CN110616212A - Application of bacillus licheniformis LCCC10150 in production of beta-glucanase and tobacco fermentation - Google Patents
Application of bacillus licheniformis LCCC10150 in production of beta-glucanase and tobacco fermentation Download PDFInfo
- Publication number
- CN110616212A CN110616212A CN201910913656.5A CN201910913656A CN110616212A CN 110616212 A CN110616212 A CN 110616212A CN 201910913656 A CN201910913656 A CN 201910913656A CN 110616212 A CN110616212 A CN 110616212A
- Authority
- CN
- China
- Prior art keywords
- bacillus licheniformis
- fermentation
- glucanase
- lccc
- beta
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 241000194108 Bacillus licheniformis Species 0.000 title claims abstract description 50
- 241000208125 Nicotiana Species 0.000 title claims abstract description 49
- 235000002637 Nicotiana tabacum Nutrition 0.000 title claims abstract description 49
- 238000000855 fermentation Methods 0.000 title claims abstract description 48
- 230000004151 fermentation Effects 0.000 title claims abstract description 48
- 101710130006 Beta-glucanase Proteins 0.000 title claims abstract description 39
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 14
- 102000004190 Enzymes Human genes 0.000 claims abstract description 45
- 108090000790 Enzymes Proteins 0.000 claims abstract description 45
- 239000007788 liquid Substances 0.000 claims description 19
- 238000000034 method Methods 0.000 claims description 18
- 239000000243 solution Substances 0.000 claims description 14
- 239000001963 growth medium Substances 0.000 claims description 13
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 10
- VWDWKYIASSYTQR-UHFFFAOYSA-N sodium nitrate Chemical compound [Na+].[O-][N+]([O-])=O VWDWKYIASSYTQR-UHFFFAOYSA-N 0.000 claims description 10
- 239000006228 supernatant Substances 0.000 claims description 9
- 238000004440 column chromatography Methods 0.000 claims description 7
- 238000010828 elution Methods 0.000 claims description 7
- 239000007974 sodium acetate buffer Substances 0.000 claims description 6
- 238000011033 desalting Methods 0.000 claims description 5
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 5
- 229910000396 dipotassium phosphate Inorganic materials 0.000 claims description 5
- 239000012153 distilled water Substances 0.000 claims description 5
- 229910052564 epsomite Inorganic materials 0.000 claims description 5
- 235000013312 flour Nutrition 0.000 claims description 5
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 claims description 5
- 229910000359 iron(II) sulfate Inorganic materials 0.000 claims description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 5
- 238000011081 inoculation Methods 0.000 claims description 4
- 238000009630 liquid culture Methods 0.000 claims description 4
- 239000002609 medium Substances 0.000 claims description 4
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 3
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 3
- 238000005119 centrifugation Methods 0.000 claims description 3
- 238000004090 dissolution Methods 0.000 claims description 2
- 239000002054 inoculum Substances 0.000 claims description 2
- 230000001376 precipitating effect Effects 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 13
- 229920001503 Glucan Polymers 0.000 abstract description 3
- 230000002255 enzymatic effect Effects 0.000 abstract description 3
- 238000000354 decomposition reaction Methods 0.000 abstract description 2
- 238000011031 large-scale manufacturing process Methods 0.000 abstract 1
- 238000011068 loading method Methods 0.000 description 4
- 239000002244 precipitate Substances 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- 241000894006 Bacteria Species 0.000 description 3
- 229920005654 Sephadex Polymers 0.000 description 3
- 239000012507 Sephadex™ Substances 0.000 description 3
- 244000005700 microbiome Species 0.000 description 3
- 238000002703 mutagenesis Methods 0.000 description 3
- 231100000350 mutagenesis Toxicity 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 239000000779 smoke Substances 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 238000012870 ammonium sulfate precipitation Methods 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 238000010612 desalination reaction Methods 0.000 description 2
- 230000003301 hydrolyzing effect Effects 0.000 description 2
