Kocuria rootrophila ZD-11 and application of Kocuria rootrophila ZD-11 in cellulose degradation aroma production

Abstract

The invention belongs to the technical field of microorganism application, and particularly relates to a Kocuria rootrophila ZD-11 strain and application thereof in cellulose degradation and aroma production. The invention discloses a Kocuria rhizophila ZD-11, which is classified and named as Kocuria rhizophila, and is preserved in the China Center for Type Culture Collection on June 16, 2025, the preservation address is Wuhan University, Wuhan, China, and the preservation number is CCTCC No: M 20251390. The Kocuria rootrophila strain ZD-11 can grow on a sodium carboxymethyl cellulose culture medium, can degrade cellulose in tobacco leaves and generate aroma substances, can be applied to improvement of tobacco combustion performance, reduction of generation of harmful substances and improvement of aroma quality and taste of cigarettes, and has good application value in the cigarette production process.

Description

Technical Field

[0001] This invention belongs to the field of microbial application technology, and specifically relates to a cellulose-degrading aroma-producing fungus. Background Technology

[0002] Cellulose is a widely distributed polysaccharide in nature, composed of glucose linked by β-1,4-glycosidic bonds. It is one of the main components of plant cell walls and an important part of tobacco leaf structure. During tobacco combustion, cell wall substances such as cellulose in tobacco leaves produce various harmful substances such as lower aldehydes, catechols, and methanol. Furthermore, tobacco stems, due to their relatively high content of cell wall substances such as cellulose, pectin, and lignin, and lower content of total sugars and nicotine, produce a pungent, burning odor and a woody aroma when burned, reducing the sensory quality of tobacco. Therefore, finding a method to effectively degrade cellulose and improve tobacco quality has become an important research direction in the tobacco industry.

[0003] With the popularization of the concept of "reducing tar and harm," the tobacco industry often reduces the aroma components of tobacco while controlling tar content, thus affecting the smoking experience. Currently, there are various methods to improve tobacco aroma and smoking quality, mainly including variety improvement and cultivation technique optimization, but adding flavorings remains the primary means. Flavoring tobacco leaves can supplement pleasant aromas, giving cigarettes unique flavor characteristics, allowing the aromas of different types and grades of tobacco to blend harmoniously, improving taste, reducing smoke irritation, and thus enhancing overall quality.

[0004] With the continuous development of microbial technology, utilizing microorganisms to degrade cellulose and produce aroma has become an important direction for improving cigarette quality. Related research shows that many microorganisms, including bacteria, fungi, and yeasts, can degrade cellulose and produce aroma substances, which can be used to improve cigarette quality. For example, patent CN110616176A discloses a flavor-producing bacterium, *Bacillus bellis* JXQ21, isolated from tobacco leaves. This strain ferments tobacco extracts, and the resulting tobacco flavoring has good compatibility with tobacco, can soften the smoke, and improve the taste of cigarettes. Patent CN112043002A discloses a microorganism, *Bacillus bellis* B-3, that degrades cellulose in tobacco flavorings. This bacterium can be applied to tobacco flavoring raw materials, effectively reducing the cellulose content and improving the aroma quality of the raw materials. Summary of the Invention

[0005] To address the aforementioned problems, this invention proposes a strain of rhizotrophic coccidia ZD-11 and its application in cellulose degradation and aroma production.

[0006] The technical solution of this invention is implemented as follows:

[0007] To improve the combustion performance of tobacco and enhance the aroma quality and taste of cigarettes, this invention discloses a cellulose-degrading aroma-producing bacterium, ZD-11, isolated from Taorong-type Daqu (a type of Chinese liquor).

[0008] On the one hand, the present invention provides a cellulose-degrading aroma-producing fungus, *Kocuria rhizophila* ZD-11, which is classified as *Kocuria rhizophila* and was deposited on June 16, 2025, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC No: M 20251390.

[0009] Secondly, this invention applies for protection of a cellulose-degrading aroma-producing fungal agent, comprising the aforementioned cellulose-degrading aroma-producing fungus *Coprinus rhizophilus* ZD-11.

