CN116694536B

CN116694536B - Arthrobacter chlorophenol BJY-3 and application thereof in degradation of nicotine

Info

Publication number: CN116694536B
Application number: CN202310934712.XA
Authority: CN
Inventors: 于涛; 费玥; 李海涛; 李�杰; 李力群; 郭春生; 云晶; 那宝丹; 张善林
Original assignee: Inner Mongolia Kunming Cigarettes Co ltd
Current assignee: Inner Mongolia Kunming Cigarettes Co ltd
Priority date: 2023-07-28
Filing date: 2023-07-28
Publication date: 2023-10-10
Anticipated expiration: 2043-07-28
Also published as: CN116694536A

Abstract

The application discloses Arthrobacter chlorophenol BJY-3 and application thereof in degrading nicotine, and belongs to the field of microorganisms. The strain is preserved in China general microbiological culture Collection center (CGMCC) with a preservation number of CGMCC No.25122. The strain BJY-3 obtained by separation has the capability of degrading nicotine, can enrich strain resources for degrading nicotine, screens genes related to high-efficiency degradation of nicotine, provides a new gene source for transgenic tobacco and engineering strains, improves the quality of tobacco products, can be used for treating nicotine in tobacco waste and planting soil, and has important economic, social and ecological benefits.

Description

Arthrobacter chlorophenol BJY-3 and application thereof in degradation of nicotine

Technical Field

The application relates to the technical field of microorganisms, in particular to Arthrobacter chlorophenol BJY-3 and application thereof in efficiently degrading nicotine.

Background

Nicotine is a pyridine alkaloid, also called Nicotine, and has chemical structure name of 1-methyl-2- (3-pyridyl) -pyrrolidine, and chemical formula of C ₁₀ H ₁₄ N ₂ . It has high toxicity, can penetrate biological film, has stable chemical structure, is an important precursor of tobacco specific carcinogen nitrosamines (TSNAs), is not easy to degrade in the environment, and seriously pollutes the environment. Can be mutually dissolved with water in any proportion, and is a great threat to human life and ecological environment.

Nicotine is harmful to human health and is also a major cause of smoking addiction. When smoking, nicotine can be absorbed into arterial blood circulation rapidly, and enter into other tissues in vivo to affect human nervous system, cardiovascular system and respiratory system. Furthermore, metabolites of nicotine in the liver of humans, including cotinine, nornicotine, nicotinic acid, and the like, can significantly increase the risk of cancer in humans. Meanwhile, tobacco waste produced by the tobacco planting industry and the tobacco industry contains a large amount of nicotine, and 300 ten thousand tons of tobacco waste are produced every year according to incomplete statistics, wherein the nicotine content is about 3% -5%. The nicotine can be deposited to soil along with rainwater, and causes great harm to industry, planting industry and environment.

For the difficult-to-degrade nicotine substances generated in the tobacco industry, scientists begin to screen microorganisms, particularly bacteria, which degrade nicotine from the last fifty years, in an effort to provide microbial strain resources for improving tobacco product quality and reducing environmental hazards.

Disclosure of Invention

In order to solve the problems, the application aims to provide a degrading bacterium capable of efficiently degrading nicotine, which can grow by using nicotine as a carbon-nitrogen source, so as to be applied to degrading nicotine in tobacco products, tobacco industry waste and tobacco planting places, reduce harm of smoking to human health and reduce damage of tobacco industry and planting industry to environment.

In one aspect, the application provides a strain of pseudoArthrobacter chlorophenol, which is pseudoArthrobacter chlorophenolPseudarthrobacter chlorophenolicus) BJY-3, deposited in China general microbiological culture Collection center (CGMCC) No.25122, and date of deposit: 2022, 6, 20, deposit address: the institute of microorganisms of national academy of sciences of China, no. 1, no. 3, north Chen West Lu, the Korean region of Beijing.

Wherein, the Arthrobacter chlorophenol BJY-3 obtained by screening is gram-positive bacteria, and the bacterial colony on the LB solid medium is light yellow, round, smooth in surface and opaque.

In another aspect, the application also provides a bacterial preparation comprising the Arthrobacter chlorophenol BJY-3.

Optionally, the bacterial preparation can be a liquid bacterial preparation or a solid bacterial preparation prepared by utilizing the Arthrobacter chlorophenol BJY-3, and can also be a bacterial composition bacterial preparation compounded with other degradable nicotine or bacterial strains with other degradation functions.

