Method for reducing phenol precursor by fermenting tobacco leaves through compound microorganisms, lactobacillus helveticus and application of lactobacillus helveticus

Abstract

The invention belongs to the technical field of microorganisms and fermentation, and discloses a method for reducing phenol precursors by fermenting tobacco leaves through compound microorganisms, lactobacillus helveticus and application of the lactobacillus helveticus. According to the invention, primarily-baked tobacco leaves, tobacco stems and the like are used as fermentation raw materials, after solid-state fermentation metabolism control, the phenol content in combustion smoke of the tobacco leaves can be effectively reduced, and meanwhile, the tobacco leaves have typical aroma characteristics and are strong in absorbability. The phenol content in the combustion smoke of the cigarette prepared by the method is relatively reduced by more than 90%.

Description

Technical Field

[0001] The invention belongs to the field of microorganisms and fermentation technology, in particular to a method for reducing phenol precursors by fermenting tobacco leaves with composite microorganisms, Lactobacillus helveticus and its application. Background Art

[0002] With tightening global tobacco control policies and rising consumer health awareness, "tar reduction and harm reduction" has become a research hotspot in the tobacco industry. Among the many harmful components produced by tobacco combustion, phenols, particularly phenol as the most toxic representative of these phenols, pose particular health risks due to their significant biotoxicity. Phenolics are primarily formed through complex physical and chemical transformations of sugars, nitrogen-containing compounds, and polyphenols in tobacco leaves under high-temperature combustion conditions. To reduce the content of harmful gases like phenol in cigarette smoke, it is crucial to prioritize reducing the levels of phenol combustion precursors, such as sugars and cellulose, in the tobacco leaf raw materials used in cigarettes.

[0003] In the area of tobacco quality improvement, the exploration of safe and efficient technologies for degrading sugars and cellulose has become a focus of academic research. Ideally, these technologies should be able to convert sugars and cellulose in tobacco leaves and stems into water-soluble reducing sugars and other small molecules that enhance tobacco quality, thereby improving both leaf quality and safety. Microbial fermentation, with its advantages of high efficiency, ease of operation, mild reaction conditions, and significant cost-effectiveness, has become a hot topic in tobacco quality improvement research in recent years.

[0004] Cigarettes are mainly composed of tobacco, filter, cigarette paper and additives. The harmful substances in the smoke after combustion are mainly generated under high temperature and oxygen-deficient conditions by tobacco leaves, cigarette paper and additives. At present, the methods for reducing the release of phenol-like harmful substances in cigarette combustion smoke mainly include: improving auxiliary additives to reduce, reducing by improving tobacco leaves or tobacco, reducing by using filter, etc. The current methods for improving tobacco leaves or tobacco to reduce harmful components mainly involve improving tobacco planting technology, adding beneficial substances to tobacco to remove harmful components in combustion smoke, etc. There are no reports on the method of using pile culture solid-state fermentation to reduce harmful components such as phenol in combustion smoke that is unique to the present invention. Summary of the Invention

[0005] The purpose of the present invention is to overcome the deficiencies in the prior art and to provide a method for reducing phenol precursors by fermenting tobacco leaves with a composite microorganism, as well as Lactobacillus helveticus and its application.

[0006] The technical solution adopted by the present invention to solve its technical problem is:

[0007] A strain of Lactobacillus helveticus capable of fermenting tobacco leaves to reduce phenol precursors, wherein the name of the Lactobacillus helveticus is TK824, the classification name is Lactobacillus helveticus, the preservation number is CGMCC No. 28261, the preservation date is August 28, 2023, and the preservation unit is the General Microbiology Center of the China Culture Collection Administration, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0008] The method of fermenting tobacco leaves using the composite microorganism of Lactobacillus helveticus to reduce phenol precursors comprises fermenting tobacco raw materials with Aspergillus and Lactobacillus helveticus as fermentation strains.