[0010] Preferably, the above-mentioned method for cultivating the cellulose-degrading aroma-producing fungus agent involves inoculating the cellulose-degrading aroma-producing fungus *Coprinus rhizophilus* ZD-11 into LB liquid medium for fermentation culture at 35-37℃ and 150-180 r / min for 12-24 h, centrifuging at 3000-4000 r / min for 10-15 min, discarding the supernatant to obtain the bacterial cells, and resuspending the bacterial cells in sterile water to obtain the cellulose-degrading aroma-producing fungus agent.

[0011] The inoculum size of the above-mentioned rhizotrophic cocci ZD-11 was 5-10% v / v; the ratio of bacterial cells to sterile water was 1:4.

[0012] The LB liquid medium formula is: 1 g / L peptone, 0.5 g / L yeast extract, and 1 g / L NaCl.

[0013] Thirdly, this invention applies to protect the use of the above-mentioned cellulose-degrading aroma-producing rhizotrophic cocci ZD-11 or the above-mentioned cellulose-degrading aroma-producing fungal agent in the degradation of cellulose and aroma production.

[0014] Preferably, the cellulose mentioned above is the cellulose found in cigarettes.

[0015] Fourthly, this invention applies to protect the application of the above-mentioned cellulose-degrading aroma-producing fungus ZD-11 or the above-mentioned cellulose-degrading aroma-producing fungus agent in improving tobacco combustion performance and enhancing the aroma quality and taste of cigarettes.

[0016] The aforementioned cellulose-degrading aroma-producing bacterium *Coprinus rhizogenes* ZD-11 or the aforementioned cellulose-degrading aroma-producing bacterium inoculum can produce aroma substances, significantly improving the aroma quality and taste of cigarettes.

[0017] Fifthly, this invention claims a method for improving the combustion performance of tobacco and enhancing the aroma quality and taste of tobacco leaves, the steps of which are: spraying the above-mentioned cellulose degradation aroma-producing fungicide onto tobacco leaves, and fermenting to obtain tobacco leaves with improved combustion performance, aroma quality and taste.

[0018] Preferably, the concentration of the aroma-producing fungal agent used during the above spraying is 200 mL / kg.

[0019] Preferably, the fermentation temperature is 30-35℃ and the time is 24-48h.

[0020] The specific steps are as follows:

[0021] (1) Fermentation of the strain: The cellulosic aroma-producing strain ZD-11 was inoculated into LB liquid medium at an inoculation amount of 10% (v / v); the culture conditions were: 37℃, 180r / min, and shaker culture for 12h.

[0022] (2) Solid-state fermentation of tobacco leaves: Centrifuge the fermentation liquid obtained in step (1) at 3000 r / min for 10 min, discard the supernatant, take the bacterial cells and resuspend them in sterile water (the ratio of bacterial cells to sterile water is 1:4), spray them onto the tobacco leaves at 200 mL / kg, and ferment at 30-35℃ for 24-48 h to obtain tobacco leaves after solid-state fermentation of aroma-producing fungi;

[0023] (3) The aroma substances of tobacco leaf samples after solid-state fermentation with aroma-producing fungi and those without aroma-producing fungi were analyzed by GC-MS, and cigarettes were rolled for smoking.

[0024] The present invention has the following beneficial effects:

[0025] This application isolated and screened a cellulose-degrading aroma-producing fungus, *Coprinus rhizophilus* ZD-11. Strain ZD-11 can degrade cellulose and produce aromatic substances. When the aroma-producing fungus agent was sprayed onto tobacco leaves for solid-state fermentation, aroma-producing fungus samples were obtained. GC-MS analysis revealed a significant increase in aroma substance content compared to the blank sample without the fungus solution, and the cigarettes produced from it showed good smoking performance. This fungus can be used to improve tobacco combustion performance, reduce the formation of harmful substances, and enhance the aroma quality and taste of cigarettes, demonstrating significant application value in cigarette production. Attached Figure Description

[0026] 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.

[0027] Figure 1 This is a photograph of a plate of Kocuria sp. ZD-11 strain.

[0028] Figure 2 This is a photomicrograph of Kocuria sp. ZD-11.

[0029] Figure 3 This is a gene sequence diagram of Kocuria sp. ZD-11.

[0030] Figure 4 Phylogenetic tree diagram of Kocuria sp. ZD-11 strain.

[0031] Figure 5 This is a Congo red staining plate image of Kocuria sp. ZD-11 strain.

[0032] Figure 6 Comparative diagram of tobacco cellulose after treatment with Kocuria sp. ZD-11 strain.

