Method for improving tobacco leaf quality through fermentation of lactobacillus plantarum

Abstract

The invention discloses a method for improving tobacco leaf quality by utilizing lactobacillus plantarum fermentation, and relates to the technical field of biological fermentation, the method comprises the following steps: step 1, inoculating lactobacillus plantarum into an MRS culture medium for activation culture; step 2, inoculating the activated lactobacillus plantarum into a tobacco powder fermentation culture medium, and simulating a tobacco leaf fermentation environment for culture to realize adaptive domestication of the strain to a target environment; step 3, centrifuging the fermentation liquor to remove supernate, resuspending with sterile water, and adjusting the concentration of thalli to obtain a high-density and high-activity lactobacillus plantarum biological agent; step 4, adding a lactobacillus plantarum biological agent into sterile water, uniformly mixing, uniformly spraying on the tobacco leaves, sealing, and fermenting; and drying the treated tobacco leaves to obtain the fermented tobacco leaves. The lactobacillus plantarum can be metabolized to generate various flavor substances harmonious with smoke in the tobacco leaf fermentation process. After the tobacco leaves are fermented by lactobacillus plantarum, the quality is obviously improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological fermentation, and particularly relates to a method for improving the quality of tobacco leaves by fermentation with lactobacillus plantarum. BACKGROUND

[0002] The aging process of tobacco leaves, i.e. fermentation of tobacco leaves, is carried out under specific temperature and humidity conditions, which significantly affects the physicochemical properties of tobacco leaves, thereby significantly improving the aroma, color and overall quality of tobacco leaves, and is an indispensable part of cigarette manufacturing. Tobacco aging technology is divided into natural aging and artificial aging. Artificial aging can accelerate the process, but often leads to an imbalance in the proportion of tobacco components, so its application has gradually decreased. In contrast, natural aging can bring better fermentation results, but this process takes a long time, occupies a large space and requires a lot of funds, thereby increasing the production cost. In addition, tobacco leaves are susceptible to mold and insect damage during long-term storage, causing economic losses to tobacco companies.

[0003] During the aging process, the microbial community on the surface of tobacco leaves is active, which absorbs the nutrients of tobacco leaves during the metabolic process and secretes various enzymes to degrade macromolecular substances such as proteins, cellulose, starch, etc. into small molecular substances such as amino acids, alcohols, esters, organic acids, etc., or activate the metabolism of other enzyme systems on the surface of tobacco leaves, which ultimately affects the smoking quality of cigarettes. By simulating the natural aging process, specific microorganisms are inoculated on tobacco leaves for fermentation, which can optimize the proportion of chemical components of tobacco leaves, increase tobacco aroma and smoke, reduce irritation, improve the taste of smoking, and significantly improve the quality of tobacco leaves.

[0004] Currently, the microorganisms used for tobacco fermentation are mostly derived from the surface of tobacco leaves or soil, some of which may be conditional pathogenic bacteria, posing a safety hazard. Lactic acid bacteria, as a food-grade safe microorganism, have been widely used in the food industry, such as yogurt, fruit juice, steamed buns, etc., to create unique flavors. Lactic acid bacteria can also inhibit the growth of pathogenic bacteria and fungi by secreting metabolic products such as lactic acid and bacteriocin. Therefore, the application of food-derived lactic acid bacteria in tobacco fermentation can not only shorten the fermentation period, improve the quality of tobacco leaves and smoking quality, but also reduce the potential risk of smoking, showing great potential in the cigarette manufacturing industry.

[0005] Lactobacillus plantarum, an important lactic acid bacterium, is a Gram-positive bacterium with diverse morphologies, appearing as straight or curved rods, and often existing singly, in pairs, or in chains. This species is a facultative anaerobe, with an optimal growth temperature of 30-35℃. It can grow in various environments and ferments in gluconate to produce lactic acid, while also exhibiting the ability to inhibit pathogenic bacteria. This study selected Lactobacillus plantarum strains with aroma-producing properties screened from food to explore their impact on the sensory quality of tobacco leaves under specific conditions, aiming to improve tobacco quality and provide a scientific basis for the application of Lactobacillus plantarum in tobacco production. Summary of the Invention

[0006] 1. The technical problem to be solved: Current technology lacks a safe method for tobacco fermentation to improve tobacco quality and smoking quality while reducing the potential risks of smoking.

[0007] To address the aforementioned technical problems, this invention provides a method for improving tobacco leaf quality through fermentation using Lactobacillus plantarum.