In another aspect, the application also provides the use of the Arthrobacter chlorophenol BJY-3 strain, and/or the bacterial preparation in microbial degradation of nicotine.

In another aspect, the application also provides application of the Arthrobacter chlorophenol BJY-3 strain and/or the bacterial preparation in microbial degradation of tobacco products containing nicotine, tobacco industrial waste and/or tobacco planting places.

In another aspect, the application also provides a bacterial degradation product obtained by treating tobacco products and/or tobacco industry waste with the Arthrobacter chlorophenol BJY-3 strain and/or bacterial preparation.

In another aspect, the application also provides a method for degrading nicotine by microorganisms, which comprises the step of treating tobacco products, tobacco industry waste and/or soil of tobacco planting places by utilizing the Arthrobacter chlorophenol BJY-3 strain and/or the bacterial preparation.

In one embodiment, the manner of processing includes: preparing the strain and/or the microbial agent into suspension with an OD value of 2.0-3.0, spraying the tobacco product or soaking the tobacco product in the suspension for 5-60 seconds, and then taking out.

In one embodiment, the inoculation amount of the strain and/or the bacterial preparation is 0.01% -20%.

In one embodiment, the method further comprises the step of culturing for 1-30 days at the temperature of 28-32 ℃, the relative humidity of 20-50% and the oxygen content of 18-25% after treatment.

In one embodiment, the tobacco product comprises tobacco leaf, redried tobacco leaf, tobacco sheet, cut tobacco, cigarette, cigar; the tobacco industry waste comprises tobacco stems and tobacco dust.

It is understood that the term "cut tobacco" as used herein refers to cut, powder or granular goods made from tobacco leaves, redried tobacco leaves or tobacco sheets.

On the other hand, the application also provides application of the chlorophenol pseudoarthrobacterium strain BJY-3, the bacterial preparation, the bacterial degradation product and/or the method in preparing combustible cigarettes, heating non-combustible cigarettes and/or tobacco tar.

Alternatively, the BJY-3 strain and/or bacterial formulation and/or bacterial degradation product may be used in the preparation of combustible cigarettes and/or heated non-combustible cigarettes at cigarette holders including, but not limited to: cut tobacco of cigarettes, filter fiber tows, cigarette paper, joint glue and the like.

Optionally, the tobacco tar is that of an electronic cigarette and/or other non-combustion type cigarette product.

In one embodiment, the strain BJY-3 of the application is obtained by the following method: collecting tobacco planting soil, tobacco waste, tobacco leaves, roots, straws, buds and other biological materials, putting the biological materials into a screening culture medium for culture to obtain a bacteria-containing culture solution, coating the culture solution on a nicotine inorganic salt solid culture medium, selecting vigorous strain for separation culture, judging the strain species by using strain 16srDNA, judging whether the strain has nicotine degradation capability or not by using high performance liquid chromatography, and finally screening to obtain the strain with high nicotine degradation capability.

The application has at least the following beneficial effects.

1. The application screens and separates the high-efficiency degradation nicotine bacterial strain chlorophenol pseudoArthrobacter BJY-3, can enrich and degrade nicotine bacterial strain resources, screens and efficiently degrades nicotine related genes, provides new gene sources for transgenic tobacco and engineering bacterial strains, improves the quality of tobacco products, and can obviously reduce the harm of smoking to human bodies.

2. The Arthrobacter chlorophenol BJY-3 obtained by screening shows high nicotine degradation capability in a nicotine culture medium, tobacco leaves and cut tobaccos, can be used in the production of tobacco products, and has the effects of reducing the content of nicotine in the tobacco products and further reducing the harm of smoking to bodies; meanwhile, the strain can be used for degrading nicotine-containing waste generated in tobacco product production, realizing waste utilization, improving the utilization rate of tobacco, or treating nicotine in tobacco planting places, thereby improving the environment, avoiding the harm of nicotine to the environment and ecology, and having important economic, social and ecological benefits.

Drawings

The accompanying drawings, which are included to provide a further understanding of the application and are incorporated in and constitute a part of this specification, illustrate embodiments of the application and together with the description serve to explain the application and do not constitute a limitation on the application.

FIG. 1 is a colony shape of BJY-3 cells in LB medium.

