Use of pantoea B-3 for degrading cellulose in tobacco

Abstract

The application discloses application of Pantoea B-3 in degrading cellulose in tobacco, and aims to solve the technical problems that cellulose in tobacco is difficult to degrade and there is lack of effective microbial method to improve tobacco quality. The Pantoea B-3 strain is isolated and screened from a cigar, and the preservation number is CCTCC NO: M 20221165. Metabolic enzyme products of the Pantoea B-3 strain can reduce the cellulose content in tobacco, improve the stimulation and bad smell in the smoking process of tobacco, and improve the safety of tobacco smoking. The process flow of the application is simple and efficient, the cost of tobacco treatment is low, the treatment period is short, the application is safe and reliable, the cellulose content in tobacco can be obviously reduced, and the quality of tobacco can be improved, and the application has good popularization and application value.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of bioengineering technology, and in particular to an application of Pantoea B-3 in degrading cellulose in tobacco. BACKGROUND

[0002] Cellulose in tobacco, together with hemicellulose, lignin, pectin and other substances, constitutes the cell wall structure of tobacco stem. The total cell wall material content in tobacco leaves is about 20-30%, and even accounts for more than 1 / 3 of the total mass in tobacco stems and tobacco sheets. Studies have shown that the content of cellulose is closely related to the quality of tobacco stems. If the content of cellulose is too high, it will produce a sharp stimulating sensation and an unpleasant odor similar to paper burning during smoking, thereby adversely affecting the quality of cigarettes. In addition, a large amount of cellulose will thermally crack to generate polycyclic aromatic hydrocarbons (PAH) at a temperature of more than 600°C, affecting the safety of tobacco consumption.

[0003] In order to improve the quality of tobacco and enhance the quality of raw materials for cigarettes, it is necessary to effectively degrade the cellulose in tobacco to keep its content within a reasonable range. In addition, the inventors have found that the structural properties of cellulose are relatively stable and it is difficult to degrade during the natural aging process of tobacco. Therefore, the research and development of new technologies for degrading cell wall materials in tobacco have broad and practical significance for improving the quality of cigarettes, saving production costs, and increasing economic benefits for enterprises.

[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 considered as admitting that the information constitutes prior art known to those skilled in the art. SUMMARY

[0005] The inventors have found that the Pantoea B-3 strain isolated and screened from cigars can selectively secrete cellulase through selective medium induction culture, and its enzyme preparation can selectively degrade cellulose in tobacco and effectively improve the quality of tobacco / tobacco. Pantoea sp.

[0006] According to one aspect of the present disclosure, Pantoea B-3 with a preservation number of CCTCC NO: M 20221165 or its biological preparation is applied in the degradation of cellulose in tobacco / tobacco.

[0007] According to another aspect of the present disclosure, Pantoea B-3 with a preservation number of CCTCC NO: M 20221165 is applied in the preparation of a biological preparation for degrading cellulose in tobacco / tobacco.

[0008] In some embodiments of the present disclosure, the above-mentioned method for preparing a biological preparation includes the following steps:

[0009] ​(1) Activating the bacteria: the Pantoea B-3 strain with the preservation number of CCTCC NO: M 20221165 is inoculated on NA solid culture medium and cultured at 25-35°C for 1-2 days;

[0010] (2) Preparing the seed liquid: the bacteria cultured in step (1) are scraped and inoculated in liquid NA culture medium, and cultured at 25-40°C with 140 r / min-200 r / min shaking for 24-72 hours to obtain the seed liquid;

[0011] (3) Expanding the culture: the seed liquid in step (2) is inoculated in the cellulase-producing selective induction liquid culture medium at a proportion of 2-4% of the weight of the culture medium, and cultured at a temperature of 25-35°C with a shaking speed of 180 r / min-220 r / min for 12-36 hours;

[0012] (4) Preparing the biological preparation: the supernatant of the culture medium after the culture in step (3) is partially filtered through a ceramic membrane, and then partially filtered through an ultrafiltration membrane. The obtained crude enzyme liquid is diluted with sterile water at a volume ratio of 1:8-10 to obtain the biological preparation.

[0013] In some embodiments of the present disclosure, the NA solid culture medium in step (1) comprises, in g / L, beef extract 2.8-3.2, protein peptone 9.0-11.0, NaCl 4.5-5.5, and agar 18-22, and has a pH of 6.5-7.0 and is sterilized at 110-125°C for 15-25 minutes.

