CN111254092B

CN111254092B - Bacterial preparation for increasing oral sweet taste and improving oral comfort during combustion and smoking of tobacco and tobacco product

Info

Publication number: CN111254092B
Application number: CN202010066237.5A
Authority: CN
Inventors: 薛磊; 李轩; 郭斌; 郭志刚; 樊雅玲; 吴成春; 潘广乐; 王芳; 王颖
Original assignee: China Tobacco Shaanxi Industrial Co Ltd
Current assignee: China Tobacco Shaanxi Industrial Co Ltd
Priority date: 2020-01-20
Filing date: 2020-01-20
Publication date: 2021-07-30
Anticipated expiration: 2040-01-20
Also published as: CN111254092A

Abstract

The invention discloses a bacterial preparation and a tobacco product for increasing oral sweet taste and improving oral comfort during tobacco combustion and smoking, wherein the bacterial preparation comprises Bacillus belgii (Bacillus velezensis) YBN3, Bacillus altitudinis Y120 and Enterobacter hopcalis (Enterobacter hormaechei) Y399, and the preservation numbers of bacterial strains in the China general microbiological culture Collection center are respectively as follows: CGMCC No.15625, CGMCC No.15626 and CGMCC No. 15627. The bacterial preparation is sprayed on the surface of the tobacco raw material, so that the oral sweet taste of the tobacco raw material during combustion and smoking can be obviously increased after fermentation, and the oral comfort is integrally improved.

Description

Bacterial preparation for increasing oral sweet taste and improving oral comfort during combustion and smoking of tobacco and tobacco product

Technical Field

The invention belongs to the technical field of microorganisms, and relates to a bacterial preparation and a tobacco product for increasing the oral sweet taste and improving the oral comfort during combustion and smoking of tobacco.

Background

Sweet is an oral taste sensation that is very important in human sensory perception. Sweet can bring pleasant satisfaction to people. Tobacco is ingested by smoking upon burning, and sweetness is one of the important oral taste characteristics of tobacco. When the tobacco is burnt, the sweet taste feeling sensed by the oral cavity can bring pleasure and comfort to consumers, and the common sweet substances in the tobacco are as follows: dihydrojuglans sigillata lactone, furfuryl alcohol, furfuryl aldehyde, furan, ketone, alcohol and other compounds (Yan Keyu, et al, 2008; Zhao Mingzhi, 2008). The oral comfort is an important component of the sensory comfort of the cigarette, and the oral comfort indexes such as the prickling, the spicy, the burning, the dry, the convergence/astringency, the residual feeling and the like of smoke in the oral cavity directly influence the preference of consumers to cigarette products.

With the increasing importance of consumers on the sensory perception of tobacco products, in order to meet consumption demands, the oral sweetness of the tobacco products is enhanced by adding some flavors and fragrances during the preparation of the tobacco products, so as to improve the smoking pleasure and satisfaction of the tobacco products. However, when the flavors and fragrances are used, the formula and the using amount of the flavors and fragrances are complex and difficult to master, and the oral sweetness of the tobacco product provided by the flavors and fragrances is not natural and mellow. Although some microorganisms for tobacco, such as aroma-providing microorganism preparations in CN102499438, CN1074467707 and CN107354109A, were developed in the previous research work, these microorganism strains only aim at improving the quality of tobacco aroma, and although the sub-items in the aroma evaluation subject also include "sweet" indexes, they refer to sweet taste feeling, such as sweet smell sensed by nasal cavity when baking cakes, but have no direct sweet taste in oral cavity, while various aromas sensed by nasal cavity and sweet taste felt by oral cavity are two completely different senses when smoking tobacco, i.e. they do not have the function of increasing oral sweet taste, and no directional research on how to increase natural oral sweet taste when smoking tobacco by using microorganisms and improve oral comfort as a whole has been reported. Therefore, the development of a new bacterial preparation for increasing the oral sweet taste and improving the oral comfort during the combustion and smoking of tobacco is of great significance for improving the quality of tobacco raw materials.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide a bacterial preparation and a tobacco product for increasing the oral sweet taste and improving the oral comfort during the combustion and smoking of tobacco.

