CN113755363A - Preparation and application of Mixta calida bacteria for degrading nicotine - Google Patents
Preparation and application of Mixta calida bacteria for degrading nicotine Download PDFInfo
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- 229960002715 nicotine Drugs 0.000 title claims abstract description 111
- SNICXCGAKADSCV-JTQLQIEISA-N (-)-Nicotine Chemical compound CN1CCC[C@H]1C1=CC=CN=C1 SNICXCGAKADSCV-JTQLQIEISA-N 0.000 title claims abstract description 108
- SNICXCGAKADSCV-UHFFFAOYSA-N nicotine Natural products CN1CCCC1C1=CC=CN=C1 SNICXCGAKADSCV-UHFFFAOYSA-N 0.000 title claims abstract description 108
- 230000000593 degrading effect Effects 0.000 title claims abstract description 22
- 238000002360 preparation method Methods 0.000 title claims abstract description 20
- 241000583653 Pantoea calida Species 0.000 title claims abstract description 19
- 239000001963 growth medium Substances 0.000 claims abstract description 44
- 238000012360 testing method Methods 0.000 claims abstract description 37
- 241000894006 Bacteria Species 0.000 claims abstract description 31
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 29
- 235000013619 trace mineral Nutrition 0.000 claims abstract description 26
- 239000011573 trace mineral Substances 0.000 claims abstract description 26
- 239000007788 liquid Substances 0.000 claims abstract description 25
- 230000015556 catabolic process Effects 0.000 claims abstract description 17
- 238000006731 degradation reaction Methods 0.000 claims abstract description 17
- 238000001914 filtration Methods 0.000 claims abstract description 16
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims abstract description 13
- 239000001110 calcium chloride Substances 0.000 claims abstract description 13
- 229910001628 calcium chloride Inorganic materials 0.000 claims abstract description 13
- 235000011148 calcium chloride Nutrition 0.000 claims abstract description 13
- 229940041514 candida albicans extract Drugs 0.000 claims abstract description 13
- 239000012153 distilled water Substances 0.000 claims abstract description 13
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 claims abstract description 13
- 229910000359 iron(II) sulfate Inorganic materials 0.000 claims abstract description 13
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 claims abstract description 13
- 229910000357 manganese(II) sulfate Inorganic materials 0.000 claims abstract description 13
- 239000012528 membrane Substances 0.000 claims abstract description 13
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- 230000000694 effects Effects 0.000 claims abstract description 10
- 239000002609 medium Substances 0.000 claims abstract description 8
- 238000009472 formulation Methods 0.000 claims abstract description 4
- 239000000203 mixture Substances 0.000 claims abstract description 4
- 241000208125 Nicotiana Species 0.000 claims description 33
- 235000002637 Nicotiana tabacum Nutrition 0.000 claims description 33
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 24
- 238000000034 method Methods 0.000 claims description 23
- 238000000855 fermentation Methods 0.000 claims description 16
- 230000004151 fermentation Effects 0.000 claims description 16
- 230000001580 bacterial effect Effects 0.000 claims description 14
- 238000012258 culturing Methods 0.000 claims description 12
- 238000009630 liquid culture Methods 0.000 claims description 12
- 238000002835 absorbance Methods 0.000 claims description 9
- 239000006228 supernatant Substances 0.000 claims description 9
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 6
- 230000003321 amplification Effects 0.000 claims description 6
- 238000002474 experimental method Methods 0.000 claims description 6
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- 238000012937 correction Methods 0.000 claims description 3
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- 239000008367 deionised water Substances 0.000 claims description 3
- 229910021641 deionized water Inorganic materials 0.000 claims description 3
- 238000007865 diluting Methods 0.000 claims description 3
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- 239000008223 sterile water Substances 0.000 claims description 3
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- 238000001035 drying Methods 0.000 claims description 2
- 238000002156 mixing Methods 0.000 claims description 2
- 239000000575 pesticide Substances 0.000 abstract description 5
- 230000000877 morphologic effect Effects 0.000 abstract description 3
- 230000000813 microbial effect Effects 0.000 abstract description 2
- 241000282414 Homo sapiens Species 0.000 description 3
- 239000013043 chemical agent Substances 0.000 description 3
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- 230000006838 adverse reaction Effects 0.000 description 1
