A fermentation method for reducing TSNAs in tobacco leaves using Bacillus pumilus 05-5402

Abstract

The present invention discloses a fermentation method for reducing TSNAs in tobacco leaves by using Bacillus pumilus 05-5402, which is a liquid fermentation method, wherein the baked tobacco leaves are ground into powder, added into an inorganic salt basic medium to prepare a tobacco medium for a liquid fermentation system, and the Bacillus pumilus 05-5402 bacterial solution is added into the tobacco medium at the beginning of fermentation, and the mixture is placed in a 25-28°C constant temperature shaking incubator for fermentation for 5-10 days to degrade TSNAs in the tobacco leaves; the preservation number of the Bacillus pumilus 05-5402 strain is CGMCC No. 7418. The present invention selects Bacillus pumilus 05-5402 that can significantly reduce tobacco-specific nitrosamines to prepare bacterial solution by fermentation and applies it to the liquid fermentation process of tobacco leaves, combines the control of temperature and fermentation time during the fermentation process, and realizes efficient and directional degradation of tobacco-specific nitrosamines, which is environmentally friendly, easy to operate, and has a high value for promotion and application.

Description

Technical Field

[0001] The invention belongs to the technical field of tobacco industry, and particularly relates to a fermentation method for reducing TSNAs in tobacco leaves by utilizing Bacillus pumilus 05-5402. Background Art

[0002] Tobacco-specific nitrosamines (TSNAs) are a general term for a class of nicotine, a class of N-nitrosamine-based compounds that only exist in tobacco and smoke. Currently, a total of eight TSNAs have been isolated and identified. The ones that have been studied more in depth include N-nitrosonornicotine (NNN), 4-(N-methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), N-nitrosoanatabine (NAT) and N-nitrosoanabasine (NAB). These substances are highly carcinogenic and pose a great threat to human health.

[0003] As early as 1962, the potential carcinogenic effect of TSNAs was first reported internationally, which can cause tumors in the human mouth, esophagus, stomach, lungs, pancreas, liver and other parts. Among them, NNN and NNK are highly carcinogenic to rodents. NNK is identified as a Class Ⅰ carcinogen (IARC Group Ⅰ) by the International Agency for Research on Cancer (IARC). It can cause DNA methylation in animal and human tissues, increasing the possibility of causing tumors. The reduction of TSNAs content in tobacco leaves is of great significance to improving tobacco quality and reducing the harm of TSNAs to human health and living environment. Therefore, reducing the content of tobacco-specific nitrosamines is crucial to improving tobacco quality and safety, and has become a research hotspot in recent years.

[0004] Studies have shown that the content of tobacco-specific nitrosamines (TSNAs) in mature and harvested fresh tobacco leaves is extremely low, and they are mainly produced and accumulated during the curing process of tobacco leaves. Previous researchers have explored methods to reduce nitrosamines (TSNAs) in tobacco leaves in many aspects, most of which involve the process of flue-cured tobacco and cigarettes, such as using heat exchange curing rooms during the curing and curing process to avoid the contact of gases produced by open flame combustion with tobacco leaves, thereby reducing the TSNAs content in tobacco leaves during the curing process; or by chemical methods, adding nitrite inhibitors such as VC and VE or other substances that can react with the decomposition products of nitrites during the curing process of tobacco leaves to reduce the precursors of TSNAs synthesis to achieve the reduction of TSNAs content; and by physical technology, microwave radiation technology or adding zeolites loaded with metal oxides to tobacco shreds to achieve the reduction of TSNAs content in cigarettes. While reducing TSNAs, the above methods are often accompanied by defects such as high cost, high pollution, and limited degradation effect.

[0005] The use of microorganisms to reduce TSNAs in tobacco leaves has the advantages of low cost, good ecological compatibility, and no secondary pollution. Therefore, reducing TSNAs in tobacco leaves by inoculating strains is an ideal way. However, there are not many reports on the use of microorganisms to reduce TSNAs. The modes of action are all spraying, spraying or coating, that is, during the drying, baking, fermentation and other curing or silk-making processes of tobacco leaves, the fermentation agents containing special strains are sprayed to inoculate the strains into tobacco leaves or tobacco shreds, and then conventional tobacco leaf curing or silk-making is performed. The effect of reducing TSNAs is not obvious, and most of the research focuses on burley tobacco and cigar tobacco, and there are few reports on flue-cured tobacco. Summary of the invention

[0006] Aiming at the technical problem that the existing technology of using microorganisms by spraying, sprinkling or coating has an insignificant effect on reducing tobacco-specific nitrosamines (TSNAs), the present invention provides a fermentation method for reducing tobacco leaf TSNAs by using Bacillus pumilus 05-5402.

[0007] The technical solution of the present invention:

[0008] A fermentation method for reducing TSNAs in tobacco leaves by using Bacillus pumilus 05-5402 is a liquid fermentation method. The baked tobacco leaves are ground into powder, added into an inorganic salt basic culture medium to prepare a tobacco culture medium for a liquid fermentation system, and at the beginning of fermentation, Bacillus pumilus 05-5402 bacterial liquid is added into the tobacco culture medium, and the tobacco leaves are placed at 25-28°C for 5-10 days to perform liquid fermentation of the tobacco leaves, so as to degrade TSNAs in the tobacco leaves. The preservation number of the Bacillus pumilus 05-5402 strain is CGMCC No.7418.

