CN110616210A - Application of bacillus licheniformis LCCC10161 in production of alpha-amylase and tobacco fermentation - Google Patents
Application of bacillus licheniformis LCCC10161 in production of alpha-amylase and tobacco fermentation Download PDFInfo
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- CN110616210A CN110616210A CN201910912477.XA CN201910912477A CN110616210A CN 110616210 A CN110616210 A CN 110616210A CN 201910912477 A CN201910912477 A CN 201910912477A CN 110616210 A CN110616210 A CN 110616210A
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- bacillus licheniformis
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- amylase
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- 241000194108 Bacillus licheniformis Species 0.000 title claims abstract description 60
- 238000000855 fermentation Methods 0.000 title claims abstract description 46
- 230000004151 fermentation Effects 0.000 title claims abstract description 46
- 241000208125 Nicotiana Species 0.000 title claims abstract description 44
- 235000002637 Nicotiana tabacum Nutrition 0.000 title claims abstract description 44
- 102000004139 alpha-Amylases Human genes 0.000 title claims abstract description 37
- 108090000637 alpha-Amylases Proteins 0.000 title claims abstract description 37
- 229940024171 alpha-amylase Drugs 0.000 title claims abstract description 37
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 16
- 102000004190 Enzymes Human genes 0.000 claims abstract description 35
- 108090000790 Enzymes Proteins 0.000 claims abstract description 35
- 229940088598 enzyme Drugs 0.000 claims abstract description 33
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 33
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 28
- 239000001963 growth medium Substances 0.000 claims description 19
- 238000000034 method Methods 0.000 claims description 19
- 239000000243 solution Substances 0.000 claims description 18
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 14
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 14
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 14
- 239000007853 buffer solution Substances 0.000 claims description 14
- 239000011780 sodium chloride Substances 0.000 claims description 14
- 239000007788 liquid Substances 0.000 claims description 12
- 239000006228 supernatant Substances 0.000 claims description 11
- 238000011081 inoculation Methods 0.000 claims description 8
- 238000009630 liquid culture Methods 0.000 claims description 8
- 229920002271 DEAE-Sepharose Polymers 0.000 claims description 6
- 229920005654 Sephadex Polymers 0.000 claims description 6
- 239000012507 Sephadex™ Substances 0.000 claims description 6
- 238000005571 anion exchange chromatography Methods 0.000 claims description 6
- 239000012153 distilled water Substances 0.000 claims description 6
- 238000001641 gel filtration chromatography Methods 0.000 claims description 6
- 238000011068 loading method Methods 0.000 claims description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 6
- 238000000502 dialysis Methods 0.000 claims description 5
- 239000000843 powder Substances 0.000 claims description 5
- 239000007836 KH2PO4 Substances 0.000 claims description 4
- 239000001888 Peptone Substances 0.000 claims description 4
- 108010080698 Peptones Proteins 0.000 claims description 4
- 244000046052 Phaseolus vulgaris Species 0.000 claims description 4
- 235000010627 Phaseolus vulgaris Nutrition 0.000 claims description 4
- 240000008042 Zea mays Species 0.000 claims description 4
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 4
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 4
- 239000001110 calcium chloride Substances 0.000 claims description 4
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 4
- 229940041514 candida albicans extract Drugs 0.000 claims description 4
- 235000005822 corn Nutrition 0.000 claims description 4
- 238000011033 desalting Methods 0.000 claims description 4
- 235000013312 flour Nutrition 0.000 claims description 4
- 239000002609 medium Substances 0.000 claims description 4
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 4
- 235000019319 peptone Nutrition 0.000 claims description 4
- 238000001556 precipitation Methods 0.000 claims description 4
- 238000000746 purification Methods 0.000 claims description 4
- 239000012138 yeast extract Substances 0.000 claims description 4
- 229910000396 dipotassium phosphate Inorganic materials 0.000 claims description 3
- 238000011049 filling Methods 0.000 claims description 3
- 230000003213 activating effect Effects 0.000 claims description 2
- 238000005119 centrifugation Methods 0.000 claims description 2
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 2
- 238000002523 gelfiltration Methods 0.000 claims description 2
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 claims description 2
