Application of enterobacter hormaechei XW-01 strain in tobacco quality improvement

Abstract

The invention relates to application of enterobacter hormaechei XW-01 in tobacco quality improvement. The XW-01 and a preparation thereof can be used for reducing the content of cellulose, total sugar and nicotine in cigars, increasing the tension value, improving the sensory quality (such as irritation, offensive odor and the like) of tobacco stems and improving the quality of cigarettes. The preparation method of the microbial preparation comprises the steps of strain activation, seed solution preparation, amplification culture, microbial agent preparation and the like. In addition, the invention further discloses a tobacco leaf processing method which comprises the steps of adjusting the water content of the tobacco leaves, spraying the microbial preparation, then sealing and fermenting, drying, balancing the water and the like. The tobacco leaf treatment cost is low, the treatment period is short, safety and reliability are achieved, the cellulose content in the tobacco leaves can be effectively reduced, the tobacco leaf quality is effectively improved, and the wide and practical significance is achieved for improving the cigarette quality and increasing the economic benefits of enterprises.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of tobacco processing, in particular to the application of Enterobacter cloacae XW-01 in improving the quality of tobacco. BACKGROUND

[0002] Cellulose is a linear polysaccharide formed by multiple glucose units connected by β-1,4-glucosidic bonds. As the main structural material of the cell wall in plant cells, cellulose forms a three-dimensional network structure with hemicellulose, pectin and lignin in the form of microfibrils through hydrogen bonds and covalent bonds in the tissue of cigar tobacco leaves. This organizational structure directly or indirectly affects the burnability and mechanical strength of cigar tobacco leaves, and the high combustion of cellulose will produce a pungent and irritating taste, which will seriously affect the sensory quality of cigar. The cellulose content in cigar tobacco leaves varies depending on the growth position. According to research, the cellulose content in lower leaves is between 10-15%, in middle leaves is between 5-10%, and in upper leaves is between 8-12%. The enrichment of cellulose (15%) in cigar tobacco leaves leads to a number of negative effects during the burning process: ① Sensory impact: During high-temperature pyrolysis, cellulose and lignin pyrolysis generate guaiacol, producing woody and grassy flavors that mask the natural aroma of cigar; ② Burnability: Cellulose is difficult to burn completely, which can cause the cigar to burn unevenly and go out during the burning process; ③ Health risks: During cigar burning under high-temperature and anaerobic conditions (such as cigar smoldering), CO (10-50 mg per stick) is produced, and through decarboxylation and dehydration, carcinogens such as formaldehyde (0.1-1 mg per stick) and polycyclic aromatic hydrocarbons (10-100 ng per stick) are produced.

[0003] On the one hand, as the audience group of cigar expands, there is an increasing demand for the quality and safety of cigar tobacco, and on the other hand, cigar manufacturers also have an urgent need for the availability of cigar tobacco. Therefore, research and development of new cellulose degradation technology in the processing of cigar tobacco have wide and practical significance for improving the quality and industrial availability of cigar.

[0004] The information disclosed in this BACKGROUND section is only for the purpose of enhancing the understanding of the background of the present disclosure and should not be taken as admitting that such information is prior art known to those of ordinary skill in the art. SUMMARY

[0005] The present application relates to the technical field of tobacco processing, in particular to the application of Enterobacter cloacae XW-01 in improving the quality of tobacco.

[0006] In one aspect of the present disclosure, Enterobacter hormaechei (CCTCC NO: M20242595) Enterobacter hormaechei application of XW-01 or its metabolic enzyme product in at least one of the following (1)~(7): (1) degrading cellulose in tobacco leaves or preparing a preparation for degrading cellulose in tobacco leaves; (2) degrading total sugar in tobacco leaves or preparing a preparation for degrading total sugar in tobacco leaves; (3) degrading nicotine in tobacco leaves or preparing a preparation for degrading nicotine in tobacco leaves; (4) improving the tensile value of tobacco leaves or preparing a preparation for improving the tensile value of tobacco leaves; (5) improving the sensory quality of tobacco leaves or preparing a preparation for improving the sensory quality of tobacco leaves, the sensory quality including at least one of aroma quality, odor, aroma amount, softness, irritability, aftertaste, strength, and concentration; (6) improving the quality of cigarettes or preparing a preparation for improving the quality of cigarettes; (7) preparing cellulase.

