Enterobacter hormaechei XW-01 as well as microbial preparation and application thereof

Abstract

The invention discloses enterobacter hormaechei XW-01 and a microbial preparation thereof, the strain (preservation number is CCTCC NO: M 20242595) can efficiently degrade protein, total sugar and nicotine in tobacco leaves, sensory indexes such as aroma and aroma quantity are remarkably improved, and protease activity is remarkably improved when the enterobacter hormaechei XW-01 and wheat bran act synergistically. The microbial preparation is obtained through the processes of strain activation, fermentation culture and microbial agent preparation, and when the microbial preparation is applied to tobacco leaf treatment, through the steps of water content adjustment, preparation spraying, sealed fermentation, low-temperature drying and the like, the technical breakthrough that the protein content is reduced, the tar release amount is reduced, and the comprehensive quality grade is improved is achieved. Compared with a traditional method, the technology has the advantages that the treatment period is shortened, the cost is greatly reduced, the COD value of wastewater is greatly reduced, the cigarette quality and the production environmental protection property are remarkably improved, and the method has a wide industrial application prospect.

Description

Technical Field

[0001] This invention relates to the field of bio-fermentation engineering technology, and in particular to a Enterobacter holmieae XW-01, its microbial preparations, and their applications. Background Technology

[0002] Cigars are made from pure natural tobacco as the base material, with zero added flavorings and entirely hand-rolled. Their quality is constructed by the synergistic effect of the chemical components of the tobacco leaves and the sensory experience, and presents the synergistic characteristics of the three-layer structure of the wrapper (appearance and texture and combustion carrier), binder (structural support and aroma transition), and filler (smoke concentration and flavor core). The intensity and complexity of cigar smoke are highly dependent on the dynamic balance of tobacco protein. High-quality tobacco leaves require strict control of protein content within the range of ≤10%. High protein content will damage quality through three mechanisms: First, it reduces the calorific value of combustion and exacerbates char deposition, leading to frequent flameouts and combustion gaps during smoking, significantly weakening ignition smoothness and combustion persistence. Second, during fermentation, excessive protein undergoes pyrolysis to generate excessive ammonia, hydrogen sulfide, and other irritating gases, masking core aromas such as caramel sweetness and woody notes, resulting in a cloudy and fragmented aroma. Third, it accelerates the cell wall fibrosis process, reducing the flexibility of tobacco leaves by more than 60%, easily causing structural defects such as wrapper brittleness and voids in the filler during rolling, directly affecting the integrity of the cigar's appearance and ash retention. This demonstrates the global regulatory role of protein content on the combustion stability, aroma purity, and physical properties of cigars, constituting a core parameter for the quality management of high-end cigars.

[0003] Current industrial production primarily relies on exogenous enzyme-catalyzed degradation technology. This involves adding neutral / acidic proteases or complex enzyme preparations to hydrolyze macromolecular proteins in tobacco leaves into polypeptides, oligopeptides, and free amino acids. The degradation products then participate in Maillard reactions to generate aroma compounds, effectively reducing astringency, bitterness, and irritation, while enhancing the aroma's complexity and finesse. However, this technology faces three major bottlenecks: First, there is an imbalance between degradation efficiency and selectivity. Enzymatic hydrolysis is significantly affected by fluctuations in the pH, temperature, humidity, and ion concentration of the tobacco substrate, resulting in insufficient targeted degradation rates of key proteins such as proteases and glutenins. Furthermore, non-specific hydrolysis easily destroys flavor precursors. Second, enzyme activity stability is insufficient. During the 6-12 month fermentation cycle of cigar tobacco leaves, existing enzyme preparations are easily deactivated under high temperature and humidity conditions, making it difficult to maintain sustained catalytic efficiency. Third, industrial applicability is limited. Enzyme preparations are mainly applied topically, making it difficult to penetrate the internal tissues of tobacco leaves. This results in incomplete degradation of deep proteins, and residual enzyme proteins may pose a risk of microbial contamination during tobacco storage. All three factors collectively restrict the large-scale application of this technology.