[0014] In some embodiments of the present disclosure, the liquid NA culture medium in step (2) comprises, in g / L, beef extract 2.8-3.2, protein peptone 9.0-11.0, NaCl 4.5-5.5, and has a pH of 6.5-7.0 and is sterilized at 110-125°C for 15-25 minutes.

[0015] In some embodiments of the present disclosure, the expansion culture medium in step (3) comprises carboxymethylcellulose sodium 15.0 g / L, protein peptone 5.0 g / L, yeast powder 10.0 g / L, KH2PO4 1.5 g / L, MgSO4•7H2O 0.25 g / L, NaCl 5.0 g / L, and has a pH of 7.5.

[0016] According to still another aspect of the present disclosure, the Pantoea B-3 with the preservation number of CCTCC NO: M 20221165 is applied to the preparation of cellulase.

[0017] According to another aspect of the present disclosure, a tobacco leaf processing method is provided, comprising the following steps:

[0018] (1) The biological agent is uniformly added to the tobacco leaves / tobacco shreds in a mass ratio of 1:2-4 with the tobacco leaves / tobacco shreds;

[0019] (2) The moisture content of the tobacco leaves / tobacco shreds is adjusted to 24-26%, and the tobacco leaves / tobacco shreds are sealed and placed for 30-40 h under the conditions of a temperature of 34-36 ℃ and a humidity of 70-80%;

[0020] (3) The tobacco leaves / tobacco shreds are dried at 70-80 ℃ for 10-20 min, and the tobacco leaves / tobacco shreds are obtained.

[0021] The one or more technical solutions provided in the embodiments of the present application have at least the following any technical effect or advantage:

[0022] 1. Pantoea (Pantoea) Pantoea sp. ) B-3, the metabolites of which can effectively reduce the content of cellulose in tobacco, improve the irritation and bad smell in the smoking process of tobacco, and improve the safety of tobacco smoking.

[0023] 2. A simple biological agent processing method for tobacco is designed, which has the technical advantages of simple and efficient process flow, low processing cost, short processing period, safety and reliability, can obviously reduce the content of cellulose in tobacco and improve the quality of tobacco leaves / tobacco shreds, and thus has good popularization and application value in the field of cigarette manufacturing technology. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 The regression variable analysis of the cellulase enzyme activity of the strain B-3 in an embodiment of the present application is carried out. DETAILED DESCRIPTION

[0025] In order to better understand 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.

[0026] In the following examples, the instruments and equipment are conventional unless otherwise specified; the reagents and culture media are commercially available unless otherwise specified; and the test methods are conventional unless otherwise specified.

[0027] The Pantoea B-3 strain involved in the following examples is classified and named as Pantoea sp., which has been preserved in the China Center for Type Culture Collection on July 25, 2022, with a preservation number of CCTCC NO: M 20221165, and the screening and identification process thereof is described in patent application CN202211161758.4. Pantoea

[0028] Example One: Preparation of Pantoea B-3 Strain Biological Agent

[0029] ​The B-3 strain screened is used to degrade cellulose in tobacco shreds, and the strain is first prepared into a biological preparation for easy use, and the preparation process is as follows:

[0030] (1) Activate the strain: the preserved Pantoea B-3 strain is inoculated on NA solid medium (containing beef extract 3.0, peptone 10.0, NaCl 5.0, agar 20, and pH=7.0, sterilized at 121°C for 20 min) by point inoculation, and cultured at 30°C for 2 days.

[0031] (2) Prepare the seed liquid: scrape the cultured bacteria in step (1) in a clean bench, inoculate in an 18x180 test tube containing 5mL liquid NA medium (containing beef extract 3.0, peptone 10.0, NaCl 5.0, pH 6.8, sterilized at 121°C for 20 min), and shake culture at 30°C, 140-200 r / min (optimum 170 r / min) for 36h to obtain the seed liquid.

[0032] (3) Scale up the culture: inoculate the seed liquid in step (2) into a cellulase-producing special liquid medium (medium composition: carboxymethyl cellulose sodium (CMC-Na) 15.0 g / L, peptone 5.0 g / L, yeast powder 10.0 g / L, KH2PO4 1.5 g / L, MgSO4•7H2O 0.25 g / L, NaCl 5.0 g / L, and pH=7.5) at 3% of the medium weight, and shake culture at 34°C and 200 r / min for 24h to make the OD 600 of the bacterial liquid reach 2.3.