- 239000012535 impurity Substances 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 230000001953 sensory effect Effects 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 238000004659 sterilization and disinfection Methods 0.000 description 2
- FYGDTMLNYKFZSV-URKRLVJHSA-N (2s,3r,4s,5s,6r)-2-[(2r,4r,5r,6s)-4,5-dihydroxy-2-(hydroxymethyl)-6-[(2r,4r,5r,6s)-4,5,6-trihydroxy-2-(hydroxymethyl)oxan-3-yl]oxyoxan-3-yl]oxy-6-(hydroxymethyl)oxane-3,4,5-triol Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1[C@@H](CO)O[C@@H](OC2[C@H](O[C@H](O)[C@H](O)[C@H]2O)CO)[C@H](O)[C@H]1O FYGDTMLNYKFZSV-URKRLVJHSA-N 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
- 241000304886 Bacilli Species 0.000 description 1
- 244000063299 Bacillus subtilis Species 0.000 description 1
- 235000014469 Bacillus subtilis Nutrition 0.000 description 1
- 229920002498 Beta-glucan Polymers 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 235000019418 amylase Nutrition 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 235000019504 cigarettes Nutrition 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- ZPAREOVLTARDFK-UHFFFAOYSA-N diethyl sulfate hydrochloride Chemical compound Cl.S(=O)(=O)(OCC)OCC ZPAREOVLTARDFK-UHFFFAOYSA-N 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 239000003205 fragrance Substances 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 229910052744 lithium Inorganic materials 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000003672 processing method Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 238000003860 storage 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
- C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
- C12N9/244—Endo-1,3(4)-beta-glucanase (3.2.1.6)
-
- 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)
- C12Y302/01006—Endo-1,3(4)-beta-glucanase (3.2.1.6)
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biomedical Technology (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
The invention provides an application of Bacillus licheniformis (LCCC 10150) in beta-glucanase production and tobacco fermentation, the Bacillus licheniformis (LCCC 10150) can produce beta-glucanase with high enzymatic activity, the Bacillus licheniformis (LCCC 10150) is used for producing the beta-glucanase, a new way is provided for the production source of the beta-glucanase, and the large-scale production of the beta-glucanase can be realized; the bacillus licheniformis with beta-glucanase with high enzyme activity is applied to the fermentation of tobacco leaves, the decomposition of glucan 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 LCCC10150 in production of beta-glucanase and tobacco fermentation, and belongs 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.
The beta-glucanase is a general name of a plurality of enzymes for catalyzing and hydrolyzing beta-glucan, and is one of the main enzymes used in tobacco leaf fermentation. The most commonly used enzyme-producing strains for the international industrial production of glucan: bacillus licheniformis can be used for producing enzyme preparation of food. Therefore, the screening of the bacillus licheniformis with high enzyme activity has important significance.
The prior art CN103820356B discloses a Bacillus licheniformis strain with high beta-glucanase enzyme yield. The strain is prepared by mixing an original strain CCTCC NO: m2013538, obtained by ultraviolet-lithium chloride-diethyl sulfate composite mutagenesis. The beta-glucanase enzyme activity produced by the strain is up to 8000-10000u/ml, which is 2.5 times of that of the original strain. The strain can obtain the beta-glucanase with high enzyme activity only by mutagenesis, and the requirement on mutagenesis conditions is strictly controlled. At present, the beta-glucanase with high enzyme activity is obtained by screening the existing bacillus licheniformis strains, and the application of the bacillus licheniformis strains in tobacco leaf fermentation is not reported at all.
Disclosure of Invention
The invention aims to provide application of Bacillus licheniformis (LCCC 10150) in production of beta-glucanase and tobacco fermentation, wherein the Bacillus licheniformis (LCCC 10150) can produce the beta-glucanase with high yield and is very suitable for production of the beta-glucanase and tobacco fermentation.