[0004] To improve the quality of cigar tobacco leaves, effectively degrading the protein content and maintaining it within a reasonable range is a crucial step. As the understanding of the importance of protein degradation technology deepens, researching and developing new cigar tobacco protein degradation technologies has broad and practical significance for improving cigar quality, enhancing smoking experience, and increasing economic benefits for businesses.

[0005] The information disclosed in this background section is intended only to enhance the understanding of the background technology of this disclosure and should not be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a strain of Enterobacter holmieae XW-01, its microbial preparation and its application. This strain and its metabolites can efficiently degrade proteins, total sugars and nicotine in tobacco leaves, which has broad and practical significance for improving cigar quality and increasing the economic benefits of enterprises.

[0007] According to one aspect of this disclosure, a strain of Enterobacter holmie ( ) was isolated and screened. Enterobacter hormaechei XW-01 was deposited on November 20, 2024, at the China Center for Type Culture Collection (address: Wuhan University, Wuhan, China, 430072, China), with accession number CCTCC NO: M 20242595.

[0008] According to another aspect of this disclosure, a microbial preparation is provided containing Enterobacter holmieae XW-01 and / or its metabolic enzyme products, or containing Enterobacter holmieae XW-01 metabolic enzyme products induced by wheat bran.

[0009] In some embodiments of this disclosure, the above-mentioned Enterobacter holmie XW-01 or biological agent is applied in at least one of the following (1) to (6): (1) Reduce the protein content in tobacco leaves or prepare reagents to reduce the protein content in tobacco; (2) Reduce the total sugar and / or nicotine content of tobacco leaves or prepare reagents to reduce the total sugar and / or nicotine content of tobacco leaves; (3) Improve the sensory quality of tobacco leaves or prepare a reagent to improve the sensory quality of tobacco leaves, wherein the sensory quality includes at least one of aroma, off-flavor, aroma quantity, mellowness, sweetness, irritation, cleanliness, aftertaste, and combustibility; (4) Use in combination with wheat bran or prepare a reagent for use in combination with wheat bran to improve protease activity; (5) Improve cigarette quality or prepare reagents to improve cigarette quality; (6) Preparation of protease.

[0010] According to another aspect of this disclosure, a method for preparing a microbial preparation is provided, comprising the following steps: (1) Activation of strain: Enterobacter holmie XW-01 strain was inoculated onto NA solid medium by spot inoculation and cultured at 33~37 ℃ for 48~96 h; (2) Seed culture preparation: scrape the bacterial cells obtained from the previous step, inoculate them into NA liquid culture medium, and culture them in a shaker at 33~37℃ and 160~200 r / min for 10~15 h to obtain the seed culture. (3) Expanded culture: Inoculate the seed culture into the fermentation medium at an inoculation rate of 2-4%, and culture in a shaker at a temperature of 33-37℃ and a rotation speed of 160-200r / min for 46-50 h; (4) Preparation of microbial agent: Centrifuge the fermentation broth from the previous step and take its supernatant.

[0011] In some embodiments of this disclosure, the fermentation medium contains: wheat bran 8~30 g / L, glucose 8~12 g / L, and NaCl 3~7 g / L.

[0012] In some embodiments of this disclosure, the tobacco leaf processing method includes the following steps: (1) Adjust the moisture content of the tobacco leaves to be treated to 25-30%; (2) Spray the microbial preparation onto the tobacco leaves at 20-30% of the weight of the tobacco leaves to be treated, then seal the leaves and ferment them at 43-47°C and 70-80% humidity for 8-12 days. (3) After fermentation, the product is dried and the moisture content is balanced.

