims description 2
- 238000011049 filling Methods 0.000 claims description 2
- 238000001556 precipitation Methods 0.000 claims description 2
- 238000011033 desalting Methods 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 10
- 229920002472 Starch Polymers 0.000 abstract description 5
- 239000008107 starch Substances 0.000 abstract description 5
- 235000019698 starch Nutrition 0.000 abstract description 5
- 230000015556 catabolic process Effects 0.000 abstract description 2
- 238000006731 degradation reaction Methods 0.000 abstract description 2
- 238000011031 large-scale manufacturing process Methods 0.000 abstract description 2
- 229940088598 enzyme Drugs 0.000 description 27
- 239000000126 substance Substances 0.000 description 6
- 230000008859 change Effects 0.000 description 4
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- 239000000779 smoke Substances 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 238000012258 culturing Methods 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 230000004913 activation Effects 0.000 description 2
- 238000005349 anion exchange Methods 0.000 description 2
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- 238000005185 salting out Methods 0.000 description 2
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- 239000000725 suspension Substances 0.000 description 2
- BFSVOASYOCHEOV-UHFFFAOYSA-N 2-diethylaminoethanol Chemical compound CCN(CC)CCO BFSVOASYOCHEOV-UHFFFAOYSA-N 0.000 description 1
- 108010011619 6-Phytase Proteins 0.000 description 1
- 239000004382 Amylase Substances 0.000 description 1
- 102000013142 Amylases Human genes 0.000 description 1
- 108010065511 Amylases Proteins 0.000 description 1
- 241000228212 Aspergillus Species 0.000 description 1
- 102100026189 Beta-galactosidase Human genes 0.000 description 1
- 238000009631 Broth culture Methods 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
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- 229920002271 DEAE-Sepharose Polymers 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 108010059881 Lactase Proteins 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 108010059820 Polygalacturonase Proteins 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- 108010059993 Vancomycin Proteins 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 238000012870 ammonium sulfate precipitation Methods 0.000 description 1
- 235000019418 amylase Nutrition 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 108010005774 beta-Galactosidase Proteins 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
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- 238000010612 desalination reaction Methods 0.000 description 1
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- 108010093305 exopolygalacturonase Proteins 0.000 description 1
- 239000003205 fragrance Substances 0.000 description 1
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- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
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- 229940116108 lactase Drugs 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 235000010298 natamycin Nutrition 0.000 description 1
- 239000004311 natamycin Substances 0.000 description 1
- 229960003255 natamycin Drugs 0.000 description 1
- NCXMLFZGDNKEPB-FFPOYIOWSA-N natamycin Chemical compound O[C@H]1[C@@H](N)[C@H](O)[C@@H](C)O[C@H]1O[C@H]1/C=C/C=C/C=C/C=C/C[C@@H](C)OC(=O)/C=C/[C@H]2O[C@@H]2C[C@H](O)C[C@](O)(C[C@H](O)[C@H]2C(O)=O)O[C@H]2C1 NCXMLFZGDNKEPB-FFPOYIOWSA-N 0.000 description 1
- 238000013386 optimize process Methods 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
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- 238000003672 processing method Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000001742 protein purification Methods 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 108010038851 tannase Proteins 0.000 description 1
- 229960003165 vancomycin Drugs 0.000 description 1
- MYPYJXKWCTUITO-LYRMYLQWSA-N vancomycin Chemical compound O([C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=C2C=C3C=C1OC1=CC=C(C=C1Cl)[C@@H](O)[C@H](C(N[C@@H](CC(N)=O)C(=O)N[C@H]3C(=O)N[C@H]1C(=O)N[C@H](C(N[C@@H](C3=CC(O)=CC(O)=C3C=3C(O)=CC=C1C=3)C(O)=O)=O)[C@H](O)C1=CC=C(C(=C1)Cl)O2)=O)NC(=O)[C@@H](CC(C)C)NC)[C@H]1C[C@](C)(N)[C@H](O)[C@H](C)O1 MYPYJXKWCTUITO-LYRMYLQWSA-N 0.000 description 1
- MYPYJXKWCTUITO-UHFFFAOYSA-N vancomycin Natural products O1C(C(=C2)Cl)=CC=C2C(O)C(C(NC(C2=CC(O)=CC(O)=C2C=2C(O)=CC=C3C=2)C(O)=O)=O)NC(=O)C3NC(=O)C2NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(CC(C)C)NC)C(O)C(C=C3Cl)=CC=C3OC3=CC2=CC1=C3OC1OC(CO)C(O)C(O)C1OC1CC(C)(N)C(O)C(C)O1 MYPYJXKWCTUITO-UHFFFAOYSA-N 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
- C12N9/2411—Amylases
- C12N9/2428—Glucan 1,4-alpha-glucosidase (3.2.1.3), i.e. glucoamylase
-
- A—HUMAN NECESSITIES