[0007] According to another aspect of the present disclosure, a preparation method of a microbial preparation is provided, comprising the following steps: (1) strain activation: Enterobacter hormaechei XW-01 with a preservation number of CCTCC NO: M20242595 is inoculated on NA solid culture medium by point inoculation method and cultured at 28~32 ℃ for 24~72 h; (2) seed liquid preparation: the bacterial cells obtained from the above step are scraped and inoculated in NA liquid culture medium, and cultured at 28~32 ℃ in a shaker at 160~200 r / min for 10~15 h to obtain a seed liquid; (3) expansion culture: the seed liquid is inoculated in a fermentation medium at an inoculation amount of 2~4%, and cultured at a temperature of 28~32 ℃ and a rotation speed of 160~200 r / min in a shaker for 10~14 h; the fermentation medium contains fructose 9 g / L, tryptone 15 g / L, and MnSO4 7 g / L; (4) microbial agent preparation: the fermentation liquid is centrifuged, and the supernatant is obtained.

[0008] According to still another aspect of the present disclosure, a method for processing cigar tobacco leaves is provided, comprising the following steps: (1) adjusting the water content of the tobacco leaves to be processed to 30~40%; (2) spraying the microbial preparation to the tobacco leaves according to 20~30% of the mass of the tobacco leaves to be processed, and then sealing, fermenting at 33~37 ℃ and 70~80% humidity for 3~5 d; (3) after fermentation, drying and balancing the moisture content, the processing is completed.

[0009] The one or more technical solutions provided in the embodiments of the present application have at least any of the following technical effects or advantages: 1. Effectively reduce cellulose content: The cellulose content of the fermented cigar tobacco after the fermentation of the optimized Enterobacter cloacae XW-01 extracellular crude enzyme solution is significantly reduced. After a certain period of treatment, the cellulose content of the cigar tobacco is greatly reduced, which helps to reduce the production of foul gas or the occurrence of extinguishing phenomenon during the combustion of tobacco leaves, and significantly improves the sensory quality of the cigar.

[0010] 2. Improve the quality of cigar tobacco: With the reduction of cellulose content in cigar tobacco, the unpleasant odor and irritation that may be caused by high cellulose content are effectively reduced, and the aroma quality and taste of cigar tobacco are significantly improved. According to the evaluation of professional smoking team, the unpleasant indicators such as irritation, bitterness and foul gas of the treated cigar tobacco are reduced, while the strength and fineness are improved, and the overall quality is significantly improved.

[0011] 3. Simple operation and easy popularization: The preparation process of the fermentation microbial preparation of Enterobacter cloacae XW-01 is simple and efficient, the fermentation cost is low, and compared with the stacking fermentation, the treatment period is shorter and the safety is higher, which can effectively reduce the cellulose content in cigar tobacco and effectively improve the industrial usability of cigar tobacco, and has good popularization and application value in the field of cigar fermentation production. BRIEF DESCRIPTION OF DRAWINGS

[0012] Figure 1 It is a phylogenetic tree of XW-01 strain based on 16S rDNA gene sequence in the embodiment of the application.

[0013] Figure 2 It is a cellulase activity determination result graph of XW-01 strain after induction treatment in the embodiment of the application.

[0014] Figure 3 It is a cellulose content determination result graph of tobacco leaves treated with microbial agent in the embodiment of the application.

[0015] Figure 4 It is a total sugar content determination result graph of tobacco leaves treated with microbial agent in the embodiment of the application.

[0016] Figure 5 It is a nicotine content determination result graph of tobacco leaves treated with microbial agent in the embodiment of the application.

[0017] Figure 6 It is a tensile value determination result graph of tobacco leaves treated with microbial agent in the embodiment of the application. DETAILED DESCRIPTION

[0018] For better understanding of the technical solutions of the present application, the above technical solutions will be described in detail below in combination with the drawings of the specification and specific embodiments.

[0019] In the following examples, the instruments and equipment are conventional unless otherwise specified; the reagents and media are commercially available unless otherwise specified; the test methods are conventional unless otherwise specified.