[0013] The present invention provides Enterobacter cholerae (Cholacillus hominis) Enterobacter hormaechei XW-01 and its metabolites demonstrate significant technical advantages and economic value in optimizing cigar quality, specifically in the following aspects: 1. Highly efficient protein degradation, optimizing chemical composition. Through bran-directed induction technology, the protease activity of strain XW-01 was significantly enhanced, achieving a technological breakthrough in substantially reducing the protein content of cigar tobacco. This process effectively improves the proportion of chemical components in tobacco leaves, reduces the generation of harmful substances such as nitrogen oxides during combustion, and significantly enhances product safety and smoking health.

[0014] 2. Enhance the sensory quality of cigars in multiple dimensions Precise control of protein content directly improves the sensory characteristics of cigars: ① Enhanced aroma quality: Significantly reduces the burnt and pungent odors produced by protein pyrolysis, resulting in a richer and purer aroma; ② Optimized taste: Verified by a professional tasting team, the processed tobacco leaves have enhanced mellowness, improved smoothness, and significantly improved aftertaste comfort; ③ Overall quality leap: Through the synergistic optimization of chemical composition and sensory quality, the overall quality level of cigars is improved by an average of two grades, significantly enhancing their market competitiveness.

[0015] 3. Green bio-manufacturing system to reduce costs and increase efficiency. ① Raw material economy: Using agricultural waste such as wheat bran as inducers, the raw material cost is reduced by more than 90% compared with traditional chemical inducers, while realizing the resource utilization of waste; ② Process environmental protection: No toxic chemical reagents are added throughout the process, the metabolic products of the strains are non-biotoxic, the COD value of the fermentation waste liquid is reduced by at least 70% compared with traditional processes, and the cost of treating waste gas, wastewater, and solid waste is reduced by more than 50%; ③ Safety and compliance: It complies with the Biosafety Law and the green manufacturing standards of the tobacco industry, helping enterprises to pass ESG (Environmental, Social, and Governance) certification.

[0016] 4. It has strong process compatibility and broad prospects for industrialization. ① Ease of operation: Only a strain cultivation module (temperature 30-37℃, pH 6.0-7.0) needs to be added to the existing fermentation process, without the need for large-scale equipment modification; ② Controllable cycle: The entire process cycle from strain activation to tobacco leaf processing is ≤60 hours, perfectly matching the pace of existing production lines; ③ Significant benefits: A single production line can process up to 800 tons of tobacco leaves per year. Based on a 20% quality premium, the annual additional economic benefits exceed 10 million yuan, demonstrating industrial value for rapid replication and promotion.

[0017] This invention, through the deep integration of microbial engineering technology and tobacco processing technology, has pioneered a new path for improving the quality of cigars. While ensuring product safety, it has achieved an organic unity of economic and ecological benefits, providing key technical support for the transformation and upgrading of the tobacco industry. Attached Figure Description

[0018] Figure 1 Phylogenetic tree of strain XW-01 based on 16S rDNA gene sequence in one embodiment of the application.

[0019] Figure 2 This is an electron micrograph of strain XW-01 from one of the embodiments of the application.

[0020] Figure 3 The image shows the protein content determination results of tobacco leaves after treatment with a microbial agent in one embodiment of the application.

[0021] Figure 4The image shows the results of total sugar content determination of tobacco leaves after treatment with microbial agents in one embodiment of the application.

[0022] Figure 5 The image shows the nicotine content determination results of tobacco leaves after treatment with a microbial agent in one embodiment of the application.

[0023] Figure 6 The image shows the results of protease activity assay of strain XW-01 after induction treatment in one embodiment of the application. Detailed Implementation

[0024] To better understand the technical solution of this application, the above technical solution will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0025] Unless otherwise specified, the instruments and equipment involved in the following embodiments are all conventional instruments and equipment; unless otherwise specified, the reagents and culture media involved are all commercially available conventional reagents and culture media; unless otherwise specified, the experimental methods involved are all conventional methods.

[0026] The following examples involve a strain of Enterobacter holmie, XW-01, which belongs to the Enterobacter holmie family (Enterobacter holmie). Enterobacter hormaechei ), taxonomically named: Enterobacter hormaechei XW-01, deposited at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, accession number CCTCC NO: M 20242595, deposited on November 20, 2024.

