Application of Aspergillus terreus in reducing nicotine content

Abstract

This invention discloses Aspergillus terreus Aspergillus terreus Applications for reducing nicotine content. Specifically, these applications include Aspergillus terreus. Aspergillus terreus This invention relates to the method for reducing nicotine content in tobacco raw materials, tobacco waste, or tobacco processing byproducts. The method is characterized by high degradation efficiency, mild conditions, and environmental friendliness, providing a new and effective tool for biologically reducing nicotine content in tobacco leaves. It shows promising application prospects in improving tobacco quality and reducing harmful substances and tar content.

Description

Technical Field

[0001] This invention belongs to the field of microbial application and tobacco processing technology, and relates to Aspergillus terrestris. Aspergillus terreus Applications that reduce nicotine content. Background Technology

[0002] Tobacco is an important economic crop in my country. Nicotine, as its main alkaloid component, directly affects the sensory quality, usability, and safety of tobacco leaves. In recent years, due to cultivation practices (such as topping), the nicotine content in flue-cured tobacco, especially in the upper leaves, has generally been high, leading to a decline in the balance of chemical components and reduced usability. Meanwhile, from a harm reduction perspective, lowering the nicotine content in tobacco leaves has become an important research direction in the industry.

[0003] Existing methods for reducing nicotine content in tobacco leaves mainly include agricultural cultivation regulation, physicochemical treatment, and biodegradation. Among these, biodegradation has attracted attention due to its advantages such as mild conditions, good selectivity, and environmental friendliness. However, research and application of technologies utilizing tobacco endophytic fungi for efficient and specific nicotine degradation remain insufficient.

[0004] Most reported nicotine-degrading bacteria originate from soil or wastewater environments. Their adaptability, safety, and degradation efficiency in tobacco systems remain unclear. In particular, research and applications of efficient degradation using tobacco endophytic fungi as a natural symbiotic system are rarely reported.

[0005] Therefore, there is an urgent need to provide a new method for degrading nicotine using specific endophytic fungi. Summary of the Invention

[0006] To address the shortcomings of existing technologies and practical needs, this invention provides Aspergillus terreus. Aspergillus terreus In addition to applications for reducing nicotine content, this invention also provides a microbial fermentation preparation prepared by this method and its application in reducing nicotine content in tobacco raw materials and cigarette smoke, providing an efficient, repeatable, and highly specific nicotine biodegradation scheme.

[0007] To achieve this objective, the present invention adopts the following technical solution: In a first aspect, the present invention provides Aspergillus terreus Aspergillus terreus Applications in reducing the nicotine content in tobacco raw materials, tobacco waste, or tobacco processing byproducts.

[0008] Secondly, the present invention provides a method for degrading nicotine, the method comprising: inoculating tobacco endophytic fungi into a liquid culture medium containing nicotine for fermentation culture, and obtaining a culture with reduced nicotine content after the culture is completed; wherein the tobacco endophytic fungi are Aspergillus terreus. Aspergillus terreus .

[0009] The method of this invention has high degradation efficiency, mild conditions, and is environmentally friendly, providing a new and effective tool for reducing nicotine content in tobacco leaves through biological methods. It has good application prospects in improving tobacco quality and reducing harm and tar.

[0010] Preferably, the Aspergillus terrestris Aspergillus terreus The nucleic acid sequence of the ITS region includes the sequence shown in SEQ ID NO.1.

[0011] SEQ ID NO.1: .

[0012] Preferably, the Aspergillus terrestris Aspergillus terreus The source includes any one or a combination of at least two of the roots, stems or leaves of plants in the genus *Nicotiana*.

[0013] Preferably, the liquid culture medium comprises potato glucose broth medium.

[0014] Preferably, the step of inoculating tobacco endophytic fungi into a liquid culture medium containing nicotine for fermentation further includes adding exogenous nicotine to the liquid culture medium before inoculation.

[0015] Preferably, the exogenous nicotine is a nicotine standard or a tobacco extract.

[0016] Preferably, the fermentation culture temperature is 28-32℃ (e.g., 28℃, 30℃ or 32℃), the rotation speed is 200-240 rpm (e.g., 200 rpm, 220 rpm or 240 rpm), and the culture time is 48-72 h (e.g., 48 h, 60 h or 72 h).

