Bacterial formula preparation for increasing fruit sweetness and fragrance of tobacco and softening smoke and tobacco product

Abstract

The invention belongs to the technical field of tobacco microorganisms, and relates to a bacterial formula preparation for increasing the sweet aroma of tobacco fruits and softening smoke and a tobacco product, the bacterial formula preparation comprises bacillus altitudinis Y120, enterobacter hormaechei Y399 and bacillus velezensis YB371, and the bacillus altitudinis Y120, the enterobacter hormaechei Y399, the bacillus velezensis YB371, the bacillus velezensis YB371, the bacillus velezensis YB371, the bacillus velezensis YB371, the bacillus velezensis YB371 and the bacillus velezensis YB371 and the bacillus velezensis YB371 are used for increasing the sweet aroma of the tobacco fruits and softening the smoke. The preservation numbers of the strain in the China General Microbiological Culture Collection Center (CGMCC) are respectively CGMCC No.15626, CGMCC No.15627 and CGMCC No.30226. The invention also discloses a method for preparing the strain. The bacterial formula preparation is sprayed on the surface of a tobacco raw material, after fermentation, the fruit sweet fragrance of the tobacco raw material during combustion and smoking can be obviously improved, and smoke is softened.

Description

Technical Field

[0001] This invention belongs to the field of tobacco microbial technology and relates to a bacterial formulation and tobacco products for increasing the fruity sweetness and smoothness of tobacco smoke. Background Technology

[0002] Fruity sweetness is one of the key aroma components of tobacco leaves. It is a pleasant complex aroma of fruit and sweetness perceived through the nasal cavity during tobacco combustion, enriching the overall aroma of tobacco. As consumers increasingly value the sensory experience of tobacco products, flavorings and fragrances are often added during manufacturing to enhance the aroma and improve smoking pleasure and satisfaction. However, the formulation and dosage of flavorings and fragrances are complex and difficult to control, and the fruity sweetness imparted to tobacco products by these flavorings is not always natural or mellow. Aroma-producing microorganisms can generate aroma components through metabolism and play a crucial role in improving the quality of tobacco raw materials during tobacco fermentation. Utilizing aroma-producing microorganisms to improve the quality of tobacco raw materials has significant practical and economic value.

[0003] However, the tobacco industry currently pays little attention to and studies microorganisms that produce fruity sweetness and aroma. For example, the fruity aroma-producing microorganisms in invention CN106978352B are mainly fungi and are not suitable for direct use as microbial additives in tobacco fermentation. Invention CN111254092B is designed to increase the natural oral sweetness when smoking tobacco, rather than targeting the fruity sweetness and aroma perceived through the nasal cavity.

[0004] Therefore, developing new bacterial formulations to enhance the fruity aroma and smooth smoke during tobacco combustion is of great significance for improving the quality of tobacco raw materials. Summary of the Invention

[0005] The present invention aims to overcome the defects of the prior art and provide a bacterial formulation and tobacco product for increasing the fruity aroma and smoothing the smoke of tobacco. After fermenting tobacco raw materials with the bacterial formulation of the present invention, the fruity aroma of tobacco raw materials during combustion and smoking can be significantly increased and the smoke can be smoothed.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows:

[0007] A bacterial formulation for enhancing the fruity aroma and smoothness of tobacco smoke, comprising *Bacillus altitudinis* Y120, *Enterobacter hormaechei* Y399, and *Bacillus velezensis* YB371; wherein *Bacillus altitudinis* Y120, *Enterobacter hormaechei* Y399, and *Bacillus velezensis* YB371 are deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession numbers CGMCC No. 15626, CGMCC No. 15627, and CGMCC No. 30226, respectively; and wherein *Bacillus altitudinis* Y120, *Enterobacter hormaechei* Y399, and *Bacillus velezensis* YB371 are deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession numbers CGMCC No. 15626, CGMCC No. 15627, and CGMCC No. 30226, respectively; The deposit date of *Enterobacter altitudinis* Y120 is April 18, 2018; the deposit date of *Enterobacter hormaechei* Y399 is April 18, 2018; and the deposit date of *Bacillus velezensis* YB371 is April 1, 2024.

[0008] Preferably, the nucleotide sequences of Bacillus altitudinis Y120, Enterobacter hormaechei Y399, and Bacillus velezensis YB371 are shown in SEQ ID No. 1, SEQ ID No. 2, and SEQ ID No. 3, respectively.

[0009] Preferably, the mass ratio of Bacillus altitudinis Y120, Enterobacter hormaechei Y399, and Bacillus velezensis YB371 is (20-30):1:(30-50).