[0027] Example 1: Screening and identification of Enterobacter holmie XW-01 strain (1) Screening of strains: Weigh 2g of cigar tobacco leaf (Yunxue No. 1) fragments and place them in 50mL of sterile water. Incubate at 30℃ and 180 r•min -1 Incubate for 36 hours, then dilute the culture medium 10 times. -6 , 10 -7 , 10 -8 After dilution, 50 μL of the culture was spread onto NA solid medium. Single colonies with different morphologies and colors were picked and inoculated onto protease-selective medium (10 g skim milk powder, 20 g agar, 1 L distilled water, autoclaved at 121°C for 20 min). After incubation at 30°C for 36 h, the presence or absence of a clear zone was observed, and the ratio of the clear zone to the colony diameter (D / d value) was measured. A D / d value greater than 2.0 was used for initial screening. The D / d value reflects the protease production capacity of the strain; the higher the D / d value, the stronger the enzyme production capacity of the strain. The strains obtained from the initial screening were inoculated onto NA liquid medium and incubated at 30°C and 180 r•min. -1 Fermentation for 36 hours at 8000 rpm -1Centrifuge for 10 min, collect the supernatant and measure the enzyme activity of the protease, and then re-screen based on the enzyme activity level.

[0028] (2) Strain identification: The screened strains were inoculated into NA solid medium and cultured at 30 ℃ for 24 h. The colony morphology was observed and the cell characteristics were observed under an electron microscope. The strains were identified morphologically and physiologically and biochemically according to Bergey's Manual of Bacterial Identification and the Manual of Systematic Identification of Common Bacteria. The strains were inoculated into NA solid medium and cultured at 30 ℃ and 180 r / min for 24 h. After the culture was completed, 10 µl of bacterial solution was transferred into a PCR tube, heated at 95 ℃ for 7 min in a PCR instrument, and centrifuged at 10000 r / min and 4 ℃ for 10 min. The supernatant was used as a DNA template by pipetting. Using the genomic DNA of the strain as a template, the 16S rDNA sequence was amplified using universal primers 27F (5′-AGAGTTTGATCMTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′). The PCR amplification system consisted of 25 µL of 2×TaqMaster Mix, 2 µL each of forward and reverse primers (10 µmol / L), 2 µL of DNA template, and 19 µL of ddH2O. The PCR reaction program was as follows: 94℃ pre-denaturation for 3 min; 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 1 min, 30 cycles; 72℃ extension for 5 min; and finally, storage at 4℃. The PCR products were sequenced by Sangon Biotech (Shanghai) Co., Ltd. The sequencing results were compared with known sequences in the NCBI database using the NCBI BLAST online tool. A phylogenetic tree of the strain was constructed using Mega11.0 software (e.g., ...). Figure 1 (As shown).

[0029] Ultimately, XW-01 was identified as Enterobacter holmieae (C. holmieae). Enterobacter hormaechei (Strain morphology as follows) Figure 2 (As shown), it has been deposited at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, with accession number CCTCCNO: M 20242595.

[0030] Example 2 Preparation of Enterobacter holmie XW-01 bacterial agent (1) Activation of strain: Enterobacter holmie XW-01 strain was inoculated onto NA solid medium by spot inoculation and cultured at 35℃ for 48~96 h.

[0031] (2) Seed culture preparation: scrape the bacterial cells cultured in step (1), inoculate them into NA liquid culture medium, and culture them in a shaker at 35 ℃ and 180 r / min for 12 h to obtain the seed culture.

[0032] (3) Preparation of liquid fermentation medium for protease production: CK: Glucose 10.0 g / L, NaCl 5.0 g / L; T1: Wheat bran 10.0 g / L, glucose 10.0 g / L, NaCl 5.0 g / L; T2: Wheat bran 15.0 g / L, glucose 10.0 g / L, NaCl 5.0 g / L; T3: Wheat bran 20.0 g / L, glucose 10.0 g / L, NaCl 5.0 g / L; T4: Wheat bran 25.0 g / L, glucose 10.0 g / L, NaCl 5.0 g / L; T5: Wheat bran 30.0 g / L, glucose 10.0 g / L, NaCl 5.0 g / L.