- 229930013930 alkaloid Natural products 0.000 description 1
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Classifications
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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
- 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
-
- A—HUMAN NECESSITIES
- A62—LIFE-SAVING; FIRE-FIGHTING
- A62D—CHEMICAL MEANS FOR EXTINGUISHING FIRES OR FOR COMBATING OR PROTECTING AGAINST HARMFUL CHEMICAL AGENTS; CHEMICAL MATERIALS FOR USE IN BREATHING APPARATUS
- A62D3/00—Processes for making harmful chemical substances harmless or less harmful, by effecting a chemical change in the substances
- A62D3/02—Processes for making harmful chemical substances harmless or less harmful, by effecting a chemical change in the substances by biological methods, i.e. processes using enzymes or microorganisms
-
- A—HUMAN NECESSITIES
- A62—LIFE-SAVING; FIRE-FIGHTING
- A62D—CHEMICAL MEANS FOR EXTINGUISHING FIRES OR FOR COMBATING OR PROTECTING AGAINST HARMFUL CHEMICAL AGENTS; CHEMICAL MATERIALS FOR USE IN BREATHING APPARATUS
- A62D2101/00—Harmful chemical substances made harmless, or less harmful, by effecting chemical change
- A62D2101/20—Organic substances
- A62D2101/26—Organic substances containing nitrogen or phosphorus
Abstract
The invention discloses a preparation method and application of a nicotine degrading Mixta calida bacterium, relating to the technical field of microbial pesticides, wherein the preparation method of the nicotine degrading Mixta calida bacterium comprises the following steps: NiuJ1 bacteria were inoculated into test tube nicotine liquid medium (5 mL per tube) with the following formulation: K2HPO413.3g, KH2PO44.0g, yeast extract 1.0g, trace element solution 10mL, distilled water to 1000mL, pH 7.0, sterilizing the culture medium with high-pressure steam at 121 deg.C for 20min, adding a certain amount of nicotine (filtering with 0.22 μm filter membrane), and adding trace element solution: MnSO4 & 7H2O0.4g, CaCl2 & 2H2O0.2g, FeSO4 & 7H2O0.2g using 0.1mol/LHCl constant volume to 100 mL; NiuJ1 was inoculated onto the medium and shake-cultured at 37 ℃ for 12h at 220rpm to obtain test tube seeds. After the activity of the strain is determined by a multi-batch degradation activity test, the strain is subjected to morphological, physiological, biochemical and molecular identification, and is determined to be a gram-negative bacterium of the Mixta calida bacterium, and the bacterium has extremely high degradation activity on nicotine.
Description
Technical Field
The invention relates to the technical field of microbial pesticides, in particular to preparation and application of a bacterium Mixta calida for degrading nicotine.
Background
Nicotine (nicotine), also known as nicotine, is a main alkaloid extracted from tobacco, belongs to a highly toxic compound, has good water solubility, is not easily degraded in the environment, not only pollutes the environment and breaks ecological balance, but also seriously threatens human health, and has various adverse reactions such as teratogenesis, carcinogenesis and the like. The contamination of the environment with nicotine mainly results from the use as a pesticide, the large amounts of high-concentration waste generated in tobacco production and processing activities. During the past decade, nicotinoids have been one of the fastest growing, most widely used insecticides in crop protection. 670 million tons of tobacco are reported to be produced annually worldwide, with china being the largest tobacco-producing country (39.6%), and the tobacco industry is expected to produce 300,274t of tobacco waste annually. The nicotine has great utilization value in the pharmaceutical industry, the tobacco industry, the agricultural industry and the chemical industry, creates great economic benefits and brings about various harms. Nicotine is extremely toxic and in large amounts it inhibits the central nervous system, causing respiratory arrest and cardioplegia. China is a big tobacco country, nicotine is an important factor for measuring the quality of tobacco, and due to the problems of planting methods, the nicotine content of upper tobacco leaves is generally high, the industrial availability is reduced, and the capital loss of thousands of yuan per year is caused. How to control nicotine of tobacco within a usable range has become one of the hot topics to be solved by the national tobacco industry. The use of the nicotine-type pesticide and the production of tobacco not only affect the health of human beings, but also pollute the environment. In conclusion, nicotine poses a great threat to the ecological environment and human health on which people live, and attention must be paid to the use of nicotine pesticides and the treatment of tobacco waste.