[0033] (4) Prepare the biological preparation: collect the supernatant after the enzyme-producing medium in step (3) is scaled up, and partially filter the supernatant through a ceramic membrane (filtration precision 0.2μm, ceramic membrane outer diameter 12mm, inner diameter 8mm, length 300mm), and then partially filter it through a 1812 ultrafiltration membrane (molecular weight cut-off 5000 daltons, filtration area 0.4m 2 ), to obtain a crude enzyme liquid, and then dilute it with sterile water at a dilution ratio of 1:9 to obtain the biological preparation.

[0034] (5) Pantoea B-3 biological preparation treatment of tobacco shreds: add the Pantoea B-3 biological preparation in step (4) to the tobacco shreds in a mass ratio of 1:3 by spraying, then adjust the moisture content of the tobacco shreds to 24-26%, seal the tobacco shreds in a sealed bag, and place them at a temperature of 35°C and a humidity of 75% for 36h to form the biological enzyme-treated tobacco shreds. Dry the tobacco shreds at 75°C for 15min to inactivate the biological preparation.

[0035] Example 2: Pantoea B-3 strain culture condition optimization

[0036] 1. A multi-factor multi-gradient combination test was set up, respectively: culture temperature (30℃, 32℃, 34℃, 36℃); culture time: (12 h, 24 h, 36 h, 48 h); shaking speed (160 r / min, 180 r / min, 200 r / min, 220 r / min). As shown in Table 1.

[0037] Table 1 Test design

[0038]

[0039] 2. Cellulase activity determination: filter paper activity (FPA) method was used to determine cellulase activity. Excel was used to design orthogonal test and data analysis, and R language was used to draw the graph.

[0040] 3. The results are as follows: Figure 1 It can be seen that in the Pantoea B-3 strain cellulase production induction test, when the culture temperature is 34℃, the culture time is 24 h, and the shaking speed is 200 r / min, the strain produces the highest cellulase activity, which is 22.61 U / mL.

[0041] Example 3: Tobacco fermentation cellulose degradation effect and sensory evaluation results

[0042] 1. Tobacco treatment: the original tobacco is marked as 1#; the tobacco added with equal amount of sterile water in the tobacco processing process is marked as 2#; the tobacco added with Pantoea B-3 for selective induction of cellulase production is marked as 3#.

[0043] 2. Tobacco sensory quality evaluation: QB2583-2003 is the standard followed for determining cellulase activity, and cellulase activity is defined as the amount of enzyme required to produce 1 mg of glucose per minute at 50℃ and pH=5.0; YC / T138-1998 standard is referred to for tobacco sensory quality evaluation.

[0044] (1) Sample rolling: before rolling, the tobacco treated by various fermentation processes was balanced to 14-16% moisture content according to GB / T 16447-2004 requirements.

[0045] If there is no special requirement, it is uniformly prepared as follows: length: 90mm, diameter: 15mm (or circumference: 47.1mm); cigarette weight: 4.05-4.15g; after the completion of cigarette rolling, the sample moisture content was balanced to 13.0-15.0% according to GB15269-2011 requirements.

[0046] (2) See Table 2 for tobacco sensory quality index scoring scale.​

[0047] Table 2 Sensory quality index score scale of tobacco leaf

[0048] .

[0049] (3) Evaluation requirements:

[0050] ① The sensory panelists should not eat spicy or stimulating food and should not drink alcohol before sensory evaluation.

[0051] ② The sensory panelists should be in good physical condition, and the evaluation environment should be quiet, ventilated, odorless, and undisturbed.

[0052] ③ The fire source generally requires a butane gas lighter or an alcohol lamp, and a wax-free and odorless match can also be used as a fire source.

[0053] ④ The standard sample should be used to calibrate the sensory panelists before evaluation.

[0054] ⑤ The evaluation method of cigar is the local circulation method, mainly evaluating the third to two-thirds of the cigarette.

[0055] ⑥ During the sensory evaluation process, light fruits or mineral water can be used to adjust and clean the oral cavity.

[0056] ⑦ At least 5 minutes should be paused between different evaluation tasks to rest the oral cavity and nasal cavity and ensure their sensitivity.

[0057] ⑧ If the body is not comfortable and the sensory function is not normal during the evaluation process, the panelist should withdraw from the evaluation.

[0058] ⑨ The sensory evaluation meeting organizer prepares the samples, standard samples, and "Cigar Tobacco Sensory Evaluation Form" (see Table 3) for the panelists, and the panelists record the data and fill in the evaluation opinions according to the requirements of the evaluation form. The scale value is recorded in the scale value column, and the scale value is divided into five grades with a gradient of 0.5.

[0059] ⑩ When evaluating aroma, odor, and taste, if the sample has characteristics beyond the given indicators, describe the aroma and taste characteristics in other columns and record the scale value.