[0008] 2. Technical Solution: A method for improving tobacco quality using Lactobacillus plantarum fermentation includes the following steps: Step 1: Inoculate Lactobacillus plantarum into MRS medium for activation culture; Step 2: Inoculate the activated Lactobacillus plantarum into the tobacco fermentation medium and cultivate it in a manner that simulates the tobacco fermentation environment, so as to achieve the adaptation and domestication of the strain to the target environment; Step 3: Centrifuge the fermentation broth to remove the supernatant, resuspend it in sterile water, adjust the cell concentration, and obtain a high-density, high-activity Lactobacillus plantarum biological agent. Step 4: Add the Lactobacillus plantarum biological agent to sterile water and mix well. Spray evenly onto the tobacco leaves, seal well, and then ferment. Dry the treated tobacco leaves to obtain fermented tobacco leaves.

[0009] Preferably, the *Lactobacillus plantarum* is screened from dairy products and deposited at the China Industrial Microbial Culture Collection Center, with accession number CICC 24936.

[0010] Furthermore, the preparation method of MRS medium is as follows: Take 10 g of casein peptone, 8 g of beef extract powder, 4 g of yeast extract powder, 20 g of glucose, 1 g of Tween 80, 5 g of sodium acetate, 2 g of diammonium hydrogen citrate, 2 g of K2HPO4, 0.2 g of MgSO4·7H2O, and 0.04 g of MnSO4·H2O into an Erlenmeyer flask, add 1000 mL of distilled water and heat until completely dissolved, adjust the pH value to 5.7±0.2, and autoclave (101 kPa, 121 °C) for 15 minutes.

[0011] Furthermore, in step 1, the *Lactobacillus plantarum* is inoculated into MRS medium and activated for 1-2 days at a temperature of 37°C and a shaking speed of 180 rpm.

[0012] Furthermore, the formula for the tobacco powder fermentation medium in step 2 is as follows: tobacco powder 3.0 g / L, yeast powder 5.0 g / L, NaCl 10 g / L, pH=7.0.

[0013] Furthermore, in step 2, the activated Lactobacillus plantarum was inoculated into the tobacco fermentation medium and fermented under the following conditions: 35-38℃, 150 rpm in a shaker for 24 hours.

[0014] Furthermore, in step 3, the centrifugation speed for collecting the cells in the fermentation broth is 8000-12000 rpm, and the centrifugation time is 5-15 min.

[0015] Furthermore, in step 3, the bacterial cell concentration was measured using a spectrophotometer, and the bacterial cell concentration was adjusted to 10. 8 cfu / mL.

[0016] Furthermore, in step 4, the amount of Lactobacillus plantarum biological agent added is 0.05-0.1% of the weight of tobacco leaves, and the amount of sterile water is 10% of the weight of tobacco leaves.

[0017] Furthermore, in step 4, the tobacco leaves are fermented under sealed conditions at 35-38℃ and RH 50-60% for 24-36 hours.

[0018] Furthermore, in step 4, the moisture content of the dried tobacco leaves is 12% w / w, and the drying temperature is 40℃.

[0019] It should be noted that, in addition to the *Lactobacillus plantarum* strain with accession number CICC 24936, other purchased or isolated *Lactobacillus plantarum* strains can also be used in this invention. Besides enhancing aroma, improving taste, and improving cigarette quality, these strains may also bring certain characteristics different from those of *Lactobacillus plantarum* strain with accession number CICC 24936. For other *Lactobacillus plantarum* strains, those skilled in the art can use conventional techniques to adjust the fermentation process.

[0020] 3. Beneficial effects: During the fermentation of tobacco leaves, *Lactobacillus plantarum* can metabolize and generate various aroma compounds that harmonize with the smoke. After fermentation by *Lactobacillus plantarum*, the content of aroma compounds in tobacco leaves increases, and the total amount of acidic, alcoholic, and ester-based aroma compounds increases significantly. Sensory evaluation results show that the aroma of treated tobacco leaves is improved, the aroma quantity and concentration increase, the taste is improved, the sweetness is increased, the off-flavors are reduced, and the quality is significantly improved. This invention utilizes Lactobacillus plantarum to ferment tobacco leaves. The operation is simple, easy to implement, and low in cost, making it suitable for industrial application. Detailed Implementation

[0021] The present invention will now be described in detail.