At present, although nicotine can be degraded by traditional agricultural cultivation technology improvement, physical and chemical means and other ways, the methods have certain limitations and disadvantages, the operability and adaptability are not ideal, and in recent years, with the enhancement of environmental awareness, the application of chemical agents is gradually limited, and except that chemical agents with low toxicity, low residue and high selectivity are continuously explored, people pay more attention to the development of biocontrol agents to be used as supplements or substitutes for the chemical agents. Therefore, the research on the degradation of nicotine by microorganisms and enzymes is naturally a major and focused issue.
Disclosure of Invention
The invention aims to provide preparation and application of a bacterium Mixta calida for degrading nicotine, so as to solve the problems in the background technology.
In order to achieve the purpose, the invention provides the following technical scheme:
preparation and application of a bacterium for degrading nicotine Mixta calida, wherein the preparation method of the bacterium for degrading nicotine Mixta calida comprises the following steps: NiuJ1 bacteria were inoculated into test tube nicotine liquid medium (5 mL per tube) with the following formulation: K2HPO413.3g, KH2PO44.0g, yeast extract 1.0g, trace element solution 10mL, distilled water to 1000mL, pH 7.0, sterilizing the culture medium with high-pressure steam at 121 deg.C for 20min, adding a certain amount of nicotine (filtering with 0.22 μm filter membrane), and adding trace element solution: MnSO4 & 7H2O0.4g, CaCl2 & 2H2O0.2g, FeSO4 & 7H2O0.2g using 0.1mol/LHCl constant volume to 100 mL; inoculating NiuJ1 onto the culture medium, and shake-culturing at 37 deg.C for 12h at 220rpm to obtain test tube seeds;
transferring the test tube seeds into a fresh nicotine liquid medium test tube, and carrying out shake culture at 37 ℃ for 12h at the rotating speed of 220rpm to obtain a bacterial liquid with strong activity.
As a further scheme of the invention: the preparation method of the nicotine degrading Mixta calida bacteria further comprises the following steps: the test tube seeds are inoculated into 250mL of triangular flask liquid culture medium (100mL per bottle), and the formula of the liquid culture medium is as follows: K2HPO413.3g, KH2PO44.0g, yeast extract 1.0g, trace element solution 10mL, distilled water to 1000mL, pH 7.0, sterilizing the culture medium with high-pressure steam at 121 deg.C for 20min, adding a certain amount of nicotine (filtering with 0.22 μm filter membrane), and adding trace element solution: MnSO4 & 7H2O0.4g, CaCl2 & 2H2O0.2g, FeSO4 & 7H2O0.2g using 0.1mol/LHCl constant volume to 100 mL; culturing at 37 deg.C for 24h with shaking at 220 rpm;
the fermentation culture was centrifuged at 10000rpm/min for 20 minutes, and the supernatant was collected and filtered through a 0.22 μm filter to obtain a sterile fermentation broth.