[0010] Preferably, the bacterial formulation containing Bacillus altitudinis Y120, Enterobacter hormaechei Y399, and Bacillus velezensis YB371 can be prepared as wet cells, bacterial liquid, powder, or lyophilized powder.

[0011] A tobacco product that has a sweet fruity aroma and a mild smoke when burned and smoked is obtained by spraying the bacterial formulation preparation onto the surface of tobacco raw materials, fermenting it, and then drying it.

[0012] Preferably, the tobacco raw material is one or a mixture of several of the following: single-grade raw tobacco leaves, cigarette blends, and cigarette leaf groups.

[0013] Preferably, the single-grade raw tobacco leaves are flue-cured tobacco, burley tobacco, aromatic tobacco or sun-cured tobacco, and the cigarette blending material is stem or reconstituted tobacco.

[0014] This invention provides a bacterial formulation for enhancing the fruity aroma and smooth smoke of tobacco during smoking, from the perspective of increasing the fruity aroma and smooth smoke of tobacco during smoking. The bacterial formulation contains Bacillus saltitudinis Y120, Enterobacter hormaechei Y399, and Bacillus velezensis YB371 strains. The results showed that Bacillus altitudinis Y120, when applied alone to tobacco raw materials, significantly increased the sweetness in the mouth during smoking; Enterobacter hormaechei Y399, when applied alone to tobacco raw materials, significantly improved oral comfort; and Bacillus velezensis YB371, when applied alone to tobacco raw materials, significantly increased the amount and quality of aroma during smoking, enhancing the fruity sweetness of the tobacco. It is evident that although all are bacterial fermentations of tobacco raw materials, different strains have different effects on the tobacco raw materials when combined in different formulations. The results of this invention show that, after fermenting tobacco with the bacterial formulation of this invention, on the one hand, the fruity sweetness of the tobacco raw material can be significantly increased from a sensory perspective during combustion and smoking, and the smoke is softened. On the other hand, the content of ketone compounds related to nasal sweetness in the smoke, especially 6-ethyl-5,6-dihydro-2H-pyran-2-one, (s)-4-hydroxymethyldihydrofuran-2-one, and 2,5-diethyl-3-hexanone, which are related to fruity sweetness, is significantly increased compared with the control sample, and the content of neophytadiene, which is related to softened smoke, is also increased.

[0015] The beneficial effects of this invention are:

[0016] This invention relates to a bacterial formulation applied to the fermentation of tobacco raw materials, which enhances the fruity and sweet aroma during the combustion and smoking of the tobacco, and softens the smoke. This bacterial formulation is not only easy to use and produces a naturally sweet aroma, but its application in tobacco production can improve tobacco utilization efficiency and reduce tobacco processing costs. Furthermore, this bacterial formulation has good versatility, effectively enhancing the fruity and sweet aroma and softening the smoke when burned and smoked from various tobacco raw materials. Attached Figure Description

[0017] Figure 1 This is an aroma style characteristic diagram of the smoke from fermented tobacco raw materials, which is used to enhance the sweet aroma and mellow smoke of tobacco and to produce a bacterial formulation for tobacco products according to the present invention.

[0018] Figure 2 This is a characteristic diagram of the flue gas from the fermented tobacco raw material of the present invention.

[0019] Figure 3 This is a chromatogram of the chemical composition of the flue gas from the fermented tobacco raw material of the present invention. Detailed Implementation

[0020] The related technologies of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0021] like Figures 1 to 3 As shown, this invention isolated three strains from tobacco leaves: Bacillus altitudinis Y120, Enterobacter hormaechei Y399, and Bacillus velezensis YB371, and preserved them as follows:

[0022] The depositary institution is the China General Microbiological Culture Collection Center (CGMCC). The accession numbers for Bacillus altitudinis Y120, Enterobacter hormaechei Y399, and Bacillus velezensis YB371 are CGMCC No. 15626, CGMCC No. 15627, and CGMCC No. 30226, respectively. The deposit date for Bacillus altitudinis Y120 is April 18, 2018; the deposit date for Enterobacter hormaechei Y399 is April 18, 2018; and the deposit date for Bacillus velezensis YB371 is April 1, 2024.

[0023] The 16S rRNA gene sequence of Bacillus altitudinis Y120 is shown in SEQ ID No. 1, the 16S rRNA gene sequence of Enterobacter hormaechei Y399 is shown in SEQ ID No. 2, and the 16S rRNA gene sequence of Bacillus velezensis YB371 is shown in SEQ ID No. 3.

