Aroma-producing bacterium ZL-5 for converting CO2 in microbial electro-synthesis system and application of aroma-producing bacterium ZL-5

Abstract

The invention belongs to the technical field of microbial engineering, and particularly relates to an aroma-producing bacterium ZL-5 for converting CO2 in a microbial electrosynthesis system and application of the aroma-producing bacterium ZL-5, the aroma-producing bacterium ZL-5 is classified and named as Paenibacillus pastensis, and the preservation number is CCTCC NO: M 20251367; after the strain ZL-5 is fermented by a microbial electrosynthesis system, the aroma substances of the obtained aroma-producing microbial agent are obviously increased through GC-MS detection, and the aroma-producing microbial agent can be used for perfuming and increasing the aroma of cigarettes in cigarette production and has good application value in cigarette production; in the aspect of smelling fragrance, the richness of the cigarette fragrance is obviously increased, smoking results show that the irritation of the cigarette is reduced, the fragrance is increased, and the overall quality is obviously improved; according to the method provided by the invention, aroma substances are generated while environmental pollution caused by CO2 emission is reduced, and the method has relatively good application value in the aspects of improving the cigarette quality and protecting the environment.

Description

Technical Field

[0001] This invention belongs to the field of microbial engineering technology, and specifically relates to an aroma-producing fungus that converts CO2 in a microbial electrosynthesis system. Background Technology

[0002] Microbial fermentation technology, as an important direction for the development of green and natural fragrances, has advantages such as natural and environmentally friendly biosynthetic pathways, flexible regulation of microbial metabolism, natural aroma of products, and the ability to effectively compensate for the sensory defects of artificial fragrances.

[0003] During tobacco processing and storage, the natural aroma components in tobacco leaves are easily affected by factors such as temperature, humidity, and oxidation, gradually volatilizing or degrading, resulting in significant loss of aroma substances. Low-quality tobacco leaves, in particular, have a lower content of aroma substances to begin with, and the heat loss during processing often leads to sensory defects such as a monotonous aroma and insufficient aroma intensity. Furthermore, high-end cigarette products have extremely high requirements for richness and harmony of aroma, which cannot be met solely by the aroma substances in the tobacco leaves themselves. Therefore, how to effectively compensate for the loss of aroma in tobacco leaves and reconstruct a harmonious aroma system has become a pressing technical challenge for the tobacco industry.

[0004] Microbial fermentation technology can improve the sensory defects of low-quality tobacco leaves through aroma compensation and enhance the style characteristics of high-end cigarettes through aroma blending. Therefore, screening for highly efficient aroma-producing microorganisms has become a research hotspot in the field of tobacco biotechnology. Patent 201510139997.3 discloses an aroma-producing bacterium, GXY35, which can ferment a culture medium containing tobacco dust components and produce p-vinylguaiacol as an aroma substance, which can be used to enhance tobacco aroma, refine and smooth the smoke, and improve the taste of cigarettes.

[0005] With the increasingly severe global climate change problem, the reduction and resource utilization of carbon dioxide (CO2) emissions have become key technological requirements for achieving carbon neutrality. Microbial electrosynthesis systems (MES), as an emerging bioelectrochemical technology, utilize autotrophic microorganisms capable of fixing CO2 as catalysts to convert CO2 into biofuels and chemicals through the Wood-Ljungdahl metabolic pathway, achieving simultaneous CO2 conversion and energy recovery. Depending on the inoculated microorganisms, bioelectrosynthesis systems can be divided into pure strain and mixed microbial bioelectrosynthesis systems. Mixed microbial bioelectrosynthesis is more economical to operate, but has lower product selectivity and is more difficult to separate and purify. In recent years, studies have shown that some microorganisms can efficiently reduce CO2 to simple organic compounds such as acetic acid and ethanol using electrical energy, but reports on the electrosynthesis of aroma substances are scarce. Therefore, the use of pure strains in microbial electrosynthesis systems to convert CO2 into aroma is of great significance for promoting CO2 resource utilization and improving the quality of cigarette products. Summary of the Invention

[0006] To address the aforementioned problems, this invention proposes a fragrance-producing fungus ZL-5 that converts CO2 in a microbial electrosynthesis system and its applications.

