Lignin-degrading bacterium and application thereof

Abstract

The application discloses a lignin-degrading bacterium and application thereof, and belongs to the technical field of microorganism breeding.The lignin-degrading bacterium is Pseudomonas parafulva ZY-03, which is preserved in the General Microbiological Culture Collection Center of China, and the preservation number is CGMCC No.24422.The Pseudomonas parafulva ZY-03 is used to prepare a bacterium agent and bacterium powder, and the bacterium agent and bacterium powder are used in tobacco raw materials such as tobacco stems, reconstituted tobacco leaves, reconstituted tobacco leaf coating liquid and low-grade tobacco leaves, so that lignin is degraded, the effect of flavoring is achieved, and the product quality is improved.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a lignin-degrading bacterium and its applications. Background Technology

[0002] Cellulose, hemicellulose, lignin, and pectin are components of tobacco cell walls. These substances, especially lignin, have varying degrees of influence on the smoking quality and safety of cigarette products. Lignin is rich in benzene rings, a major source of harmful substances such as polycyclic aromatic hydrocarbons and aromatic amines in tobacco tar. Furthermore, the content of lignin remains relatively stable during tobacco processing, making it a relatively stable compound in tobacco. The high lignin content in tobacco stems leads to insufficient aroma and a strong off-flavor when burned, causing a pungent and irritating cough, which significantly affects the smoking quality of cigarettes. Studies have found that lignin also has a significant impact on phenols in reconstituted tobacco smoke. The content of phenols and other phenolic substances produced by the pyrolysis of reconstituted tobacco increases with increasing lignin content. Catechols and alkylcatechols contained in the pyrolysis products can cause astringency and have carcinogenic activity.

[0003] Lignin is mainly composed of three different hydroxycinnamic acid alcohols, which polymerize into three monomers: p-hydroxyphenyl (H) lignin, guaiacol (G) lignin, and syringyl (S) lignin. Traditional physical and chemical methods have low lignin removal rates from biomass, and the resulting hydrolysis products significantly inhibit subsequent bioconversion and easily pollute the environment. Biological degradation of lignin offers advantages such as mild conditions, environmental friendliness, and the absence of subsequent fermentation inhibitors. However, lignin's complex structure and high molecular weight make it a natural barrier against degradation in plants. This allows it to resist hydrolysis by many common microbial enzymes, enabling it to be degraded only by specific fungi or bacteria. Furthermore, tobacco contains nicotine and other substances that easily inactivate enzymes, further complicating the biodegradation of lignin in tobacco raw materials.

[0004] Microbial degradation of lignocellulose has received relatively little attention in the tobacco industry. Fungi are generally recognized as microorganisms with a higher capacity for lignin degradation than other fungal species. Among fungi, white-rot fungi, brown-rot fungi, and soft-rot fungi have been extensively studied. Generally, molds are more capable than bacteria in degrading lignocellulose. However, due to their slow growth, poor thermal stability of related enzymes, and complex structures of their lignocellulose-degrading proteases, research on lignocellulose biodegradation using molds as the primary model strain has long failed to develop a feasible industrial degradation pathway. Bacteria are characterized by rapid growth, simple structure, and the production of mostly extracellular enzymes. However, existing bacterial methods for lignin degradation are time-consuming and inefficient. Therefore, how to safely and effectively reduce lignin in tobacco raw materials has become an important problem that needs to be solved. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a lignin-degrading bacterium and its application. This strain is isolated from the surface of tobacco leaf fragments, can tolerate the complex environment of tobacco itself, and efficiently degrades lignin. The abundant enzymes produced by the strain have an aroma-enhancing effect.

[0006] The technical problem to be solved by the present invention is achieved through the following technical solution:

[0007] A lignin-degrading bacterium, namely *Pseudomonas parafulva* ZY-03, is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 24422 and deposit date of February 23, 2022.