[0020] Example 1, screening and identification of Enterobacter hormaechei XW-01 strain (1) Strain screening: 2 g of crushed tobacco leaves (Yunxue No. 1) was weighed into 50 mL of sterile water, and cultured at 30 °C, 180 r•min -1 for 36 h. After the culture solution was diluted 10 -6 , 10 -7 , 10 -8 times, 50 μL was taken and spread on NA solid medium. Single colonies with different morphologies and colors were picked and inoculated into protease selective medium (10 g of skim milk powder, 20 g of agar, 1 L of distilled water, 121 °C high-pressure sterilization for 20 min), and cultured at 30 °C for 36 h. The presence or absence of transparent circles was observed, and the ratio of transparent circle diameter to colony diameter (D / d value) was measured. The D / d value greater than 2.0 was used as the standard for preliminary screening. The D / d value can reflect the ability of the strain to produce protease, and the greater the D / d value, the stronger the ability of the strain to produce enzyme. The strains obtained by preliminary screening were inoculated into NA liquid medium, and cultured at 30 °C, 180 r•min -1 for 36 h, and centrifuged at 8000 r•min -1 for 10 min. The supernatant was taken to measure the protease enzyme activity. The enzyme activity was used as the standard for re-screening.

[0021] (2) Identification of bacterial species: The selected strain XW-01 was inoculated into NA solid culture medium and cultured at 30°C for 24 h. The morphological characteristics of the colonies were observed and the bacterial characteristics were observed under an electron microscope. The strain was identified morphologically and physiologically according to the Bergey's Manual of Bacterial Identification and the Manual of Identification of Common Bacterial Systems. The strain was inoculated into NA solid culture medium and cultured at 30°C and 180 r / min for 24 h. After the culture was completed, 10 µl of the bacterial solution was transferred to a PCR tube, placed in a PCR instrument, heated at 95°C for 7 min, centrifuged at 10,000 r / min and 4°C for 10 min, and the supernatant was aspirated with a pipette as a DNA template. Using genomic DNA from 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× Taq Master Mix, 2 µL of each upstream and downstream primer (10 µmol / L), 2 µL of DNA template, and 19 µL of ddH₂O. The PCR reaction procedure was as follows: 94°C pre-denaturation for 3 min; 30 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 1 min; and finally, 5 min of extension at 72°C; followed by storage at 4°C. PCR products were sequenced by Sangon Biotech (Shanghai) Co., Ltd. and 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 shown).

[0022] Finally, XW-01 was identified as Enterobacter hallii ( Enterobacter hormaechei ), has been deposited in China Center for Type Culture Collection, address: Wuhan University, deposit number: CCTCC NO: M 20242595, deposit date: November 20, 2024.

[0023] Example 2: Preparation of Enterobacter hallii XW-01 bacterial agent (1) Strain activation: The E. coli XW-01 strain was inoculated into NA solid culture medium by the spot inoculation method and cultured in a 30°C incubator for 48 h.

[0024] (2) Preparation of seed solution: scrape the cultured bacteria in step (1), inoculate into NA liquid culture medium, and shake in a shaker at 30°C and 180 rpm for 12 h to obtain seed solution.

[0025] (3) Microbial fermentation liquid preparation: the seed liquid obtained in step (2) was inoculated into NA medium and cellulase-producing liquid medium (fructose 9 g / L, tryptone 15 g / L, MnSO4 7 g / L) at an inoculation amount of 3%, and cultured at a temperature of 30°C and a shaking speed of 180 r / min for 48 h.

[0026] (4) Preparation of extracellular crude enzyme liquid: the fermentation liquid in step (4) was centrifuged at 4°C and 8000 r / min for 10 min, and the supernatant after centrifugation was obtained: T1: extracellular crude enzyme liquid of NA culture solution; T2: extracellular crude enzyme liquid of optimized culture solution.

[0027] The cellulase enzyme activity of each crude enzyme liquid was determined by DNS method, and the results are shown in Figure 2 . As can be seen from the figure, the cellulase activity of Enterobacter cloacae XW-01 after optimization of the culture medium increased by nearly one time compared with NA medium, and the highest cellulase activity after optimization was 16.232 U / ml.

[0028] Example 3, Fermentation treatment of cigar tobacco (1) Preparation of fermentation preparation The crude enzyme preparation obtained in Example 1 and distilled water were used to prepare the following fermentation preparations (by volume percentage): CK: distilled water; T1: extracellular crude enzyme liquid of NA culture solution, 80% distilled water; T2: extracellular crude enzyme liquid of optimized culture solution, 80% distilled water.