- A24—TOBACCO; CIGARS; CIGARETTES; SIMULATED SMOKING DEVICES; SMOKERS' REQUISITES
- A24B—MANUFACTURE OR PREPARATION OF TOBACCO FOR SMOKING OR CHEWING; TOBACCO; SNUFF
- A24B15/00—Chemical features or treatment of tobacco; Tobacco substitutes, e.g. in liquid form
- A24B15/18—Treatment of tobacco products or tobacco substitutes
- A24B15/20—Biochemical treatment
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01003—Glucan 1,4-alpha-glucosidase (3.2.1.3), i.e. glucoamylase
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- Health & Medical Sciences (AREA)
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- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
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- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Biomedical Technology (AREA)
- Medicinal Chemistry (AREA)
- Tropical Medicine & Parasitology (AREA)
- Mycology (AREA)
- Virology (AREA)
- Botany (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Molecular Biology (AREA)
- Enzymes And Modification Thereof (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention provides an application of Aspergillus niger (Aspergillus niger) LCCC30112 in production of glucoamylase and fermentation of tobacco leaves, wherein the Aspergillus niger (Aspergillus niger) LCCC30112 can produce glucoamylase with high enzyme activity, and the Aspergillus niger (Aspergillus niger) LCCC30112 is used for producing glucoamylase, so that a new way is provided for a production source of the glucoamylase, and large-scale production of the glucoamylase can be realized; the Aspergillus niger with glucoamylase producing high enzyme activity is applied to the fermentation of the tobacco leaves, so that the degradation of starch in the tobacco leaves can be accelerated, the fermentation period of the tobacco leaves is obviously shortened, and the quality of the tobacco leaves is improved.
Description
Technical Field
The invention relates to application of Aspergillus niger LCCC30112 in production of glucoamylase tobacco fermentation, and belongs to the technical field of microbial application.
Background
The glucoamylase can hydrolyze alpha-1.4 glycosidic bond and alpha-1.6 glycosidic bond in starch, and gradually hydrolyze glucose from non-reducing end in substrate molecule in hydrolysis process, and can be used for degrading starch in tobacco leaves. The fermentation of tobacco leaves is a process for promoting the physical and chemical properties of the tobacco leaves to be deeply changed under certain temperature and humidity conditions, and is a primary processing method for improving the product quality in the cigarette industry.
For the fermentation of tobacco leaves, natural fermentation and artificial fermentation are mostly adopted at present. The natural fermentation is mainly carried out by means of the change of natural climate, and is mainly characterized in that the temperature rise in spring of one year is utilized to promote the activity of enzymes in the tobacco leaves during the storage period of the tobacco leaves, so that the tobacco leaves are subjected to a slow fermentation process to achieve the purpose of improving the quality of the tobacco leaves. The artificial fermentation is a method for accelerating the change of the tobacco leaves by utilizing artificial conditions (namely suitable temperature and humidity) suitable for the change of the internal quality of the tobacco leaves. However, both methods have the disadvantage that the fermentation progresses slowly.
Aspergillus niger (Aspergillus niger) belongs to Aspergillus filamentous fungi, and is widely used in the fermentation industry due to its strong protein secretion ability, and the strains produce the most enzymes, such as amylase, protease, pectinase, cellulase, lactase, phytase, tannase, penicillin, natamycin, vancomycin, and the like. More than 20 kinds of enzyme using strains for food industry are judged by food safety expert committee such as FAO/WHO/JECFA, wherein Aspergillus niger is the first high-living strain with permitted use and safety. In the prior art, reports of Aspergillus niger with high enzyme activity screened from the existing strains and application thereof are not found.
Disclosure of Invention
The invention aims to provide application of Aspergillus niger (LCCC 30112) in production of glucoamylase and tobacco fermentation, wherein the Aspergillus niger (LCCC 30112) can be used for high-yield production of glucoamylase and is very suitable for production of glucoamylase and tobacco fermentation.
In one aspect, the invention provides the use of Aspergillus niger (Aspergillus niger) LCCC30112 for the production of glucoamylase, Aspergillus niger (Aspergillus niger) LCCC30112 is capable of producing glucoamylase with high enzymatic activity, providing a new source for the production source of glucoamylase.