[0009] The fermentation method comprises the following steps:

[0010] Step 1: crushing tobacco leaves: grinding the roasted tobacco leaves into tobacco leaf powder to obtain fermentation raw materials;

[0011] Step 2, preparing bacterial liquid: activating the Bacillus pumilus 05-5402 strain in a preserved state to obtain a bacterial liquid containing a fermentation agent, wherein the preservation number of the Bacillus pumilus 05-5402 strain is CGMCC No.7418;

[0012] Step 3, tobacco leaf fermentation: adding the tobacco leaf powder to an inorganic salt basal culture medium to prepare a tobacco culture medium, adding a bacterial solution prepared with the Bacillus pumilus 05-5402 strain to the tobacco culture medium at the beginning of fermentation, sealing the medium, and performing liquid fermentation of the tobacco leaves at a fermentation temperature of 25-28° C. and a fermentation time of 5-10 days.

[0013] The OD of the bacterial solution 600 is 2.0.

[0014] The inoculation amount of the bacterial solution is 1% of the total volume of the tobacco culture medium.

[0015] The tobacco culture medium for the liquid fermentation system is prepared according to the ratio of tobacco leaf powder (g): inorganic salt culture medium (g) = 1:9.

[0016] The strain activation is to inoculate the preserved Bacillus pumilus 05-5402 strain on LB solid culture medium, culture at 28°C for 10-12 hours until a single colony grows, select a single colony and transfer it to LB liquid culture medium, culture at 28°C for 24-48 hours to obtain a fermentation agent, and then adjust the OD value of the fermentation agent to OD 600 =2.0, and obtain bacterial solution.

[0017] The components of the LB liquid culture medium are calculated in g / L: 5 g yeast extract powder, 10 g peptone, and 10 g NaCl; the LB solid culture medium is prepared by adding agar powder to the liquid culture medium at a ratio of 15 g / L.

[0018] The liquid fermentation of the tobacco leaves is carried out in a constant temperature shaking incubator, with a fermentation temperature of 25-28° C., a fermentation time of 5-10 days, and a rotation speed of 160 rpm / min.

[0019] The components of the inorganic salt basic culture medium are calculated in g / L: K2HPO4 0.2, KH2PO4 0.8, MgSO4 0.2, CaSO4·H2O 0.1, Na2MoO4 0.0033, FeSO4·7H2O 0.005.

[0020] The baked tobacco leaves are baked cinnabar tobacco leaves.

[0021] Beneficial effects of the present invention:

[0022] The present application uses a specially selected strain of Bacillus pumilus 05-5402 (with a deposit number of CGMCC No. 7418) to reduce TSNAs in tobacco leaves for the first time by liquid fermentation, which greatly improves the removal efficiency of TSNAs in tobacco leaves. At the beginning of fermentation, a bacterial solution prepared with Bacillus pumilus 05-5402 strain is added to a liquid fermentation system formed by tobacco leaf powder and inorganic salt basic culture medium according to a certain inoculation amount. Different from the solid-state fermentation method of spraying fermentation agents on tobacco leaves or tobacco shreds, the present application adds the bacterial liquid to a liquid inorganic salt culture medium containing tobacco leaf powder for liquid fermentation. Unlike conventional culture media that contain sufficient nutrients required for strain growth, the inorganic salt basal culture medium used in the present application only contains inorganic salt components without exogenous carbon and nitrogen sources. Therefore, the present application ferments in liquid mode without adding any carbon and nitrogen sources, and only uses the chemical components of the tobacco leaf powder as the only nutrient source for the strain in the liquid fermentation system, so as to utilize as many compounds in the tobacco leaves as possible, thereby improving the TSNAs degradation rate in the tobacco leaves. At the same time, based on the self-reproduction characteristics of the 05-5402 strain, the present application provides a suitable environment for the strain to survive by regulating the fermentation conditions to ferment at 25-28°C for 5-10 days during the liquid fermentation process, thereby improving the survival rate and activity of the inoculated strain, and achieving efficient and directional degradation of tobacco-specific nitrosamines by Bacillus pumilus 05-5402. The TSNAs content of tobacco leaves can be reduced by 13.99% to 31.99%, significantly reducing the harm of tobacco TSNAs to human health and the living environment. In addition, the strain has a low cost of use, and the operation of applying the bacterial liquid to tobacco leaves is convenient. The purpose of reducing TSNAs can be achieved during the fermentation process, and the TSNAs content in tobacco leaves can be efficiently and directional degraded with good stress resistance. The method has the advantages of being environmentally friendly and easy to operate, and has a high value for promotion and application.

[0023] Control the OD of bacterial solution 600 A value of 2.0 means that the bacteria is in the logarithmic growth phase, with vigorous growth, strong reproduction, stronger activity, and higher utilization rate of TSNAs.