[0007] The technical solution of this invention is implemented as follows:

[0008] On the one hand, this application protects a fragrance-producing fungus ZL-5 that converts CO2 in a microbial electrosynthesis system. Its taxonomic name is Paenibacillus pasadenensis, and it was deposited on June 12, 2025, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO:M 20251367.

[0009] Secondly, this invention applies to protect an aroma-producing fungus agent comprising the aforementioned aroma-producing fungus ZL-5.

[0010] Thirdly, this invention applies for protection of the preparation method of the above-mentioned aroma-producing fungal agent, which involves centrifuging the seed liquid of the above-mentioned aroma-producing fungus ZL-5, discarding the supernatant to obtain the bacterial cells, inoculating the bacterial cells into a microbial electrosynthesis system containing a CO2 fermentation medium, and the fermentation broth obtained after the system is running is the aroma-producing fungal agent.

[0011] Specifically, the seed culture of the aroma-producing Bacillus pasadenae ZL-5 (250 mL) was centrifuged to obtain bacterial cells, which were then inoculated into the cathode chamber of an electrosynthesis system containing CO2 fermentation medium. The system was operated under the following conditions: an applied voltage of 3.0 V, a fermentation temperature of 37 °C, and a pH of 7.0. The reaction device was run continuously for one week, and the aroma-producing agent was collected from the sampling port using a syringe.

[0012] Preferably, the amount of seed liquid used is 250-500 mL; the operating voltage is 3.0-3.5 V.

[0013] The above-mentioned method for preparing the seed liquid involves inoculating aroma-producing fungus ZL-5 into an enrichment medium, and then culturing it under shaking conditions at 37°C, 160 r / min, and pH 7.0 until the logarithmic phase, after which fermentation is stopped, thus obtaining the seed liquid of aroma-producing fungus ZL-5.

[0014] The enrichment medium was prepared by dissolving 20 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl and 2 g / L agar in deionized water, adjusting the pH to 7.0, and then sterilizing at 121°C for 20 min.

[0015] Preferably, the CO2 fermentation medium contains 0.5-1 g / L L-cysteine, 0.5-1 g / L KH2PO4, and 1-1.5 g / L NH4Cl.

[0016] Preferably, the operating temperature is 35-37℃, the pH is 7.0-7.5, and the operating time is 5-7 days.

[0017] Fourthly, this invention applies to protect the application of the above-mentioned aroma-producing fungus ZL-5 or the above-mentioned aroma-producing fungus agent in the conversion of CO2 to enhance the aroma of cigarettes.

[0018] Fifthly, this invention applies for protection of a method for enhancing the aroma of tobacco leaves by converting CO2 using a microbial electrosynthesis system, the steps of which are: centrifuging the above-mentioned aroma-producing agent to remove the bacterial cells and then mixing it with tobacco shreds to obtain tobacco leaves with enhanced aroma.

[0019] Preferably, the centrifugation speed is 7500-8000 r / min and the time is 10-15 min; the volume ratio of aroma-producing agent to tobacco is 1:10, and the mixing and conversion time is 6-8 h; the tobacco is equilibrated for 24 h at a temperature of 22℃ and a relative humidity of 60%, and then rolled into a sample.

[0020] The present invention has the following beneficial effects:

[0021] This application isolated and screened a fragrance-producing bacterium, *Bacillus persadenia* ZL-5, which can generate aroma by electrosynthesizing CO2. After fermentation in a microbial electrosynthesis system, the resulting fragrance-producing agent showed a significant increase in aroma substances as detected by GC-MS. It can be used for flavoring cigarettes, enhancing their aroma and demonstrating good application value in cigarette production. Sensory evaluation and cigarette tasting revealed a significant increase in the richness of cigarette aroma, a reduction in cigarette irritation, and an increase in aroma, resulting in a significant improvement in overall quality. The method provided in this application generates aroma substances while reducing CO2 emissions, demonstrating good application value in both improving cigarette quality and protecting the environment. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 This is a simplified diagram of a microbial electrosynthesis system.