[0009] Further, the following steps are included:

[0010] (1) Spray the Lactobacillus helveticus agent evenly on the surface of the tobacco raw material after primary baking, and stir it up and down evenly; stir it up and down again after an interval of 20-28 hours;

[0011] (2) The Aspergillus spore suspension is evenly sprayed on the upper layer of the tobacco raw material pile treated in step (1). After 6-10 hours, 3-5% of the total mass of the tobacco raw material is evenly sprayed on the upper layer. After continuing to ferment for 40-50 hours, the upper layer is turned upside down once until the fermentation ends.

[0012] Furthermore, in the step (1), the amount of Lactobacillus helveticus added is 0.2-0.8% of the total mass of the tobacco raw material, and the number of viable bacteria of the Lactobacillus helveticus agent is (1-8)×(10 8 ~10 9 )cFu / g.

[0013] Furthermore, in the step (2), the amount of Aspergillus added is 0.1-0.4% of the total mass of the tobacco raw material, and the number of viable bacteria in the Aspergillus spore suspension is (1-8)×(10 6 ~10 7 )cFu / mL.

[0014] Furthermore, in the steps (1) and (2), the turning up and down is to mix the tobacco raw materials in the upper layer and the tobacco raw materials in the lower layer of the pile evenly, rather than turning the lower layer to the upper layer.

[0015] Furthermore, the tobacco raw materials include tobacco leaves and tobacco stems.

[0016] Furthermore, in step (2), the fermentation temperature is 15-40° C., and the fermentation time is 7-20 days.

[0017] Furthermore, after the fermented tobacco raw material is prepared into cigarettes, the phenol content in the combustion smoke is relatively reduced by more than 90%.

[0018] The use of Lactobacillus helveticus as described above in fermenting tobacco leaves to reduce phenol precursors.

[0019] The advantages and positive effects achieved by the present invention are:

[0020] 1. The method of the present invention uses Aspergillus and Lactobacillus helveticus as fermentation strains, based on mixed bacterial synergistic fermentation and metabolic control technology, and uses primary flue-cured tobacco leaves, tobacco stems, etc. as fermentation raw materials. After solid-state fermentation, cigarette products are prepared, and the phenol content in the combustion flue gas is relatively reduced by more than 90%.

[0021] 2. The present invention uses Aspergillus and Lactobacillus helveticus for mixed fermentation. Among them, Lactobacillus helveticus has been screened through multiple rounds. The combination of this strain with different Aspergillus strains, through proprietary microbial colonization control technology (fermentation metabolism control technology), achieves the characteristics of fast strain proliferation, high enzyme activity such as amylase, and high temperature resistance, so as to achieve the purpose of rapidly degrading phenol combustion precursors such as sugars and cellulose in tobacco leaves.

[0022] 3. The fermentation metabolism control conditions described in the method of the present invention are optimized based on the clarification of the synergistic metabolic network and interaction mechanism of Aspergillus and Lactobacillus helveticus. KEGG enrichment analysis found that the synergistic metabolic pathways are mainly metabolic pathways such as the biosynthesis of phenylpropanoids, phenylpropanoid biosynthesis, and tyrosine metabolism.

[0023] 4. The present method utilizes Aspergillus and Lactobacillus helveticus as fermentation strains, employing mixed fermentation and metabolic control technology. Using pre-cured tobacco leaves and stems as fermentation feedstock, this solid-state fermentation metabolic control effectively reduces the phenol content in tobacco smoke, while maintaining a typical aroma profile and enhancing smokability. Cigarettes produced using this method exhibit a relative reduction of phenol content in smoke by over 90%. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the combustion pyrolysis GC-MS spectrum of the sample in Example 1 of the present invention;

[0025] Figure 2 This is the combustion pyrolysis GC-MS spectrum of the sample of Comparative Example 1 of the present invention;

[0026] Figure 3 This is the combustion pyrolysis GC-MS spectrum of the sample of Comparative Example 2 of the present invention;

[0027] Figure 4This is a colony morphology diagram of Lactobacillus helveticus TK824 in the present invention. DETAILED DESCRIPTION

[0028] The present invention will be further described below with reference to the following examples. The following examples are descriptive rather than restrictive, and the scope of protection of the present invention cannot be limited by the following examples.

[0029] The various experimental operations involved in the specific embodiments are all routine techniques in the field. For parts not specifically annotated in this document, ordinary technicians in this field can refer to various commonly used reference books, scientific literature or related instructions, manuals, etc. before the filing date of this invention to implement them.