As a further scheme of the invention: also comprises the following preparation steps:
the method comprises the following steps: the test tube species culture is carried out, and the formula of a culture medium is as follows: K2HPO413.3g, KH2PO44.0g, yeast extract 1.0g, trace element solution 10mL, distilled water to 1000mL, pH 7.0, sterilizing the culture medium with high-pressure steam at 121 deg.C for 20min, adding a certain amount of nicotine (filtering with 0.22 μm filter membrane), and adding trace element solution: MnSO4 & 7H2O0.4g, CaCl2 & 2H2O0.2g, FeSO4 & 7H2O0.2g using 0.1mol/LHCl constant volume to 100 mL; inoculating the gram-negative bacteria of NiuJ1 to a culture medium, and culturing at 37 ℃ for 12h to obtain test tube seeds;
step two: liquid amplification culture is carried out, and the formula of a culture medium is as follows: K2HPO413.3g, KH2PO44.0g, yeast extract 1.0g, trace element solution 10mL, distilled water to 1000mL, pH 7.0, sterilizing the culture medium with high-pressure steam at 121 deg.C for 20min, adding a certain amount of nicotine (filtering with 0.22 μm filter membrane), and adding trace element solution: MnSO4 & 7H2O0.4g, CaCl2 & 2H2O0.2g, FeSO4 & 7H2O0.2g using 0.1mol/LHCl constant volume to 100 mL; the test tube seeds are inoculated into 500mL of triangular flask liquid culture medium, 200mL of each bottle is filled, and the shaking culture is carried out at 37 ℃ for 12h at the rotating speed of 220 rpm.
As a further scheme of the invention: the method also comprises a test for degrading nicotine by NiuJ1 bacteria, and comprises the following procedures:
the first process is as follows: preparing a bacterial liquid for test, and inoculating and culturing the NiuJ1 strain according to the liquid amplification culture method.
And a second process: and preparing a control reagent, wherein in the experiment for detecting the degradation of nicotine by NiuJ1 bacteria, the control adopts a nicotine culture medium with a corresponding amount as a blank control.
And the third flow, the test method comprises the following two steps,
(1) and mixing the sterile fermentation liquor of the test tube species in a ratio of 1: inoculating 1 proportion into 50mL of triangular flask liquid culture medium, loading 10mL of culture medium in each bottle, centrifuging 1mL of culture solution inoculated with nicotine-degrading bacteria at 10000rpm for 10min at different culture time, taking the culture solution added with nicotine and not inoculated with degrading bacteria as a control, treating by the same method, and taking supernatant to measure the light absorption value of nicotine in each fermentation liquid under the wavelength of 259nm of an enzyme labeling instrument; if the OD value is too large, diluting the supernatant with 0.05mol/L hydrochloric acid; substituting the measured absorbance value into a nicotine standard curve to obtain an equation to convert the concentration of the residual nicotine, thereby obtaining the nicotine degradation rate of the bacterium; nicotine degradation rate = (initial nicotine content-nicotine content in fermentation broth)/initial nicotine content X100%;
(2) the experiment for degrading nicotine in tobacco leaves by NiuJ1 bacterial liquid is carried out by weighing 5g of modulated tobacco leaf fragments (with the water content of 15% -17%) and placing the tobacco leaf fragments into a triangular flask, adding equivalent bacterial suspension (10% proportion inoculation) into an experimental group, adding equivalent sterile water into a control group, adjusting the water content of the materials to 45% -50%, sealing, fermenting for 7d at 37 ℃, taking out the tobacco leaves, drying and crushing at 64 ℃, and measuring the nicotine content in the powder by a hydrochloric acid extraction and decoloration method; and (4) comparing with a control group, calculating the nicotine degradation rate of the tobacco leaves, and repeating for 3 times, wherein 3 bottles are used for each time.