In order to achieve the purpose, the technical scheme of the invention is as follows:

a bacterial formulation for increasing oral sweetness and improving oral comfort when smoking tobacco, comprising Bacillus belgii (Bacillus velezensis) YBN3, Bacillus altitudinis (Bacillus altitudinis) Y120 and Enterobacter hopcalis (Enterobacter hormaechei) Y399; the Bacillus belgii (Bacillus velezensis) YBN3, the Bacillus altitudinis (Bacillus altitudinis) Y120 and the Enterobacter huoshi (Enterobacter hormaechei) Y399 are all preserved in the general microbiological center of the China Committee for culture Collection of microorganisms at 18 th 4 th 2018, and the preservation numbers are respectively as follows: CGMCC No.15625, CGMCC No.15626 and CGMCC No.15627, and the preservation address is No.3 of Xilu No.1 of Beijing area of rising area.

Preferably, the nucleotide sequences of the Bacillus belgii (Bacillus velezensis) YBN3, Bacillus altitudinis (Bacillus altitudinis) Y120 and Enterobacter hutchii (Enterobacter hormaechei) Y399 are shown as SEQ ID No.1, SEQ ID No.2 and SEQ ID No.3, respectively.

Preferably, the mass ratio of the Bacillus belgii (Bacillus velezensis) YBN3 to the Bacillus altitudinis (Bacillus altitudinis) Y120 to the Enterobacter hophillea (Enterobacter hormaechei) Y399 is (0.1-10): (0.1-10): 1.

preferably, the bacterial preparation comprising Bacillus belief (Bacillus velezensis) YBN3, Bacillus altitudinis (Bacillus altitudinis) Y120 and Enterobacter hophaticus (Enterobacter hormaechei) Y399 may be prepared as a wet cell, a bacterial solution, a powder or a freeze-dried powder.

A tobacco product with sweet taste and comfortableness in oral cavity during combustion and smoking is prepared by spraying the bacterial preparation on the surface of tobacco raw material, fermenting, and oven drying.

Preferably, the tobacco raw material is one or a mixture of several of single-grade raw material tobacco leaves, cigarette blending materials and cigarette leaf groups.

Preferably, the single-grade raw material tobacco leaves are flue-cured tobacco, burley tobacco, aromatic tobacco or sun-cured tobacco, and the cigarette blend is cut stems or reconstituted tobacco.

The invention discloses a bacterial preparation for increasing the oral sweet taste of tobacco during combustion and suction and improving the oral comfort from the perspective of increasing the oral sweet taste of the tobacco during combustion and suction, wherein the bacterial preparation contains Bacillus subtilis YBN3, Bacillus altitudinis Y120 and Enterobacter hollandii Y399 bacterial strains. Research results show that when Bacillus subtilis YBN3 and Bacillus altitudinis Y120 respectively and independently act on tobacco raw materials, the function of the Bacillus subtilis YBN3 and the Bacillus altitudinis Y120 is obviously to increase the oral sweet taste of tobacco during burning and smoking; and the Enterobacter hollandii (Enterobacter hormaechei) Y399 can obviously improve the oral comfort when acting on the tobacco raw material alone. It can be seen that although the bacteria ferment the tobacco material, the effect of different strains on the tobacco material is different. The research result of the invention shows that after the combined bacterial preparation is adopted to ferment the tobacco, on one hand, the contents of chemical components for increasing the oral sweet taste, such as 2-acetylfuran, dihydroactinidiolide, 5-methylfurfural and the like in the tobacco are greatly increased, and on the other hand, the oral sweet taste of the tobacco raw material during combustion and smoking can be obviously increased from the sensory angle, and the oral comfort is integrally improved. Compared with the prior art, the invention uses the bacterial preparation for the first time to increase the sweet taste of the mouth when the tobacco is burnt and smoked and improve the comfort of the mouth as a whole.

The bacterial preparation is applied to the fermentation of tobacco raw materials, so that the quality of the oral sweet taste of the tobacco raw materials during combustion and smoking is obviously improved, and the oral comfort is integrally improved. The bacterial preparation has simple application method and natural oral sweet taste, is used for tobacco production, and can improve tobacco utilization efficiency and reduce tobacco processing cost; the bacterial preparation has better universality, better increases the oral sweet taste when various tobacco raw materials are burnt and smoked, and integrally improves the oral comfort.

