CN103224894B - Enterobacter mori, and biotransformation method for fermented wheat bran extract used for cigarette and application - Google Patents
Enterobacter mori, and biotransformation method for fermented wheat bran extract used for cigarette and application Download PDFInfo
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- CN103224894B CN103224894B CN201210117406.9A CN201210117406A CN103224894B CN 103224894 B CN103224894 B CN 103224894B CN 201210117406 A CN201210117406 A CN 201210117406A CN 103224894 B CN103224894 B CN 103224894B
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- wheat bran
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- type tobacco
- fermentation
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Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses Enterobacter mori, and a biotransformation method for a fermented wheat bran extract used for cigarette and an application, which belongs to the biology technical field and cigarette flavor technical field. The invention provides a strain of (Enterobacter mori) VL4-3CCTCC M2012020. The biotransformation method for fermented wheat bran extract used for cigarette by the Enterobacter mori comprises the following steps: 1)backflow extraction; 2)inclined plane culture; 3) seed culture solution preparation; 4) fermentation; and 5) extract preparation. The invention also provides an application of the Enterobacter mori in biotransformation to prepare the fermented wheat bran extract used for cigarette. According to the invention, the process is simple, environmental protection and harmlessness performances are provided, the raw material cost is low, the product is natural, and the fermented wheat bran extract has industrial prospect. The fermented wheat bran extract for cigarette can be directly used for perfuming on the cigarette and coordinating with flue gas, the fermented wheat bran extract has thick and sweet fragrance, thereby the flue gas is softened, the fragrance is enriched, and the adsorption can be improved.
Description
Technical field
The invention belongs to biological technical field and tobacco aromatics using technical field, be specifically related to the mulberry enterobacteria that a strain screens from vanilla plant, carry out the method that fermentation type tobacco wheat bran medicinal extract is prepared in bio-transformation with this mulberry enterobacteria, and the application of the fermentation type tobacco wheat bran medicinal extract that makes of the method in cigarette.
Background technology
In prior art, essence spice for cigarette is the indispensable auxiliary material of production of cigarettes, is also the effective means of the low harmful compensating aroma of perfect, low Jiao of cigarette product style.At present, China's tobacco aromatics using is mainly natural perfume or the chemically composite perfume directly extracting from tobacco or other plant, natural perfume is subject to the restriction of plant resources finiteness, chemical industry synthetic perfume exists fragrance single, the defect that preparation cost is too high, and some spices still can not obtain with synthetic.
Wheat bran is the outermost epidermis of wheat, is the byproduct obtaining after wheat processing flour, and mainly, as feed, economic worth is not high.The nutritive ingredient that Testa Tritici contains a large amount of needed by human, as quite abundant vitamin-E and vitamin B group, vitamin-E is relevant with the reproductive function of animal, is the nutrient substance that prevents infertility and human body cell aging, can also be anticancer.Support one's family that to be that E has water insoluble, be dissolved in fat and fatsolvent, heat-resisting, in the time of anaerobic, be heated to 200 DEG C of impregnable features; In wheat bran, contain the organic nitrogen source of the protein of 15% left and right; Contain the essential growth hormone of microorganism growth such as VitB1, riboflavin, nicotinic acid; Rich cellulose and hemicellulose; In addition, also contain a-amylase, beta-amylase, oxydase, peroxidase etc., these are all that microorganism is necessary.Measure according to modern scientific research, its para-amino benzoic acid content is the highest in plant, and para-amino benzoic acid is body cell growth and divides one of integral part of necessary material folic acid, and has the effect of the fur of reply color.In fact wheat bran as Chinese medicine simply, has moistening lung, skin care, cancer-resisting, and invigorating the spleen and regulating the stomach, crow sends out solid sending out, and clears up the effects such as stomach.But directly from wheat bran, extract and make medicinal extract and cannot be used for perfuming cigarette, inharmonious because of itself and flue gas, the gas weight of mixing, pungency is large, and cigarette odor-absorbing is had to larger negative impact.
Microorganism can produce huge high active enzyme system in breeding, as polysaccharide hydrolysis enzyme, protease, esterification enzyme, redox enzymes and lyases etc., in enzymatic action, under the synergy of the interior complicated metabolism of chemical action and microbe, can make raw material decompose, degraded, oxidation, reduction, polymerization, coupling, the effects such as conversion, form complicated low molecular compound, comprising various perfume compounds, as alcohols, aldehydes, ketone, acids, lipid, phenols, pyrans class, pyridines and terpenes etc., these aroma substances are also tobacco or the aroma component for the additive of tobacco undoubtedly.But it is low and be not easy to control in prior art, to produce the yield of tobacco aromatics using with microbial fermentation processes.Prior art has no the tobacco extract that makes with the wheat bran report for cigarette.