[0030] A strain of Lactobacillus helveticus capable of fermenting tobacco leaves to reduce phenol precursors, wherein the name of the Lactobacillus helveticus is TK824, the classification name is Lactobacillus helveticus, the preservation number is CGMCC No. 28261, the preservation date is August 28, 2023, and the preservation unit is the General Microbiology Center of the China Culture Collection Administration, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0031] The method of fermenting tobacco leaves using the composite microorganism of Lactobacillus helveticus to reduce phenol precursors comprises fermenting tobacco raw materials with Aspergillus and Lactobacillus helveticus as fermentation strains.

[0032] Preferably, the method comprises the following steps:

[0033] (1) Spray the Lactobacillus helveticus agent evenly on the surface of the tobacco raw material after primary baking, and stir it up and down evenly; stir it up and down again after an interval of 20-28 hours;

[0034] (2) The Aspergillus spore suspension is evenly sprayed on the upper layer of the tobacco raw material pile treated in step (1). After 6-10 hours, 3-5% of the total mass of the tobacco raw material is evenly sprayed on the upper layer. After continuing to ferment for 40-50 hours, the upper layer is turned upside down once until the fermentation ends.

[0035] Preferably, in the step (1), the amount of Lactobacillus helveticus added is 0.2-0.8% of the total mass of the tobacco raw material, and the number of viable bacteria of the Lactobacillus helveticus agent is (1-8)×(10 8 ~10 9 )cFu / g.

[0036] Preferably, in step (2), the amount of Aspergillus added is 0.1-0.4% of the total mass of the tobacco raw material, and the number of viable bacteria in the Aspergillus spore suspension is (1-8)×(10 6 ~10 7 )cFu / mL.

[0037] Preferably, in steps (1) and (2), the turning up and down is to mix the tobacco raw materials in the upper layer and the tobacco raw materials in the lower layer of the pile evenly, rather than turning the lower layer to the upper layer.

[0038] Preferably, the tobacco raw materials include tobacco leaves and tobacco stems.

[0039] Preferably, in step (2), the fermentation temperature is 15-40°C and the fermentation time is 7-20 days.

[0040] Preferably, after the fermented tobacco raw material is prepared into cigarettes, the phenol content in the combustion smoke thereof is relatively reduced by more than 90%.

[0041] The use of Lactobacillus helveticus as described above in fermenting tobacco leaves to reduce phenol precursors.

[0042] Specifically, the relevant preparation and detection are as follows:

[0043] A strain of Lactobacillus helveticus capable of fermenting tobacco leaves to reduce phenol precursors, wherein the name of the Lactobacillus helveticus is TK824, the classification name is Lactobacillus helveticus, the preservation number is CGMCC No. 28261, the preservation date is August 28, 2023, and the preservation unit is the General Microbiology Center of the China Culture Collection Administration, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0044] The relevant screening method of the described Lactobacillus helveticus TK824 is:

[0045] Sample source: Commercially available pickled mustard greens, purchased from Beitang Vegetable Market, Tianjin, China.

[0046] The culture medium used was:

[0047] MRS solid medium: 20.0 g / L glucose, 10.0 g / L peptone, 10.0 g / L beef extract powder, 5.0 g / L yeast extract powder, 3.02 g / L sodium acetate, 1.16 g / L potassium dihydrogen phosphate, 0.05 g / L magnesium sulfate, 2.0 g / L triammonium citrate, 0.03 g / L manganese sulfate, 1.0 mL Tween 80, 2.0 g / L agar. Sterilize at 121°C for 15 min.

[0048] MRS liquid medium: 20.0 g / L glucose, 10.0 g / L peptone, 10.0 g / L beef extract powder, 5.0 g / L yeast extract powder, 3.02 g / L sodium acetate, 1.16 g / L potassium dihydrogen phosphate, 0.05 g / L magnesium sulfate, 2.0 g / L ammonium citrate tribasic, 0.03 g / L manganese sulfate, 1.0 mL Tween 80. Sterilize at 121°C for 15 min.