[0024] Figure 2 This is a colony morphology diagram of Bacillus pasadenae ZL-5.

[0025] Figure 3 This is an optical microscope image of Bacillus pasadenae ZL-5, magnified 10*100.

[0026] Figure 4 Phylogenetic tree of Bacillus pasadenae ZL-5.

[0027] Figure 5 A cathode electron microscope image of the electrosynthesis and conversion of CO2 by Bacillus pasadenae ZL-5.

[0028] Figure 6 A graph showing the current density of CO2 conversion via electrosynthesis by Bacillus pasadenae ZL-5.

[0029] Figure 7 GC-MS image of Bacillus pasadenae ZL-5 fermentation broth compared to blank control sample.

[0030] Figure 8 Sensory evaluation results of tobacco shreds treated with Bacillus pasadenae ZL-5 fermentation broth. Detailed Implementation

[0031] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0032] Unless otherwise specified, the experimental methods used in the following experimental examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0033] In the embodiments of the present invention, the solid culture medium involved is vacuum-sealed using a vacuum bag before being placed in the incubator.

[0034] Example 1: Isolation and identification of Bacillus pasadenae ZL-5

[0035] (1) Isolation of Pasadena Bacillus ZL-5

[0036] The Pasadena spp. ZL-5 of this invention was isolated from tobacco plants (December 30, 2024, Zhengzhou, Henan Province). The specific steps were as follows: freshly picked tobacco leaves were chopped and placed in an appropriate amount of sterile water. After thorough shaking, 1 ml of the mixture was diluted with sterile water to a concentration of 1:10. -1 times, 10 -2 times, 10 -3 times, 10 -4 times, 10 -5 times, 10 -6 times, 10 -7 Different dilutions of the bacterial suspension were evenly spread on LB agar plates, sealed with sealing film, and placed in a constant temperature incubator for cultivation. The cultivation temperature was 37℃, and the cultivation time was 2-3 days. After single colonies grew on the plates, the target strain was transferred to new LB agar plates, and the colonies were purified multiple times using the streak plating method.

[0037] (2) Molecular biological identification of Bacillus pasadenae ZL-5

[0038] Total DNA was extracted from the purified strain using the Ezup column-based bacterial genomic DNA extraction kit. PCR amplification of 16S rDNA was performed using universal primers for bacterial 16S rDNA (synthesized by Shanghai Sangon Biotech Co., Ltd., with the forward primer sequence 27F: AGAGTTTGATCMTGGCTCAG and the reverse primer sequence 1492R: GGTTACCTTGTTACGACTT).

[0039] PCR reaction system (25 μL): 10×PCR Buffer 2.5 μL, dNTPs (2.5 mmol / L each) 2 μL, primer 27F (10 μmol / L) 1 μL, primer 1492R (10 μmol / L) 1 μL, Taq DNA polymerase (5 U / μL) 0.2 μL, template DNA 1 μL, ddH2O 17.3 μL.

[0040] PCR reaction conditions: 95℃ pre-denaturation for 5 min; followed by 95℃ denaturation for 30 s, 55℃ annealing for 30 s, and 72℃ extension for 90 s, repeated 35 times; final extension at 72℃ for 10 min. The purification and sequencing of the obtained PCR amplification products were performed by Shanghai Sangon Biotech Co., Ltd., and BLAST analysis was conducted to construct a phylogenetic tree.

[0041] See the colony morphology diagram of strain ZL-5. Figure 2 ,Depend on Figure 2 It can be seen that strain ZL-5 appears milky white on the plate; under an optical microscope, the strain appears rod-shaped. Figure 3 The phylogenetic tree of this strain was constructed using MEGA11. Figure 4 The strain ZL-5 showed 100% similarity to Paenibacillus pasadenensis. Based on the analysis of colony morphology and physiological and biochemical characteristics, the strain ZL-5 was identified as Paenibacillus pasadenensis and named Paenibacillus pasadenensis ZL-5.