[0033] (4) Fermentation broth preparation: The *Enterobacter holmieae* XW-01 strain seed culture from step (2) was inoculated into the special fermentation medium from step (3) at an inoculation rate of 4%. The culture was then incubated at 35 ℃ and a shaking speed of 180 r / min for 48 h. OD 600 =2.0; (5) Preparation of extracellular crude enzyme solution: Centrifuge the fermentation broth in step (4) at 4 ℃ and 8000 r / min for 10 min, and take the supernatant as the extracellular crude enzyme solution.

[0034] Example 3: Cigar Tobacco Leaf Processing When treating tobacco leaves with the various microbial agents prepared in Example 2, the specific steps are as follows: The crude enzyme solution obtained at 20% was sprayed evenly at 25% of the tobacco leaf weight, sealed in a sealed bag, and fermented for 10 days at 45℃ and 75% humidity to obtain cigar tobacco leaves treated with extracellular crude enzyme solution. After the fermentation, the leaves were placed in an oven and dried at 80℃ for 10 minutes. After drying, they were placed in a constant temperature and humidity chamber to balance the moisture content for 24 hours. Then, sensory quality evaluation, protein content determination, total sugar content determination, and nicotine content determination were performed.

[0035] Method for protease activity assay: Protease activity assay shall be performed in accordance with GB / T 23527-2009.

[0036] Protein content determination method: Protein content was determined using the protein content kit from Beijing Solarbio Science & Technology Co., Ltd., according to its instructions.

[0037] Total sugar content was determined according to the tobacco industry standard YC / T 159-2002 / 2019 "Determination of water-soluble sugars in tobacco and tobacco products - continuous flow method".

[0038] Nicotine content determination method: Nicotine content was determined according to tobacco industry standard YC / T 217-2007 "Determination of potassium in tobacco and tobacco products - Continuous flow method".

[0039] Depend on Figure 3 It was found that compared with the control group, the protein content of tobacco leaves treated with the extracellular crude enzyme solution of *Enterobacter holmieae* XW-01 induced by wheat bran was reduced. The T5 treatment showed the best degradation effect, reducing the protein content by 24.2%. This indicates that the protease produced by *Enterobacter holmieae* XW-01 induced by wheat bran can effectively reduce the protein content in cigars. Furthermore, the protein content of tobacco leaves treated with the extracellular crude enzyme solution of *Enterobacter holmieae* XW-01 induced by wheat bran was also reduced. Figure 4 and Figure 5 It can be seen that the total sugar and nicotine content also decreased slightly.

[0040] Example 4: Optimization of induction conditions for protease production by Enterobacter holmie XW-01 Glucose (10 g / L) was used as the carbon source, and NaCl (5 g / L) was used as the inorganic salt. Different concentrations of wheat bran (10 g / L, 15 g / L, 20 g / L, 25 g / L, and 30 g / L) were added sequentially as the nitrogen source. The protease activity assay results are as follows: Figure 6 As shown.

[0041] As shown in the figure, the protease activity of Enterobacter holmieae XW-01 gradually increased with the increase of wheat bran concentration, reaching the highest value of 19.81 U / mL at a concentration of 30 g / L.

[0042] Example 5: Protein degradation effect and sensory evaluation of cigar tobacco leaves during fermentation Cigar tobacco leaves were fermented according to the method in Example 2. The tobacco leaves sprayed with an equal amount of sterile water were denoted as CK0. The tobacco leaves treated with extracellular crude enzyme solutions induced by different liquid fermentation media (CK, T1, T2, T3, T4, T5) in Example 2 were denoted as CK, T1, T2, T3, T4 and T5 in sequence.