FIG. 2 is a view of BJY-3 cells by gram stain under an electron microscope.

FIG. 3 is a graph of the blue degradation circle produced by BJY-3 cells in nicotine inorganic salt medium.

FIG. 4 shows the sequencing of BJY-3 cell 16srDNA and analysis of the detected genus.

FIG. 5 is a high performance liquid chromatogram of nicotine 0h in a BJY-3 cell degradation inorganic salt medium.

FIG. 6 is a high performance liquid chromatogram of nicotine 13h in a BJY-3 cell degradation inorganic salt medium.

FIG. 7 is a high performance liquid chromatogram of nicotine in tobacco prior to degradation of BJY-3 cells.

FIG. 8 is a high performance liquid chromatogram of nicotine content in tobacco leaves after 6 days of BJY-3 thallus treatment.

FIG. 9 is a bar graph showing the area change of the liquid chromatogram peak of the nicotine content in tobacco leaves before (FIG. 7) and after (FIG. 8) the treatment of the tobacco leaves with BJY-3 bacteria.

FIG. 10 is a plot of nicotine content change in tobacco after 21 days of tobacco shred treatment with BJY-3 bacteria.

Detailed Description

In order to more clearly illustrate the general concept of the present application, the following detailed description is given by way of example. In the following description, numerous specific details are set forth in order to provide a more thorough understanding of the present application. It will be apparent, however, to one skilled in the art that the application may be practiced without one or more of these details. In other instances, well-known features have not been described in detail in order to avoid obscuring the application.

In the following embodiments, unless specified otherwise, the reagents or apparatus used are conventional products available commercially without reference to the manufacturer. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer.

Example 1 screening and identification of strains.

And (I) collecting biological materials.

The soil of the tobacco planting field is separated from tobacco leaves, roots, straws, buds and tobacco waste to contain a large amount of nicotine, so that various nicotine-degrading bacteria can be presumably separated from the soil.

The collection location and material.

1. Soil, roots, straws and leaves in tobacco planting places are collected in Harbin, binxi county of Heilongjiang province.

2. Tobacco buds, roots, straws and leaves are collected in tobacco fields in Paeonia suffruticosa river city of Heilongjiang province.

3. Sichuan Shish 370225, tobacco leaves and tobacco waste.

Acquisition requirements.

1. Each sampling point is spaced about 20 meters apart.

2. Taking a soil sample of about one table tennis ball size at each point, taking soil with depths of 5cm, 10cm and 15cm, and collecting tobacco leaves, straws and the like in the soil, wherein the soil is required to be as complete as possible.

3. The tobacco waste collection method is the same as that of the soil of the tobacco planting field.

And secondly, separating to obtain the pseudomonas aeruginosa BJY-3 capable of efficiently degrading nicotine and chlorophenol.

Using an inorganic salt medium, nicotine is added as the sole carbon and nitrogen source, and a strain that can grow using nicotine is isolated. Finally, 9 different strains are obtained by separation, wherein BJY-3 has high nicotine degradation activity.

The specific method is as follows.

1) And (5) preparing and sterilizing a culture medium.

A. Configuration of LB Medium.

Tryptone 10 g, yeast extract 5 g, sodium chloride l 0g, distilled water to a constant volume of 1L, and natural pH. Autoclaving at 121℃for 20 min.

B. Preparing nicotine inorganic salt culture medium.

According to the data in Table 1, media were prepared, pure water was set to 1L, pH was natural, autoclaved at 121℃for 20min (nicotine was added through 0.22 μm filter), and solid media was added with 2.0% agar on the basis of liquid media.

TABLE 1 Nicotine mineral salts Medium formulation

Note that: agar was added to the solid medium.

2) And (5) enrichment culture.

A. The majority of the microorganisms in the experimental material (2 g each of the minced plant tissue and soil samples) was collected with LB medium for 3-4 days (30 ℃ C., 160 rpm).

B. Stress is carried out by using nicotine inorganic salt culture medium with high concentration of nicotine, the temperature is 30 ℃, the rpm is 160, the liquid is changed once every 7 days, and the process is repeated for 6 times.

3) And (5) coating and screening bacteria.