[0042] Example 2: Activation of Bacillus pasadenae ZL-5

[0043] Select the isolated Bacillus pasadenae ZL-5, take one loopful and inoculate it onto the enrichment medium for culture. The culture conditions are pH 7.0, temperature 37℃, and rotation speed 160 r / min. Stop fermentation when the bacterial solution is in the logarithmic growth phase to obtain activated Bacillus pasadenae ZL-5 bacterial solution.

[0044] The enrichment medium was prepared by dissolving 20 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl and 2 g / L agar in deionized water, adjusting the pH to 7.0, and then sterilizing at 121°C for 20 min.

[0045] Example 3: Persadena spp. ZL-5 produces aroma through electrosynthesis of CO2.

[0046] 250 mL of activated bacterial solution was centrifuged to collect the bacterial cells and then infused into a cathode chamber containing 2.5 L of CO2 fermentation medium, with a headspace of approximately 2.5 L. The fermenter inlet was connected to a CO2 gas cylinder, and CO2 gas was introduced for 30 min to remove oxygen from the cathode liquid. Subsequently, a peristaltic pump was connected to the fermenter inlet, continuously pumping CO2 collected in a gas sampling bag into the fermenter at 50 rpm. The fermenter outlet was connected to another sampling bag via a conduit. The entire system operated at a cathode applied voltage of 3.0 V, a fermentation temperature of 37 °C, and an initial fermentation pH of 7.0, and was connected to a high-performance battery detection system. The entire device was run continuously for one week. Fermentation broth containing aroma-producing bacteria was collected from the sampling port using a syringe, and the aroma compounds in the product were analyzed by GC-MS. Table 1 shows a comparison of the changes in aroma compounds between the fermentation broth and the blank control sample. A simplified diagram of the microbial electrosynthesis system is shown below. Figure 1 Cathodic electron microscope image of Bacillus pasadenae ZL-5 converting CO2 via electrosynthesis is shown below. Figure 5 The cathode current density during fermentation is shown in the figure. Figure 6 The GC-MS comparison results are shown in [link to GC-MS comparison]. Figure 7 .

[0047] Table 1 Comparison of aroma compounds in aroma-producing fungal fermentation broth and blank sample

[0048]

[0049]

[0050] From Table 1 and Figure 7 It was found that, compared with the control group, the fermentation broth of strain ZL-5 showed an increase in the content of aroma compounds such as phenylethanol, isoamyl alcohol, 2-methylbutanol, and 3-hydroxy-2-butanone. Among these, the increases in phenylethanol and isoamyl alcohol were more significant, with chromatographic peak areas increasing by 193600230 and 151273221, respectively. These results indicate that strain ZL-5 can effectively increase the content of aroma compounds and has a significant aroma-enhancing effect.

[0051] Depend on Figure 5 It can be seen that strain ZL-5 exhibited good growth under experimental conditions and successfully accumulated on the cathode surface, forming a bacterial film.

[0052] Depend on Figure 6 It can be seen that strain ZL-5 maintained a high current density (0.4–1.45 A / m) throughout the system operation. 2 This indicates that strain ZL-5 has a high electron transfer efficiency.

[0053] GC-MS sample pretreatment method: Take 10 mL of sample, centrifuge at 12000 r / min for 30 min, collect the supernatant and mix it with an equal volume of CH2Cl2, then transfer it to a separatory funnel for extraction. Gently shake to thoroughly mix the two phases, and after standing for 10 minutes to separate the layers, collect the lower organic phase. Repeat the extraction process 5 times, combine all organic phases, add anhydrous sodium sulfate to dry, let stand overnight, filter, remove the CH2Cl2 solvent using a rotary evaporator, dissolve the residue in 1 mL of chromatographic grade CH2Cl2, filter through a 0.45 μm organic filter membrane, and finally use the sample for GC-MS analysis. All the above operations were performed under light-protected conditions.