As a further scheme of the invention: the method also comprises the following specific steps of: weighing 0.03g of tobacco leaf sample, adding the tobacco leaf sample into a 100mL triangular flask, and adding 0.25 of activated carbon and 25mL of 0.5N hydrochloric acid; oscillating for 5min, filtering into a volumetric flask (100mL), washing the triangular flask with deionized water, combining filtrates, and shaking to constant volume; adding 0.5N25mL hydrochloric acid into a 100mL volumetric flask, and shaking up with constant volume to serve as a blank reference; the absorbance values were determined separately at 3 wavelengths (236 nm; 259 nm; 282nm) with blank reference, with the reading adjusted to zero on the spectrophotometer instrument; the calculation formula of nicotine content is as follows: nicotine% (% of nicotine) (% of nicotine 1.059 × [ a 259-0.5 (a236+ a282) ] × 100 × 100/[ W (1-water%) × 34.3 × 1000], where 1.059 is the correction coefficient, W is the dry weight, and 34.3 is the absorption coefficient of nicotine (absorbance of a solution containing 1g of nicotine in 1000 mL).
Compared with the prior art, the invention has the beneficial effects that:
after the activity of the strain is determined by a multi-batch degradation activity test, the strain is subjected to morphological, physiological and biochemical and molecular identification and determined to be a gram-negative bacterium of the Mixta calida, and the strain has the main morphological characteristics that: the bacterial colony on the LB plate culture medium is round and full, the surface is moist and smooth, and the bacterial lawn is milky white. Gram-negative bacteria in the form of rod-shaped bacteria and flagella are observed under a scanning electron microscope, and have extremely high degradation activity on nicotine.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
In the embodiment of the invention, the preparation and application of the nicotine degrading Mixta calida bacteria are as follows: NiuJ1 bacteria were inoculated into test tube nicotine liquid medium (5 mL per tube) with the following formulation: K2HPO413.3g, KH2PO44.0g, yeast extract 1.0g, trace element solution 10mL, distilled water to 1000mL, pH 7.0, sterilizing the culture medium with high-pressure steam at 121 deg.C for 20min, adding a certain amount of nicotine (filtering with 0.22 μm filter membrane), and adding trace element solution: MnSO4 & 7H2O0.4g, CaCl2 & 2H2O0.2g, FeSO4 & 7H2O0.2g using 0.1mol/LHCl constant volume to 100 mL; inoculating NiuJ1 onto the culture medium, and shake-culturing at 37 deg.C for 12h at 220rpm to obtain test tube seeds;
transferring the test tube seeds into a fresh nicotine liquid medium test tube, and carrying out shake culture at 37 ℃ for 12h at the rotating speed of 220rpm to obtain a bacterial liquid with strong activity.
The preparation method of the nicotine degrading Mixta calida bacteria further comprises the following steps: the test tube seeds are inoculated into 250mL of triangular flask liquid culture medium (100mL per bottle), and the formula of the liquid culture medium is as follows: K2HPO413.3g, KH2PO44.0g, yeast extract 1.0g, trace element solution 10mL, distilled water to 1000mL, pH 7.0, sterilizing the culture medium with high-pressure steam at 121 deg.C for 20min, adding a certain amount of nicotine (filtering with 0.22 μm filter membrane), and adding trace element solution: MnSO4 & 7H2O0.4g, CaCl2 & 2H2O0.2g, FeSO4 & 7H2O0.2g using 0.1mol/LHCl constant volume to 100 mL; culturing at 37 deg.C for 24h with shaking at 220 rpm;
the fermentation culture was centrifuged at 10000rpm/min for 20 minutes, and the supernatant was collected and filtered through a 0.22 μm filter to obtain a sterile fermentation broth.