[0024] This invention relates to a bacterial formulation for enhancing the fruity aroma and smoothness of tobacco smoke, comprising Bacillus altitudinis Y120, Enterobacter hormaechei Y399, and Bacillus velezensis YB371. The mass ratio of Bacillus altitudinis Y120, Enterobacter hormaechei Y399, and Bacillus velezensis YB371 is (20-30):1:(30-50).

[0025] Bacterial formulations containing Bacillus altitudinis Y120, Enterobacter hormaechei Y399, and Bacillus velezensis YB371 can be prepared as wet cells, bacterial solutions, powders, or lyophilized powders.

[0026] Tobacco raw materials may be single-grade raw tobacco leaves (flue-cured tobacco, burley tobacco, aromatic tobacco or sun-cured tobacco), cigarette blends (stems, reconstituted tobacco leaves, etc.) or cigarette leaf groups, or mixtures of the above tobacco components.

[0027] Example

[0028] Example 1

[0029] Isolation, screening, and culture of Bacillus altitudinis Y120, Enterobacter hormaechei Y399, and Bacillus velezensis YB371:

[0030] 1. The steps for isolating strains from fresh tobacco leaves are as follows: Fresh tobacco leaves are chopped and mixed, 10g is weighed and added to 90mL of sterile water, and then diluted stepwise. 0.25mL of each dilution suspension is spread on modified LB solid medium plates and incubated at 37℃ for 24h. Single colonies with good growth are picked, purified and stored at 4℃.

[0031] The modified LB liquid medium consisted of: 1% tryptone, 0.5% yeast extract, and 0.25% NaCl; pH 7.0.

[0032] The modified LB solid medium consists of: 1% tryptone, 0.5% yeast extract, 0.25% NaCl, and 1% agar; pH 7.0. The modified LB solid medium can be prepared into plates or slants, etc.

[0033] Unless otherwise specified, all percentages in this invention refer to mass percentages.

[0034] 2. Perform enzyme activity tests on all pure culture strains obtained from screening.

[0035] Protease activity test: The strain was inoculated onto the protease selection medium and cultured. If a transparent hydrolysis zone appeared around the strain, it indicated that the strain could produce protease. The size of the hydrolysis zone was measured and recorded.

[0036] Amylase activity test: The strain was inoculated onto amylase selection medium and cultured. Iodine solution was added to the plate to cover the entire medium. If an unstained transparent hydrolysis zone appeared around the strain, it indicated that the strain could produce amylase. The size of the hydrolysis zone was measured and recorded.

[0037] The enzyme activity tests of Bacillus altitudinis Y120, Enterobacter hormaechei Y399, and Bacillus velezensis YB371 described in this invention show that they all have the ability to produce both protease and amylase simultaneously.

[0038] The protease screening medium consisted of: 1% casein, 0.5% yeast extract, 0.4% NaCl, 1% agar, and pH 7.2.

[0039] The amylase screening medium consisted of: 1% soluble starch, 0.5% peptone, 0.5% NaCl, 0.5% beef extract, 1% agar, and pH 7.0.

[0040] Unless otherwise specified, all percentages in this invention refer to mass percentages.

[0041] 3. Preliminary tobacco fermentation tests were conducted on all the pure culture strains obtained from the screening. Based on the physicochemical properties of tobacco and the evaluation results of sensory quality changes during tobacco burning, the strains finally screened and identified were Bacillus altitudinis Y120, Enterobacter hormaechei Y399, and Bacillus velezensis YB371. Among them, Bacillus altitudinis Y120, when applied alone to tobacco raw materials, significantly increases the sweetness in the mouth during tobacco combustion and smoking; Enterobacter hormaechei Y399, when applied alone to tobacco raw materials, significantly improves oral comfort; and Bacillus velezensis YB371, when applied alone to tobacco raw materials, significantly increases the amount of aroma during tobacco smoking, enhances the aroma quality, and adds a fruity sweetness to the tobacco flavor.

[0042] 4. The obtained Bacillus altitudinis Y120, Enterobacter hormaechei Y399, and Bacillus velezensis YB371 were cultured as follows: Bacillus altitudinis Y120, Enterobacter hormaechei Y399, and Bacillus velezensis YB371 were each inoculated into 100 mL of modified LB liquid medium and cultured at 25℃~40℃ with shaking on a shaker at 100rpm~200rpm for 12h~48h to obtain Bacillus altitudinis Y120, Enterobacter hormaechei Y399, and Bacillus velezensis YB371, respectively. Culture medium of velezensis YB371; centrifuge the culture medium of each strain at 4000r / min~18000r / min for 5min~20min at 4℃, discard the supernatant to obtain each wet cell, which can be further processed into powder or lyophilized powder and stored in a refrigerator at 4℃ for later use.