In another aspect, the present invention provides a method for producing glucoamylase using Aspergillus niger (Aspergillus niger) LCCC30112, comprising the steps of inoculating activated Aspergillus niger (Aspergillus niger) LCCC30112 in a liquid medium for fermentation culture, and centrifuging the fermentation broth to obtain a supernatant.
Preferably, the Aspergillus niger LCCC30112 is inoculated in an amount of 105～107cfu/ml, wherein the culture is aeration culture, and the aeration quantity is 1: 0.5-1.5V/V.min, the temperature is 20-40 ℃, and the time is 2-4 d;
preferably, the inoculum size is 106cfu/ml, ventilation 1: 1.0V/V.min, the temperature is 30 ℃, and the time is 3 d;
preferably, the liquid medium contains: 2g/L of cane sugar, 4g/L of corn starch, 1g/L of soybean meal, 0.5g/L of sodium nitrate and 0.05g/L, pH of monopotassium phosphate are 5.0-6.0, and the loading amount of the liquid culture medium is 60-80%;
preferably, the pH is 5.5, and the filling amount of the liquid culture medium is 75%;
preferably, the centrifugation temperature is 0-4 ℃, the rotation speed is 5000-7000 r/min, and the time is 10-20 min;
preferably, Aspergillus niger (LCCC 30112) is activated and cultured in a PDA culture medium for 2-5 days and then inoculated;
preferably, the PDA medium contains: 200g/L of potato, 20g/L of glucose and 15g/L of agar.
Further, the method also comprises the steps of adding ammonium sulfate into the supernatant for precipitation, and obtaining crude enzyme after dialysis and desalination.
Preferably, the saturation degree of the ammonium sulfate is 75-85%;
preferably, the saturation of ammonium sulfate is 80%;
preferably, after the supernatant is added with ammonium sulfate for dissolution, 7000-9000 r/min is centrifuged for 20-40 min, and the precipitate is dialyzed by deionized water;
preferably, the centrifugation is carried out for 30min at 8000 r/min.
Further, the method may further comprise a step of purifying the crude enzyme using a Sepharose Fast Flow anion exchange chromatography column and a Sephadex G-50 gel filtration chromatography column.
Preferably, the specific steps of the purification are as follows:
(1) loading the crude enzyme to a Sepharose Fast Flow anion exchange chromatography column, eluting with Tris-HCl buffer solution at the Flow rate of 1.5-2.5 mL/min, collecting active components, dialyzing with deionized water, and freeze-drying to obtain enzyme powder;
(2) dissolving the enzyme powder by using a citric acid-disodium hydrogen phosphate buffer solution, loading the enzyme powder to a Sephadex G-50 gel filtration chromatography column, eluting by using a Tris-HCl buffer solution at the flow rate of 1.5-2.5 mL/min, collecting active components, dialyzing by using deionized water, and freeze-drying to obtain the purified enzyme powder.
In another aspect, the invention also provides the use of Aspergillus niger (Aspergillus niger) LCCC30112 or the method in tobacco leaf fermentation.
In another aspect, the invention also provides a tobacco leaf fermentation method, Aspergillus niger (Aspergillus niger) LCCC30112 is inoculated in tobacco leaves for fermentation.
Preferably, the Aspergillus niger LCCC30112 is inoculated in an amount of 105～107cfu/g, the fermentation temperature is 20-40 ℃, the humidity is 40-70%, and the time is 5-10 d.
The invention has the beneficial effects that:
the invention discloses Aspergillus niger LCCC30112 capable of producing glucoamylase with high enzyme activity, which provides a new way for the production source of glucoamylase and can realize the large-scale production of glucoamylase; the Aspergillus niger with glucoamylase producing high enzyme activity is applied to the fermentation of the tobacco leaves, so that the degradation of starch in the tobacco leaves can be accelerated, the fermentation period of the tobacco leaves is obviously shortened, and the quality of the tobacco leaves is improved.
Detailed Description
The present invention is described in detail with reference to specific examples, which are provided to facilitate the understanding of the technical solutions of the present invention by those skilled in the art, and the implementation or use of the present invention is not limited by the description of the present invention.
In the present invention, the raw materials and equipment used are commercially available or commonly used in the art, if not specified. The Aspergillus niger strain used in the invention is purchased from Liaoning province microorganism strain preservation center, and the number is as follows: LCCC 30112.
The methods in the examples are conventional in the art unless otherwise specified.
Example 1Aspergillus niger LCCC30112 fermentation production of glucoamylase
Aspergillus niger LCCC30112 fermentation process research
Through single-factor multi-level, multi-factor multi-level and other formula tests, the liquid enzyme production process of the Aspergillus niger LCCC30112 is determined as follows:
the preserved strain is transferred to an activation culture medium and cultured for 3d at 30 ℃ for standby.