- 239000002054 inoculum Substances 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 15
- 229920002472 Starch Polymers 0.000 abstract description 6
- 235000019698 starch Nutrition 0.000 abstract description 6
- 239000008107 starch Substances 0.000 abstract description 6
- 230000015556 catabolic process Effects 0.000 abstract description 2
- 238000006731 degradation reaction Methods 0.000 abstract description 2
- 230000002255 enzymatic effect Effects 0.000 abstract description 2
- 238000011031 large-scale manufacturing process Methods 0.000 abstract description 2
- 230000008859 change Effects 0.000 description 6
- 238000012258 culturing Methods 0.000 description 3
- 230000000813 microbial effect Effects 0.000 description 3
- 239000000779 smoke Substances 0.000 description 3
- 230000001954 sterilising effect Effects 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 239000004382 Amylase Substances 0.000 description 2
- 102000013142 Amylases Human genes 0.000 description 2
- 108010065511 Amylases Proteins 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 235000019418 amylase Nutrition 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 241000228245 Aspergillus niger Species 0.000 description 1
- 240000006439 Aspergillus oryzae Species 0.000 description 1
- 235000002247 Aspergillus oryzae Nutrition 0.000 description 1
- 241000193744 Bacillus amyloliquefaciens Species 0.000 description 1
- 241000193749 Bacillus coagulans Species 0.000 description 1
- 244000063299 Bacillus subtilis Species 0.000 description 1
- 235000014469 Bacillus subtilis Nutrition 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 229920001353 Dextrin Polymers 0.000 description 1
- 239000004375 Dextrin Substances 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 241000193385 Geobacillus stearothermophilus Species 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 239000012880 LB liquid culture medium Substances 0.000 description 1
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 229920002684 Sepharose Polymers 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 238000012870 ammonium sulfate precipitation Methods 0.000 description 1
- 229940054340 bacillus coagulans Drugs 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- GUBGYTABKSRVRQ-QUYVBRFLSA-N beta-maltose Chemical compound OC[C@H]1O[C@H](O[C@H]2[C@H](O)[C@@H](O)[C@H](O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@@H]1O GUBGYTABKSRVRQ-QUYVBRFLSA-N 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 235000019504 cigarettes Nutrition 0.000 description 1
- 238000010612 desalination reaction Methods 0.000 description 1
- 235000019425 dextrin Nutrition 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000003205 fragrance Substances 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- 229910021645 metal ion Inorganic materials 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 229920001542 oligosaccharide Polymers 0.000 description 1
- 150000002482 oligosaccharides Chemical class 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
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- 238000003672 processing method Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000005185 salting out Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
Classifications
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- 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
- C12N9/2414—Alpha-amylase (3.2.1.1.)
-
- 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)
- C12Y302/01001—Alpha-amylase (3.2.1.1)
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- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Medicinal Chemistry (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
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- Enzymes And Modification Thereof (AREA)
Abstract
The invention provides an application of Bacillus licheniformis (LCCC 10161) in producing alpha-amylase and tobacco fermentation, the Bacillus licheniformis (LCCC 10161) can produce alpha-amylase with high enzymatic activity, the Bacillus licheniformis (LCCC 10161) is used for producing the alpha-amylase, a new way is provided for the production source of the alpha-amylase, and the large-scale production of the alpha-amylase can be realized; the bacillus licheniformis with the alpha-amylase 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 bacillus licheniformis LCCC10161 in tobacco fermentation for producing alpha-amylase, belonging to the technical field of microbial application.
Background
The tobacco leaf fermentation is one of the important links in the tobacco leaf processing process, and the tobacco leaf fermentation process promotes the deep change of the physical and chemical properties of the tobacco leaves under certain temperature and humidity conditions, so that the method 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.