[0029] (2) Test treatment The cigar tobacco to be treated was taken, and each fermentation preparation was uniformly sprayed (in the form of spray) according to 35% of the dry weight of the cigar tobacco, and was sealed with double-layer sealed bags, and was fermented at 35°C and 70% RH for 4 d to obtain the cigar tobacco treated with extracellular crude enzyme liquid. The cellulose content was determined to judge the degradation effect, the moisture content was balanced at 26°C and 80% RH for 24 h, and then the tensile force was determined, and the cigar tobacco was rolled for sensory evaluation.

[0030] Cellulose content determination method: the cellulose content was detected by the cellulose content kit of Enzyme-linked Biotechnology Co., Ltd. according to the instruction manual, and the results are shown in Figure 3 .

[0031] Total sugar content determination method: the total sugar content was determined according to the Tobacco Industry Standard YC / T 159-2002 / 2019 "Determination of Water-soluble Sugar in Tobacco and Tobacco Products by Continuous Flow Method", and the results are shown in Figure 4 .

[0032] Nicotine content determination method: according to the tobacco industry standard YC / T 217-2007 "Tobacco and Tobacco Products Potassium Determination Continuous Flow Method" to determine the nicotine content, the results are as follows Figure 5 .

[0033] Tensile value determination method: cut the tobacco leaves into 2*5cm squares, use baosheng technology texture analyzer to replace the tensile detection module, detect the tensile value of tobacco leaves, 5 replicates for each treatment, the results are as follows Figure 6 .

[0034] It can be seen from Figure 3 that compared with the control group, the cellulose content of the tobacco leaves treated by Enterobacter cloacae XW-01 extracellular crude enzyme liquid is reduced, among which the degradation effect of T2 treatment is the best, which is reduced by 28.9%, indicating that the cellulase produced by Enterobacter cloacae XW-01 can effectively reduce the cellulose content in cigar tobacco; in addition, from Figure 4 and Figure 5 , it can be seen that the total sugar and nicotine content also decreases slightly; and after fermentation by Enterobacter cloacae XW-01 extracellular crude enzyme liquid, the tensile value of cigar tobacco is improved to a certain extent compared with before fermentation (as shown in Figure 6 ), from 0.936N to 1.534N, which greatly improves the usability in the process of cigar rolling.

[0035] Example 4, sensory evaluation of fermented cigar tobacco According to the method in example 3, the cigar tobacco was fermented, wherein the tobacco sprayed with equal amount of sterile water was marked as CK, and the extracellular crude enzyme liquid produced by the induction culture of different liquid fermentation media (T1, T2) in example 3 was sprayed and treated, which was marked as T1 and T2 respectively.

[0036] Sensory evaluation method: after the extracellular crude enzyme liquid treatment, the cigar was rolled into a cigarette, and after the moisture was balanced at 26±1℃, relative humidity 80±2% for 24h, the sensory quality of aroma quality, offensive odor, aroma amount, softness, irritation, aftertaste, strength, concentration of 8 aspects was evaluated by the organization of experts (experts according to the current cigar evaluation standard to evaluate cigar, among which aroma quality is the quality of aroma, aroma amount is the amount of aroma in smoke, offensive odor is the smell in smoke that makes people feel unpleasant, irritation is the stimulation of smoke to oral cavity and throat, aftertaste refers to the quality of residual taste in the mouth after smoke, strength refers to the satisfaction of physiological strength of evaluation, concentration refers to the concentration of smoke after discharge, softness refers to the overall feeling when the smoke enters the mouth). The evaluation results are shown in Table 1.

[0037] Table 1 Sensory evaluation score table of fermented cigar .

[0038] From Table 1, the use of the optimized medium (T1, T2) induced by Enterobacter hormaechei XW-01 extracellular crude enzyme solution treatment of cigar tobacco, after the test verification, achieved the following significant technical effects: 1. Significantly reduce the cellulose content: the cellulose content of the treated cigar tobacco is effectively reduced (in the example, reduced by 28.9%), which improves the processing performance of the tobacco leaves and reduces the extinguishing phenomenon during the rolling process.

[0039] 2. Overall improve the sensory quality of cigar: Taking the optimal T2 treatment scheme as an example: the total score of sensory evaluation is the highest (49.5 points, significantly improved compared with 41.5 points before treatment).