In one aspect, the invention provides an application of Bacillus licheniformis (LCCC 10150) in producing beta-glucanase. The Bacillus licheniformis (Bacillus licheniformis) LCCC10150 can produce beta-glucanase with high enzymatic activity, and provides a new source for producing the beta-glucanase.
In another aspect, the present invention also provides a method for producing beta-glucanase by using Bacillus licheniformis (LCCC 10150), which comprises the step of inoculating the Bacillus licheniformis (LCCC 10150) into a liquid medium for fermentation culture to obtain a fermentation broth.
Inoculation amount of Bacillus licheniformis (Bacillus licheniformis) LCCC10150 105～107cfu/ml, the culture is shaking culture, and the rotating speed is100-200 r/min, 20-40 ℃ and 1-3 d;
preferably, the inoculum size is 106cfu/ml, the rotating speed is 160r/min, the temperature is 35 ℃, and the time is 2 d;
preferably, the liquid medium contains NaNO3 0.2g、K2HPO4 0.1g、KCl 0.05g、MgSO4·7H2O 0.05g、FeSO4·7H20.01g of O, 5g of oat flour and 100mL of distilled water, wherein the pH value of the culture medium is 6.5-7.0;
preferably, the pH is 6.7.
Further, the method also comprises the steps of centrifuging the fermentation liquor, and adding ammonium sulfate into the supernatant for precipitation to obtain a crude enzyme solution.
Preferably, the rotating speed of the centrifugation is 8000-12000 rpm, and the time is 8-12 min;
preferably, the supernatant is added with 30-50% of saturated ammonium sulfate for full dissolution and then centrifuged, and the supernatant is added with 60-80% of saturated ammonium sulfate and centrifuged to obtain the crude enzyme.
The method also comprises the step of desalting the crude enzyme solution by using a SephadexG-25 column and purifying by using a SephadexG 100 column chromatography in sequence.
Preferably, when desalting is carried out by a SephadexG-25 column, eluting with 45-55 mmol/L sodium acetate buffer solution with the pH of 5-5.5 at the elution speed of 55-65 ml/h;
and/or eluting with 45-55 mmol/L sodium acetate buffer solution with pH of 5-5.5 at an elution speed of 10-15 mL/h during SephadexG 100 column chromatography.
In another aspect, the invention provides a tobacco leaf fermentation method, wherein Bacillus licheniformis (LCCC 10150) is inoculated into tobacco leaves for fermentation.
Preferably, the inoculation amount of Bacillus licheniformis (LCCC 10150) is 105～107cfu/g, the fermentation temperature is 20-40 ℃, the humidity is 50-70%, and the time is 7-14 d.
The invention has the beneficial effects that:
the invention discloses Bacillus licheniformis (LCCC 10150) capable of producing beta-glucanase with high enzyme activity, provides a new way for the production source of the beta-glucanase, and applies the Bacillus licheniformis capable of producing the beta-glucanase with high enzyme activity to tobacco leaf fermentation, so that the decomposition of glucan in 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. Bacillus licheniformis (Bacillus licheniformis) used in the invention is purchased from Liaoning province microorganism strain preservation center and is numbered LCCC 10150.
The methods in the examples are conventional in the art unless otherwise specified.
Example 1Liquid strain culture and enzyme production process
The liquid strain culture process comprises the following steps:
by optimizing and researching the culture medium conditions, liquid loading amount, culture temperature, culture time and other multi-factor culture conditions, the following culture process of the first-stage liquid strain is obtained:
the formula of the liquid culture medium is as follows: NaNO3 0.2g、K2HPO4 0.1g、KCl 0.05g、MgSO4·7H2O 0.05g、FeSO4·7H20.01g of O, 5g of oat flour and 100mL of distilled water, and the pH value of the culture medium is 6.7.
The liquid loading amount is as follows: 120mL in a 500mL triangular bottle.
And (3) sterilization conditions: 1atm, 30 min.