[0043] Sensory evaluation method: Cigars treated with extracellular crude enzyme solution were rolled into tobacco and equilibrated at 22±1℃ and 60±2% relative humidity for 48 h. Then, sensory evaluation experts were organized to assess the quality of the tobacco in nine aspects: aroma, off-flavors, aroma intensity, smoothness, sweetness, irritation, cleanliness, aftertaste, and combustibility. The evaluation standards or specifications referred to GB 15269.4-2011 "Cigars" and YC / T 138-1998 "Sensory Evaluation Methods for Tobacco and Tobacco Products". The evaluation results are shown in Table 1.

[0044] Table 1 Sensory Evaluation Scores of Cigars After Fermentation .

[0045] As shown in Table 1, cigars treated with T5 exhibit reduced harshness, increased aroma, a cleaner and more pleasant aftertaste, and better combustibility, achieving the highest overall score among all treatment options. This indicates that the T5 treatment process has significant advantages in improving cigar quality, maximizing the optimization of sensory quality, and is one of the effective means to improve cigar quality.

[0046] Although some preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of the invention.

[0047] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from the spirit and scope of its inventive concept. Therefore, if these modifications and variations of this invention fall within the scope of the claims of this application and their equivalents, this invention is also intended to include these modifications and variations.

Claims

1. A type of Enterobacter cholerae ( Enterobacter hormaechei XW-01, characterized in that, Its accession number is CCTCC NO: M 20242595.

2. A microbial preparation comprising the Enterobacter holmieae XW-01 of claim 1 and / or its metabolic enzyme products, or comprising the Enterobacter holmieae XW-01 of claim 1 induced by wheat bran.

3. The use of the Enterobacter holmie XW-01 of claim 1 or the biological agent of claim 2 in at least one of the following (1) to (6): (1) Reduce the protein content in tobacco leaves or prepare reagents to reduce the protein content in tobacco leaves; (2) Reduce the total sugar and / or nicotine content of tobacco leaves or prepare reagents to reduce the total sugar and / or nicotine content of tobacco leaves; (3) Improve the sensory quality of tobacco leaves or prepare a reagent to improve the sensory quality of tobacco leaves, wherein the sensory quality includes at least one of aroma, off-flavor, aroma quantity, mellowness, sweetness, irritation, cleanliness, aftertaste, and combustibility; (4) Used in combination with wheat bran or prepared as a reagent for use with wheat bran to improve protease activity; (5) Improve cigarette quality or prepare reagents to improve cigarette quality; (6) Preparation of protease.

4. The method for preparing the microbial preparation according to claim 2, characterized in that, Includes the following steps: (1) Activation of strain: The Enterobacter holmie strain XW-01 described in claim 1 is inoculated onto NA solid medium and cultured at 33~37 ℃ for 48~96 h; (2) Seed culture preparation: scrape the bacterial cells obtained from the previous step, inoculate them into NA liquid culture medium, and culture them in a shaker at 33~37℃ and 160~200 r / min for 10~15 h to obtain the seed culture. (3) Large-scale culture: Inoculate the seed liquid into the fermentation medium at an inoculation rate of 2-4%, and culture it in a shaker at a temperature of 33-37℃ and a rotation speed of 160-200r / min for 46-50 h; (4) Preparation of microbial agent: Centrifuge the fermentation broth from the previous step and take its supernatant.

5. The preparation method according to claim 4, characterized in that, The fermentation medium contains: wheat bran 8~30 g / L, glucose 8~12 g / L, and NaCl 3~7 g / L.

6. A method for processing tobacco leaves, characterized in that, Includes the following steps: (1) Adjust the moisture content of the tobacco leaves to be treated to 25-30%; (2) Spray the microbial preparation described in claim 2 onto the tobacco leaves at 20-30% of the weight of the tobacco leaves to be treated, then seal the leaves and ferment them at 43-47°C and 70-80% humidity for 8-12 days. (3) After fermentation, the product is dried and the moisture content is balanced.

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