[0017] Thirdly, the present invention provides a microbial fermentation preparation for degrading nicotine, said microbial fermentation preparation being prepared from a fermentation culture obtained by the method for degrading nicotine described in the first aspect.

[0018] Preferably, the dosage form of the microbial fermentation preparation includes any one of the following: liquid inoculant (the fermentation broth is directly filtered to remove bacteria and then packaged), solid inoculant (the fermentation broth is mixed with sterile peat carrier for adsorption and then dried), or freeze-dried powder (the bacterial cells are collected by centrifugation, mixed with a freeze-drying protectant, and then freeze-dried and ground into powder).

[0019] Fourthly, the present invention provides the application of the method for degrading nicotine as described in the second aspect or the microbial fermentation preparation for degrading nicotine as described in the third aspect in reducing the nicotine content in tobacco raw materials, tobacco waste or tobacco processing by-products.

[0020] Compared with the prior art, the present invention has the following beneficial effects: The method of this invention can efficiently and specifically degrade nicotine in a liquid fermentation system, showing great application potential. The method is mild and environmentally friendly, providing a new and operable biotechnological solution for targeted and controllable reduction of nicotine content in tobacco raw materials in tobacco agriculture and processing. It has direct significance for improving the coordination of chemical components in tobacco leaves and enhancing their quality. Attached Figure Description

[0021] Figure 1 Microscopic morphological images of endophytic fungi isolated from tobacco plants, 2 replicates; Figure 2 This is a diagram showing the fermentation culture status of tobacco endophytic fungi in liquid culture medium according to the present invention, with 2 parallel replicates; Figure 3 This is a chromatogram of the high performance liquid chromatography (HPLC) results for nicotine standards. Figure 4This is a high-performance liquid chromatography (HPLC) result of the extract of tobacco endophytic fungi fermentation broth according to the present invention. Detailed Implementation

[0022] To further illustrate the technical means and effects of this invention, the following description, in conjunction with embodiments and accompanying drawings, provides a further explanation of the invention. It is understood that the specific embodiments described herein are merely illustrative of the invention and not intended to limit it.

[0023] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0024] Example 1 This embodiment uses commercially available Aspergillus terrestris (… Aspergillus terreus ATCC 10020 reduces nicotine content.

[0025] Aspergillus terrestris was picked from fresh potato dextrose agar (containing streptomycin at a concentration of 10 mg / L). Aspergillus terreus The mycelium of strain ATCC 10020 was added to 100 mL of liquid culture medium, placed into a 250 mL Erlenmeyer flask, sealed with a film, and cultured on a shaker at 30℃ and 220 rpm for 72 h.

[0026] The liquid culture medium formula is: potato glucose broth (25 g / L), autoclaved at 121°C for 20 min, with streptomycin added at a concentration of 10 mg / L.

[0027] The liquid culture medium was treated with nicotine by adding 1 mg of nicotine standard to the liquid culture medium and then fermenting it with endophytic fungi.

[0028] After fermentation, the culture medium was filtered, freeze-dried, reconstituted with methanol, and filtered through a membrane. Nicotine residues were then detected by HPLC.

[0029] Preparation of standard solution: Weigh 1 mg of nicotine standard accurately, add 1 mL of methanol to dissolve, and the solution is ready.

[0030] Preparation of sample solution: Aspergillus terrestris ( Aspergillus terreus Fermentation broth of strain ATCC 10020 was used to remove mycelium. 10 mL of the bacterial solution was freeze-dried. The dried product was dissolved in 1.5 mL of methanol and filtered through a 0.22 μm filter membrane to obtain the final product.

[0031] Nicotine standard and Aspergillus terrestris ( Aspergillus terreusChromatographic detection conditions for the fermentation broth extract of strain ATCC 10020: The instrument used was an Agilent HPLC-1260 Infinity high-performance liquid chromatograph; the column was C18 (4.6 mm × 250 mm, 5 μm); the mobile phase consisted of acetonitrile / ammonium formate solution (10% acetonitrile, solvent was 0.02 mol / L ammonium formate solution) as A and acetonitrile as B, with gradient elution as specified in Table 1; the flow rate was 1 mL / min; the column temperature was 35℃; and the detection wavelength was 260 nm. The gradient elution table is shown in Table 1.