The culture conditions are as follows: the temperature is 30-40 ℃, and the rotating speed of the shaking table is 100-200 r/min.
And (3) culture period: 12-14 h.
The quality standard of the strain is as follows:
sensory index: color of the culture medium: milky white. The bacteria liquid is layered after being placed for 1h, and precipitates are visible at the bottom and are 40% high.
Microscopic conditions: the middle rod is tidy and robust, and the content in the rod can be seen to form spores.
Viable count: 15 to 20 hundred million/mL.
pH of the solution: 6.5 to 7.0.
The production process of the enzyme:
the strain enzyme production process is obtained by optimizing and researching multi-factor culture conditions such as culture medium conditions, liquid loading amount, culture temperature, culture time and enzyme production relation and the like:
the formula of the liquid culture medium is as follows: NaNO3 0.2g、K2HPO4 0.1g、KCl 0.05g、MgSO4·7H2O 0.05g、FeSO4·7H20.01g of O, 5g of oat flour and 100mL of distilled water, and the pH value of the culture medium is 6.7.
The liquid loading amount is as follows: 70-75%.
And (3) sterilization conditions: 1atm, 30 min.
The culture conditions are as follows: the temperature is 35 ℃ and the rotating speed is 100-200 r/min.
And (3) culture period: 24-60 h.
And (3) quality standard of culture solution:
sensory index: color of the culture medium: milky white. The bacteria liquid is layered after standing for 10min, and precipitates are visible at the bottom and are 40% high.
Microscopic conditions: few intact bacilli exist, almost all are spores.
pH of the solution: 7.2 to 7.4
The enzyme activity of the fermentation liquor is measured as follows: is more than or equal to 1.0U/mL.
Example 2Preparation of beta-glucanase Using Bacillus licheniformis (LCCC 10150) LCCC10150
The preparation method for producing beta-glucanase by utilizing Bacillus licheniformis (Bacillus licheniformis) LCCC10150 comprises the following steps:
(1) crude extraction of beta-glucanase:
bacillus licheniformis (Bacillus licheniformis) LCCC10150 is inoculated in an amount of 106cfu/ml is inoculated in a liquid culture medium at the temperature of 35 ℃ at 160r/min,and (5) performing fermentation culture for 2 d.
The liquid medium contains NaNO3 0.2g、K2HPO4 0.1g、KCl 0.05g、MgSO4·7H2O 0.05g、FeSO4·7H20.01g of O, 5g of oat flour and 100mL of distilled water, and the pH value of the culture medium is 6.7.
Centrifuging the fermented supernatant at 10000rpm for 10min to remove thallus and impurities, adding 40% saturated ammonium sulfate into the supernatant, standing overnight, centrifuging, removing precipitate, adding 70% saturated ammonium sulfate into the supernatant, and centrifuging to obtain crude enzyme.
According to the fermentation characteristics of microorganisms, during the fermentation process of the microorganisms, extracellular enzymes such as amylase, protease and saccharifying enzyme can be produced while producing beta-glucanase, the enzymes are very beneficial to saccharifying and hydrolyzing polysaccharides in tobacco, and the stability of crude enzyme liquid is found to be higher than that of pure enzyme, probably because the activity of the enzyme is protected by the impure protein in the crude enzyme liquid to a certain extent.
(2) Beta-glucanase purification
In order to study the enzymatic properties of the beta-glucanase, the crude enzyme after ammonium sulfate precipitation needs to be further purified, and the process flow is as follows:
removing bacteria and impurities from fermentation liquor by centrifugation, ammonium sulfate fractional precipitation, SephadexG-25 desalination, SephadexG 100 column chromatography and purified enzyme.
Desalting on a Sephadex G-25 column: the 70% saturated ammonium sulfate precipitate was dissolved in 50ml of 50mmol/L sodium acetate buffer (pH 5.3), and then 25m1 was desalted on a Sephadex G-25 column (50X 3cm) and eluted with 50mmol/L sodium acetate buffer (pH 5.3) at an elution rate of 60 ml/h. 3m1 was collected from each tube, and the protein concentration was measured to collect protein-containing fractions.