[0043] Example 2

[0044] In practical implementation, Bacillus altitudinis Y120, Enterobacter hormaechei Y399, and Bacillus velezensis YB371 are mixed in a mass ratio of (20-30):1:(30-50) to obtain the bacterial formulation of this invention. The bacterial formulation can be prepared as wet cells, bacterial solution, powder, or lyophilized powder.

[0045] 1. The Bacillus altitudinis Y120, Enterobacter hormaechei Y399, and Bacillus velezensis YB371 obtained in Example 1 were mixed in a mass ratio of 20:1:30, which is called bacterial formulation preparation 1.

[0046] 2. The Bacillus altitudinis Y120, Enterobacter hormaechei Y399, and Bacillus velezensis YB371 obtained in Example 1 were mixed in a mass ratio of 30:1:50, which is called bacterial formulation preparation 2.

[0047] 3. The Bacillus altitudinis Y120, Enterobacter hormaechei Y399, and Bacillus velezensis YB371 obtained in Example 1 were mixed in a mass ratio of 25:1:40, which is called bacterial formulation preparation 3.

[0048] 4. The Bacillus altitudinis Y120, Enterobacter hormaechei Y399, and Bacillus velezensis YB371 obtained in Example 1 were mixed in a mass ratio of 20:1:50, which is called bacterial formulation preparation 4.

[0049] Example 3

[0050] The bacterial formulation prepared in Example 2 was used for the fermentation of tobacco raw materials: the bacterial formulation prepared in Example 2 was sprayed onto the surface of the tobacco raw materials, and fermented at a constant temperature and humidity of 20℃~45℃ and 30%~90% for 20h~80h. After fermentation, the raw materials were dried to a moisture content of 10%~13%, thus obtaining the tobacco raw materials fermented with the bacterial formulation. The tobacco raw materials can be single-grade raw tobacco leaves (flue-cured tobacco, burley tobacco, aromatic tobacco, or sun-cured tobacco), cigarette blends (stems, reconstituted tobacco leaves, etc.), cigarette leaf groups, or mixtures of the above tobacco components.

[0051] 1. The bacterial formulation 1 prepared in Example 2 was used for the fermentation of tobacco raw materials: The bacterial formulation 1 prepared in Example 2 was sprayed onto the surface of the tobacco raw materials, and fermented at a constant temperature and humidity of 20°C and 90% for 80 hours. After fermentation, the raw materials were dried until the moisture content of the tobacco raw materials was 13%, thus obtaining the tobacco raw materials fermented with the bacterial formulation. The tobacco raw materials were stems.

[0052] 2. The bacterial formulation 2 prepared in Example 2 was used for the fermentation of tobacco raw materials: The bacterial formulation 2 prepared in Example 2 was sprayed onto the surface of the tobacco raw materials, and fermented at a constant temperature and humidity of 45°C and 30% for 20 hours. After fermentation, the raw materials were dried until the moisture content of the tobacco raw materials was 10%, thus obtaining the tobacco raw materials fermented with the bacterial formulation. The tobacco raw materials were reconstituted tobacco leaves.

[0053] 3. The bacterial formulation 3 prepared in Example 2 was used for the fermentation of tobacco raw materials. The bacterial formulation 3 prepared in Example 2 was sprayed onto the surface of the tobacco raw materials, and fermented at a constant temperature and humidity of 37°C and 60% for 50 hours. After fermentation, the raw materials were dried until the moisture content of the tobacco raw materials was 12%, thus obtaining the tobacco raw materials fermented with the bacterial formulation. The tobacco raw materials were tobacco leaves.

[0054] Example 4

[0055] The tobacco raw materials (a mixture of tobacco components such as tobacco leaves and reconstituted tobacco leaves) fermented with the bacterial formulation of the present invention obtained in Example 3 were used as experimental samples. Under the same conditions, the tobacco raw materials were treated directly with the same amount of sterile water under the same conditions without inoculation with the bacterial formulation for fermentation, serving as control samples. Cigarettes were then rolled. Sensory evaluation was conducted by 11 sensory evaluation personnel with tobacco industry qualifications, referring to the tobacco industry standard "Sensory Evaluation Method for Chinese-style Cigarette Style" YC / T 497-2014. The aroma style and smoke characteristics of the smoke were detected, and the average results were taken (the score range for each indicator is 0-10 points, with 0.5 points as the scoring unit, and the average value is taken). The chemical components causing the smoke were analyzed by gas chromatography-mass spectrometry (GC / MS).