[0032] Table 1 Determination: HPLC was used to analyze Aspergillus terreus (… Aspergillus terreus The components of the fermentation broth extract of strain ATCC 10020 were detected. The results showed that the peak time of the blank control (nicotine standard) was 15.539 min, while the fermentation broth of tobacco endophytic fungi did not detect nicotine at the corresponding retention time within the allowable error time range, indicating that nicotine had been effectively degraded.

[0033] Example 2 Isolation and purification of endophytic fungi from Yunyan 87.

[0034] Material processing: Tobacco plant samples harvested from the field were divided into six parts: upper leaves, middle leaves, lower leaves, tobacco stems, coarse roots, and fibrous roots. The tobacco plant samples were healthy Yunyan 87 tobacco plants from Xundian, Yunnan Province, and the plants were in the field mature stage (two weeks after topping).

[0035] Material disinfection: The upper leaves, middle leaves, and lower leaves were soaked in 75% alcohol for 30 seconds, then in 0.1% mercuric chloride for 15 seconds, and rinsed three times with sterile water. The tobacco stems were wiped with 75% alcohol to remove the epidermis, leaving the cortex and stele intact. The coarse roots and fibrous roots were soaked in 75% alcohol for 60 seconds, then in 0.1% mercuric chloride for 20 seconds, and rinsed three times with sterile water. The sterile water from the last rinse was streaked on a plate as a control to check whether the sample disinfection was thorough.

[0036] Inoculation, isolation, and purification: Tobacco leaves were ground with sterile water. The grinding liquid and residue were evenly spread on the surface of potato dextrose agar medium (containing streptomycin at a concentration of 10 mg / L). Tobacco stems were cut into 5-8 mm pieces with an inoculation knife and spread on the above medium surface. Coarse roots were cut into 3-5 mm segments with the cut side down and spread on the above medium surface. Fibrous roots were ground with sterile water. The grinding liquid and residue were evenly spread on the above medium surface. All materials were incubated at 28°C for 7 days, and the colony growth was observed. Due to the different reproduction rates of different fungi, faster-growing strains were prioritized for isolation and purification. Slower-growing strains were isolated and purified after stabilization. Three inoculation purifications were performed until a single colony was obtained. After screening, one tobacco endophytic fungus, 001f-17, was obtained.

[0037] Morphological identification: Microscopic observation showed that the surface of the tobacco endophytic fungus 001f-17 colonies was flat or had shallow radial wrinkles, velvety or occasionally flocculent, cinnamon-colored to sandy brown, and the reverse side of the colonies and the matrix were dull yellow to brown. Figure 1 ).

[0038] Molecular identification: Genomic DNA of the isolated strain was amplified by PCR and sequenced using universal primers ITS1 / ITS4. The obtained sequences were compared with public databases, and ITS-specific sequences of one functional strain were obtained through sequence analysis. The obtained sequence (SEQ ID NO.1) was BLAST-aligned with Aspergillus terreus in the NCBI database. Aspergillus terreus The sequence similarity of multiple model strains of this strain was greater than 99%. Simultaneously, this strain exhibited typical Aspergillus terreus colony morphology on culture medium (colon surface flat or with shallow radial wrinkles, velvety or occasionally flocculent, cinnamon to sandy brown, colony reverse side and matrix dull yellow to brown). Based on these molecular biological and morphological characteristics, strain 001f-17 was identified as Aspergillus terreus. Aspergillus terreus ).

[0039] ITS1 (SEQ ID NO. 2):TCCGTAGGTGAACCTGCGG.

[0040] ITS4 (SEQ ID NO. 3):TCCTCCGCTTATTGATATGC.

[0041] Example 3 Nicotine degradation fermentation culture and degradation effect detection (HPLC analysis).

[0042] Aspergillus terrestris was picked from fresh potato dextrose agar (containing streptomycin at a concentration of 10 mg / L). Aspergillus terreusThe mycelia of strain 001f-17 were added to 100 mL of liquid culture medium, transferred to a 250 mL Erlenmeyer flask, sealed with a film, and cultured on a shaker at 30℃ and 220 rpm for 72 h. Figure 2 ).

[0043] The liquid culture medium formula is: potato glucose broth (25 g / L), autoclaved at 121°C for 20 min, with streptomycin added at a concentration of 10 mg / L.

[0044] The liquid culture medium was treated with nicotine by adding 1 mg of nicotine standard to the liquid culture medium and then fermenting it with endophytic fungi.