Drawings

FIG. 1 is a graph showing the oral taste characteristics of tobacco raw material smoke after fermentation with the bacterial formulation of the present invention.

FIG. 2 is a graph showing the oral comfort of tobacco raw material smoke after fermentation with the bacterial formulation of the present invention.

Detailed Description

The present invention will be described in further detail with reference to the accompanying drawings.

The invention separates three strains of Bacillus velezensis YBN3, Bacillus altitudinis Y120 and Enterobacter hopcalis Y399 from tobacco leaves, and preserves the strains as follows:

the preservation date is as follows: 18 months 4 in 2018; the preservation unit: china general microbiological culture Collection center; the accession numbers of Bacillus belgii (Bacillus velezensis) YBN3, Bacillus altitudinis (Bacillus altitudinis) Y120 and Enterobacter huoshi (Enterobacter hormoneyi) Y399 are respectively as follows: CGMCC No.15625, CGMCC No.15626 and CGMCC No. 15627.

Wherein, the 16S rRNA gene sequence of the Bacillus velezensis YBN3 is shown as SEQ ID No.1, the 16S rRNA gene sequence of the Bacillus altitudinis Y120 is shown as SEQ ID No.2, and the 16S rRNA gene sequence of the Enterobacter hollandii Y399 is shown as SEQ ID No. 3.

The invention relates to a bacterial preparation for increasing oral sweetness and improving oral comfort during tobacco combustion and smoking, which comprises Bacillus belgii (Bacillus velezensis) YBN3, Bacillus altitudinis Y120 and Enterobacter hollandii (Enterobacter hormaechei) Y399. The mass ratio of Bacillus velezensis (Bacillus velezensis) YBN3 to Bacillus altitudinis (Bacillus altitudinis) Y120 to Enterobacter hophatschei (Enterobacter hormoneyi) Y399 is (0.1-10): (0.1-10): 1.

the bacterial preparation comprising Bacillus velezensis YBN3, Bacillus altitudinis Y120 and Enterobacter huoshi (Enterobacter hormaechei) Y399 can be prepared into wet cells, bacterial solutions, powders or freeze-dried powders.

The tobacco material can be single grade tobacco (flue-cured tobacco, burley tobacco, aromatic tobacco or sun-cured tobacco), cigarette blend (cut stem, reconstituted tobacco, etc.) or cigarette leaf group, or mixture of the above tobacco components.

Example 1

Isolation, selection and culture of Bacillus velezensis YBN3, Bacillus altitudinis Y120 and Enterobacter hollisae Y399:

1. the method for separating the bacterial strain for increasing the oral sweet taste and improving the oral comfort during the combustion and the suction of the tobacco from the fresh tobacco leaves comprises the following steps: cutting fresh tobacco leaves, uniformly mixing, weighing 10g, adding into 90mL of sterile water, diluting step by step, coating 0.25mL of suspension with each dilution degree on an improved LB solid culture medium flat plate, culturing at the constant temperature of 37 ℃ for 24h, selecting a single colony with good growth vigor, purifying and storing in a refrigerator at 4 ℃.

The components of the improved LB liquid culture medium are as follows: tryptone 1%, yeast extract 0.5%, NaCl 0.25%; the pH was 7.0.

The components of the improved LB solid culture medium are as follows: tryptone 1%, yeast extract 0.5%, NaCl 0.25%, agar 1%; pH7.0; the improved LB solid culture medium can be made into a flat plate or a slant plate, etc.

All percentages in the present invention are by mass unless otherwise specified.

2. All the pure culture strains obtained by screening were subjected to enzyme production activity test.

Protease activity assay: the strain is inoculated on a protease screening culture medium for culture, if a transparent hydrolysis ring appears around the strain, the strain is shown to be capable of producing protease, and the size of the hydrolysis ring is measured and recorded.