Coating the last culture solution on a nicotine solid culture medium, selecting a strain with vigorous growth vigor for independent culture, separating and purifying the strain, inoculating the strain into the nicotine culture medium, performing shaking culture at 30 ℃ and 160rpm for 24h, detecting the content of nicotine in the culture medium by a high performance liquid chromatograph (High Performance Liquid Chromatography, HPLC), and screening the strain with nicotine degradation capability.

The strain BJY-3 with outstanding nicotine degradation capability is finally obtained through screening and is derived from tobacco waste in the city of Sichuan Shish 370225.

4) Morphology observation and degradation circle observation of strain BJY-3.

As shown in FIG. 1, strain BJY-3 was pale yellow, round, smooth in surface and opaque in colony on LB solid medium.

As shown in FIG. 2, the gram-staining result of strain BJY-3 showed that it was blue after staining and was a gram-positive bacterium.

As shown in FIG. 3, a circular hole of about 1.1 cm a diameter was formed in the middle of the nicotine solid medium using oxford cup, and 150. Mu.L of bacteria solution was added to the hole, and a blue degradation ring was observed in the medium.

And thirdly, determining the genus of the fungus.

The 16SrDNA sequencing was used, and the relationship between the strain BJY-3 and the standard strain Arthrobacter chlorophenol Pseudarthrobacter chlorophenolicus A was analyzed and detected, and the specific phylogenetic tree is shown in FIG. 4.

The specific steps are as follows.

1. Extracting genome DNA of the strain.

2. The target gene amplification is carried out by using a 16SrDNA universal primer, wherein,

the forward primer is: 5'-TGGCGAACGGGTGAGTAATACAT-3';

the reverse primer is as follows: 5'-GCGGTTAGGCTAACTACTTCTGG-3'.

The PCR reaction system is shown in Table 2.

TABLE 2 PCR System of 16srDNA

Note that: the total volume was 50. Mu.L.

Example 2 Nicotine decomposing Activity of Arthrobacter chlorophenol BJY-3.

The chlorophenol pseudoArthrobacter BJY-3 is inoculated into the nicotine inorganic salt culture medium, tobacco leaves and tobacco shreds shown in table 1 respectively, and the nicotine content is detected by high performance liquid chromatography HPLC to confirm the activity of degrading the nicotine of BJY-3.

Wherein, the high performance liquid chromatography condition for detecting nicotine content is as follows: mobile phase: pure methanol: 0.1% aqueous diethylamine = 40:60. column temperature: 40 ℃. Sample injection amount: 20. Mu.L. Flow rate: 0.8 mL/min.

Degradation of nicotine by strain BJY-3.

Inoculating the strain BJY-3 into a nicotine inorganic salt culture medium with the concentration of 500mg/L at 5 mL at 30 ℃ and 160rpm, culturing, taking a proper amount of culture solution before adding the strain and after adding the strain every 1-2 hours, centrifuging the culture solution at 12000 rpm for 10 min, and loading the supernatant into a liquid phase sample bottle through an organic filter membrane of 0.22 nm for high performance liquid chromatography analysis. The results are shown in FIG. 5, FIG. 6 and Table 3.

FIGS. 5 and 6 are high performance liquid chromatograms of the strains BJY-3 after treating 500mg/L of nicotine inorganic salt medium for 0h and 13h, respectively, and it is apparent from the results of FIGS. 5, 6 and 3 that 500mg of nicotine is almost degraded after treating the nicotine inorganic salt medium for 13h in Table 1 with the strain BJY-3.

TABLE 3 degradation effect of strain BJY-3 on nicotine in inorganic salt medium

Note that: h: hours.

And (II) degrading tobacco leaves, cut tobaccos, tobacco stems and soil of tobacco planting places by the strain BJY-3.

1) And (5) sample pretreatment.

a. Respectively taking a proper amount of tobacco leaves (provided by an inner Mongolia cigarette factory), tobacco shreds (provided by the inner Mongolia cigarette factory), tobacco wastes (provided by the inner Mongolia cigarette factory, specifically waste tobacco powder and tobacco stems of picked tobacco leaves are mixed in a mass ratio of 1:1 and crushed to 100 meshes), and tobacco planting soil (provided by the inner Mongolia cigarette factory, specifically a soil sample collected from the tobacco planting area of the karst mountain in the red mountain area of the inner Mongolia, namely, the tobacco planting area of the karst mountain, sterilizing 6 h by using an ultraviolet lamp, and continuously turning over during the period to ensure a sterilizing effect.

b. After sterilization, the sample bags are placed in sterile sampling bags, a certain amount of sterile water is added, and the sample bags are left for 24 hours to balance the water (the step can be omitted).