[0040] Key indicators are improved: the irritability is significantly reduced (score 6.5 vs. 4.5 before treatment), the offensive odor is significantly reduced (score 6.0 vs. 4.0 before treatment), the aftertaste is cleaner and more comfortable, the softness is improved (score 7.0 vs. 6.0 before treatment), and the strength is moderately increased (score 7.0 vs. 5.5 before treatment). The aroma quality, aroma amount and concentration remain stable. The T2 treatment scheme can maximize the optimization of the aroma quality and taste of cigar and reduce the adverse feelings.

[0041] 3. Verify the practicality and superiority of the technical scheme: the T2 treatment scheme has the best effect, which fully proves that the Enterobacter hormaechei XW-01 fermentation preparation and its application method of the present application have significant advantages in effectively degrading cellulose and improving the quality of cigar tobacco, and also have the characteristics of simple operation, low cost, short cycle, safety and environmental protection, and high popularization and application value.

[0042] In summary, the T2 treatment scheme successfully achieves effective degradation of cellulose in cigar tobacco (reduced by 28.9%), significantly improves the sensory quality (the total score is increased to 49.5 points, and the key adverse indicators are significantly improved), and has environmental protection and economy, which fully verifies its technical superiority and industrial application potential; the treated cigar has reduced irritability, increased strength, clean and comfortable aftertaste, improved softness, and the highest comprehensive score, indicating that the T2 treatment process is an effective means to optimize the sensory quality of cigar and improve the quality.

[0043] Although some preferred embodiments of the present application have been described, those skilled in the art can make additional changes and modifications to these embodiments once they know the basic inventive concept. Therefore, the appended claims are intended to include the preferred embodiments and all changes and modifications falling within the scope of the present application.

[0044] It will be apparent to those skilled in the art that various modifications and variations can be made to the disclosed embodiments without departing from the spirit or scope of the application. Thus, it is intended that the present application cover the modifications and variations of this application provided they come within the scope of the appended claims and their equivalents.

Claims

1. Enterobacter hallii with a deposit number of CCTCC NO: M20242595 ( Enterobacter hormaechei ) Use of XW-01 or its metabolite products in at least one of the following (1) to (7): (1) Degrading cellulose in tobacco leaves or preparing a preparation for degrading cellulose in tobacco leaves; (2) Degrading total sugar in tobacco leaves or preparing a preparation for degrading total sugar in tobacco leaves; (3) Degrading nicotine in tobacco leaves or preparing preparations for degrading nicotine in tobacco leaves; (4) Improving the tensile strength of tobacco leaves or preparing preparations that improve the tensile strength of tobacco leaves; (5) Improving the sensory qualities of tobacco leaves or preparing preparations for improving the sensory qualities of tobacco leaves, wherein the sensory qualities include at least one of aroma quality, miscellaneous odors, aroma quantity, softness, pungency, aftertaste, strength, and concentration; (6) Improving the quality of cigarettes or preparing preparations that improve the quality of cigarettes; (7) Preparation of cellulase.

2. A method for preparing a microbial preparation, characterized in that: The steps include: (1) Activation of bacteria: Enterobacter hallii XW-01 with the deposit number CCTCC NO: M20242595 was inoculated on NA solid culture medium by the spot inoculation method and cultured at 28-32°C for 24-72 hours; (2) Seed solution preparation: scrape the bacterial cells obtained in the previous step, inoculate them into NA liquid medium, and culture them in a shaking incubator at 28-32°C and 160-200 rpm for 10-15 h to obtain the seed solution; (3) Expanding the culture: inoculate the seed solution into a fermentation medium at an inoculum rate of 2-4%, and culture in a shaker at a temperature of 28-32°C and a rotation speed of 160-200 r / min for 10-14 h; the fermentation medium contains 9 g / L fructose, 15 g / L tryptone, and 7 g / L MnSO4; (4) Preparation of bacterial agent: Centrifuge the fermentation liquid from the previous step and take the supernatant.

3. A method for processing cigar tobacco leaves, characterized in that: The following steps are involved: (1) Adjust the moisture content of the tobacco leaves to be processed to 30-40%; (2) Spray the microbial preparation described in claim 2 onto the tobacco leaves in an amount of 20-30% by mass of the tobacco leaves to be treated, seal the leaves, and ferment them at 33-37°C and 70-80% humidity for 3-5 days; (3) After fermentation is completed, it is dried and the moisture is balanced.

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