[0024] The inoculation amount is controlled to be the preferred inoculation amount commonly used in the field of microorganisms, specifically, the bacterial liquid accounts for 1% of the total volume of the tobacco leaf culture medium.

[0025] The tobacco culture medium for the liquid fermentation system was prepared according to the ratio of tobacco leaf powder (g): inorganic salt basic culture medium (g) = 1:9.

[0026] The steps and conditions for controlling the activation of Bacillus pumilus 05-5402 are as follows: inoculating the Bacillus pumilus 05-5402 strain in a preserved state on an LB solid culture medium, culturing at 28° C. for 10 to 12 hours until a single colony grows; selecting a single colony and transferring it to an LB liquid culture medium, culturing at 28° C. for 24 to 48 hours to obtain a fermentation agent; then adding an appropriate amount of sterile deionized water to the fermentation agent, testing the bacterial solution until the absorbance value is 2.0, and obtaining the OD 600 The bacterial solution has a value of 2.0.

[0027] The LB solid medium contains the following ingredients in g / L: 5g yeast extract, 10g peptone, and 10g NaCl; the LB solid medium is based on the above liquid medium and agar powder is added at a ratio of 15g / L. LB, a universal medium for the growth of microorganisms, is used to ensure the normal growth of the strain.

[0028] The liquid fermentation system is placed in a constant temperature shaking incubator for liquid fermentation, with a fermentation temperature of 25-28°C, a fermentation time of 5-10 days, and a rotation speed of 160rpm / min. The purpose of shaking is to keep the bacteria at a normal reproduction rate, so that the bacteria can grow and reproduce at a suitable temperature and fermentation time, have high activity, and improve the degradation rate of TSNAs in tobacco leaves.

[0029] The inorganic salt basic culture medium contains the following components in g / L: K2HPO4 0.2, KH2PO4 0.8, MgSO4 0.2, CaSO4·H2O 0.1, Na2MoO4 0.0033, FeSO4·7H2O 0.005. It contains only inorganic salt components, ensuring that it does not contain nutrients such as carbon source and nitrogen source required for bacterial reproduction and growth.

[0030] The cured tobacco leaves are cured cinnabar tobacco leaves, and can also be used for other cured tobacco leaves. DETAILED DESCRIPTION

[0031] The present invention is further described below in conjunction with the embodiments, but the present invention is not limited in any way. Any changes or improvements made based on the teachings of the present invention belong to the protection scope of the present invention.

[0032] A fermentation method for reducing TSNAs in tobacco leaves by using Bacillus pumilus 05-5402 is a liquid fermentation method. The tobacco leaves after baking are ground into tobacco leaf powder and added into an inorganic salt basic culture medium to prepare a tobacco culture medium. At the beginning of fermentation, a bacterial liquid prepared with Bacillus pumilus 05-5402 strain is added into the tobacco culture medium. The tobacco leaf powder is used as the only nutrient source of the strain in the liquid fermentation system to carry out liquid fermentation of the tobacco leaves. The fermentation temperature is 25-28°C and the fermentation time is 5-10 days. The preservation number of the Bacillus pumilus 05-5402 strain is CGMCC No.7418.

[0033] The fermentation method comprises the following steps:

[0034] Step 1: crushing tobacco leaves: grinding the roasted tobacco leaves into tobacco leaf powder to obtain fermentation raw materials.

[0035] In order to select a suitable particle size so that the tobacco leaf powder can fully contact with the bacteria, the tobacco leaf powder is sieved through a 50-mesh sieve.

[0036] The baked tobacco leaves are conventionally baked tobacco leaves, such as cinnabar tobacco leaves. The conventionally baked cinnabar tobacco leaves are fresh tobacco leaves harvested by tying tobacco leaves with tobacco rods for baking. The baking process is divided into three stages according to the change of tobacco leaf color: yellowing, color fixation, and dry tendons. The total curing time is 7 to 10 days. The cinnabar tobacco is a high-quality flue-cured tobacco variety, for example, the improved variety of Yunyan 87 - cinnabar No. 2, which has a unique glutinous rice aroma and a low nicotine content.

[0037] Step 2, preparation of bacterial liquid: activating the Bacillus pumilus 05-5402 strain in a preserved state to obtain a bacterial liquid containing a fermentation agent, wherein the preservation number of the Bacillus pumilus 05-5402 strain is CGMCC No.7418.

[0038] The strain activation is to streak the Bacillus pumilus 05-5402 strain in the preserved state on the LB solid culture medium, place it in a constant temperature incubator at 28°C and culture it for 10 to 12 hours until a single colony grows; select a single colony and transfer it to the LB liquid culture medium, place it in a constant temperature shaking incubator at 28°C and 150rpm / min and culture it for 24 to 48 hours to obtain a fermentation agent, then add an appropriate amount of sterile deionized water to the fermentation agent, and measure the absorbance value of the bacterial solution at 600nm at this time, adjust the absorbance value to 2.0, and obtain OD 600 The bacterial solution has a value of 2.0.