[0033] Figure 7 The total ion chromatograms are for the control and the aroma-producing sample from Kocuria sp. ZD-11 strain.

[0034] Figure 8 Sensory evaluation diagram of tobacco leaves treated with Kocuria sp. ZD-11 strain. Detailed Implementation

[0035] 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.

[0036] 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.

[0037] Example 1: Enrichment culture of strain ZD-11

[0038] The Tao Rong type Daqu powder, which will be obtained from Mianchi County, Henan Province in March 2024, was pulverized. 25g of Daqu powder was placed in an Erlenmeyer flask containing 100mL of sodium carboxymethyl cellulose liquid culture medium. The Erlenmeyer flask was cultured in a shaker at 37℃ for 12h to obtain a microbial enrichment culture medium.

[0039] (Note: The formula for sodium carboxymethyl cellulose liquid culture medium is: sodium carboxymethyl cellulose 20.0 g / L, dipotassium hydrogen phosphate 2.5 g / L, disodium hydrogen phosphate 2.5 g / L, pH natural.)

[0040] Example 2: Screening of strain ZD-11

[0041] The enriched bacterial culture was serially diluted. 150 μL of each diluted culture was evenly spread onto a sodium carboxymethyl cellulose (CMC) solid medium plate using the spread plate method and incubated at 37°C for 2-3 days until single colonies appeared. Single colonies were streaked and cultured for 2-3 days before being transferred to Congo red solid medium for further cultivation. The presence of a clear zone around the colony was observed. The strain with the strongest cellulose degradation ability was selected based on the ratio of the clear zone diameter (D) to the colony diameter (d) (D / d). The Congo red staining results for this strain are shown below. Figure 5 As shown, its average D / d value is 7.1, which is higher than the average D / d value of other aroma-producing fungi.

[0042] (Note: The formula for sodium carboxymethyl cellulose solid medium is: sodium carboxymethyl cellulose 20.0 g / L, dipotassium hydrogen phosphate 2.5 g / L, disodium hydrogen phosphate 2.5 g / L, agar 2%, pH natural.)

[0043] (Note: Congo red cellulose solid medium: CMC-Na 1.5%, KH2PO4 0.1%, NH4NO3 0.1%, MgSO4·7H2O 0.05%, agar 2%.)

[0044] Example 3: Identification of strain ZD-11

[0045] (1) Morphological identification

[0046] First, colonies on solid culture media were observed for morphological analysis, recording key information such as their shape, size, color, and surface characteristics. Next, the isolated and purified strains were stained with crystal violet, ensuring standardized staining procedures and uniform staining results. After staining, the stained strains were observed under high magnification using an optical microscope to carefully identify their cell structure, arrangement, and other microscopic features, while simultaneously taking photographs for recording.

[0047] The colony morphology of strain ZD-11 is as follows Figure 1 As shown, the colonies are round and raised, pale yellow in color, 2-3 mm in diameter, with neat edges and a smooth surface; Figure 2 Microscopic images show that the bacterial cells of this strain are spherical (approximately 2.5 μm in radius), lack flagella, and do not produce spores.

[0048] (2) Molecular biological identification

[0049] Total DNA was extracted from strain ZD-11 using the Ezup column-based bacterial genomic DNA extraction kit. Genomic PCR amplification was performed using universal primers for bacterial 16S rDNA (synthesized by Shanghai Sangon Biotech Co., Ltd., with the forward primer sequence 5'-AGTTTGATCMTGGCTCAG-3' and the reverse primer sequence 5'-GGTTACCTTGTTACGACTT-3'). The gene sequence of Kocuria sp. ZD-11 is shown below. Figure 3 As shown. The purification and sequencing of the obtained PCR products were performed by Shanghai Sangon Biotech Co., Ltd. Blast analysis was conducted on the obtained sequences, and a phylogenetic tree was constructed. Figure 4 The results showed that strain ZD-11 had 100% similarity to Kocuria rhizophila. Based on the analysis of colony morphology and physiological and biochemical characteristics, strain ZD-11 was preliminarily identified as Kocuria rhizophila.