Alpha-amylase is one of the main enzymes used in tobacco leaf fermentation by hydrolyzing alpha-1, 4-glucosidic bonds in a starch molecular chain to cut the starch chain into short-chain dextrin, oligosaccharide and a small amount of maltose and glucose. The alpha-amylase can be produced by microbial fermentation, and can also be extracted from plants and animals. At present, the alpha-amylase is produced on a large scale by a microbial fermentation method in industrial production. Useful α -amylase producing bacteria are: bacillus subtilis, bacillus licheniformis, bacillus stearothermophilus, bacillus coagulans, bacillus amyloliquefaciens, aspergillus oryzae, aspergillus niger and the like.
The prior art CN104357357B discloses a Bacillus licheniformis (Bacillus licheniformis) HWyb1401 for producing alpha-amylase, and the preservation number is CGMCC No. 8718. The Bacillus licheniformis (Bacillus licheniformis) HWyb1401 disclosed by the invention has a high-temperature resistance characteristic, can survive at 70 ℃, metabolizes alpha-amylase, and has the enzyme activity of 70U/mL in fermentation liquor at 55 ℃. The invention provides a new bacillus licheniformis strain, which has high temperature resistance and does not relate to the content of screening alpha-amylase with high enzyme activity in the existing strain.
Disclosure of Invention
The invention aims to provide application of Bacillus licheniformis LCCC10161 in production of alpha-amylase and tobacco fermentation, wherein the Bacillus licheniformis LCCC10161 can produce the alpha-amylase at high yield and is very suitable for production of the alpha-amylase and tobacco fermentation.
In one aspect, the invention provides the application of the bacillus licheniformis LCCC10161 in the production of alpha-amylase, and the bacillus licheniformis LCCC10161 can produce the alpha-amylase with high enzyme activity, thereby providing a new source for the production source of the alpha-amylase.
In another aspect, the present invention provides a method for producing alpha-amylase by using Bacillus licheniformis (LCCC 10161), which comprises the steps of inoculating the activated Bacillus licheniformis (LCCC 10161) into a liquid culture medium for fermentation culture, and centrifuging the fermentation liquid to obtain a supernatant.
Preferably, the inoculation amount of Bacillus licheniformis (LCCC 10161) is 105～107cfu/ml, wherein the culture is shaking culture, the rotating speed is 150-250 r/min, the temperature is 20-45 ℃, and the time is 3-5 d;
preference is given toOf 10 (a)6cfu/ml, the rotating speed is 200r/min, the temperature is 36 ℃, and the time is 4 d;
preferably, the liquid medium contains: 0.8g of bean cake powder, 2.0g of corn flour, 1.0g of NaCl and K2HPO40.01g、KH2PO4 0.01g、CaCl20.3g of distilled water and 100mL of distilled water, wherein the pH value is 7.5-8.5, and the content of a liquid culture medium is 8-15%;
preferably, the pH is 8.0, and the filling amount of the liquid culture medium is 10%;
preferably, Bacillus licheniformis (Bacillus licheniformis) LCCC10161 is subjected to activated culture for 15-25 days and then inoculated;
preferably, the culture medium used for activating the Bacillus licheniformis (Bacillus licheniformis) LCCC10161 is LB culture medium;
preferably, the LB medium contains 10g/L peptone, 5g/L yeast extract, 10g/L sodium chloride;
preferably, the centrifugation temperature is 0-4 ℃, the rotation speed is 8000-12000 rpm, and the time is 20-40 min.
Further, the method also comprises the steps of adding ammonium sulfate into the supernatant for precipitation, and obtaining crude enzyme liquid after dialysis and desalination;
preferably, the saturation degree of the ammonium sulfate is 40-60%;
preferably, the supernatant is dissolved by adding the ammonium sulfate, centrifuged, and precipitated with ddH2Dissolving O, and desalting by dialysis with an acetic acid buffer solution of 15-25 mmol/L, pH 5.8.8-6.2.
Further, the method comprises a step of purifying the crude enzyme solution by using a DEAE Sepharose Fast Flow anion exchange chromatography column and a Sephadex G-75 gel filtration chromatography column.