[0039] The LB liquid culture medium contains the following components (in g / L): 5 g yeast extract, 10 g peptone, and 10 g NaCl; the LB solid culture medium is added with agar powder at a ratio of 15 g / L based on the above liquid culture medium.

[0040] Step 3, tobacco leaf fermentation: adding the tobacco leaf powder to the inorganic salt basic culture medium to prepare a tobacco culture medium, adding a bacterial solution prepared with the Bacillus pumilus 05-5402 strain to the tobacco culture medium at the beginning of fermentation, sealing the medium, and performing liquid fermentation of the tobacco leaves at a fermentation temperature of 25-28° C. and a fermentation time of 5-10 days.

[0041] The bacterial liquid is applied to the liquid fermentation system from the beginning of fermentation, so that the bacterial strain grows and reproduces using the chemical substances in the tobacco leaves as the only nutrient source.

[0042] The components of the inorganic salt basic culture medium are calculated in g / L: K2HPO4 0.2, KH2PO4 0.8, MgSO4 0.2, CaSO4·H2O 0.1, Na2MoO4 0.0033, and FeSO4·7H2O 0.005.

[0043] The tobacco culture medium for the liquid fermentation system was prepared according to the ratio of tobacco leaf powder (g): inorganic salt basic culture medium (g) = 1:9. 600 =2.0, and the amount of the bacterial solution used each time is 1% of the total volume of the tobacco culture medium.

[0044] When preparing the tobacco leaf culture medium, the step of sterilizing the tobacco leaf culture medium in a high pressure steam sterilizer at 120° C. for 15 minutes to obtain a sterile tobacco leaf culture medium is also included, ensuring that only the added 05-5402 bacteria exist during the fermentation process and play a role in degrading TSNAs.

[0045] When liquid fermentation is carried out, the liquid fermentation system is placed in a constant temperature shaking incubator at 25-28°C, with a rotation speed of 160 rpm / min, and the fermentation time is 5-10 days.

[0046] Example 1

[0047] Example 1 relates to a fermentation method for reducing TSNAs in tobacco leaves using Bacillus pumilus 05-5402, which is a liquid fermentation method, and the liquid fermentation system is fermented at 28° C. for 5 days.

[0048] The liquid fermentation method comprises the following steps:

[0049] Step 1: crushing tobacco leaves: grind the roasted tobacco leaves into tobacco leaf powder, and then pass it through a 50-mesh sieve to obtain fermentation raw materials.

[0050] The baked tobacco leaves are conventionally baked red sand tobacco leaves, which are grown in Yanhe Town, Yuxi City, Yunnan Province. The middle tobacco leaves are picked after maturity, and the tobacco leaves are collected after baking at the test base, ground into powder and used for fermentation. The baking process is divided into three stages according to the change of tobacco leaf color: yellowing, color fixing, and dry tendons, and the total curing time is 7 to 10 days.

[0051] Step 2, preparation of bacterial solution: streak the Bacillus pumilus 05-5402 strain obtained through the separation, screening and purification process stored in the glycerol tube on the LB solid culture medium, and then culture it in a 28°C constant temperature incubator overnight until a single colony grows; select a single colony and transfer it to 5ml LB liquid culture medium, place it in a constant temperature shaking incubator at 28°C and 150rpm / min and culture it for 48h to obtain a fermentation agent, then add an appropriate amount of sterile deionized water and measure the absorbance of the bacterial solution at 600nm, adjust the absorbance to 2.0, and obtain the bacterial solution.

[0052] The deposit number of the Bacillus pumilus 05-5402 strain is CGMCC No.7418.

[0053] Step 3: Tobacco leaf fermentation. The fermentation process is as follows:

[0054] 5.00 g of tobacco leaf powder was put into a triangular flask, 45 ml of inorganic salt basal culture medium was added to make the volume to 50 ml to prepare tobacco leaf culture medium, and the culture medium was sterilized in a 120° C. high-pressure steam sterilizer for 15 min to obtain a sterile tobacco leaf culture medium; in the experimental group, 500 μl of the prepared bacterial solution was added to the tobacco culture medium in the clean bench, and in the control group, 500 μl of sterile deionized water was added, and the mixture was sealed with a sealing film, placed in a 28° C. constant temperature shaking incubator, and shaken and cultured at a speed of 160 rpm / min for 5 days. The tobacco leaf powder was used as the sole nutrient source of the strain, and liquid fermentation of tobacco leaves was carried out to determine the role of the strain in the degradation of tobacco leaf TSNAs.

[0055] At the end of the experiment, the fermentation liquid of the experimental group and the control group were transferred to sterile 50ml centrifuge tubes, centrifuged at 4500rpm / min for 10min at room temperature, the supernatant was discarded, the precipitate was collected, and the precipitate was washed once with sterile deionized water. The obtained tobacco leaf powder precipitate and bacterial cell mixture was placed in a 55℃ oven to completely evaporate the water and dry. The content of TSNAs in the fermented tobacco leaf powder was determined, as shown in Table 1.