[0060] ⑪ The overall evaluation of the sample is described by the group leader after organizing the discussion based on the statistical results.

[0061] ⑫ Samples with serious quality defects are not evaluated, including:

[0062] ---- There is obvious odor or mold;

[0063] ---- The burning is extinguished.

[0064] 3. The results are as follows: as shown in Table 3, the cellulose content of the 3# treatment is lower than that of the 1# and 2# treatments, and the degradation rate of cellulose in the cut tobacco reaches 50.98%, indicating that the content of cellulose in the cut tobacco can be effectively reduced after treatment with the Pantoea B-3 bacterial agent.

[0065] Table 3. Cellulose content detection results after treatment

[0066] .

[0067] As shown in Table 4, after the cut tobacco is treated with the Pantoea B-3 enzyme preparation (3#), the aroma quality is better, the aroma amount is increased, the offensive odor is reduced, the aftertaste is improved, and the overall sensory quality score of the cut tobacco is significantly higher than that of the blank group (1#) and the control group (2#).

[0068] Table 4. Sensory quality evaluation score table of treated cut tobacco

[0069] .

[0070] 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 be interpreted as including all the preferred embodiments and all the changes and modifications falling within the scope of the present application.

[0071] Obviously, those skilled in the art can make various modifications and variations to the present application without departing from the spirit and scope of the application. Thus, if these modifications and variations fall within the scope of the claims of the present application and their equivalent technologies, the present application also intends to include these modifications and variations.

Claims

1. Pantoea agglomerans (ATCC 29827) with the preservation number CCTCC NO: M 20221165 Pantoea sp. ) B-3 or a biological preparation thereof in degrading cellulose in tobacco leaves / tobacco shreds.

2. Pantoea sp. CCTCC NO: M 20221165 Pantoea sp. ) B-3 for use in the preparation of a biological agent that degrades cellulose in tobacco leaves / tobacco shreds.

3. Use according to claim 2, characterized in that, The method comprises the following steps: (1) activating the bacterial strain: inoculate the Pantoea B-3 strain with the preservation number CCTCC NO: M 20221165 on NA solid culture medium, and culture at 25-35°C for 1-2 days; (2) preparing the seed liquid: scrape the bacterial body cultured in step (1), inoculate in liquid NA culture medium, and culture at 25-40°C with 140-200 r / min shaking for 24-72 hours to obtain the seed liquid; (3) expanding the culture: inoculate the seed liquid in step (2) in the cellulase-producing selective induction liquid culture medium at 2-4% of the weight of the culture medium, and culture at 25-35°C with 180-220 r / min shaking for 12-36 hours; (4) preparing the biological preparation: take the supernatant of the culture medium after the culture in step (3), partially filter the supernatant through a ceramic membrane, and then partially filter through an ultrafiltration membrane, dilute the obtained crude enzyme liquid with sterile water at a volume ratio of 1:8-12 to obtain the biological preparation.

4. Use according to claim 3, characterized in that, In the step (1), the NA solid culture medium comprises, in g / L, beef extract 2.8-3.2, protein peptone 9.0-11.0, NaCl 4.5-5.5, and agar 18-22, with pH 6.5-7.0, and sterilized at 110-125°C for 15-25 minutes.

5. Use according to claim 3, characterized in that, In the step (2), the liquid NA culture medium comprises, in g / L, beef extract 2.8-3.2, protein peptone 9.0-11.0, NaCl 4.5-5.0, and pH 6.5-7.0, and sterilized at 110-125°C for 15-25 minutes.

6. Use according to claim 3, characterized in that, In the step (3), the expansion culture medium comprises carboxymethyl cellulose sodium 15.0 g / L, protein peptone 5.0 g / L, yeast powder 10.0 g / L, KH2PO4 1.5 g / L, MgSO4·7H2O 0.25 g / L, NaCl 5.0 g / L, and pH=7.

5.

7. Application of the Pantoea B-3 with the preservation number CCTCC NO: M 20221165 in preparing cellulase.

8. A tobacco leaf / tobacco shred processing method, comprising the following steps: (1) adding the biological preparation in claim 2 to the tobacco leaf / tobacco shred at a mass ratio of 1:2-4; (2) then adjusting the moisture content of the tobacco leaf / tobacco shred to 24-26%, and placing the tobacco leaf / tobacco shred in a sealed state at a temperature of 34-36°C and a humidity of 70-80% for 30-40 hours; (3) drying the tobacco leaf / tobacco shred at 70-80°C for 10-20 minutes, and the process is completed.

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