Amylase production activity test: inoculating the strain to amylase screening culture medium for culture, dripping iodine solution on a flat plate to make the flat plate fully spread on the whole culture medium, observing the occurrence of undyed transparent hydrolysis ring around the strain to show that the strain can produce amylase, and measuring and recording the size of the hydrolysis ring.

The activity tests of the Bacillus belgii (Bacillus velezensis) YBN3, Bacillus altitudinis Y120 and Enterobacter hopcalis (Enterobacter hormaechei) Y399 for producing enzyme show that the Bacillus belgii (Bacillus velezensis) YBN3, the Bacillus altitudinis Y120 and the Enterobacter hopcalis (Enterobacter hormaechei) Y399 all have the capability of producing protease and amylase simultaneously.

The protease screening culture medium comprises the following components: 1% of casein, 0.5% of yeast extract, 0.4% of NaCl, 1% of agar and 7.2% of pH;

the amylase screening culture medium comprises the following components: 1% of soluble starch, 0.5% of peptone, 0.5% of NaCl, 0.5% of beef extract, 1% of agar and pH 7.0;

3. All pure culture strains obtained by screening are subjected to a tobacco fermentation preliminary test, and are classified according to the physicochemical properties of tobacco and the results of evaluation of the oral sweetness and the oral comfort of the tobacco, and finally screened and determined strains are Bacillus belief (YBN 3), Bacillus altitudinis (Y120) and Enterobacter hollandii (Y399). When Bacillus subtilis YBN3 and Bacillus altitudinis (Bacillus velezensis) Y120 respectively and independently act on tobacco raw materials, the main function classification is that the oral sweet taste can be obviously increased during the burning and smoking of tobacco; enterobacter holtzeri (Enterobacter hormaechei) Y399, when acting alone on tobacco raw materials, has a main functional classification as being capable of obviously improving the oral comfort.

4. The obtained Bacillus belgii (Bacillus velezensis) YBN3, Bacillus altitudinis (Bacillus altitudinis) Y120 and Enterobacter huoshi (Enterobacter hormaechei) Y399 were cultured by the following method: respectively inoculating Bacillus subtilis YBN3, Bacillus altitudinis Y120 and Enterobacter hopcalis (Enterobacter hormaechei) Y399 to 100mL of improved LB liquid culture medium, and carrying out shaking culture on a constant temperature shaking table at the temperature of 25-40 ℃ and the speed of 100-200 rpm for 12-48 h to respectively obtain culture solutions of the Bacillus subtilis YBN3, the Bacillus altitudinis Y120 and the Enterobacter hopcalis (Enterobacter hormaechei) Y399; centrifuging the obtained culture solution of each strain at the temperature of 4 ℃ at 4000 r/min-18000 r/min for 5 min-20 min, discarding the supernatant to obtain each wet thallus, and further preparing powder or freeze-dried powder which is stored in a refrigerator at the temperature of 4 ℃ for later use.

Example 2

In specific implementation, Bacillus belief (Bacillus velezensis) YBN3, Bacillus altitudinis (Bacillus altitudinis) Y120 and Enterobacter hophallus (Enterobacter hormaechei) Y399 are mixed according to the mass ratio of (0.1-10): (0.1-10): 1, the bacterial preparation of the invention can be obtained. The bacterial preparation can be prepared into wet thallus, bacterial liquid, powder or freeze-dried powder.

1. The Bacillus belius (Bacillus velezensis) YBN3, the Bacillus altitudinis (Bacillus altitudinis) Y120 and the Enterobacter hophaticus (Enterobacter hormaechei) Y399 obtained in the example 1 are proportioned according to the mass ratio of 0.1:0.1:1 and are called as a bacterial preparation 1;

2. the Bacillus belius (Bacillus velezensis) YBN3, the Bacillus altitudinis (Bacillus altitudinis) Y120 and the Enterobacter hophallus (Enterobacter hormaechei) Y399 obtained in the example 1 are proportioned according to the mass ratio of 3:5:1 and are called as a bacterial preparation 2;

3. the Bacillus belius (Bacillus velezensis) YBN3, the Bacillus altitudinis (Bacillus altitudinis) Y120 and the Enterobacter hophallus (Enterobacter hormaechei) Y399 obtained in the example 1 are proportioned according to the mass ratio of 6:8:1 and are called as a bacterial preparation 3;

4. the Bacillus belgii (Bacillus velezensis) YBN3, Bacillus altitudinis Y120 and Enterobacter hophallii (Enterobacter hormaechei) Y399 obtained in example 1 were mixed in a mass ratio of 10:10:1, and the mixture was designated as bacterial preparation 4.