[0056] Table 1 TSNAs content of tobacco leaves after 5 days of liquid fermentation at 28℃ after inoculation of bacteria

[0057]

[0058] Example 2

[0059] Example 2 relates to a fermentation method for reducing TSNAs in tobacco leaves using Bacillus pumilus 05-5402, which is a liquid fermentation method, and the liquid fermentation system is controlled to ferment at 28° C. for 7 days.

[0060] On the basis of Example 1, Example 2 placed the tobacco leaf culture medium inoculated with Bacillus pumilus 05-5402 in a constant temperature shaking incubator at 28°C and cultured at a speed of 160 rpm / min for 7 days as the experimental group. 500 μl of sterile deionized water was added to the control group, and other experimental conditions were the same as those in Example 1.

[0061] At the end of the experiment, the fermentation liquid was transferred to a sterile 50 ml centrifuge tube and centrifuged at room temperature at 4500 rpm / min for 10 min. The supernatant was discarded, the precipitate was collected, and the precipitate was washed once with sterile deionized water. The obtained mixture of tobacco leaf precipitate and bacterial cells was placed in a 55°C oven to completely evaporate the water, and the content of tobacco-specific nitrosamines (TSNAs) after fermentation was determined as shown in Table 2.

[0062] Table 2 TSNAs content of tobacco leaves after 7 days of liquid fermentation at 28℃ after inoculation

[0063]

[0064] Example 3

[0065] Example 3 relates to a fermentation method for reducing TSNAs in tobacco leaves using Bacillus pumilus 05-5402, which is a liquid fermentation method, and the liquid fermentation system is controlled to ferment at 28° C. for 10 days.

[0066] On the basis of Example 1, Example 3 placed the tobacco leaf culture medium inoculated with Bacillus pumilus 05-5402 in a constant temperature shaking incubator at 28°C and cultured at a speed of 160 rpm / min for 10 days as the experimental group. 500 μl of sterile deionized water was added to the control group, and other experimental conditions were the same as those in Example 1.

[0067] At the end of the experiment, the fermentation liquid was transferred to a sterile 50 ml centrifuge tube and centrifuged at 4500 rpm / min for 10 min at room temperature. The supernatant was discarded, the precipitate was collected, and the precipitate was washed once with sterile deionized water. The obtained mixture of tobacco leaf precipitate and bacterial cells was placed in a 55°C oven to completely evaporate the water, and the content of tobacco-specific nitrosamines (TSNAs) after fermentation was determined as shown in Table 3.

[0068] Table 3 TSNAs content of tobacco leaves after 10 days of liquid fermentation at 28℃ after inoculation

[0069]

[0070] Example 4

[0071] Example 4 relates to a fermentation method for reducing TSNAs in tobacco leaves using Bacillus pumilus 05-5402, which is a liquid fermentation method, and the liquid fermentation system is controlled to ferment at 25° C. for 5 days.

[0072] Using the method of Example 1, Example 4 added 500 μl of the prepared bacterial solution to the sterilized tobacco leaf culture medium in a clean bench. The difference was that after sealing, it was placed in a 25°C constant temperature shaking incubator and shaken at a speed of 160 rpm / min for 5 days as the experimental group. The control group added 500 μl of sterile deionized water, and the other experimental conditions were the same as those of Example 1.

[0073] At the end of the experiment, the fermentation liquid was transferred to a sterile 50 ml centrifuge tube, centrifuged at room temperature at 4500 rpm / min for 10 min, the supernatant was discarded, the precipitate was collected, and the precipitate was washed once with sterile deionized water. The obtained tobacco leaf precipitate and bacterial cell mixture was placed in a 55°C oven to completely evaporate the water, and the content of tobacco-specific nitrosamines (TSNAs) in the prepared tobacco leaves was determined as shown in Table 4.

[0074] Table 4 TSNAs content of tobacco leaves after 25℃ liquid fermentation for 5 days after inoculation

[0075]

[0076] Example 5

[0077] Example 5 relates to a fermentation method for reducing TSNAs in tobacco leaves using Bacillus pumilus 05-5402, which is a liquid fermentation method, and the liquid fermentation system is controlled to ferment at 25° C. for 7 days.

[0078] Using the method of Example 1, Example 5 added 500 μl of the prepared bacterial solution to the sterilized tobacco leaf culture medium in a clean bench. The difference was that after sealing, it was placed in a 25°C constant temperature shaking incubator and shaken at a speed of 160 rpm / min for 7 days as the experimental group. The control group added 500 μl of sterile deionized water, and the other experimental conditions were the same as those of Example 1.

[0079] At the end of the experiment, the fermentation liquid was transferred to a sterile 50 ml centrifuge tube, centrifuged at room temperature at 4500 rpm / min for 10 min, the supernatant was discarded, the precipitate was collected, and the precipitate was washed once with sterile deionized water. The obtained tobacco leaf precipitate and bacterial cell mixture was placed in a 55°C oven to completely evaporate the water, and the content of tobacco-specific nitrosamines (TSNAs) in the prepared tobacco leaves was measured as shown in Table 5.