Also comprises the following preparation steps:
the method comprises the following steps: the test tube species culture is carried out, and the formula of a culture medium is as follows: K2HPO413.3g, KH2PO44.0g, yeast extract 1.0g, trace element solution 10mL, distilled water to 1000mL, pH 7.0, sterilizing the culture medium with high-pressure steam at 121 deg.C for 20min, adding a certain amount of nicotine (filtering with 0.22 μm filter membrane), and adding trace element solution: MnSO4 & 7H2O0.4g, CaCl2 & 2H2O0.2g, FeSO4 & 7H2O0.2g using 0.1mol/LHCl constant volume to 100 mL; inoculating the gram-negative bacteria of NiuJ1 to a culture medium, and culturing at 37 ℃ for 12h to obtain test tube seeds;
step two: liquid amplification culture is carried out, and the formula of a culture medium is as follows: K2HPO413.3g, KH2PO44.0g, yeast extract 1.0g, trace element solution 10mL, distilled water to 1000mL, pH 7.0, sterilizing the culture medium with high-pressure steam at 121 deg.C for 20min, adding a certain amount of nicotine (filtering with 0.22 μm filter membrane), and adding trace element solution: MnSO4 & 7H2O0.4g, CaCl2 & 2H2O0.2g, FeSO4 & 7H2O0.2g using 0.1mol/LHCl constant volume to 100 mL; the test tube seeds are inoculated into 500mL of triangular flask liquid culture medium, 200mL of each bottle is filled, and the shaking culture is carried out at 37 ℃ for 12h at the rotating speed of 220 rpm.
The method also comprises a test for degrading nicotine by NiuJ1 bacteria, and comprises the following procedures:
the first process is as follows: preparing a bacterial liquid for test, and inoculating and culturing the NiuJ1 strain according to the liquid amplification culture method.
And a second process: and preparing a control reagent, wherein in the experiment for detecting the degradation of nicotine by NiuJ1 bacteria, the control adopts a nicotine culture medium with a corresponding amount as a blank control.
The third flow, the test method,
1. sterile fermentation broth of the test tube species was mixed at a ratio of 1: inoculating 1 proportion into 50mL of triangular flask liquid culture medium, loading 10mL of culture medium in each bottle, centrifuging 1mL of culture solution inoculated with nicotine-degrading bacteria at 10000rpm for 10min at different culture time, taking the culture solution added with nicotine and not inoculated with degrading bacteria as a control, treating by the same method, and taking supernatant to measure the light absorption value of nicotine in each fermentation liquid under the wavelength of 259nm of an enzyme labeling instrument; if the OD value is too large, diluting the supernatant with 0.05mol/L hydrochloric acid; substituting the measured absorbance value into a nicotine standard curve to obtain an equation to convert the concentration of the residual nicotine, thereby obtaining the nicotine degradation rate of the bacterium; nicotine degradation rate = (initial nicotine content-nicotine content in fermentation broth)/initial nicotine content X100%.
2. An experiment for degrading nicotine in tobacco leaves by NiuJ1 bacterial liquid is carried out, 5g of modulated tobacco leaf fragments (the water content is 15% -17%) are weighed and placed into a triangular flask, an equal amount of bacterial suspension (10% proportion inoculation) is added into an experimental group, an equal amount of sterile water is added into a control group, the water content of materials is adjusted to 45% -50%, sealing is carried out, after 7d fermentation at 37 ℃, tobacco leaves are taken out, dried and crushed at 64 ℃, and the nicotine content in the powder is measured by a hydrochloric acid extraction and decoloration method; comparing with the control group, calculating the nicotine degradation rate of the tobacco leaves, repeating for 3 times, and 3 bottles each time;
the method also comprises the following specific steps of:
weighing 0.03g of tobacco leaf sample, adding the tobacco leaf sample into a 100mL triangular flask, and adding 0.25 of activated carbon and 25mL of 0.5N hydrochloric acid; oscillating for 5min, filtering into a volumetric flask (100mL), washing the triangular flask with deionized water, combining filtrates, and shaking to constant volume; adding 0.5N25mL hydrochloric acid into a 100mL volumetric flask, and shaking up with constant volume to serve as a blank reference; the blank reference is used for adjusting the reading to be zero on a spectrophotometer instrument, and the absorbance values are respectively measured at 3 wavelengths (236 nm; 259 nm; 282 nm); the calculation formula of nicotine content is as follows: nicotine% (% of nicotine) (% of nicotine 1.059 × [ a 259-0.5 (a236+ a282) ] × 100 × 100/[ W (1-water%) × 34.3 × 1000], where 1.059 is the correction coefficient, W is the dry weight, and 34.3 is the absorption coefficient of nicotine (absorbance of a solution containing 1g of nicotine in 1000 mL).