Example 3

The bacterial preparation prepared in example 2 was used for the fermentation of tobacco raw material: spraying the bacterial preparation prepared in the embodiment 2 on the surface of a tobacco raw material, fermenting for 20-80 h at a constant temperature and humidity under the conditions that the temperature is 20-55 ℃ and the relative humidity is 30-90%, and drying after fermentation until the water content is 11-15%, thus obtaining the tobacco raw material fermented by the bacterial preparation. The tobacco material can be single grade tobacco (flue-cured tobacco, burley tobacco, aromatic tobacco or sun-cured tobacco), cigarette blend (cut stem, reconstituted tobacco, etc.), cigarette leaf group, or mixture of the above tobacco components.

1. The bacterial preparation 1 prepared in example 2 was used for the fermentation of tobacco raw material: spraying the bacterial preparation 1 prepared in the embodiment 2 on the surface of a tobacco raw material, fermenting for 80 hours at a constant temperature and humidity under the conditions that the temperature is 20 ℃ and the relative humidity is 90%, drying after fermentation until the moisture content of the tobacco raw material is 15%, and thus obtaining the tobacco raw material fermented by the bacterial preparation. The tobacco material is tobacco leaf.

2. The bacterial preparation 2 prepared in example 2 was used for the fermentation of tobacco raw material: spraying the bacterial preparation 2 prepared in the embodiment 2 on the surface of a tobacco raw material, fermenting for 20 hours at a constant temperature and humidity under the conditions that the temperature is 55 ℃ and the relative humidity is 30%, drying after fermentation until the moisture content of the tobacco raw material is 11%, and thus obtaining the tobacco raw material fermented by the bacterial preparation. The tobacco raw material is reconstituted tobacco.

3. The bacterial preparation 3 prepared in the embodiment 2 is used for fermenting tobacco raw materials, the bacterial preparation 3 prepared in the embodiment 2 is sprayed on the surface of the tobacco raw materials, the tobacco raw materials are fermented for 50 hours at a constant temperature and humidity under the conditions that the temperature is 38.5 ℃ and the relative humidity is 50%, and the tobacco raw materials are dried after fermentation until the moisture content of the tobacco raw materials is 13% to obtain the tobacco raw materials fermented by the bacterial preparation. The tobacco material is cut stems.

Example 4

The tobacco material (mixture containing tobacco components such as tobacco leaves and reconstituted tobacco leaves) fermented by the bacterial preparation of the invention obtained in example 3 was used as an experimental sample, and under the same conditions, the tobacco material was directly treated with the same amount of sterile water as a control sample without being inoculated with the bacterial preparation for fermentation, and then rolled into cigarettes. Carrying out sensory quality inspection by 11 sensory evaluation personnel qualified for the sensory evaluation technology of cigarettes in the tobacco industry according to tobacco industry standard YC/T496-,

(the taste characteristics of the oral cavity are 'sour, sweet and bitter' with the score range of 0-5, and the score unit is divided by 0.5, the average value is taken, the higher the score is, the better each taste degree is, the comfort index of the oral cavity with the score unit of 0.5 with the score range of 0-10, the average value is taken, the higher the score is, the lighter each discomfort degree is); the sweetening chemical components causing the tobacco to have an increased oral sweetness are analyzed by gas chromatography-mass spectrometry (GC/MS).

As shown in figure 1, the oral sweet taste of the tobacco raw material treated by the bacterial preparation is obviously increased when the tobacco raw material is combusted and smoked, which is 29.20% higher than that of a control sample, and the oral sweet taste of the tobacco is natural. The bitterness of the oral cavity is reduced by 5.69 percent compared with the control sample; the oral sourness is improved by 6.17% compared with the control sample, and the change is not large.