[0080] Table 5 TSNAs content of tobacco leaves after 7 days of liquid fermentation at 25℃ after inoculation of bacteria

[0081]

[0082] Example 6

[0083] Example 6 relates to a fermentation method for reducing TSNAs in tobacco leaves using Bacillus pumilus 05-5402, which is a liquid fermentation method, and the liquid fermentation system is controlled to ferment at 25° C. for 10 days.

[0084] Using the method of Example 1, Example 6 added 500 μl of the prepared bacterial solution to the sterilized tobacco leaf culture medium in a clean bench. The difference was that after sealing, it was placed in a 25°C constant temperature shaking incubator and shaken at a speed of 160 rpm / min for 10 days as the experimental group. The control group added 500 μl of sterile deionized water, and the other experimental conditions were the same as those of Example 1.

[0085] At the end of the experiment, the fermentation liquid was transferred to a sterile 50 ml centrifuge tube, centrifuged at room temperature at 4500 rpm / min for 10 min, the supernatant was discarded, the precipitate was collected, and the precipitate was washed once with sterile deionized water. The obtained tobacco leaf precipitate and bacterial cell mixture was placed in a 55°C oven to completely evaporate the water, and the content of tobacco-specific nitrosamines (TSNAs) in the prepared tobacco leaves was measured as shown in Table 6.

[0086] Table 6 TSNAs content of tobacco leaves after 10 days of liquid fermentation at 25℃ after inoculation of bacteria

[0087]

[0088] Comparative Example 1

[0089] Comparative Example 1 relates to a fermentation method for reducing TSNAs in tobacco leaves using Bacillus pumilus 05-5402, which is a liquid fermentation method, and the liquid fermentation system is controlled to ferment at 28° C. for 14 days.

[0090] On the basis of Example 1, the tobacco leaf culture medium inoculated with Bacillus pumilus 05-5402 was placed in a constant temperature shaking incubator at 28°C and shaken at a speed of 160 rpm / min for 14 days as the experimental group. 500 μl of sterile deionized water was added to the control group, and other experimental conditions were the same as in Example 1.

[0091] At the end of the experiment, the fermentation liquid was transferred to a sterile 50 ml centrifuge tube and centrifuged at room temperature at 4500 rpm / min for 10 min. The supernatant was discarded, the precipitate was collected, and the precipitate was washed once with sterile deionized water. The obtained mixture of tobacco leaf precipitate and bacterial cells was placed in a 55°C oven to completely evaporate the water, and the content of tobacco-specific nitrosamines (TSNAs) after fermentation was determined as shown in Table 7.

[0092] Table 7 TSNAs content of tobacco leaves after 14 days of liquid fermentation at 28℃ after inoculation

[0093]

[0094] Comparative Example 2

[0095] Comparative Example 2 relates to a fermentation method for reducing TSNAs in tobacco leaves using Bacillus pumilus 05-5402, which is a liquid fermentation method, and the liquid fermentation system is controlled to ferment at 28° C. for 21 days.

[0096] On the basis of Example 1, the tobacco leaf culture medium inoculated with Bacillus pumilus 05-5402 was placed in a constant temperature shaking incubator at 28°C and shaken at a speed of 160 rpm / min for 21 days as the experimental group. 500 μl of sterile deionized water was added to the control group, and other experimental conditions were the same as in Example 1.

[0097] At the end of the experiment, the fermentation liquid was transferred to a sterile 50 ml centrifuge tube and centrifuged at 4500 rpm / min for 10 min at room temperature. The supernatant was discarded, the precipitate was collected, and the precipitate was washed once with sterile deionized water. The obtained mixture of tobacco leaf precipitate and bacterial cells was placed in a 55°C oven to completely evaporate the water, and the content of tobacco-specific nitrosamines (TSNAs) after fermentation was determined as shown in Table 8.

[0098] Table 8 TSNAs content of tobacco leaves after 21 days of liquid fermentation at 28℃ after inoculation

[0099]

[0100] Comparative Example 3

[0101] Comparative Example 3 relates to a fermentation method for reducing TSNAs in tobacco leaves using Bacillus pumilus 05-5402, which is a liquid fermentation method, and the liquid fermentation system is controlled to ferment at 35° C. for 7 days.

[0102] The method of Example 1 was used to add 500 μl of the prepared bacterial solution to the sterilized tobacco leaf culture medium in a clean bench. The difference was that after sealing, it was placed in a 35°C constant temperature shaking incubator and shaken at 160 rpm / min for 7 days as the experimental group. 500 μl of sterile deionized water was added to the control group, and other experimental conditions were the same as those in Example 1.

[0103] At the end of the experiment, the fermentation liquid was transferred to a sterile 50 ml centrifuge tube, centrifuged at room temperature at 4500 rpm / min for 10 min, the supernatant was discarded, the precipitate was collected, and the precipitate was washed once with sterile deionized water. The obtained tobacco leaf precipitate and bacterial cell mixture was placed in a 55°C oven to completely evaporate the water, and the content of tobacco-specific nitrosamines (TSNAs) in the prepared tobacco leaves was determined as shown in Table 9.