Preferably, the specific steps of the crude enzyme solution purification are as follows:
(1) loading the crude enzyme solution to a Sepharose Fast Flow anion exchange chromatography column, eluting with an acetic acid buffer solution A until A280 is unchanged, and then eluting with an acetic acid buffer solution containing 0.1mol/L NaCl at the Flow rate of 0.8-1.2 mL/min to obtain a collection solution;
(2) and (3) loading the collected liquid to a Sephadex G-75 gel filtration chromatographic column, eluting with an acetic acid buffer solution A containing 0.2mol/L NaCl at the flow rate of 0.2-0.4 mL/min, and concentrating the collected active components to the density of 1.1-1.2G/mL.
On the other hand, the invention also provides application of the Bacillus licheniformis (Bacillus licheniformis) LCCC10161 or the method in tobacco leaf fermentation.
On the other hand, the invention also provides a tobacco leaf fermentation method, which is to inoculate Bacillus licheniformis (LCCC 10161) in tobacco leaves for fermentation.
Preferably, the inoculation amount of Bacillus licheniformis (LCCC 10161) is 105～107cfu/g, the fermentation temperature is 20-45 ℃, the humidity is 50-70%, and the time is 5-10 d.
The invention has the beneficial effects that:
the invention discloses Bacillus licheniformis (Bacillus licheniformis) LCCC10161 capable of producing alpha-amylase with high enzymatic activity, which provides a new way for the production source of the alpha-amylase and can realize the large-scale production of the alpha-amylase; the bacillus licheniformis with the alpha-amylase 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 Bacillus licheniformis (Bacillus licheniformis) strain used by the invention is purchased from Liaoning province microorganism strain preservation center, and the serial numbers are as follows: LCCC 10161.
The methods in the examples are conventional in the art unless otherwise specified.
Example 1Utilizing Bacillus licheniformis LCCC10161 production of alpha-amylase
The culture condition is optimized for improving the enzyme yield of LCCC10161 Bacillus licheniformis. Mainly inspects the influence of the seed age, the inoculation amount, the most suitable liquid filling amount, the fermentation temperature and the initial pH of the culture medium on the enzyme production.
The optimal culture conditions for LCCC10161 Bacillus licheniformis enzyme production are finally determined by experiments as follows: the temperature is 36 ℃, the seed age is 20h, and the inoculation amount is 10 percent (10 percent)6cfu/ml), the liquid loading of the triangular flask is 10 percent, the maximum enzyme production time is 4d, the optimum pH value is 8.0, and the speed is 200 r/min.
The culture medium selected during strain activation:
(1) the solid strain adopts LB solid culture medium. Each L contains 10g of peptone, 5g of yeast extract, 10g of sodium chloride, and 16-20g of agar, and has pH of 7.0-7.2. Sterilizing at 121 deg.C under 0.1MPa for 30 min.
The culture conditions are as follows: culturing at 33 + -1 deg.C for 24 h.
(2) The liquid strain is prepared from LB liquid culture medium containing peptone 1.0g, yeast extract 0.5g, NaCl 1.0g, starch 0.1g, and distilled water 100mL, with pH of 7.0-7.2, 0.1MPa, 121 deg.C, and sterilizing for 30 min.
The culture conditions are as follows: culturing at 33 + -1 deg.C for 20 h.
The culture medium selected during strain culture:
liquid culture medium: 0.8g of bean cake powder, 2.0g of corn flour, 1.0g of NaCl and K2HPO4 0.01g、KH2PO40.01g、CaCl20.3, 100mL of distilled water, 10 percent of liquid culture medium in a triangular flask, and sterilizing for 30min at the temperature of 121 ℃ and 0.1 MPa.
Inoculating strains: the flame ring is used for aseptic inoculation, and the inoculation amount is 10 percent (10 percent)6cfu/ml)。
The culture conditions are as follows: culturing at 36 + -1 deg.C and shaking at rotation speed of 200r/min for 96 h.
In order to obtain higher enzyme activity, the liquid fermentation culture medium of the bacillus licheniformis LCCC10161 is optimized. Mainly considers the selection and concentration determination of carbon source, nitrogen source and metal ions. The optimal enzyme production culture medium of the bacillus licheniformis LCCC10161 is finally determined to be 0.8 percent of bean cake powder, 2.0 percent of corn flour, 1 percent of NaCl and K through experiments2HPO4 0.01％、KH2PO40.01％、CaCl2 0.3％。
The enzyme activity of the fermentation supernatant reaches 102U/mL by using the optimal culture conditions and the optimal enzyme production culture medium.