As shown in figure 2, the overall oral comfort of the tobacco raw material smoke treated by the bacterial preparation is obviously better than that of a control sample, the discomfort and the prickle, the hot feeling, the burning feeling, the dry feeling, the convergence/astringency feeling and the residual feeling of the experimental sample smoke in the oral cavity are obviously reduced compared with the control sample, and the scores of various oral comfort indexes are obviously higher than those of the control sample.

TABLE 1 sweetening chemical composition (. mu.g/g) of tobacco material for smoking after fermentation with the bacterial preparation of the invention

Sweetening chemical ingredient	Control sample	Experimental sample	The increase rate%
				5-methylfurfural	1.064	5.343	402.28
Furfuryl alcohol	1.945	4.061	108.86
				Furfural	5.329	10.743	101.60
2-acetylfuran	0.489	0.916	87.13
				2-furanones	0.14	0.151	7.69
Dibutyl phthalate	0.3	0.495	65.16
				Dihydroactinidiolide	0.261	0.358	37.08
Methyl cyclopentenolone	0.058	0.075	28.73
				Hexadecanoic acid	7.21	8.525	18.24
Geraniol	0.121	0.122	1.21
				Phenylethanolic acid	0.06	0.079	31.75
Phenylacetaldehyde	2.115	2.3	8.73
				Total up to	19.092	33.169	73.73

As can be seen from Table 1, the total amount of the sweetening chemical components responsible for the increased oral sweetness of tobacco during the combustion puff was significantly increased to 73.73% in the test sample compared to the control sample. Wherein, for example, 2-acetylfuran is increased by 87.13%, dihydroactinidiolide is increased by 28.73%, 5-methylfurfural is increased by 402.28%, and the content of the other tobacco sweetening chemical components is increased by 1.21-108.86%.

Sequence listing

<110> tobacco industry, Limited liability company in Shaanxi

<120> a bacterial preparation for increasing sweetness of the mouth and improving comfort of the mouth during smoking of tobacco and a tobacco product