[0008] A method for isolating and culturing lignin-degrading bacteria, comprising the following steps:

[0009] S1 Sampling and Enrichment Culture: Take tobacco leaf fragments and place them in an Erlenmeyer flask. Add sterile physiological saline and culture with shaking at room temperature. Then transfer to YPD solid medium for further enrichment culture.

[0010] S2 seed culture: Pick a single colony and inoculate it into YPD liquid medium. Incubate at 22-37℃ and shaker speed of 100-250r / min for 12-72h to obtain seed culture;

[0011] Preparation of S3 inoculum or inoculum powder: Centrifuge the seed culture at 8000-12000 r / min at 4℃ for 10-20 min, remove the supernatant, and reconstitute with sterile water to an OD value of 1.8-2.1 to obtain *Pseudomonas paraxanthii* inoculum; or reconstitute with sterile water to prepare a strain concentration of (1-2)×10⁻⁶. 9 CFU / mL bacterial suspension was pre-frozen and vacuum dried to prepare Pseudomonas paraxanthii bacterial powder.

[0012] Preferably, in the above technical solution, the YPD liquid culture medium is composed of 10 g / L yeast powder, 20 g / L peptone and 20 g / L glucose, and is prepared by autoclaving at 121°C for 15 min.

[0013] Preferably, in the above technical solution, the seed culture conditions are 30℃, shaking speed 120r / min, and culture for 48h.

[0014] Preferably, the pre-freezing conditions for the bacterial powder are: -80℃ for 8-12 hours; vacuum drying conditions are -50℃ and 0.4-2 Pa; the concentration of the bacterial strain in the powder is determined by the dilution plating method, and the concentration should be within (2-8)×10⁻⁶. 8 CFU / g.

[0015] Preferably, in the above technical solution, the protective agent contains 1-3% glycerol, 1-5% sorbitol, 1-3% sodium ascorbate and 2%-5% glucose.

[0016] The application of a lignin-degrading bacterium or a bacterial agent or powder prepared by the above-mentioned isolation and culture method in enhancing the aroma of tobacco.

[0017] Preferably, in the above technical solution, the lignin-degrading bacteria ZY-03 can degrade lignin and produce enzymes with aroma-enhancing functions.

[0018] Preferably, in the above technical solution, depending on the state of the tobacco raw material, the tobacco raw material is fermented using a lignin-degrading bacteria agent or a bacterial powder. This involves applying 5%-15% by weight of the agent or 2-10% by weight of the bacterial powder to the tobacco raw material, fermenting at 30-40°C for 8-96 hours.

[0019] Preferably, in the above technical solution, the tobacco raw material is one or more of tobacco stems, reconstituted tobacco leaves, reconstituted tobacco leaf coating liquid, low-grade tobacco leaves, and shredded tobacco.

[0020] The above-described technical solution of the present invention has the following beneficial effects:

[0021] This application utilizes the *Pseudomonas paraxanthum* ZY-03 to prepare inoculants and powders for use in tobacco raw materials such as tobacco stems, reconstituted tobacco leaves, reconstituted tobacco leaf coating liquid, and low-grade tobacco leaves, in order to achieve the effects of degrading lignin and enhancing aroma, while improving product quality. Attached Figure Description

[0022] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments of the invention and, together with their description, serve to explain the principles of the invention.

[0023] Figure 1 Image showing the growth of Pseudomonas paraxanthizobium ZY-03 in a lignin-degrading enzyme containing aniline blue for 3 days.

[0024] Figure 2 Figure 1 shows the growth of Pseudomonas paraxanthizobium ZY-03 after 7 days of qualitative culture with lignin-degrading enzymes containing aniline blue. Detailed Implementation

[0025] Various exemplary embodiments of the present invention will now be described in detail. It should be noted that, unless otherwise specifically stated, the relative arrangement, numerical expressions, and values ​​of the components and steps set forth in these embodiments do not limit the scope of the invention.