2) Culturing and extracting the strain.

A. Strain BJY-3 was inoculated in 100 mL of LB medium at 30℃and 160rpm for 24h at an inoculum size of 2%.

B. Poured into 50 mL centrifuge tubes and centrifuged at 8000 rpm for 3 min to retain the pellet.

C. 10-20. 20 mL sterile water is added to the centrifuge tube and washed with shaking.

D. Centrifuging at 8000 rpm for 3 min, retaining the precipitate, and repeating the washing twice.

E. And adding a certain amount of sterile water to enable the OD value of the bacterial liquid to reach 2.0-3.0, and obtaining BJY-3 suspension bacterial liquid.

3) Bacterial strain for treating tobacco leaves, cut tobacco, tobacco waste and soil of tobacco planting land

A. Tobacco leaf treatment: soaking 30 g tobacco leaves in BJY-3 suspension of bacteria solution for 30 s, taking out, drying, placing in a 30 deg.C incubator, and culturing under conditions of relative humidity of 30% and oxygen content of 21%.

B. Treating tobacco shreds, tobacco wastes and tobacco planting soil: taking 10mL of BJY-3 suspension bacterial liquid, respectively weighing 250g of dried tobacco shred, tobacco waste and soil sample, spraying the bacterial liquid into the tobacco shred, the tobacco waste and the soil sample, and then placing the tobacco shred, the tobacco waste and the soil sample in a 30 ℃ incubator for culture under the conditions of 30% of relative humidity and 21% of oxygen content.

Degradation effect.

1. The strain BJY-3 has the effect of degrading nicotine in tobacco leaves.

The degradation effect of the strain BJY-3 on nicotine in tobacco leaves is shown in Table 4.

TABLE 4 degradation effect of the strain BJY-3 on nicotine in tobacco

Time	Nicotine content (mass ratio m/m)	Degradation rate
			0d	3.80%
5d	3.62%	4.74%
			10d	3.51%	7.63%
15d	3.32%	12.63%

Note that: d: and (3) days.

As can be seen from Table 4, the tobacco leaves had an initial nicotine content (mass ratio m/m) of about 3.80%, and after 15 days of treatment with strain BJY-3, the tobacco leaves had a nicotine content (mass ratio m/m) of about 3.32% and a degradation rate of 12.63%.

The content change condition of nicotine in tobacco leaves before and after the degradation of BJY-3 thalli is analyzed by high performance liquid chromatography, and the obtained results are shown in figures 7-9. Fig. 7 is a high performance liquid chromatogram of nicotine in tobacco leaves before degradation by using BJY-3 bacteria, fig. 8 is a high performance liquid chromatogram of nicotine content in tobacco leaves after 6 days of treatment by using BJY-3 bacteria, fig. 9 is a liquid chromatogram peak area change of nicotine content in tobacco leaves before and after treatment by using BJY-3 bacteria in fig. 7 and 8, and comparison of fig. 7, 8 and 9 shows that the nicotine content of tobacco leaves is obviously reduced before and after treatment by using BJY-3 bacteria, which indicates that the BJY-3 bacteria obtained by screening has degradation effect on nicotine in tobacco leaves.

2. The strain BJY-3 has the effect of degrading nicotine in tobacco shreds.

The effect of strain BJY-3 on the degradation of nicotine in tobacco is shown in Table 5.

TABLE 5 degradation effect of Strain BJY-3 on nicotine in tobacco

Time	Nicotine content (mass ratio m/m)	Degradation rate
			0d	1.94%
7d	1.82%	6.19%
			14d	1.69%	12.89%
21d	1.54%	20.62%

Note that: d: and (3) days.

As shown in Table 5, the tobacco shred has an initial nicotine content (mass ratio m/m) of about 1.94%, and after 21 days of treatment with strain BJY-3, the nicotine content (mass ratio m/m) is about 1.54%, and the degradation rate is 20.62%.

The degradation data of the nicotine in the cut tobacco is drawn into a line graph to analyze the change trend, and the obtained result is shown in fig. 10. As can be seen from FIG. 10, the nicotine content in the tobacco shreds is reduced in 21 days after the tobacco shreds are treated by BJY-3 thalli, which shows that BJY-3 thalli obtained by screening of the application has a degrading effect on the nicotine in tobacco leaves.