<160> 3

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1379

<212> DNA

<213> Bacillus belgii YBN3

<400> 1

ctcaccgact tcgggtgtta caaactctcg tggtgtgacg ggcggtgtgt acaaggcccg 60

ggaacgtatt caccgcggca tgctgatccg cgattactag cgattccagc ttcacgcagt 120

cgagttgcag actgcgatcc gaactgagaa cagatttgtg ggattggctt aacctcgcgg 180

tttcgctgcc ctttgttctg tccattgtag cacgtgtgta gcccaggtca taaggggcat 240

gatgatttga cgtcatcccc accttcctcc ggtttgtcac cggcagtcac cttagagtgc 300

ccaactgaat gctggcaact aagatcaagg gttgcgctcg ttgcgggact taacccaaca 360

tctcacgaca cgagctgacg acaaccatgc accacctgtc actctgcccc cgaaggggac 420

gtcctatctc taggattgtc agaggatgtc aagacctggt aaggttcttc gcgttgcttc 480

gaattaaacc acatgctcca ccgcttgtgc gggcccccgt caattccttt gagtttcagt 540

cttgcgaccg tactccccag gcggagtgct taatgcgtta gctgcagcac taaggggcgg 600

aaacccccta acacttagca ctcatcgttt acggcgtgga ctaccagggt atctaatcct 660

gttcgctccc cacgctttcg ctcctcagcg tcagttacag accagagagt cgccttcgcc 720

actggtgttc ctccacatct ctacgcattt caccgctaca cgtggaattc cactctcctc 780

ttctgcactc aagttcccca gtttccaatg accctccccg gttgagccgg gggctttcac 840

atcagactta agaaaccgcc tgcgagccct ttacgcccaa taattccgga caacgcttgc 900

cacctacgta ttaccgcggc tgctggcacg tagttagccg tggctttctg gttaggtacc 960

gtcaaggtgc cgccctattt gaacggcact tgttcttccc taacaacaga gctttacgat 1020

ccgaaaacct tcatcactca cgcggcgttg ctccgtcaga ctttcgtcca ttgcggaaga 1080

ttccctactg ctgcctcccg taggagtctg ggccgtgtct cagtcccagt gtggccgatc 1140

accctctcag gtcggctacg catcgtcgcc ttggtgagcc gttacctcac caactagcta 1200

atgcgccgcg ggtccatctg taagtggtag ccgaagccac cttttatgtc tgaaccatgc 1260

ggttcaaaca accatccggt attagccccg gtttcccgga gttatcccag tcttacaggc 1320

aggttaccca cgtgttactc acccgtccgc cgctaacatc agggagcaag ctcccatct 1379

<210> 2

<211> 1363

<212> DNA

<213> Bacillus altitudinis Y120

<400> 2

accgacttcg ggtgttgcaa actctcgtgg tgtgacgggc ggtgtgtaca aggcccggga 60

acgtattcac cgcggcatgc tgatccgcga ttactagcga ttccagcttc acgcagtcga 120

gttgcagact gcgatccgaa ctgagaacag atttgtggga ttggctaaac cttgcggtct 180

cgcagccctt tgttctgtcc attgtagcac gtgtgtagcc caggtcataa ggggcatgat 240

gatttgacgt catccccacc ttcctccggt ttgtcaccgg cagtcacctt agagtgccca 300

actgaatgct ggcaactaag atcaagggtt gcgctcgttg cgggacttaa cccaacatct 360

cacgacacga gctgacgaca accatgcacc acctgtcact ctgtccccga agggaaagcc 420

ctatctctag ggttgtcaga ggatgtcaag acctggtaag gttcttcgcg ttgcttcgaa 480

ttaaaccaca tgctccaccg cttgtgcggg cccccgtcaa ttcctttgag tttcagtctt 540

gcgaccgtac tccccaggcg gagtgcttaa tgcgttagct gcagcactaa ggggcggaaa 600

ccccctaaca cttagcactc atcgtttacg gcgtggacta ccagggtatc taatcctgtt 660

cgctccccac gctttcgctc ctcagcgtca gttacagacc agagagtcgc cttcgccact 720

ggtgttcctc cacatctcta cgcatttcac cgctacacgt ggaattccac tctcctcttc 780

tgcactcaag tttcccagtt tccaatgacc ctccccggtt gagccggggg ctttcacatc 840

agacttaaga aaccgcctgc gagcccttta cgcccaataa ttccggacaa cgcttgccac 900

ctacgtatta ccgcggctgc tggcacgtag ttagccgtgg ctttctggtt aggtaccgtc 960

aaggtgcaag cagttactct tgcacttgtt cttccctaac aacagagctt tacgatccga 1020

aaaccttcat cactcacgcg gcgttgctcc gtcagacttt cgtccattgc ggaagattcc 1080

ctactgctgc ctcccgtagg agtctgggcc gtgtctcagt cccagtgtgg ccgatcaccc 1140

tctcaggtcg gctacgcatc gtcgccttgg tgagccgtta cctcaccaac tagctaatgc 1200

gccgcgggtc catctgtaag tgacagccga aaccgtcttt catccttgaa ccatgcggtt 1260

caaggaacta tccggtatta gctccggttt cccggagtta tcccagtctt acaggcaggt 1320

tacccacgtg ttactcaccc gtccgccgct aacatccggg agc 1363

<210> 3

<211> 1357

<212> DNA

<213> Enterobacter hollisae Y399

<400> 3

ctacttcttt tgcaacccac tcccatggtg tgacgggcgg tgtgtacaag gcccgggaac 60

gtattcaccg tggcattctg atccacgatt actagcgatt ccgacttcat ggagtcgagt 120

tgcagactcc aatccggact acgacgcact ttatgaggtc cgcttgctct cgcgaggtcg 180