The activating culture medium adopts a PDA culture medium: potato 200g/L, glucose 20g/L, agar 15g/L, natural pH, and sterilizing at 121 deg.C for 30 min.
Preparing a seed solution:
transferring Aspergillus niger LCCC30112 strain to activation culture medium, culturing for 4 days until large amount of spores are produced, repeatedly washing spores with 1% physiological saline, collecting spore suspension, inoculating to seed liquid culture medium, inoculating 1% (with concentration of 10%) of the spore suspension8cfu/mL), culturing at 30 deg.C and 180r/min for 3d, and inoculating to fermentation broth culture medium with inoculum size of 106cfu/mL。
The culture medium of the fermentation liquid and the culture medium of the seed liquid are as follows: 2g/L of cane sugar, 4g/L of corn starch, 1g/L of soybean meal, 0.5g/L of sodium nitrate, 0.05g/L of monopotassium phosphate and pH 5.5. Sterilization of a fermentation tank culture medium: 121 ℃ and 60 min.
Inoculation: the sterile inoculation is carried out by utilizing a flame ring, and the inoculation amount is 1 percent.
The culture conditions are as follows: the culture temperature is 30 ℃, the culture medium is inoculated into a fermentation tank with 75% of liquid culture medium, and the culture is carried out for 72 hours in a ventilation mode, wherein the ventilation rate is 1: 1 (V/V.min).
The liquid enzyme production process of the Aspergillus niger LCCC30112 is determined by adopting formula tests such as single-factor multi-level and multi-factor multi-level orthogonal tests: the culture medium formula comprises 2g/L of sucrose, 4g/L of corn starch, 1g/L of soybean meal, 0.5g/L of sodium nitrate, 0.05g/L of monopotassium phosphate, pH5.5, and culturing for 72 h. The optimized process improves the enzyme activity of the glucoamylase to 986.7U/mL.
Example 2Separating and purifying glucoamylase:
(1) ammonium sulfate precipitation
Centrifuging the fermentation broth at 4 deg.C and 6000r/min for 15min to remove mycelium to obtain supernatant;
slowly adding ammonium sulfate with saturation of 80% into the supernatant, salting out and precipitating, centrifuging at 8000r/min for 30min, and dialyzing the precipitate with deionized water to obtain crude enzyme solution.
(2) DEAE Sepharose Fast Flow anion exchange chromatography
The crude enzyme solution was applied to a DEAE anion exchange column (16 mm. times.100 mm) for enzyme protein purification. And (2) balancing by using 10mmol of Tris-HCl buffer solution (pH 8.4 and containing 2mmol of calcium chloride), eluting by using the Tris-HCl buffer solution at the flow rate of 2mL/min, collecting components with enzyme activity peaks, dialyzing by using deionized water, and freeze-drying to obtain enzyme powder.
(3) Superdex-50 gel filtration chromatography
And dissolving the freeze-dried enzyme powder in 3mL of citric acid-disodium hydrogen phosphate buffer solution (pH 4.5), eluting by using a Superdex-50 chromatographic column at the elution speed of 2mL/min, collecting an active part, dialyzing by using deionized water, and freeze-drying to obtain the purified enzyme.
After crude enzyme liquid fermented by the Aspergillus niger LCCC30112 liquid is separated and purified by ammonium sulfate salting out, a DEAE-52 anion exchange column and a Superdex-50 chromatographic column, enzyme protein is obviously purified, the enzyme activity is improved to 21000U/mL from 986.7U/mL of fermentation supernatant, and the purification multiple is 21.2 times.
Example 3Application of Aspergillus niger (LCCC 30112) in tobacco leaf fermentation
Inoculating Aspergillus niger LCCC30112 to tobacco leaves, wherein the inoculation amount of Aspergillus niger LCCC30112 is 105～107cfu/g, the fermentation temperature is 20-40 ℃, the humidity is 40-70%, and the time is 5-10 d.
Or adding fermentation supernatant of Aspergillus niger LCCC30112 obtained in example 2, or crude enzyme thereof, or purified glucoamylase prepared finally into tobacco leaf, and fermenting, wherein the enzyme activity of the added glucoamylase in the tobacco leaf is 10-20U/mg, and the fermentation conditions are as follows: the temperature is 20-40 ℃, the humidity is 40-70%, and the time is 5-10 d.