[0104] Table 9 TSNAs content of tobacco leaves after 7 days of liquid fermentation at 35℃ after inoculation

[0105]

[0106] Comparative Example 4

[0107] Comparative Example 4 relates to a fermentation method for reducing TSNAs in tobacco leaves using Bacillus pumilus 05-5402, which is a liquid fermentation method, and the liquid fermentation system is controlled to ferment at 35° C. for 14 days.

[0108] The method of Example 1 was used to add 500 μl of the prepared bacterial solution to the sterilized tobacco leaf culture medium in a clean bench. The difference was that after sealing, it was placed in a 35°C constant temperature shaking incubator and cultured at a speed of 160 rpm / min for 14 days as the experimental group. 500 μl of sterile deionized water was added to the control group, and the other experimental conditions were the same as those in Example 1.

[0109] At the end of the experiment, the fermentation liquid was transferred to a sterile 50 ml centrifuge tube and centrifuged at 4500 rpm / min for 10 min at room temperature. The supernatant was discarded, the precipitate was collected, and the precipitate was washed once with sterile deionized water. The obtained mixture of tobacco leaf precipitate and bacterial cells was placed in a 55°C oven to completely evaporate the water, and the content of tobacco-specific nitrosamines (TSNAs) after fermentation was determined as shown in Table 10.

[0110] Table 10 TSNAs content of tobacco leaves after inoculation at 35℃ for 14 days

[0111]

[0112] Comparative Example 5

[0113] Comparative Example 5 relates to a fermentation method for reducing TSNAs in tobacco leaves using Bacillus pumilus 05-5402, which is a liquid fermentation method, and the liquid fermentation system is controlled to ferment at 35° C. for 21 days.

[0114] The method of Example 1 was used to add 500 μl of the prepared bacterial solution to the sterilized tobacco leaf culture medium in a clean bench. The difference was that after sealing, it was placed in a 35°C constant temperature shaking incubator and cultured at a speed of 160 rpm / min for 21 days as the experimental group. 500 μl of sterile deionized water was added to the control group, and other experimental conditions were the same as those in Example 1.

[0115] At the end of the experiment, the fermentation liquid was transferred to a sterile 50 ml centrifuge tube and centrifuged at 4500 rpm / min for 10 min at room temperature. The supernatant was discarded, the precipitate was collected, and the precipitate was washed once with sterile deionized water. The obtained mixture of tobacco leaf precipitate and bacterial cells was placed in a 55°C oven to completely evaporate the water, and the content of tobacco-specific nitrosamines (TSNAs) after fermentation was determined as shown in Table 11.

[0116] Table 11 TSNAs content of tobacco leaves after inoculation at 35℃ for 21 days

[0117]

[0118] Comparative Example 6

[0119] Comparative Example 6 relates to a fermentation method for reducing TSNAs in tobacco leaves using Bacillus pumilus 05-5402, which is a solid-state fermentation method. The solid-state fermentation system is controlled to ferment at 28° C. for 7 days with a water content of 50%.

[0120] Similar to the liquid fermentation method, the flue-cured tobacco used in the solid fermentation process is cinnabar tobacco grown in Yanhe Town, Yuxi City, Yunnan Province. The middle tobacco leaves are picked after maturity, collected after being baked at the experimental base, and ground into powder for fermentation.

[0121] The solid-state fermentation operation is as follows:

[0122] Weigh 5g tobacco leaves into a fermentation bottle, add appropriate amount of water to adjust the moisture content of the whole fermentation system to 50%, seal it with sealing film and sterilize it in a high pressure steam sterilizer at 115℃ for 20min. After sterilization, wait for the tobacco leaves to cool to room temperature and add 2mlOD 600 = 2.0 bacterial solution (the bacterial solution is the same as in Example 1), the control group was added with an equal amount of sterile deionized water, three replicates were set for each treatment, and then the fermentation bottle was placed in a constant temperature incubator at 28°C for 7 days. After the fermentation was completed, the tobacco leaf powder was placed in a 65°C oven for drying, and then the TSNAs content was detected.

[0123] Table 12 TSNAs content of tobacco leaves after inoculation with 50% moisture content at 28°C for 7 days

[0124]

[0125] Comparative Example 7

[0126] Comparative Example 7 relates to a fermentation method for reducing TSNAs in tobacco leaves using Bacillus pumilus 05-5402, which is a solid-state fermentation method. The solid-state fermentation system is controlled to ferment at 28° C. for 7 days with a water content of 70%.