Example 2The steps of separating and purifying the alpha-amylase comprise:
(1) ammonium sulfate precipitation
Ammonium sulfate fractional precipitation: collecting 20mL of supernatant of each strain sample, adding ammonium sulfate to increase the concentration of ammonium sulfate from 30% (w/v) to 80% (w/v) by 5% (w/v), shaking, dissolving, standing for 20min, centrifuging at 10000r/min at 4 deg.C for 10min, removing supernatant, precipitating with same small amount of ddH2Dissolving O to obtain a crude enzyme solution; the crude enzyme solution was dialyzed with 20mmol/L, pH 6.0.0 acetic acid buffer solution for 12 hours to remove a large amount of ammonium sulfate component from the enzyme solution, and the activity of the desalted crude enzyme solution was measured by changing the dialysis buffer twice.
When the concentration of the ammonium sulfate measured by the method is 40-60% of the saturation degree of the ammonium sulfate, the extraction of the alpha-amylase is facilitated.
(2) DEAE Sepharose Fast Flow anion exchange chromatography
Loading the dialyzed crude enzyme solution onto DEAE Sepharose Fast Flow column (2.6cm × 20cm) balanced with acetic buffer solution A (20mmol/L, pH6.0 acetic buffer solution), eluting enzyme protein with acetic buffer solution A to A280After the change, the elution is carried out by using an acetic acid buffer solution A containing 0.1mol/L NaCl at the flow rate of 1.0mL/min, and a tube I is collected for 10 min. The collected samples were assayed for activity and the active test tube pools were pooled.
(3) Sephadex G-75 gel filtration chromatography
Subjecting the collected solution of the previous step to Sephadex G-75 gel filtration chromatography (1.6cm × 60cm), eluting with acetic acid buffer solution A containing 0.2mol/L NaCl at flow rate of 0.3mL/min, and collecting in 1 tube for 5 min. And performing activity determination on the collected sample, collecting an active part, dialyzing for desalting, and freeze-drying and concentrating.
After the fermentation supernatant enzyme liquid of the bacillus licheniformis LCCC10161 is centrifuged, the electrophoresis-grade pure enzyme is obtained through three steps of ammonium sulfate fractional precipitation (40-60%), DEAE Sepharose Fast Flow anion exchange chromatography and SephadexG-75 gel filtration chromatography, the specific activity of the enzyme is improved to 217.8U/mg, and the purification multiple is 18 times.
And (3) carrying out SDA-PAGE gel electrophoresis on the enzyme activity parts purified in each step. The alpha-amylase achieves electrophoretic purity after being purified by three steps of ammonium sulfate fractional salting-out, ion exchange chromatography and gel filtration chromatography, and the amylase activity dyeing result shows that a unique reversed white band appears at a corresponding position. The enzyme protein obtained by separation and purification is proved to have the biological activity of amylase.
Example 3Application of Bacillus licheniformis (Bacillus licheniformis) LCCC10161 in tobacco leaf fermentation
Inoculating Bacillus licheniformis LCCC10161 into tobacco leaf, wherein the inoculation amount of the Bacillus licheniformis LCCC10161 is 105～107cfu/g, the fermentation temperature is 20-45 ℃, the humidity is 50-70%, and the time is 5-10 d.
Or adding the fermentation supernatant of the bacillus licheniformis LCCC10161 obtained in the example 2, or the crude enzyme solution thereof, or the purified alpha-amylase prepared finally into tobacco leaves for fermentation, wherein the enzyme activity of the alpha-amylase in the tobacco leaves is 10-20U/mg, and the fermentation conditions are as follows: the temperature is 20-45 ℃, the humidity is 50-70%, and the time is 5-10 days.