[0045] After fermentation, the culture medium was filtered, freeze-dried, reconstituted with methanol, and filtered through a membrane. Nicotine residues were then detected by HPLC.

[0046] Preparation of standard solution: Weigh 1 mg of nicotine standard accurately, add 1 mL of methanol to dissolve, and the solution is ready.

[0047] Preparation of sample solution: The mycelium of the endophytic fungal fermentation broth was filtered off, 10 mL of the bacterial broth was taken and freeze-dried, the dried product was dissolved in 1.5 mL of methanol and filtered through a 0.22 μm filter membrane to obtain the sample solution.

[0048] Chromatographic detection conditions for nicotine standards and endophytic fungal fermentation broth extracts: The instrument used was an Agilent HPLC-1260 Infinity high-performance liquid chromatograph; the column was C18 (4.6 mm × 250 mm, 5 μm); the mobile phase consisted of acetonitrile / ammonium formate solution (10% acetonitrile, solvent was 0.02 mol / L ammonium formate solution) as A and acetonitrile as B, with gradient elution as specified in Table 1; the flow rate was 1 mL / min; the column temperature was 35℃; and the detection wavelength was 260 nm. The gradient elution table is shown in Table 1.

[0049] Table 1 Determination: The components of the tobacco endophytic fungal fermentation broth extract were determined by HPLC, and the results are as follows. Figure 3 and Figure 4 As shown, the blank control (nicotine standard) had a peak elution time of 15.539 min, while the fermentation broth of tobacco endophytic fungi did not detect nicotine at the corresponding retention time within the allowable error time range, indicating that nicotine had been effectively degraded.

[0050] In summary, the method of this invention has high degradation efficiency, mild conditions, and is environmentally friendly, providing a new and effective tool for reducing nicotine content in tobacco leaves through biological methods. It has good application prospects in improving tobacco quality and reducing harm and tar content.

[0051] The applicant declares that the detailed method of the present invention is illustrated by the above embodiments, but the present invention is not limited to the above detailed method, that is, it does not mean that the present invention must rely on the above detailed method to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. Aspergillus terreus Aspergillus terreus Applications in reducing the nicotine content in tobacco raw materials, tobacco waste, or tobacco processing byproducts.

2. A method for degrading nicotine, characterized in that, The method for degrading nicotine includes: inoculating tobacco endophytic fungi into a liquid culture medium containing nicotine for fermentation culture, and obtaining a culture with reduced nicotine content after the culture is completed; the tobacco endophytic fungi are Aspergillus terreus. Aspergillus terreus.

3. The method for degrading nicotine according to claim 2, characterized in that, Aspergillus terreus Aspergillus terreus The nucleic acid sequence of the ITS region includes the sequence shown in SEQ ID NO.

1.

4. The method for degrading nicotine according to claim 2 or 3, characterized in that, Aspergillus terreus Aspergillus terreus The source includes any one or a combination of at least two of the roots, stems or leaves of plants in the genus *Nicotiana*.

5. The method for degrading nicotine according to any one of claims 2-4, characterized in that, The liquid culture medium includes potato glucose broth medium.

6. The method for degrading nicotine according to any one of claims 2-5, characterized in that, The method of inoculating tobacco endophytic fungi into a liquid culture medium containing nicotine for fermentation further includes adding exogenous nicotine to the liquid culture medium before inoculation.

7. The method for degrading nicotine according to claims 2-6, characterized in that, The exogenous nicotine is a nicotine standard or a tobacco extract.

8. The method for degrading nicotine according to any one of claims 2-7, characterized in that, The fermentation culture was carried out at a temperature of 28-32℃, a rotation speed of 200-240 rpm, and a culture time of 48-72 h.

9. A microbial fermentation preparation for degrading nicotine, characterized in that, The microbial fermentation preparation is prepared from a fermentation culture obtained by the method for degrading nicotine according to any one of claims 2-8; Preferably, the dosage form of the microbial fermentation preparation includes any one of liquid inoculum, solid inoculum, or lyophilized powder.

10. The method for degrading nicotine according to any one of claims 2-8 or the microbial fermentation preparation for degrading nicotine according to claim 9, used to reduce the nicotine content in tobacco raw materials, tobacco waste or tobacco processing by-products.

Images