Summary of the invention
For the above-mentioned deficiency of prior art, the technical problem to be solved in the present invention is to provide the methods and applications of a kind of mulberry enterobacteria and bio-transformation fermentation type tobacco wheat bran medicinal extract thereof, strain mulberry enterobacteria (Enterobacter mori) VL4-3CCTCC NO:M2012020 who screens from vanilla plant, prepare fermentation type tobacco wheat bran medicinal extract and the fermentation type tobacco of preparation wheat bran medicinal extract is applied in cigarette with its bio-transformation, have with flue gas and coordinate, increase mellow sweet fragrance, make flue gas soft, enrich fragrance, stimulate little, assorted gas is few, improve the advantage that tobacco inhales taste.
The invention provides strain mulberry enterobacteria (Enterobacter mori) VL4-3, be preserved in Wuhan University's Chinese Typical Representative culture collection center on February 16th, 2012, preserving number is CCTCC NO:M2012020, the 16SrRNA sequence of the bacterial strain VL4-3 of described mulberry enterobacteria, its sequence total length 1414bp, reaches 99.608% with the similarity of mulberry enterobacteria.
A strain mulberry enterobacteria as above carries out bio-transformation and prepares the method for fermentation type tobacco wheat bran medicinal extract and carry out according to following steps:
(1) refluxing extraction: dry wheat bran and water are mixed 1:8～50 in mass ratio, and refluxing extraction was filtered after 1～3 hour, got wheat bran filtrate, sterilizing 25 minutes at 115 DEG C;
(2) slant culture: mulberry enterobacteria CCTCC NO:M2012020 is inoculated on potato dextrose agar slant medium, under 25～35 DEG C, pH5～9 condition, leaves standstill to cultivate after 24～72 hours and obtain slant culture, be placed in 4 DEG C of refrigerators;
(3) prepare seed culture fluid: with the slant culture of gained in step (2), be inoculated into seed culture medium, at 25～35 DEG C, shaking culture 12～36h under rotating speed 100～150rpm/min condition, transfers continuously after 1～4 time and makes seed culture fluid;
(4) fermentation: the seed culture fluid of gained in step (3) is formed to fermented liquid in 2～20% wheat bran filtrates that are inoculated in above-mentioned steps (1) by volume, the tinning amount of described fermented liquid is tank volume 10～40%, in pH6～10,25～35 DEG C of temperature, rotating speed 100～200rpm/min, not lucifuge condition bottom fermentation 24～72 hours;
(5) prepare medicinal extract: be that 95% ethanol mixes 1:3～4 by volume by the fermented liquid of gained in step (4) and weight percent, precipitation spend the night after suction filtration, by suction filtration liquid concentrating under reduced pressure, except desolventizing, gained tawny medicinal extract is fermentation type tobacco wheat bran medicinal extract; Described fermentation type tobacco is between 1.2100～1.3500 by the density of wheat bran medicinal extract.
Described wheat bran is Testa Tritici.In described slant medium: potato immersion liquid 200mL/L, glucose 20g/L, agar 13g/L.In described seed culture medium: maltose 50g/L, peptone 10g/L, sodium-chlor 5g/L.
A strain mulberry enterobacteria as above carries out the fermentation type tobacco application of wheat bran medicinal extract in cigarette that bio-transformation prepares: in perfuming cigarette operation, add fermentation type tobacco wheat bran medicinal extract in the ratio of pipe tobacco gross weight 0.05%～0.1%.
Advantage of the present invention is: the present invention adopts mulberry enterobacteria CCTCC NO:M2012020, described mulberry enterobacteria taking Testa Tritici as natural, sole carbon source, nitrogenous source, bio-transformation substrate, produce through fermentation energy the fermented type wheat bran tobacco extract that natural tobacco is used, method technological process is simple, environmentally friendly, environmentally safe, raw materials cost is very cheap, product is natural, has industrial applications prospect; Gained fermented type wheat bran tobacco extract can be directly used in the perfuming of cigarette, have advantages of with flue gas coordinate, increase mellow sweet fragrance, make flue gas soft, enrich fragrance, stimulate little, assorted gas few, improve tobacco and inhale taste, can use with other essence spice for cigarette mixing preparation, direct use wheat bran extract medicinal extract and flue gas are overcome inharmonious, assorted gas weight, pungency is large, the critical defect of cannot cigarette using.