cttctctttg tatgcgccat tgtagcacgt gtgtagccct actcgtaagg gccatgatga 240

cttgacgtca tccccacctt cctccagttt atcactggca gtctcctttg agttcccggc 300

cggaccgctg gcaacaaagg ataagggttg cgctcgttgc gggacttaac ccaacatttc 360

acaacacgag ctgacgacag ccatgcagca cctgtctcag agttcccgaa ggcaccaaac 420

catctctgct aagttctctg gatgtcaaga gtaggtaagg ttcttcgcgt tgcatcgaat 480

taaaccacat gctccaccgc ttgtgcgggc ccccgtcaat tcatttgagt tttaaccttg 540

cggccgtact ccccaggcgg tcgacttaac gcgttagctc cggaagccac gcctcaaggg 600

cacaacctcc aagtcgacat cgtttacggc gtggactacc agggtatcta atcctgtttg 660

ctccccacgc tttcgcacct gagcgtcagt ctttgtccag ggggccgcct tcgccaccgg 720

tattcctcca gatctctacg catttcaccg ctacacctgg aattctaccc ccctctacaa 780

gactctagcc tgccagtttc gaatgcagtt cccaggttga gcccggggat ttcacatccg 840

acttgacaga ccgcctgcgt gcgctttacg cccagtaatt ccgattaacg cttgcaccct 900

ccgtattacc gcggctgctg gcacggagtt agccggtgct tcttctgcgg gtaacgtcaa 960

tcgacaaggt tattaacctt atcgccttcc tccccgctga aagtacttta caacccgaag 1020

gccttcttca tacacgcggc atggctgcat caggcttgcg cccattgtgc aatattcccc 1080

actgctgcct cccgtaggag tctggaccgt gtctcagttc cagtgtggct ggtcatcctc 1140

tcagaccagc tagggatcgt cgcctaggtg agccgttacc ccacctacta gctaatccca 1200

tctgggcaca tccgatggca agaggcccga aggtccccct ctttggtctt gcgacgttat 1260

gcggtattag ctaccgtttc cagtagttat ccccctccat caggcagttt cccagacatt 1320

actcacccgt ccgccactcg tcagcgaagc agcaagc 1357

Claims

1. A bacterial formulation for increasing oral sweetness and improving oral comfort in tobacco combustion smoking, comprising Bacillus belgii (Bacillus velezensis) YBN3, Bacillus altitudinis Y120 and Enterobacter holtzeri (Enterobacter hormaechei) Y399; the Bacillus belgii (Bacillus velezensis) YBN3, the Bacillus altitudinis (Bacillus altitudinis) Y120 and the Enterobacter huoshi (Enterobacter hormaechei) Y399 are all preserved in the general microbiological center of the China Committee for culture Collection of microorganisms at 18 th 4 th 2018, and the preservation numbers are respectively as follows: CGMCC No.15625, CGMCC No.15626 and CGMCC No. 15627.

2. A bacterial formulation for increasing oral sweetness and improving oral comfort when smoked by tobacco according to claim 1, wherein the nucleotide sequences of Bacillus belgii (Bacillus velezensis) YBN3, Bacillus altitudinis Y120 and Enterobacter holtzeri (Enterobacter hormaechei) Y399 are shown in SEQ ID No.1, SEQ ID No.2 and SEQ ID No.3, respectively.

3. The bacterial formulation for increasing oral sweetness and improving oral comfort during tobacco combustion smoking according to claim 1, wherein the mass ratio of the Bacillus belius (Bacillus velezensis) YBN3, the Bacillus altitudinis (Bacillus altitudinis) Y120 and the Enterobacter hophallii (Enterobacter hormaechei) Y399 is (0.1-10): (0.1-10): 1.

4. the bacterial preparation for increasing oral sweetness and improving oral comfort during tobacco combustion and smoking according to claim 1, wherein the bacterial preparation is wet thallus, bacterial liquid, powder or freeze-dried powder.

5. A tobacco product having sweetness and comfortableness to the oral cavity when being burnt and smoked, which is characterized in that the bacterial preparation of claims 1 to 4 is sprayed on the surface of tobacco raw materials, fermented and dried to obtain the tobacco product.

6. A smoking article having sweetness and comfort in the mouth when smoked according to claim 5 wherein the tobacco material is one or a mixture of single grade tobacco leaf, cigarette blend and group of cigarette leaves.

7. A smoking article having sweetness and oral comfort when smoked by combustion in accordance with claim 6 wherein the single grade raw tobacco is flue-cured, burley, oriental or sun cured and the cigarette blend is cut rolled or reconstituted tobacco.