[0127] The flue-cured tobacco used in the fermentation process is cinnabar tobacco grown in Yanhe Town, Yuxi City, Yunnan Province. The middle tobacco leaves are picked after maturity, and the tobacco leaves are collected after being baked at the test base, and then ground into powder for fermentation. The solid-state fermentation operation is as follows:

[0128] Weigh 5g tobacco leaves into a fermentation bottle, add appropriate amount of water to adjust the moisture content of the whole fermentation system to 70%, seal it with sealing film and sterilize it in a high pressure steam sterilizer at 115℃ for 20min. After sterilization, wait for the tobacco leaves to cool to room temperature and add 2mlOD 600= 2.0 bacterial solution (the bacterial solution is the same as in Example 1), the control group was added with an equal amount of sterile deionized water, three replicates were set for each treatment, and then the fermentation bottle was placed in a constant temperature incubator at 28°C for 7 days. After the fermentation was completed, the tobacco leaf powder was placed in a 65°C oven for drying, and then the TSNAs content was detected.

[0129] Table 13 TSNAs content of tobacco leaves after inoculation with 70% moisture content at 28°C for 7 days

[0130]

[0131] Experimental results:

[0132] From the results of Tables 1 to 11, it can be seen that in the method of reducing TSNAs in tobacco leaves during liquid fermentation of tobacco leaves using Bacillus pumilus 05-5402, by controlling the fermentation conditions as follows: fermentation temperature: 25-28°C, fermentation time: 5-10 days, the TSNAs content of tobacco leaves can be reduced by 13.99% to 31.99%. The best fermentation conditions are fermentation at 28°C for 7 days, and the TSNAs degradation rate is 31.99%. On the contrary, if the liquid fermentation conditions are not properly controlled, there is no obvious degradation effect. Among them, the TSNAs content of tobacco leaves in the experimental group fermented at 35°C for 7-21 days has a low reduction, or even an increase, and the TSNAs content of tobacco leaves in the experimental group fermented at 28°C for 21 days has a low reduction. From the results of Table 2, Table 12 and Table 13, it can be seen that under the same fermentation temperature and time, under the condition of 50% water content, the 05-5402 solid-state fermentation (Comparative Example 6) has almost no degradation effect on TSNAs; under the condition of 70% water content, the TSNAs degradation rate of 05-5402 solid-state fermentation (Comparative Example 7) is 17%, while in the liquid fermentation mode (Example 2), the TSNAs content decreases by 31.99%, indicating that under the same conditions, the degradation effect of liquid fermentation is more significant.

[0133] The above description is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that are not conceived through creative work should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope defined in the claims.

Claims

1. A fermentation method for reducing TSNAs in tobacco leaves using Bacillus pumilus 05-5402, characterized in that The steps include: Step 1: crushing tobacco leaves: grinding the roasted tobacco leaves into tobacco leaf powder to obtain fermentation raw materials; Step 2, preparing bacterial liquid: activating the Bacillus pumilus 05-5402 strain in a preserved state to obtain a bacterial liquid containing a fermentation agent, wherein the preservation number of the Bacillus pumilus 05-5402 strain is CGMCC No.7418; Step 3, tobacco leaf fermentation: adding the tobacco leaf powder to an inorganic salt basal medium to prepare a tobacco medium, adding a bacterial solution prepared with the Bacillus pumilus 05-5402 strain to the tobacco medium at the beginning of fermentation, sealing the medium, and performing liquid fermentation of the tobacco leaves at a fermentation temperature of 25-28° C. and a fermentation time of 5-10 days. The OD of the bacterial solution is 600 The activation of the strain is to inoculate the Bacillus pumilus 05-5402 strain in the preserved state on LB solid culture medium, culture at 28°C for 10-12h until a single colony grows, select a single colony and transfer it to LB liquid culture medium, culture at 28°C for 24-48h to obtain a fermentation agent, and then adjust the OD value of the fermentation agent to OD with sterile deionized water. 600 =2.0, to obtain a bacterial solution, wherein the components of the LB liquid culture medium are calculated in g / L: 5 g yeast extract powder, 10 g peptone, and 10 g NaCl; The LB solid medium is prepared by adding agar powder to the liquid medium at a ratio of 15 g / L. The inorganic salt basal medium components are calculated in g / L: K2HPO4 0.2, KH2PO4 0.8, MgSO4 0.2, CaSO4·H2O 0.1, Na2MoO4 0.0033, FeSO4·7H2O0.

005.

2. The fermentation method for reducing TSNAs in tobacco leaves using Bacillus pumilus 05-5402 according to claim 1, characterized in that The inoculation amount of the bacterial solution is 1% of the total volume of the tobacco culture medium.

3. The fermentation method for reducing TSNAs in tobacco leaves using Bacillus pumilus 05-5402 according to claim 1, characterized in that The tobacco culture medium for the liquid fermentation system is prepared according to the ratio of tobacco leaf powder (g): inorganic salt culture medium (g) = 1:

9.

4. The fermentation method for reducing TSNAs in tobacco leaves using Bacillus pumilus 05-5402 according to claim 1, characterized in that The liquid fermentation of the tobacco leaves is carried out in a constant temperature shaking incubator, with a fermentation temperature of 25-28° C., a fermentation time of 5-10 days, and a rotation speed of 160 rpm / min.

5. The fermentation method for reducing TSNAs in tobacco leaves using Bacillus pumilus 05-5402 according to claim 1, characterized in that The baked tobacco leaves are baked cinnabar tobacco leaves.

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