[0026] The tobacco materials used in this application were obtained as follows: tobacco leaves and other tobacco raw materials were obtained from a cigarette factory under Henan China Tobacco Industry Co., Ltd., and provided by Henan China Tobacco Co., Ltd. Lignin testing was performed using industry standard YC / T347-2010, "Determination of Neutral Detergent Fibers, Acid Detergent Fibers and Acid-Treated Lignin in Tobacco and Tobacco Products - Detergent Method".

[0027] This application provides a lignin-degrading bacterium, Pseudomonas parafulva ZY-03, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 24422 and deposit date of February 23, 2022.

[0028] This strain, isolated from the surface of tobacco leaf fragments, is tolerant of the complex environment of tobacco and efficiently degrades lignin. The abundant enzymes produced by the strain have an aroma-enhancing effect. This *Pseudomonas paraxanthum* ZY-03 can be used to prepare inoculants and powders, which can then be applied to tobacco raw materials such as tobacco stems, reconstituted tobacco leaves, reconstituted tobacco coating solutions, and low-grade tobacco leaves to achieve lignin degradation and aroma enhancement.

[0029] The specific applications of this *Pseudomonas paraxanthizobium* ZY-03 are as follows:

[0030] (1) Seed culture:

[0031] A single colony of *Pseudomonas paraxanthum* ZY-03 with preservation number CGMCC NO.24422 was inoculated into YPD liquid medium and cultured at 22-37℃ and a shaking speed of 100-250 r / min for 12-72 h. Preferably, the culture conditions were: 30℃, shaking speed of 120 r / min, and culture for 48 h.

[0032] YPD liquid culture medium consists of 10 g / L yeast extract, 20 g / L peptone, and 20 g / L glucose, and is autoclaved at 121°C for 15 min.

[0033] (2) Preparation of inoculum or inoculum powder:

[0034] Centrifuge the seed culture at 8000-12000 r / min at 4℃ for 10-20 min, remove the supernatant, add sterile water to reconstitute, and make the OD value 1.8-2.1 to obtain the Pseudomonas paraxanthii inoculum.

[0035] Alternatively, the strain can be prepared by reconstitution with sterile water to a concentration of 1-2 × 10⁻⁶. 9A bacterial suspension with CFU / mL was pre-frozen at -80°C for 8-12 hours and then vacuum-dried at -50°C at 0.4-2 Pa to obtain bacterial powder. The concentration of the bacterial strain in the powder was determined by the dilution-coating plate method, and the concentration should be 2-8 × 10⁻⁶. 8 CFU / g.

[0036] (3) Fermentation of tobacco raw materials:

[0037] Depending on the state of the tobacco raw materials, apply 5%-15% by weight of microbial agent or 2-10% by weight of microbial powder to tobacco stems, reconstituted tobacco leaves, reconstituted tobacco leaf coating liquid, low-grade tobacco leaves, shredded tobacco, etc., and ferment at 30-40℃ for 8-96 hours.

[0038] Example 1

[0039] Enrichment culture of Pseudomonas paraxanthizobium ZY-03:

[0040] Take 10g of tobacco leaf fragments and place them in a 250mL Erlenmeyer flask. Add 100mL of sterile physiological saline to the Erlenmeyer flask and shake at 25℃ and 100r / min for 4h. Take 1mL of the liquid and inoculate it into an Erlenmeyer flask containing 50mL of YPD culture medium. Incubate at 30℃ and 150r / min for 48h.

[0041] Example 2

[0042] Microbial isolation:

[0043] The bacterial culture enriched in Example 1 was serially diluted with sterile physiological saline (dilution range: 10). -1 -10 -7 The diluted bacterial suspension was spread on YPD solid medium plates and incubated in a 30°C incubator for 24-48 hours. The differences in color and morphology of single colonies were observed. Single colonies were picked and isolated and purified on YPD solid medium plates.