[0056] like Figure 1 As shown, the aroma profile of tobacco raw materials treated with the bacterial formulation of this invention exhibits a significant increase in fruity and sweet notes during combustion and smoking, with increases of 105.56% and 64.86% respectively compared to the control sample. Meanwhile, as... Figure 2 As shown, the smoke characteristics of tobacco raw materials treated with the bacterial formulation of this invention during combustion and smoking are significantly improved, with a 13.16% improvement compared to the control sample. The aroma and off-flavors are also improved.

[0057] As shown in Table 1, the content of ketone compounds related to nasal sweetness in the tobacco smoke treated with the bacterial preparation of the present invention increased by 5.77% compared with the control sample. Among them, the content of 6-ethyl-5,6-dihydro-2H-pyran-2-one, which is related to fruity sweetness, increased by 5.72% compared with the control sample; the content of (s)-4-hydroxymethyldihydrofuran-2-one increased by 8.92% compared with the control sample; the content of 2,5-diethyl-3-hexanone increased by 13.90% compared with the control sample; and the content of neophytadiene, which is related to mild smoke, increased by 35.23% compared with the control.

[0058] Retention time Chemical component name control sample Test sample 24.84 2,3-Dihydro-3,5-Dihydroxy-6-methyl-4H-pyran-4-one 0.313 0.317 25.70 6-Ethyl-5,6-dihydro-2H-pyran-2-one 0.828 0.875 26.83 (s)-4-hydroxymethyldihydrofuran-2-one 0.131 0.142 28.28 2,5-Diethyl-3-hexanone 0.034 0.038 43.55 Megastigmatrienone A 0.119 0.147 48.91 4-(3-hydroxybutyl)-3,5,5-trimethylcyclohexyl-2-en-1-one 0.260 0.262 53.27 Neophytadiene 26.506 35.843

[0059] Table 1

[0060] In summary, the present invention is simple to use, and produces a natural, sweet fruit aroma and a mild smoke. When used in tobacco production, it can improve tobacco utilization efficiency and reduce tobacco processing costs. Furthermore, the bacterial formulation of the present invention has good versatility, effectively enhancing the sweet fruit aroma and softening the smoke when burned and smoked from various tobacco raw materials.

[0061] It should be emphasized that the above are merely preferred embodiments of the present invention and are not intended to limit the present invention in any way. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention shall still fall within the scope of the technical solution of the present invention.

Claims

1. A bacterial formulation for enhancing the fruity aroma and smoothness of tobacco smoke, characterized in that, The bacterial formulation comprises: Bacillus altitudinis Y120, Enterobacter hormaechei Y399, and Bacillus velezensis YB371; Bacillus altitudinis Y120, Enterobacter hormaechei Y399, and Bacillus velezensis YB371 are deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession numbers CGMCC No. 15626, CGMCC No. 15627, and CGMCC No. 30226, respectively; the deposit date of Bacillus altitudinis Y120 is April 18, 2018, and the accession date of Enterobacter hormaechei YB371 is... The deposit date of Bacillus velezensis YB371 is April 1, 2024.

2. The bacterial formulation for enhancing the fruity aroma and smoothness of tobacco smoke according to claim 1, characterized in that, The nucleotide sequences of Bacillus altitudinis Y120, Enterobacter hormaechei Y399, and Bacillus velezensis YB371 are shown in SEQ ID No. 1, SEQ ID No. 2, and SEQ ID No. 3, respectively.

3. The bacterial formulation for enhancing the fruity aroma and smoothness of tobacco smoke according to claim 1, characterized in that, The mass ratio of Bacillus altitudinis Y120, Enterobacter hormaechei Y399 and Bacillus velezensis YB371 is (20-30):1:(30-50).

4. The bacterial formulation for enhancing the fruity aroma and smoothness of tobacco smoke according to claim 1, characterized in that, The bacterial formulation is a wet bacterial cell, bacterial liquid, powder, or lyophilized powder.

5. A tobacco product that, when burned and smoked, has a fruity aroma and a mild smoke, characterized in that, The bacterial formulation of any one of claims 1-4 is sprayed onto the surface of tobacco raw materials, fermented, and then dried.

6. The tobacco product according to claim 5, which has a fruity aroma and mild smoke when burned and inhaled, is characterized in that... The tobacco raw material is one or a mixture of several of the following: single-grade raw tobacco leaves, cigarette blends, and cigarette leaf groups.

7. The tobacco product with a fruity aroma and mild smoke when burned and inhaled according to claim 6, characterized in that, The single-grade raw tobacco leaves are flue-cured tobacco, burley tobacco, aromatic tobacco, or sun-cured tobacco, and the cigarette blending materials are stems or reconstituted tobacco leaves.

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