In the description of the present invention, it should be noted that, unless otherwise explicitly specified or limited, the terms "mounted," "connected," and "connected" are to be construed broadly, e.g., as meaning either a fixed connection, a removable connection, or an integral connection; can be mechanically or electrically connected; they may be connected directly or indirectly through intervening media, or they may be interconnected between two elements. The specific meaning of the above terms in the present invention can be understood by those of ordinary skill in the art through specific situations.
Although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that various changes in the embodiments and/or modifications of the invention can be made, and equivalents and modifications of some features of the invention can be made without departing from the spirit and scope of the invention.
Claims (5)
1. Preparation and application of a bacterium for degrading nicotine Mixta calida are characterized in that the preparation method of the bacterium for degrading nicotine Mixta calida comprises the following steps: NiuJ1 bacteria were inoculated into test tube nicotine liquid medium (5 mL per tube) with the following formulation: K2HPO413.3g, KH2PO44.0g, yeast extract 1.0g, trace element solution 10mL, distilled water to 1000mL, pH 7.0, sterilizing the culture medium with high-pressure steam at 121 deg.C for 20min, adding a certain amount of nicotine (filtering with 0.22 μm filter membrane), and adding trace element solution: MnSO4 & 7H2O0.4g, CaCl2 & 2H2O0.2g, FeSO4 & 7H2O0.2g using 0.1mol/LHCl constant volume to 100 mL; inoculating NiuJ1 onto the culture medium, and shake-culturing at 37 deg.C for 12h at 220rpm to obtain test tube seeds;
transferring the test tube seeds into a fresh nicotine liquid medium test tube, and carrying out shake culture at 37 ℃ for 12h at the rotating speed of 220rpm to obtain a bacterial liquid with strong activity.
2. The preparation and use of a nicotine-degrading bacterium Mixta calida according to claim 1, wherein the preparation method further comprises: the test tube seeds are inoculated into 250mL of triangular flask liquid culture medium (100mL per bottle), and the formula of the liquid culture medium is as follows: K2HPO413.3g, KH2PO44.0g, yeast extract 1.0g, trace element solution 10mL, distilled water to 1000mL, pH 7.0, sterilizing the culture medium with high-pressure steam at 121 deg.C for 20min, adding a certain amount of nicotine (filtering with 0.22 μm filter membrane), and adding trace element solution: MnSO4 & 7H2O0.4g, CaCl2 & 2H2O0.2g, FeSO4 & 7H2O0.2g using 0.1mol/LHCl constant volume to 100 mL; culturing at 37 deg.C for 24h with shaking at 220 rpm;
the fermentation culture was centrifuged at 10000rpm/min for 20 minutes, and the supernatant was collected and filtered through a 0.22 μm filter to obtain a sterile fermentation broth.
3. The preparation and use of a nicotine-degrading bacterium Mixta calida according to claim 1, further comprising the following steps:
the method comprises the following steps: the test tube species culture is carried out, and the formula of a culture medium is as follows: K2HPO413.3g, KH2PO44.0g, yeast extract 1.0g, trace element solution 10mL, distilled water to 1000mL, pH 7.0, sterilizing the culture medium with high-pressure steam at 121 deg.C for 20min, adding a certain amount of nicotine (filtering with 0.22 μm filter membrane), and adding trace element solution: MnSO4 & 7H2O0.4g, CaCl2 & 2H2O0.2g, FeSO4 & 7H2O0.2g using 0.1mol/LHCl constant volume to 100 mL; inoculating the gram-negative bacteria of NiuJ1 to a culture medium, and culturing at 37 ℃ for 12h to obtain test tube seeds;
step two: liquid amplification culture is carried out, and the formula of a culture medium is as follows: K2HPO413.3g, KH2PO44.0g, yeast extract 1.0g, trace element solution 10mL, distilled water to 1000mL, pH 7.0, sterilizing the culture medium with high-pressure steam at 121 deg.C for 20min, adding a certain amount of nicotine (filtering with 0.22 μm filter membrane), and adding trace element solution: MnSO4 & 7H2O0.4g, CaCl2 & 2H2O0.2g, FeSO4 & 7H2O0.2g using 0.1mol/LHCl constant volume to 100 mL; the test tube seeds are inoculated into 500mL of triangular flask liquid culture medium, 200mL of each bottle is filled, and the shaking culture is carried out at 37 ℃ for 12h at the rotating speed of 220 rpm.