[0044] Example 3

[0045] Screening of functional bacteria and characterization of lignin-degrading enzymes:

[0046] Single colonies were inoculated onto lignin inorganic salt agar plates containing 3 g / L sodium lignin sulfonate for screening using the streak plating method. The plates were incubated at 30°C for 24-48 h. The selected target strains were then spotted onto lignin inorganic salt agar plates containing aniline blue and RB brilliant blue, respectively. These plates were incubated at 30°C for 7 days, and the clear zones of the colonies were observed. Results are as follows: Figure 1 and Figure 2 As shown.

[0047] Application Example 1

[0048] (1) Preparation of YPD culture medium:

[0049] Yeast powder 10g / L, peptone 20g / L, glucose 20g / L, autoclaved at 121℃ for 15min.

[0050] (2) Seed culture:

[0051] Single colonies of Pseudomonas paraxanthizobium ZY-03 were picked and inoculated into YPD liquid medium and cultured at 30°C and 120 r / min for 24 h.

[0052] (3) Tobacco leaf processing:

[0053] Rehydrate and shred according to standard procedures.

[0054] (4) Preparation of microbial agents:

[0055] The seed culture prepared in (2) was centrifuged at 10000 r / min and 4℃ for 10 min, the supernatant was removed, and sterile water was added to reconstitute it so that the OD value was 1.9, thus obtaining the Pseudomonas paraxanthii inoculum.

[0056] (5) Application:

[0057] Based on the mass percentage, 8% of the microbial agent was applied to the tobacco shreds, and the shreds were spread out and fermented for 72 hours at 37°C and 70% humidity.

[0058] Results analysis: The lignin content of tobacco shreds before and after fermentation was determined.

[0059] like Figure 1 and Figure 2 As shown, after culturing on lignin inorganic salt agar plates containing aniline blue and RB brilliant blue for 3 days, the aniline blue plate showed a clear transparent zone, which almost completely faded after 7 days of culture, while the RB brilliant blue plate showed no transparent zone. This indicates that the strain degrades lignin by producing lignin peroxidase Lip and manganese peroxidase MnP, and may not produce laccase.

[0060] The lignin content of tobacco shreds before and after fermentation in Application Example 1 was measured (average value of three replicates). The results are shown in Table 1. Compared with the lignin content before and after fermentation, the lignin content after fermentation decreased by 21.25%, and the lignin degradation effect was obvious.

[0061] Table 1. Lignin content (unit: %)

[0062]

[0063] Fermented and unfermented tobacco were rolled into cigarettes separately and then smoked for evaluation. The results showed that the fermented tobacco had less off-flavors, less irritation, more aroma, and a smoother smoke.

[0064] Although the present invention has been disclosed above with reference to embodiments, it is not intended to limit the present invention. Any person skilled in the art can make various different choices and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention is defined by the claims and their equivalents.

Claims

1. A lignin-degrading bacterium, characterized in that, The lignin-degrading bacterium is Pseudomonas parafulva ZY-03, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 24422. The lignin-degrading bacterium ZY-03 can degrade lignin in tobacco raw materials and produce enzymes with aroma-enhancing functions.

2. The application of a lignin-degrading bacterium or a bacterial agent or powder containing said lignin-degrading bacterium in tobacco flavoring, characterized in that, The lignin-degrading bacterium is Pseudomonas parafulva ZY-03, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 24422. The lignin-degrading bacterium ZY-03 can degrade lignin in tobacco raw materials and produce enzymes with aroma-enhancing functions.

3. The application according to claim 2, characterized in that, Depending on the state of the tobacco raw materials, apply 5%-15% by weight of microbial agent or 2-10% by weight of microbial powder to the tobacco raw materials and ferment at 30-40℃ for 8-96 hours.

4. The application according to claim 3, characterized in that, The tobacco raw materials are one or more of the following: tobacco stems, reconstituted tobacco leaves, reconstituted tobacco leaf coating liquid, low-grade tobacco leaves, and shredded tobacco.

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