[0049] Rinse the sauerkraut with 0.9% sterile saline to make a dilution of 10 -1 The mother solution was diluted in a 10-fold dilution method to obtain a dilution factor of 10 -2 ~10 -5 Take 200 μL of the bacterial solution and spread it on solid MRS medium for culture. Select an appropriate dilution based on the number of colonies growing on the medium. Take 100 μL of the sample and plate it onto MRS solid medium at 37°C for 24 hours. Then, repeatedly streak a colony until a single colony appears. Then, inoculate the selected colony into 5 mL of fresh MRS liquid medium for culture. Culture the strain in a 37°C incubator at 180 rpm for 24 hours. The resulting bacterial solution is then subcultured using the above method at a 2% inoculum size, serving as seed stock for subsequent experiments.

[0050] The colony morphology is shown in the figure Figure 4 As shown, from Figure 4 It can be seen that the colony is round, with neat edges, opaque, milky white on the front, convex in the middle, smooth and bright surface, moist texture, and easy to pick up.

[0051] Example 1

[0052] The method for fermenting tobacco leaves using the composite microorganism of Lactobacillus helveticus to reduce phenol precursors comprises the following steps:

[0053] Weigh 5000g of tobacco leaves after primary curing, add 25g of Lactobacillus helveticus (4×10 9 cFu / g viable bacteria count) was evenly sprayed on the surface of the tobacco leaf pile and stirred up and down for even mixing. After the pile was left to stand for 25 hours, it was stirred up and down again to mix evenly. Further, 10 mL of Aspergillus spore suspension (viable bacteria count of 6×10 6 cFu / mL), after standing and fermenting for 8 hours, 200 mL of water was sprayed on the upper layer of the pile, and the pile was continued to stand and ferment for 48 hours and then turned up and down to mix evenly. The fermentation environment temperature was controlled within the range of 15-40°C. The total fermentation time was 18 days. After the fermentation was terminated, it was prepared into cigarettes.

[0054] Comparative Example 1

[0055] This comparative example was not inoculated with Lactobacillus helveticus, and other conditions were the same as those in Example 1.

[0056] Weigh 5000 g of primary-cured tobacco leaves and spray 10 mL of Aspergillus spore suspension (viable count 6 × 10 6cFu / mL), after standing and fermenting for 8 hours, 200 mL of water was sprayed on the upper layer of the pile, and the pile was continued to stand and ferment for 48 hours and then turned up and down to mix evenly. The fermentation environment temperature was controlled within the range of 15-40°C. The total fermentation time was 18 days. After the fermentation was terminated, it was prepared into cigarettes.

[0057] Comparative Example 2

[0058] This comparative example was not inoculated with Aspergillus, and other conditions were the same as those in Example 1.

[0059] Weigh 5000g of tobacco leaves after primary curing, add 25g of Lactobacillus helveticus (4×10 9 cFu / g viable bacteria count) is evenly sprayed on the surface of the tobacco leaf pile, and the pile is stirred up and down for uniformity. After the pile is allowed to stand for 25 hours, the pile is stirred up and down again for uniform mixing. After standing and fermenting for 8 hours, 200 mL of water is sprayed on the upper layer of the pile. The pile is allowed to stand and ferment for another 48 hours and then stirred up and down for uniform mixing. The fermentation environment temperature is controlled within the range of 15-40°C. The total fermentation time is 18 days. After the fermentation is terminated, the pile is prepared into cigarettes.

[0060] Effect evaluation:

[0061] The present invention provides a method for reducing phenol precursors by fermenting tobacco leaves with a composite microorganism and Lactobacillus helveticus. To demonstrate the effectiveness, the research results are as follows:

[0062] The smoke components in each sample were detected and analyzed using the method of combustion pyrolysis combined with GC-MS. The results are as follows: Figure 1 、 Figure 2 and Figure 3 As shown in the figure, the phenol content in the flue gas was significantly reduced by 91.6%.