SephadexG-100 column chromatography: the protein-containing fractions (36m1) were combined and concentrated, and 15ml was applied to a Sephadex G-100 (50X 3cm) column, and eluted with the same buffer as above at an elution rate of 12 ml/h. 2mI was collected per tube and the enzyme containing fractions were collected.
As a result: the crude enzyme solution is subjected to ammonium sulfate precipitation, SephadexG-25 desalination and SephadexG-100 column chromatography to obtain the electrophoresis-grade pure enzyme, the enzyme activity yield in the purification process is 13.9 percent, the purification multiple is 6.6, the specific enzyme activity is improved from 1432U/mg of the crude enzyme to 9500U/mg, and the electrophoresis-grade purity is achieved.
Example 3Application of Bacillus licheniformis (Bacillus licheniformis) LCCC10150 in tobacco fermentation
Bacillus licheniformis (LCCC 10150) is inoculated into tobacco leaves, or the Bacillus licheniformis (LCCC 10150) is added into the tobacco leaves together with other strains, such as Bacillus subtilis or yeast, for fermentation. Wherein the inoculation amount of Bacillus licheniformis (LCCC 10150) is 105～107cfu/g, fermentation conditions are as follows: the temperature is 20-40 ℃, the humidity is 50-70%, and the time is 7-14 d.
Or adding the purified beta-glucanase of the embodiment 2 or crude enzyme liquid or fermentation liquid containing the beta-glucanase into tobacco leaves for fermentation, wherein the enzyme activity of the beta-glucanase added into the tobacco leaves is 10-20U/mg, and the fermentation conditions are as follows: the temperature is 20-40 ℃, the humidity is 50-70%, and the time is 7-14 d.
The Bacillus licheniformis (Bacillus licheniformis) LCCC10150, beta-glucanase or enzyme liquid containing the beta-glucanase is used for fermenting tobacco leaves, so that the fermentation period of the tobacco leaves is obviously shortened, and the quality of the tobacco leaves is also obviously improved.
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) LCCC10150 as a treatment group 1 and adding 15U/mg of prepared beta-glucanase enzyme powder as a treatment group 2, wherein the fermentation temperature is 35 ℃, the humidity is 55 percent, the time is 10 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 (LCCC 10150) or beta-glucanase 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 is softened, the outbreak of the smoke is increased, and the smoke quality is 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) LCCC10150 for producing beta-glucanase.
2. A method for producing beta-glucanase by utilizing Bacillus licheniformis (LCCC 10150), which is characterized by comprising the step of inoculating the Bacillus licheniformis (LCCC 10150) into a liquid culture medium for fermentation culture to obtain a fermentation liquid.
3. The method of claim 2, wherein the Bacillus licheniformis (LCCC 10150) is inoculated in an amount of 105～107cfu/ml, wherein the culture is shaking culture, the rotating speed is 100-200 r/min, the temperature is 20-40 ℃, and the time is 1-3 d;
preferably, the inoculum size is 106cfu/ml, the rotating speed is 160r/min, the temperature is 35 ℃, and the time is 2 d;
preferably, the liquid medium contains NaNO3 0.2g、K2HPO4 0.1g、KCl 0.05g、MgSO4·7H2O 0.05g、FeSO4·7H20.01g of O, 5g of oat flour and 100mL of distilled water, wherein the pH value of the culture medium is 6.5-7.0;
preferably, the pH is 6.7.
4. The method of claim 2, further comprising the step of centrifuging the fermentation broth, and precipitating the supernatant by adding ammonium sulfate to obtain a crude enzyme solution.
5. The method according to claim 4, wherein the rotation speed of the centrifugation is 8000-12000 rpm, and the time is 8-12 min;
preferably, the supernatant is added with 30-50% of saturated ammonium sulfate for full dissolution and then centrifuged, and the supernatant is added with 60-80% of saturated ammonium sulfate and centrifuged to obtain the crude enzyme.