The invention takes 2018 Chongqing B4F raw tobacco as a control group, and 10 is added6cfu/g Aspergillus niger (Aspergillus niger) LCCC30112 as treatment group 1, 15U/mg prepared glucoamylase enzyme powder as treatment group 2 were added to ferment the tobacco leaves at 50 deg.C and 55% humidity for 8 days, and the change of each chemical component in the fermented tobacco leaves is shown in Table 1.
As can be seen from Table 1, the total sugar content in tobacco leaves increased and the starch content significantly decreased after addition of Aspergillus niger LCCC30112 or glucoamylase. The total sugar generates an acidic reaction to inhibit the alkalinity of alkaline substances in the smoke, so that the acid-base balance of the smoke is moderate, the irritation is reduced, and the sugar is a precursor substance for forming aroma substances. The processed tobacco leaves have smooth smoke, the fine and greasy feeling and the comfort level of the smoke are improved, and meanwhile, the tobacco fragrance is enriched.
TABLE 1 chemical composition of tobacco leaves in each treatment
The above description is only an example of the present application, and the protection scope of the present application is not limited by these specific examples, but is defined by the claims of the present application. Various modifications and changes may occur to those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the technical idea and principle of the present application should be included in the protection scope of the present application.
Claims (10)
1. Use of Aspergillus niger LCCC30112 for the production of glucoamylase.
2. A method for producing glucoamylase by using Aspergillus niger (LCCC 30112), which is characterized in that the method comprises the steps of inoculating activated Aspergillus niger (LCCC 30112) in a liquid culture medium for fermentation culture, and centrifuging the fermentation liquid to obtain a supernatant.
3. The method of claim 2, wherein the Aspergillus niger (Aspergillus niger) LCCC30112 is inoculated in an amount of 105～107cfu/ml, wherein the culture is aeration culture, and the aeration quantity is 1: 0.5-1.5V/V.min, the temperature is 20-40 ℃, and the time is 2-4 d;
preferably, the inoculum size is 106cfu/ml, ventilation 1: 1.0V/V.min, the temperature is 30 ℃, and the time is 3 d;
preferably, the liquid medium contains: 2g/L of cane sugar, 4g/L of corn starch, 1g/L of soybean meal, 0.5g/L of sodium nitrate and 0.05g/L, pH of monopotassium phosphate are 5.0-6.0, and the loading amount of the liquid culture medium is 60-80%;
preferably, the pH is 5.5, and the filling amount of the liquid culture medium is 75%;
preferably, the centrifugation temperature is 0-4 ℃, the rotation speed is 5000-7000 r/min, and the time is 10-20 min;
preferably, Aspergillus niger (LCCC 30112) is activated and cultured in a PDA culture medium for 2-5 days and then inoculated;
preferably, the PDA medium contains: 200g/L of potato, 20g/L of glucose and 15g/L of agar.
4. The method of claim 2, further comprising the steps of adding ammonium sulfate to the supernatant for precipitation, and desalting by dialysis to obtain crude enzyme.
5. The method of claim 4, wherein the saturation of ammonium sulfate is 75-85%;
preferably, after the supernatant is added with ammonium sulfate for dissolution, 7000-9000 r/min is centrifuged for 20-40 min, and the precipitate is dialyzed by deionized water.
6. The method according to claim 4, wherein the method further comprises a step of purifying the crude enzyme using a Sepharose Fast Flow anion exchange chromatography column and a Sephadex G-50 gel filtration chromatography column in this order.
7. The method according to claim 6, wherein the specific steps of the purification are as follows:
(1) loading the crude enzyme to a Sepharose Fast Flow anion exchange chromatography column, eluting with Tris-HCl buffer solution at the Flow rate of 1.5-2.5 mL/min, collecting active components, dialyzing with deionized water, and freeze-drying to obtain enzyme powder;
(2) dissolving the enzyme powder by using a citric acid-disodium hydrogen phosphate buffer solution, loading the enzyme powder to a Sephadex G-50 gel filtration chromatography column, eluting by using a Tris-HCl buffer solution at the flow rate of 1.5-2.5 mL/min, collecting active components, dialyzing by using deionized water, and freeze-drying to obtain the purified enzyme powder.
8. Use of Aspergillus niger (Aspergillus niger) LCCC30112 or the method according to any of claims 2 to 7 for the fermentation of tobacco leaves.
9. A tobacco leaf fermentation method is characterized in that Aspergillus niger (LCCC 30112) is inoculated in tobacco leaves for fermentation.
10. The tobacco leaf fermentation method according to claim 9, wherein the amount of Aspergillus niger (Aspergillus niger) LCCC30112 inoculated is 105～107cfu/g, the fermentation temperature is 20-40 ℃, the humidity is 40-70%, and the time is 5-10 d.
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