[0063] The samples of Example 1, Comparative Example 1 and Comparative Example 2 were pyrolyzed at 600°C, and the chromatograms of the pyrolysis products were shown in FIG. Figure 1-3, wherein the phenol content in Example 1 decreased by 91.6% compared with that in Comparative Example 1 and decreased by 76.5% compared with that in Comparative Example 2. It can also be seen that there is a synergistic effect between Lactobacillus helveticus and Aspergillus in the method of the present invention, which can synergistically reduce phenol precursors. At the same time, compared with Comparative Examples 1 and 2, the content of harmful components related to phenol in Example 1, such as hydroquinone and catechol, also showed a significant downward trend. Long-term contact with these phenolic compounds may cause damage to the liver and kidneys, and there is a potential carcinogenic risk. In addition, the study also found that compared with Comparative Examples 1 and 2, other harmful components in Example 1 also produced varying degrees of degradation effects. For example, the content of substances such as scopoletin, which is neurotoxic, 3,5-dihydroxy-2methyl-4H-pyran-4-one, which may cause skin irritation, and 5-methylfurfural, which is toxic to the liver, were all reduced to varying degrees. This result shows that fermentation treatment can effectively reduce a variety of harmful substances produced during the thermal cracking of tobacco stem extract, thereby reducing its potential health risks to the human body.

[0064] In summary, the method for reducing phenol precursors by fermenting tobacco leaves with composite microorganisms and the Lactobacillus helveticus described in the present invention have good application effects and can significantly reduce the phenol content in cigarette combustion smoke.

[0065] Although the embodiments of the present invention are disclosed for illustrative purposes, those skilled in the art will understand that various substitutions, changes and modifications are possible without departing from the spirit and scope of the present invention and the appended claims. Therefore, the scope of the present invention is not limited to the contents disclosed in the embodiments.

Claims

1. A Lactobacillus helveticus strain capable of fermenting tobacco leaves to reduce phenol precursors, characterized by: The name of the Lactobacillus helveticus is: TK824, the classification name is: Lactobacillus helveticus, the preservation number is: CGMCC No. 28261, the preservation date is: August 28, 2023, and the preservation unit is: General Microbiology Center of China Culture Collection Administration, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

2. A method for reducing phenol precursors by fermenting tobacco leaves using the composite microorganism of Lactobacillus helveticus as claimed in claim 1, characterized in that: The method uses Aspergillus and Lactobacillus helveticus as fermentation strains to mix and ferment tobacco raw materials.

3. The method according to claim 2, wherein: The steps include: (1) Spray the Lactobacillus helveticus agent evenly on the surface of the tobacco raw material after primary baking, and stir it up and down evenly; stir it up and down again after an interval of 20-28 hours; (2) The Aspergillus spore suspension is evenly sprayed on the upper layer of the tobacco raw material pile treated in step (1). After 6-10 hours, 3-5% of the total mass of the tobacco raw material is evenly sprayed on the upper layer. After continuing to ferment for 40-50 hours, the upper layer is turned upside down once until the fermentation ends.

4. The method according to claim 3, wherein: In the step (1), the amount of Lactobacillus helveticus added is 0.2-0.8% of the total mass of the tobacco raw material, and the number of viable bacteria of the Lactobacillus helveticus agent is (1-8)×(10 8 ~10 9 )cFu / g.

5. The method according to claim 3, wherein: In the step (2), the amount of Aspergillus added is 0.1-0.4% of the total mass of the tobacco raw material, and the number of viable bacteria in the Aspergillus spore suspension is (1-8)×(10 6 ~10 7 )cFu / mL.

6. The method according to claim 3, wherein: In the steps (1) and (2), the up and down turning is to mix the tobacco raw materials in the upper layer and the tobacco raw materials in the lower layer of the pile evenly, rather than turning the lower layer to the upper layer.

7. The method according to claim 2, wherein: The tobacco raw materials include tobacco leaves and tobacco stems.

8. The method according to claim 3, wherein: In the step (2), the fermentation temperature is 15-40° C. and the fermentation time is 7-20 days.

9. The method according to any one of claims 2 to 8, characterized in that: After the fermented tobacco raw materials are prepared into cigarettes, the phenol content in the combustion smoke is relatively reduced by more than 90%.

10. Use of the Lactobacillus helveticus according to claim 1 in fermenting tobacco leaves to reduce phenol precursors.

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