3. The strain BJY-3 has the effect of degrading nicotine in tobacco waste.

The effect of strain BJY-3 on the degradation of nicotine in tobacco waste is shown in Table 6.

TABLE 6 degradation effect of strain BJY-3 on nicotine in tobacco waste

Time	Nicotine content (mass ratio m/m)	Degradation rate
			0d	3.06%
7d	2.71%	11.44%
			14d	1.64%	46.41%
21d	1.27%	58.50%
			28d	1.15%	62.42%

Note that: d: and (3) days.

As is clear from Table 6, the tobacco waste had an initial nicotine content (mass ratio m/m) of about 3.06%, and after 28 days of treatment with strain BJY-3, the nicotine content (mass ratio m/m) was about 1.15%, and the degradation rate was 62.42%.

4. Strain BJY-3 has a degrading effect on nicotine in a soil sample.

The effect of strain BJY-3 on the degradation of nicotine in soil samples is shown in Table 7.

Table 7 degradation effect of Strain BJY-3 on Nicotiana tabacum planting soil

Time	Nicotine content (mass ratio m/m)	Degradation rate
			0d	1.69%
7d	1.45%	14.20%
			14d	1.28%	24.26%
21d	1.03%	39.05%
			28d	0.97%	42.60%

Note that: d: and (3) days.

As is clear from Table 7, the initial nicotine content (mass ratio m/m) of the soil sample was about 1.69%, and the nicotine content (mass ratio m/m) after 28 days of treatment with strain BJY-3 was about 0.97%, and the degradation rate was 42.60%.

From the above, the application provides the chlorophenol pseudoarthrobacterPseudarthrobacter chlorophenolicus) BJY-3 pair of nicotine culture medium, tobacco product, tobacco waste and tobacco plantingThe nicotine in the soil has good degradation capability, and has important economic, social and ecological benefits in tobacco planting and production industries.

The foregoing is merely exemplary of the present application and is not intended to limit the present application. Various modifications and variations of the present application will be apparent to those skilled in the art. Any modification, equivalent replacement, improvement, etc. which come within the spirit and principles of the application are to be included in the scope of the claims of the present application.

Claims

1. A pseudoArthrobacter chlorophenol strain is characterized in that the strain is pseudoArthrobacter chlorophenol strainPseudarthrobacter chlorophenolicus) BJY-3, and is preserved in China general microbiological culture Collection center (CGMCC) No.25122.

2. A bacterial formulation comprising the pseudoArthrobacter chlorophenol BJY-3 of claim 1.

3. Use of a pseudoArthrobacter chlorophenol strain of claim 1, and/or a bacterial formulation of claim 2 for microbial degradation of nicotine.

4. Use of a strain of pseudoArthrobacter chlorophenol according to claim 1, and/or of a bacterial preparation according to claim 2 for the microbial degradation of tobacco products containing nicotine, tobacco industry waste and/or soil of tobacco planting sites.

5. A method of microbial degradation of nicotine comprising the step of treating tobacco products, tobacco industry waste and/or tobacco planting soil with a chlorophenol pseudoarthrobacter strain of claim 1, and/or a bacterial formulation of claim 2.

6. The method of claim 5, wherein the processing comprises: preparing the strain and/or the microbial agent into suspension with an OD value of 2.0-3.0, spraying the tobacco product or soaking the tobacco product in the suspension for 5-60 seconds, and then taking out; and/or the inoculation amount of the strain and/or the bacterial preparation is 0.01% -20%.

7. The method according to claim 5, further comprising the step of culturing for 1 to 30 days at a temperature of 28 to 32 ℃ and a relative humidity of 20 to 50% and an oxygen content of 18 to 25% after the treatment.

8. The method of claim 5, wherein the tobacco product comprises a reconstituted tobacco, cut tobacco, cigarette, cigar; the tobacco industry waste comprises tobacco stems and tobacco dust.

9. Use of a strain of pseudoArthrobacter chlorophenol according to claim 1, a bacterial preparation according to claim 2 and/or a method according to any one of claims 5 to 8 for the preparation of a combustible cigarette, a heated non-combustible cigarette and/or a tobacco tar.