[0050] (Note: PCR amplification reaction system (total 25μL): 12.5μL of 10X PCR Buffer, dNTP (each 10mM), Taq Plus DNA Polymerase (5U / μL) and 50mM MgSO4, 1μL primer F (10μM), 1μL primer R (10μM), 1μL template (DNA), 9.5μL ddH2O;

[0051] PCR amplification reaction conditions: 95℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 57℃ annealing for 30 s, 72℃ extension for 90 s, 30 cycles; 72℃ final extension for 10 min.

[0052] Example 4: Degradation verification experiment of strain ZD-11

[0053] The obtained cellulose-degrading aroma-producing fungus was inoculated into LB liquid medium at 10% (volume ratio) and fermented for 12 hours to obtain the aroma-producing fungus fermentation broth. After centrifugation at 3000 r / min for 10 min, the supernatant was discarded. The fungal cells were resuspended in sterile water (the ratio of fungal cells to sterile water was 1:4) and sprayed onto tobacco leaves at 200 mL / kg. After fermentation at 30℃ for 48 hours, the broth was treated at 70℃ for 30 min to inactivate the cellulase in the fermentation broth, and the tobacco leaf sample was obtained. The tobacco leaf sample was dried and ground into powder, passed through a 40-mesh sieve, and 0.25g of tobacco powder was accurately weighed and placed in a 100mL Erlenmeyer flask. 25mL of 5% acetic acid solution was added, and the mixture was shaken and extracted for 30 minutes. The mixture was filtered, and the residue was washed 2-3 times with distilled water. The residue was then transferred to a 100mL Erlenmeyer flask, and 0.25g of cellulase was added, followed by 60mL of pH 4.7 citrate buffer. The mixture was shaken at 37℃ for 24 hours. The sample was then cooled to room temperature, filtered, and the volume was adjusted to 100mL. The glucose content in the solution was determined using a flow analyzer to calculate the cellulose content. The result was compared with a blank control sample. Figure 6 As shown, by Figure 6 It can be seen that the cellulose degradation rate of tobacco leaves treated with this strain is 40.1%.

[0054] Example 5: Aroma Production Verification Experiment of Strain ZD-11

[0055] The obtained cellulose-degrading aroma-producing fungus was inoculated at 10% (v / v) into LB liquid medium and fermented for 12 h to obtain the aroma-producing fungus fermentation broth. After centrifugation at 3000 r / min for 10 min, the supernatant was discarded, and the fungal cells were resuspended in sterile water (fungus to sterile water ratio of 1:4). The broth was then sprayed onto tobacco leaves at 200 mL / kg and fermented at 30℃ for 48 h to obtain the aroma-producing fungus sample. The total ion chromatogram compared with the blank control sample is shown below. Figure 7 The changes in aroma compounds are shown in Table 1:

[0056] Table 1 Comparison of changes in aroma compounds

[0057]

[0058] From Table 1 and Figure 7 It can be seen that, compared with the control group, the aroma component content of the samples fermented by strain ZD-11 was significantly higher. Among them, the content of DDMP and 5-methylfurfural, two aroma components, in the fermented group was significantly higher than that in the control group, indicating that strain ZD-11 can increase the content of aroma substances in tobacco leaves.

[0059] Detection and analysis results:

[0060] Sample pretreatment: Dry the blank control sample and aroma-producing fungus sample at 40℃, pulverize them, and pass them through a 40-mesh sieve. Accurately weigh 25g of the blank control sample, aroma-producing fungus sample, 30g of sodium chloride, and 200mL of deionized water into a 500mL round-bottom flask at one end of a simultaneous distillation extraction apparatus. Place 60mL of dichloromethane at the other end of the apparatus and heat in a water bath at 60℃ for simultaneous distillation extraction for 150min. After extraction, dry the dichloromethane extract with anhydrous sodium sulfate, let it stand overnight at 4℃, concentrate the extract by heating in a water bath at 60℃, and make up to 1mL with dichloromethane. Filter the solution through a 0.22μm filter membrane into a 2.0mL chromatographic vial and immediately perform instrumental analysis.

[0061] Analysis conditions

[0062] Chromatographic conditions: The column was an HP-5MS quartz capillary column (60m×250μm×0.25μm); the injection port temperature was 280℃; the temperature program was as follows: initial temperature 50℃, hold for 1 min, increase to 148℃ at 4℃ / min and hold for 1 min, then increase to 172℃ at 2℃ / min and hold for 1 min; increase to 280℃ at 4℃ / min and hold for 5 min; the carrier gas was helium with a flow rate of 3.0 mL / min; splitless injection was used with a split ratio of 5:1.