[0022] The bacterial strains and culture media used in the experiments of this invention are as follows: Lactobacillus plantarum: Screened from dairy products and deposited at the China Industrial Microbial Culture Collection Center, accession number CICC24936; The preparation method of MRS medium is as follows: Take 10g of casein peptone, 8g of beef extract powder, 4g of yeast extract powder, 20g of glucose, 1g of Tween 80, 5g of sodium acetate, 2g of diammonium hydrogen citrate, 2g of K2HPO4, 0.2g of MgSO4·7H2O, and 0.04g of MnSO4·H2O into an Erlenmeyer flask, add 1000mL of distilled water and heat until completely dissolved, adjust the pH value to 5.7±0.2, and autoclave (101Kpa, 121℃) for 15 minutes; The formula for the tobacco powder fermentation medium is as follows: tobacco powder 3.0 g / L, yeast powder 5.0 g / L, NaCl 10 g / L, pH=7.0.

[0023] Example 1 Lactobacillus plantarum biological agents were prepared using the following methods: Step 1: Pick Lactobacillus plantarum from the preservation medium, inoculate it into MRS medium and activate it for 1 day at a temperature of 37°C and a shaking speed of 180 rpm. Step 2: Inoculate the activated Lactobacillus plantarum into the tobacco fermentation medium and ferment it in a shaker at 37°C and 150 rpm for 24 hours; Step 3: Centrifuge the fermentation broth at 12000 rpm for 5 min, remove the supernatant, and collect the bacterial cells; Step 4: Resuspend the bacterial cells collected in Step 3 in sterile water, determine the bacterial cell concentration using a spectrophotometer, and adjust the bacterial cell concentration to 10. 8 The cfu / mL concentration yielded the Lactobacillus plantarum biological agent.

[0024] Example 2 Lactobacillus plantarum biological agents were prepared using the following methods: Step 1: Pick Lactobacillus plantarum from the preservation medium, inoculate it into MRS medium and activate it for 2 days at a temperature of 37°C and a shaking speed of 180 rpm. Step 2: Inoculate the activated Lactobacillus plantarum into the tobacco fermentation medium and ferment it in a shaker at 35℃ and 150rpm for 24 hours; Step 3: Centrifuge the fermentation broth at 8000 rpm for 15 min, remove the supernatant, and collect the bacterial cells; Step 4: Resuspend the bacterial cells collected in Step 3 in sterile water, determine the bacterial cell concentration using a spectrophotometer, and adjust the bacterial cell concentration to 10. 8 The cfu / mL concentration yielded the Lactobacillus plantarum biological agent.

[0025] Example 3 Ferment tobacco leaves using the following methods: Step 1: Weigh 500g of re-dried and aged tobacco leaves; weigh 0.25g of the *Lactobacillus plantarum* biological agent prepared in Example 1; weigh 50g of sterile water; Step 2: Add the weighed Lactobacillus plantarum biological agent to sterile water and mix well. Spray evenly onto the tobacco leaves, seal well, and ferment at 37℃ and 50% RH for 24 hours. Step 3: Dry the processed tobacco leaves at 40℃ until the moisture content is 12% (w / w) to obtain fermented tobacco leaves; Step 4: Cut the fermented tobacco leaves into shreds and roll them into cigarettes, then have them evaluated for sensory quality by professional smokers.

[0026] Example 4 Ferment tobacco leaves using the following methods: Step 1: Weigh 500g of re-dried and aged tobacco leaves; weigh 0.5g of the *Lactobacillus plantarum* biological agent prepared in Example 1; weigh 50g of sterile water; Step 2: Add the weighed Lactobacillus plantarum biological agent to sterile water and mix well. Spray evenly onto the tobacco leaves, seal well, and ferment at 37℃ and RH60% for 36 hours. Step 3: Dry the processed tobacco leaves at 40℃ until the moisture content is 12% (w / w) to obtain fermented tobacco leaves; Step 4: Cut the fermented tobacco leaves into shreds and roll them into cigarettes, then have them evaluated for sensory quality by professional smokers.

[0027] Example 5 Ferment tobacco leaves using the following methods: Step 1: Weigh 500g of tobacco leaves; weigh 0.25g of the biological agent prepared in Example 2; weigh 50g of sterile water; Step 2: Add the weighed Lactobacillus plantarum biological agent to sterile water and mix well. Spray evenly onto the tobacco leaves, seal well, and ferment at 37℃ and 50% RH for 24 hours. Step 3: Dry the processed tobacco leaves at 40℃ until the moisture content is 12% (w / w) to obtain fermented tobacco leaves; Step 4: Cut the fermented tobacco leaves into shreds and roll them into cigarettes, then have them evaluated for sensory quality by professional smokers.