6. The method according to claim 4, further comprising the step of desalting the crude enzyme solution using SephadexG-25 column and purifying the crude enzyme solution using SephadexG 100 column chromatography in this order.
7. The method as claimed in claim 6, wherein, in the desalting of SephadexG-25 column, the elution is carried out with 45-55 mmol/L sodium acetate buffer solution with pH of 5-5.5 at an elution speed of 55-65 ml/h;
and/or eluting with 45-55 mmol/L sodium acetate buffer solution with pH of 5-5.5 at an elution speed of 10-15 mL/h during SephadexG 100 column chromatography.
8. Use of Bacillus licheniformis (LCCC 10150) 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 10150) is inoculated into tobacco leaves for fermentation.
10. The tobacco fermentation process of claim 9, wherein the inoculation amount of Bacillus licheniformis (LCCC 10150) is 105～107cfu/g, the fermentation temperature is 20-40 ℃, the humidity is 50-70%, and the time is 7-14 d.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910913656.5A CN110616212A (en) | 2019-09-25 | 2019-09-25 | Application of bacillus licheniformis LCCC10150 in production of beta-glucanase and tobacco fermentation |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910913656.5A CN110616212A (en) | 2019-09-25 | 2019-09-25 | Application of bacillus licheniformis LCCC10150 in production of beta-glucanase and tobacco fermentation |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN110616212A true CN110616212A (en) | 2019-12-27 |
Family
ID=68924601
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910913656.5A Pending CN110616212A (en) | 2019-09-25 | 2019-09-25 | Application of bacillus licheniformis LCCC10150 in production of beta-glucanase and tobacco fermentation |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110616212A (en) |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101940361A (en) * | 2010-10-11 | 2011-01-12 | 广东中烟工业有限责任公司 | Processing method of waste tobacco |
| CN103820419A (en) * | 2013-12-23 | 2014-05-28 | 湖南鸿鹰生物科技有限公司 | Preparation method of beta-glucanase with good thermal stability |
| CN105018369A (en) * | 2015-01-19 | 2015-11-04 | 云南大学 | High temperature bacterium Bacillus licheniformis and application of same in prevention and treatment of mildew of tobacco leaves |
| CN105286073A (en) * | 2015-11-19 | 2016-02-03 | 云南省烟草公司昭通市公司 | Tobacco leaf curing modulating agent and using method thereof |
| CN105969703A (en) * | 2016-07-27 | 2016-09-28 | 云南中烟工业有限责任公司 | Bacillus licheniformis and application thereof |
| CN108823190A (en) * | 2018-06-06 | 2018-11-16 | 云南中烟工业有限责任公司 | A kind of complex enzyme formulation and its preparation method and application reducing typical nitrosamine of tobacco leaves |
| CN109874919A (en) * | 2019-03-05 | 2019-06-14 | 武汉新华扬生物股份有限公司 | A kind of additive agent for feeding, preparation method and applications |
| CN112522149A (en) * | 2020-12-11 | 2021-03-19 | 湖南稼陶农业科技有限公司 | Compound microbial agent capable of preventing and treating tobacco diseases |
-
2019
- 2019-09-25 CN CN201910913656.5A patent/CN110616212A/en active Pending
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101940361A (en) * | 2010-10-11 | 2011-01-12 | 广东中烟工业有限责任公司 | Processing method of waste tobacco |
| CN103820419A (en) * | 2013-12-23 | 2014-05-28 | 湖南鸿鹰生物科技有限公司 | Preparation method of beta-glucanase with good thermal stability |
| CN105018369A (en) * | 2015-01-19 | 2015-11-04 | 云南大学 | High temperature bacterium Bacillus licheniformis and application of same in prevention and treatment of mildew of tobacco leaves |
| CN105286073A (en) * | 2015-11-19 | 2016-02-03 | 云南省烟草公司昭通市公司 | Tobacco leaf curing modulating agent and using method thereof |