[0063] Mass spectrometry conditions: EI ionization source was used with an electron energy of 70 eV and an ion source temperature of 230℃; quadrupole temperature was 150℃; transfer line temperature was 250℃; acquisition was in full scan mode with an m / Z acquisition range of 35-550.

[0064] Application example: Application and evaluation results of aroma-producing fungus ZD-11

[0065] Aroma-producing bacteria isolated from Taorong-type Daqu (a type of starter culture) were fermented to obtain an aroma-producing bacterial liquid. After centrifugation to remove the bacterial cells (8000 r / min, 10 min), the liquid was sprayed onto tobacco leaves. The leaves were then fermented simultaneously with tobacco leaves without the added aroma-producing bacterial liquid at 30℃ for 48 h. These were then rolled to prepare blank control samples and aroma-producing bacterial samples. A professional sensory evaluation team (7 members) then conducted sensory evaluations on both samples. Evaluation indicators included aroma quality, aroma quantity, off-flavors, smoothness, smoke concentration, irritation, and sweetness. The average value of the sensory evaluation results was used as the evaluation standard. The scoring results are shown in Table 2 and... Figure 8 As shown:

[0066] Table 2 Sensory evaluation scores

[0067]

[0068] From Table 2 and Figure 8It can be seen that the samples fermented with strain ZD-11 all showed improved aroma and quality compared to the control group. In terms of aroma characteristics, compared with the control, fermentation treatment with strain ZD-11 can enhance the aroma quality and quantity of cigarettes, and reduce off-flavors and irritation. In terms of taste characteristics, compared with the control group, fermentation treatment with strain ZD-11 can effectively increase the smoothness and sweetness of cigarettes, while making the overall taste of cigarettes more harmonious.

[0069] 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 cellulose-degrading aroma-producing fungus, *Coprinus rhizophilus* ZD-11, is classified as follows: Kocuria rhizophila It was deposited on June 16, 2025 at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC No: M 20251390.

2. A cellulose-degrading aroma-producing fungal agent, characterized in that: It includes the cellulose-degrading aroma-producing fungus ZD-11 as described in claim 1.

3. The method for cultivating the cellulose-degrading aroma-producing fungal agent according to claim 2, characterized in that, The steps are as follows: Inoculate the cellulose-degrading aroma-producing fungus ZD-11 described in claim 1 into LB liquid medium for fermentation. After centrifugation of the fermentation broth, discard the supernatant to obtain the bacterial cells. Resuspend the bacterial cells in a solvent to obtain the cellulose-degrading aroma-producing fungal agent.

4. The method for cultivating aroma-producing fungi of cellulose degradation according to claim 3, characterized in that: The inoculum size of *Coprinus rhizophilus* ZD-11 was 5-10% v / v; the fermentation conditions were 35-37℃ and 150-180 r / min for 12-24 h; the solvent was sterile water, and the ratio of bacteria to sterile water was 1:

4.

5. The application of the cellulose-degrading aroma-producing rhizotrophic cocciformis ZD-11 as described in claim 1 or the cellulose-degrading aroma-producing fungal agent as described in claim 2 in the degradation of cellulose and aroma production.

6. The application according to claim 5, characterized in that: The cellulose mentioned is the cellulose found in cigarettes.

7. The application of the cellulose-degrading aroma-producing fungus ZD-11 as described in claim 1 or the cellulose-degrading aroma-producing fungus agent as described in claim 2 in improving tobacco combustion performance and enhancing the aroma quality and taste of cigarettes.

8. A method for improving tobacco combustion performance and enhancing the aroma quality and taste of tobacco leaves, characterized in that, The steps are as follows: spray the cellulose degradation aroma-producing fungicide described in claim 2 onto the tobacco leaves, and ferment to obtain tobacco leaves with improved combustion performance, aroma quality and taste.

9. The method for improving tobacco combustion performance and enhancing cigarette aroma quality and taste according to claim 8, characterized in that: The concentration of the aroma-producing fungicide used during spraying is 200 mL / kg.

10. The method for improving tobacco combustion performance and enhancing cigarette aroma quality and taste according to claim 9, characterized in that: The fermentation temperature is 30-35℃ and the time is 24-48 h.

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