The invention takes 2018 Chongqing B4F raw tobacco as a control group, and 10 is added6The tobacco leaves are fermented by using cfu/g of Bacillus licheniformis (Bacillus licheniformis) LCCC10161 as a treatment group 1 and adding 15U/mg of prepared alpha-amylase powder as a treatment group 2, wherein the fermentation temperature is 36 ℃, the humidity is 55 percent, the time is 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, after Bacillus licheniformis (Bacillus licheniformis) LCCC10161 or alpha-amylase is added, the starch content in the tobacco leaves is obviously reduced, the total sugar content in the tobacco leaves is increased, the tobacco fragrance can be increased, the smoke can be softened, the outbreak of the smoke can be increased, and the smoke quality can be improved.
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 Bacillus licheniformis (Bacillus licheniformis) LCCC10161 for producing alpha-amylase.
2. A method for producing alpha-amylase by utilizing Bacillus licheniformis (LCCC 10161), which is characterized in that the method comprises the steps of inoculating the activated Bacillus licheniformis (LCCC 10161) into a liquid culture medium for fermentation culture, and centrifuging the fermentation liquid to obtain a supernatant.
3. The method of claim 2, wherein the Bacillus licheniformis (LCCC 10161) is inoculated in an amount of 105～107cfu/ml, wherein the culture is shaking culture, the rotating speed is 150-250 r/min, the temperature is 20-45 ℃, and the time is 3-5 d;
preferably, the inoculum size is 106cfu/ml, the rotating speed is 200r/min, the temperature is 36 ℃, and the time is 4 d;
preferably, the liquid medium contains: 0.8g of bean cake powder, 2.0g of corn flour, 1.0g of NaCl and K2HPO4 0.01g、KH2PO4 0.01g、CaCl20.3g of distilled water and 100mL of distilled water, wherein the pH value is 7.5-8.5, and the content of a liquid culture medium is 8-15%;
preferably, the pH is 8.0, and the filling amount of the liquid culture medium is 10%;
preferably, Bacillus licheniformis (Bacillus licheniformis) LCCC10161 is subjected to activated culture for 15-25 days and then inoculated;
preferably, the culture medium used for activating the Bacillus licheniformis (Bacillus licheniformis) LCCC10161 is LB culture medium;
preferably, the LB medium contains 10g/L peptone, 5g/L yeast extract, 10g/L sodium chloride;
preferably, the centrifugation temperature is 0-4 ℃, the rotation speed is 8000-12000 rpm, and the time is 20-40 min.
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 a crude enzyme solution.
5. The method of claim 4, wherein the saturation of ammonium sulfate is 40-60%;
preferably, the supernatant is dissolved by adding ammonium sulfate, centrifuged, and precipitated with ddH2Dissolving O, and desalting by dialysis with an acetic acid buffer solution of 15-25 mmol/L, pH 5.8.8-6.2.
6. The method according to claim 4, further comprising the step of purifying the crude enzyme solution by sequentially using a DEAE Sepharose Fast Flow anion exchange chromatography column and a Sephadex G-75 gel filtration chromatography column.
7. The method according to claim 6, wherein the specific steps of the purification are as follows:
(1) loading the crude enzyme solution to DEAE Sepharose Fast Flow anion exchange chromatography column, and eluting with acetic acid buffer solution A to A280After the solution is not changed, eluting the solution by using an acetic acid buffer solution containing 0.1mol/L NaCl at the flow rate of 0.8-1.2 mL/min to obtain a collection solution;
(2) and (3) loading the collected liquid to a Sephadex G-75 gel filtration chromatographic column, eluting with an acetic acid buffer solution A containing 0.2mol/L NaCl at the flow rate of 0.2-0.4 mL/min, and concentrating the collected active components to the density of 1.1-1.2G/mL.
8. Use of Bacillus licheniformis (LCCC 10161) or the method according to any of claims 2-7 for the fermentation of tobacco leaves.
9. A tobacco leaf fermentation method is characterized in that Bacillus licheniformis (LCCC 10161) is inoculated into tobacco leaves for fermentation.
10. The tobacco fermentation process of claim 9, wherein the inoculation amount of Bacillus licheniformis (LCCC 10161) is 105～107cfu/g, the fermentation temperature is 20-45 ℃, the humidity is 50-70%, and the time is 5-10 d.
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