[0028] Example 6 Ferment tobacco leaves using the following methods: Step 1: Weigh 500g of tobacco leaves; weigh 0.5g of the biological agent prepared in Example 2; weigh 50g of sterile water; Step 2: Add the weighed Lactobacillus plantarum biological agent to sterile water and mix well. Spray evenly onto the tobacco leaves, seal well, and ferment at 37℃ and RH60% for 36 hours. Step 3: Dry the processed tobacco leaves at 40℃ until the moisture content is 12% (w / w) to obtain fermented tobacco leaves; Step 4: Cut the fermented tobacco leaves into shreds and roll them into cigarettes, then have them evaluated for sensory quality by professional smokers.

[0029] Comparative Example 1: Ferment tobacco leaves using the following methods: Step 1: Pick Lactobacillus plantarum from the preservation medium, inoculate it into MRS medium and activate it for 1 day at a temperature of 37°C and a shaking speed of 180 rpm. Step 2: Centrifuge the fermentation broth at 8000 rpm for 15 min, remove the supernatant, and collect the bacterial cells; Step 3: Resuspend the bacterial cells collected in Step 2 in sterile water, determine the bacterial cell concentration using a spectrophotometer, and adjust the bacterial cell concentration to 10. 8 cfu / mL; Step 4: Weigh 500g of tobacco leaves; weigh 0.5g of the bacterial solution from Step 3 and mix it with 50g of sterile water, then spray it evenly onto the tobacco leaves, seal it well, and ferment it at 37℃ and 50% RH for 24 hours. Step 5: Dry the processed tobacco leaves at 40℃ until the moisture content is 12% (w / w) to obtain fermented tobacco leaves; Step 6: Cut the fermented tobacco leaves into shreds and roll them into cigarettes. Then, have professional smokers conduct a sensory quality evaluation.

[0030] Experiment 1: Detection of Aroma-Conducing Substances Instrumentation: Simultaneous distillation and extraction apparatus; 7890A / 5975C gas chromatograph / mass spectrometer (Agilent Technologies, USA); HP-5MS capillary column; R114 rotary evaporator (BüCHI, Switzerland).

[0031] Analytical Method: Tobacco leaf samples were pulverized and passed through a 60-mesh sieve to obtain tobacco dust. The tobacco dust was equilibrated in a constant temperature and humidity chamber at 22℃ and 60% humidity for 24 h. Then, 25.0 g of the equilibrated sample was accurately weighed and placed in a simultaneous distillation-extraction apparatus with a certain amount of internal standard added. Dichloromethane was used as the solvent for continuous dynamic extraction of the sample for 2 h. The obtained extract was dried and concentrated to a final volume of 1.0 mL. The concentrated solution was analyzed using gas chromatography / mass spectrometry (GC / MS). The obtained chromatograms were searched using computer spectral libraries and Wiley 275, and the relative content of each peak was calculated using the internal standard correction normalization method.

[0032] Analytical conditions: Capillary column: HP-5MS (30m×0.25mm×0.25μm); Inlet temperature: 250℃; Carrier gas: He, 1mL / min; Temperature program: Initial temperature 80℃, hold for 1 min, increase to 220℃ at 5℃ / min, hold for 3 min, then increase to 280℃ at 8℃ / min, hold for 10 min; Injection volume: 1μL, Split ratio: 20:1; Transfer line temperature: 280℃; Ionization method: EI, Ionization energy: 70eV; Ion source temperature: 230℃; Quadrupole temperature: 150℃; Mass range 35-455 aum.

[0033] The experimental results are shown in Table 1: Table 1 GC-MS Authentication Results

[0034]

[0035] Note: nd indicates not detected. The blank control group used unfermented tobacco leaves.

[0036] Experiment 2: Sensory Quality Evaluation Fifty smoker volunteers (age and gender not limited) were randomly selected to sample the cigarettes prepared in Examples 3-6. The cigarettes were evaluated for their gloss, aroma, harmony, off-flavors, irritation, and aftertaste according to GB5606.4-2005 "Cigarettes Part 4: Sensory Technical Requirements". The average value of the evaluation was recorded and the total sensory quality evaluation score was calculated.

[0037] The experimental results are shown in Table 2: Table 2 Sensory evaluation results

[0038] Note: The blank control group used unfermented tobacco leaves.