4. The preparation and use of the bacterium Mixta calida according to claim 1, further comprising the test of the bacterium NiuJ1 for degrading nicotine, comprising the following steps:
the first process is as follows: preparing a bacterial liquid for test, and inoculating and culturing the NiuJ1 strain according to the liquid amplification culture method.
And a second process: and preparing a control reagent, wherein in the experiment for detecting the degradation of nicotine by NiuJ1 bacteria, the control adopts a nicotine culture medium with a corresponding amount as a blank control.
And the third flow, the test method comprises the following two steps,
(1) and mixing the sterile fermentation liquor of the test tube species in a ratio of 1: inoculating 1 proportion into 50mL of triangular flask liquid culture medium, loading 10mL of culture medium in each bottle, centrifuging 1mL of culture solution inoculated with nicotine-degrading bacteria at 10000rpm for 10min at different culture time, taking the culture solution added with nicotine and not inoculated with degrading bacteria as a control, treating by the same method, and taking supernatant to measure the light absorption value of nicotine in each fermentation liquid under the wavelength of 259nm of an enzyme labeling instrument; if the OD value is too large, diluting the supernatant with 0.05mol/L hydrochloric acid; substituting the measured absorbance value into a nicotine standard curve to obtain an equation to convert the concentration of the residual nicotine, thereby obtaining the nicotine degradation rate of the bacterium; nicotine degradation rate = (initial nicotine content-nicotine content in fermentation broth)/initial nicotine content X100%;
(2) the experiment for degrading nicotine in tobacco leaves by NiuJ1 bacterial liquid is carried out by weighing 5g of modulated tobacco leaf fragments (with the water content of 15% -17%) into a triangular flask, adding equivalent bacterial suspension (10% proportion inoculation) into an experimental group, adding equivalent sterile water into a control group, adjusting the water content of the materials to 45% -50%, sealing, fermenting for 7d at 37 ℃, taking out the tobacco leaves, drying and crushing at 64 ℃, and determining the nicotine content in the powder by a hydrochloric acid extraction and decoloration method; the nicotine degradation rate of tobacco leaves was calculated in 3 replicates, 3 bottles each, compared to the control group.
5. The preparation and use of a nicotine-degrading bacterium Mixta calida according to claim 4, further comprising the following steps of: weighing 0.03g of tobacco leaf sample, adding the tobacco leaf sample into a 100mL triangular flask, and adding 0.25 of activated carbon and 25mL of 0.5N hydrochloric acid; oscillating for 5min, filtering into a volumetric flask (100mL), washing the triangular flask with deionized water, combining filtrates, and shaking to constant volume; adding 0.5N25mL hydrochloric acid into a 100mL volumetric flask, and shaking up with constant volume to serve as a blank reference; the blank reference is used for adjusting the reading to be zero on a spectrophotometer instrument, and the absorbance values are respectively measured at 3 wavelengths (236 nm; 259 nm; 282 nm); the calculation formula of nicotine content is as follows: nicotine% (% of nicotine) (% of nicotine 1.059 × [ a 259-0.5 (a236+ a282) ] × 100 × 100/[ W (1-water%) × 34.3 × 1000], where 1.059 is the correction coefficient, W is the dry weight, and 34.3 is the absorption coefficient of nicotine (absorbance of a solution containing 1g of nicotine in 1000 mL).
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