[0054] GC-MS Detection Method: An HP-5MS column (30m × 0.25mm × 0.25μm) was used. The injection port temperature was set to 280℃, and high-purity helium was used as the carrier gas at a flow rate of 1mL / min. The injection volume was 1μL, using splitless injection mode. The temperature program was as follows: initial temperature 50℃, hold for 4 min, then increase to 240℃ at a rate of 2℃ / min, and finally stop the temperature rise. GC-MS analysis was performed in full scan mode. The mass spectrometry conditions were as follows: transfer line temperature 280℃, ion source temperature 280℃, quadrupole temperature 150℃; ionization mode: electron impact (EI), electron energy 70eV; solvent delay time 8 min; scan mass range (m / z) 35-550.

[0055] Application example: Application and evaluation results of Bacillus pasadenae ZL-5.

[0056] Using ZL-5, an aroma-producing fungus previously isolated from tobacco plants, CO2 was converted into aroma-producing fungal liquid via a microbial electrosynthesis system. After centrifugation to remove the fungal cells (8000 r / min, 10 min), the liquid was mixed with tobacco shreds at a volume ratio of 1:10 and converted for 8 h. The tobacco shreds were then equilibrated for 24 h at a temperature of 22℃ and a relative humidity of 60%, and then rolled into samples for sensory evaluation.

[0057] Table 2 Comparison of respiratory evaluation results between the control group and the treatment group

[0058]

[0059] The evaluation results are shown in Table 2 and Figure 8 From Table 2 and Figure 8 It can be seen that, in terms of aroma, the richness of the cigarette aroma has been significantly increased; the tasting results show that the cigarette irritation has been reduced, the aroma has been increased, and the overall quality has been significantly improved.

[0060] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A fragrance-producing fungus, ZL-5, that converts CO2 in a microbial electrosynthesis system, is classified as follows: Paenibacillus pasadenensis It was deposited on June 12, 2025 at the China Center for Type Culture Collection, located at Wuhan University, Wuhan, China, with accession number CCTCC NO: M 20251367.

2. An aroma-producing fungicide, characterized in that: It includes the aroma-producing fungus ZL-5 as described in claim 1.

3. The method for preparing the aroma-producing fungal agent according to claim 2, characterized in that: After centrifuging the seed liquid of the aroma-producing fungus ZL-5 as described in claim 1, the supernatant was discarded to obtain the bacterial cells. The bacterial cells were inoculated into a microbial electrosynthesis system containing CO2 fermentation medium. The fermentation broth obtained after the system was running was the aroma-producing fungal agent.

4. The method for preparing the aroma-producing fungicide according to claim 3, characterized in that: The amount of seed solution used is 250-500 mL; the operating voltage is 3.0-3.5 V.

5. The method for preparing the aroma-producing fungal agent according to claim 4, characterized in that: The CO2 fermentation medium contains 0.5-1 g / L L-cysteine, 0.5-1 g / L KH2PO4, and 1-1.5 g / L NH4Cl.

6. The method for preparing the aroma-producing fungicide according to claim 5, characterized in that: The operating temperature is 35-37℃, the pH is 7.0-7.5, and the operating time is 5-7 days.

7. The application of the aroma-producing fungus ZL-5 according to claim 1 or the aroma-producing fungus agent according to claim 2 in the conversion of CO2 to enhance the aroma of cigarettes.

8. A method for enhancing cigarette aroma by converting CO2 using a microbial electrosynthesis system, characterized in that, The steps are as follows: centrifuge the aroma-producing fungicide described in claim 2 to remove the fungal cells, then mix it with tobacco shreds for conversion to obtain cigarettes with enhanced aroma.

9. The method for enhancing cigarette aroma by converting CO2 using a microbial electrosynthesis system according to claim 8, characterized in that: The volume ratio of the aroma-producing agent to the tobacco shreds is 1:10, and the mixing and conversion time is 6-8 hours.

10. The method for enhancing cigarette aroma by converting CO2 using a microbial electrosynthesis system according to claim 8, characterized in that: The centrifugation speed is 7500-8000 r / min and the time is 10-15 min.

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