[0039] Comparison of data in Tables 1 and 2 shows that after fermentation with Lactobacillus plantarum, the content of aroma compounds in tobacco leaves increases, and the total amount of aroma compounds such as acids, alcohols, and esters increases significantly. Sensory evaluation results indicate that the aroma of treated tobacco leaves is improved, the aroma quantity and concentration increase, the taste is improved, the sweetness is increased, the off-flavors are reduced, and the quality is significantly improved.

[0040] Although the present invention has been disclosed above with reference to preferred embodiments, these are not intended to limit the invention. Any person skilled in the art can make various changes or modifications without departing from the spirit and scope of the invention. Therefore, the scope of protection of the present invention should be defined by the scope of the claims of this application.

Claims

1. A method for improving tobacco leaf quality using Lactobacillus plantarum fermentation, characterized in that, Includes the following steps: Step 1: Inoculate Lactobacillus plantarum into MRS medium for activation culture; Step 2: Inoculate the activated Lactobacillus plantarum into the tobacco fermentation medium and cultivate it in a manner that simulates the tobacco fermentation environment, so as to achieve the adaptation and domestication of the strain to the target environment; Step 3: Centrifuge the fermentation broth to remove the supernatant, resuspend it in sterile water, adjust the cell concentration, and obtain a high-density, high-activity Lactobacillus plantarum biological agent. Step 4: Add the Lactobacillus plantarum biological agent to sterile water and mix well. Spray evenly onto the tobacco leaves, seal well, and then ferment. Dry the treated tobacco leaves to obtain fermented tobacco leaves.

2. The method for improving tobacco quality using Lactobacillus plantarum fermentation according to claim 1, characterized in that, The *Lactobacillus plantarum* strain was screened from dairy products and deposited at the China Industrial Microbial Culture Collection Center, accession number CICC 24936.

3. A method for improving tobacco quality using *Lactobacillus plantarum* fermentation according to claim 1 or 2, characterized in that, The preparation method of MRS medium is as follows: Take 10g of casein peptone, 8g of beef extract powder, 4g of yeast extract powder, 20g of glucose, 1g of Tween 80, 5g of sodium acetate, 2g of diammonium hydrogen citrate, 2g of K2HPO4, 0.2g of MgSO4·7H2O, and 0.04g of MnSO4·H2O into an Erlenmeyer flask, add 1000mL of distilled water and heat until completely dissolved. Adjust the pH value to 5.7±0.2, autoclave at 101Kpa and 121℃ for 15 minutes.

4. The method for improving tobacco quality using Lactobacillus plantarum fermentation according to claim 3, characterized in that, Step 1: The activation culture time for Lactobacillus plantarum in MRS medium is 1-2 days, the culture temperature is 37℃, and the shaking speed is 180 rpm.

5. A method for improving tobacco quality using *Lactobacillus plantarum* fermentation according to claim 1, characterized in that, The formula for the tobacco powder fermentation medium in step 2 is: 3.0 g / L tobacco powder, 5.0 g / L yeast powder, 10 g / L NaCl, pH=7.

0.

6. The method for improving tobacco quality using Lactobacillus plantarum fermentation according to claim 5, characterized in that, In step 2, the activated Lactobacillus plantarum was inoculated into the tobacco fermentation medium and fermented under the following conditions: 35℃-38℃, 150rpm in a shaker for 24 hours.

7. The method for improving tobacco quality using Lactobacillus plantarum fermentation according to claim 1, characterized in that, In step 3, the centrifugation speed for collecting the cells in the fermentation broth is 8000-12000 rpm, and the centrifugation time is 5-15 min.

8. A method for improving tobacco quality using *Lactobacillus plantarum* fermentation according to claim 1, characterized in that, In step 3, the bacterial cell concentration was measured using a spectrophotometer, and the bacterial cell concentration was adjusted to 10. 8 cfu / mL.

9. A method for improving tobacco quality using Lactobacillus plantarum fermentation according to claim 1, characterized in that, Step 4: The amount of Lactobacillus plantarum biological agent added is 0.05-0.1% of the weight of tobacco leaves, and the amount of sterile water is 10% of the weight of tobacco leaves.

10. A method for improving tobacco quality using Lactobacillus plantarum fermentation according to claim 9, characterized in that, In step 4, the tobacco leaves are fermented under sealed conditions at 35-38℃ and RH 50-60% for 24-36 hours; the moisture content of the dried tobacco leaves is 12% w / w, and the drying temperature is 40℃.

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