| CN105969703A (en) * | 2016-07-27 | 2016-09-28 | 云南中烟工业有限责任公司 | Bacillus licheniformis and application thereof |
| CN108823190A (en) * | 2018-06-06 | 2018-11-16 | 云南中烟工业有限责任公司 | A kind of complex enzyme formulation and its preparation method and application reducing typical nitrosamine of tobacco leaves |
| CN109874919A (en) * | 2019-03-05 | 2019-06-14 | 武汉新华扬生物股份有限公司 | A kind of additive agent for feeding, preparation method and applications |
| CN112522149A (en) * | 2020-12-11 | 2021-03-19 | 湖南稼陶农业科技有限公司 | Compound microbial agent capable of preventing and treating tobacco diseases |
Non-Patent Citations (2)
| Title |
|---|
| 中国食品添加剂和配料协会: "《食品添加剂手册 第3版》", 30 September 2012 * |
| 何沛等: "微生物在烟草中的研究、开发与利用进展", 《湖北农业科学》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP5765859B2 (en) | Yellow pigmentation defects Use in the production of sphingolipidoid aeromonas and gellan gum | |
| CN108642104B (en) | Bacterial cellulose, preparation method and application thereof | |
| EP3041941B1 (en) | Enzyme composition and process of preperation theroff | |
| CN107557407B (en) | Method for regulating and controlling molecular weight of schizophyllan of schizophyllum commune fermentation product | |
| JP2017006019A (en) | Novel mannanase and novel microorganism producing this | |
| US7951561B2 (en) | Method for the preparation of κ-carrageenase | |
| CN110616210A (en) | Application of bacillus licheniformis LCCC10161 in production of alpha-amylase and tobacco fermentation | |
| CN102220244B (en) | Hypocrea lixii strain and method for preparing dextran enzyme with the Hypocrea lixii strain | |
| CN106754486B (en) | Pseudomonas for high-yield trehalose synthase and fermentation enzyme production method thereof | |
| CN110616153A (en) | Application of trichoderma koningii LCCC30119 in production of cellulase and tobacco fermentation | |
| CN110616212A (en) | Application of bacillus licheniformis LCCC10150 in production of beta-glucanase and tobacco fermentation | |
| CN107365730B (en) | Bacillus subtilis strain and method for producing pullulanase by using same | |
| CN110699338A (en) | Application of aspergillus niger LCCC30112 in production of glucoamylase and fermentation of tobacco leaves | |
| KR100339715B1 (en) | Process for Preparing Pullulan Employing Waste Materials Containing High Concentration of Sugars from Food-processing Industry | |
| CN106834131B (en) | Fungus for degrading pectin and xylan in tobacco stems and application of fungus | |
| EP0258038B1 (en) | Use of cellulase preparations in the cultivation and use of cellulose-producing micro-organisms | |
| JPS5982092A (en) | Novel bacterial strain and preparation of cyclic beta-1,2-glucan using the same | |
| CN116515795B (en) | Application of Aspergillus tubingensis in preparing phytase and/or degrading phytic acid | |
| CN116555094B (en) | Polysaccharide degrading bacteria of vibrio alginolyticus and culture method and application thereof | |
| CN116515647B (en) | Aspergillus flavus and application thereof in preparing tannase and/or degrading tannin | |
| CN113862165B (en) | Method for directly producing low-molecular-weight xanthan gum by co-culture fermentation | |
| TWI792803B (en) | Method of producing natural type n-acetylglucosamine | |
| JPS61162181A (en) | Production of thermostable xylanase | |
| CN108588055B (en) | Method for producing beta-mannase by using symbiotic spore bacteria | |
| CN110129211B (en) | High-temperature-resistant aspergillus fumigatus strain 23# and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20191227 |
|
| RJ01 | Rejection of invention patent application after publication |