Biomaterial preservation information
One strain mulberry enterobacteria (Enterobacter mori) VL4-3 is preserved in Wuhan University's Chinese Typical Representative culture collection center on February 16th, 2012, and preserving number is CCTCC NO:M2012020.
Embodiment
Below in conjunction with embodiment, the specific embodiment of the present invention and Related Technical Issues are described in further detail.Mulberry enterobacteria of the present invention (Enterobacter mori) VL4-3CCTCC NO:M2012020 screened from vanilla plant, had been preserved in Wuhan University's Chinese Typical Representative culture collection center on February 16th, 2012.Its biological property is: bacterial strain is direct rod shape, is Gram-negative bacteria, facultative aerobic, has the enzymic activity of ONPG beta-galactosidase enzymes, LDC lysine decarboxylase, ADH arginine dihydrolase, ODC ornithine decarboxylase; Tryptophane desaminase, urine enzyme reaction, H2S generation, gelatine liquefication, indoles produce and are negative; Can utilize citric acid, glucose, N.F,USP MANNITOL, sorbyl alcohol, rhamnosyl, sucrose, melibiose, amygdaloside, pectinose, it is sour gentle to produce, and generates sour-sweet aroma substance.Can grow taking multiple amino acids, most of monose, polysaccharide, ester class and amino acid as sole carbon source; With cyclodextrin, methylene-succinic acid, α-one valeric acid, quinic acid, adonitol, i-erythritol, L-fucose, succinamic acid, lactulose, hydroxyl-L-PROLINE, L-Leu, L-Glutimic acid, D, VBT, γ-aminobutyric acid, D-lactobionic acid lactone, α-ketoglutaric acid, sebacic acid, gamma-hydroxybutyric acid, phenylethylamine, butanediamine, 2-monoethanolamine, can not grow on the 2,3-butanediol substratum that is sole carbon source.
The Molecular Identification of this mulberry enterobacteria (Enterobacter mori) VL4-3 is characterized as: taking the chromosomal DNA of bacterial strain to be measured as template, undertaken by synthetic primer.The amplification of 16S rRNA gene PCR.Upstream primer: 5 '-GAGTTTGATCCTGGCTCAG-3 '; Downstream primer: 5 '-AGAAAGGAGGTGATCCAGCC-3 ' (Shanghai Ying Jun biotech firm provides).After reclaiming order-checking, proofreading and correct, obtain the 16S rRNA sequence of bacterial strain L4-3, its sequence total length 1414bp.Reach 99.608% with the similarity of mulberry enterobacteria (Enterobacter mori).
The mulberry enterobacteria CCTCC NO:M2012020 adopting in the method for fermentation type tobacco wheat bran medicinal extract is prepared in mulberry enterobacteria of the present invention bio-transformation, nobody adopted this bacterial strain to carry out fermentative production tobacco aromatics using, also nobody adopts wheat bran to ferment as natural, unique carbon source, nitrogenous source, bio-transformation substrate, transforms and produces fermentation type tobacco wheat bran medicinal extract.
The present invention also carries out mulberry enterobacteria CCTCC NO:M2012020 the fermentation type tobacco wheat bran medicinal extract that bio-transformation prepares and applies in cigarette, and fermentation type tobacco, adds in the ratio of pipe tobacco gross weight 0.05%～0.1% with wheat bran medicinal extract in perfuming cigarette operation.
The applicant adopts GC/MS the present invention's " fermentation type tobacco wheat bran medicinal extract " to be carried out to the mensuration of volatility aroma component, and with (not utilizing mulberry enterobacteria CCTCC NO:M2012020 to ferment) common " wheat bran extract medicinal extract " sample as a comparison, carry out same mensuration and contrast, be summarized as follows.
(1) while distillation extraction
Fermentation type tobacco of the present invention is respectively got in the round-bottomed flask that 5g is respectively charged into distilling and extracting device with wheat bran medicinal extract and wheat bran extract medicinal extract, add 400mL water, be heated to seethe with excitement with electric mantle, in the round-bottomed flask of opposite side, add the methylene dichloride of 200mL, 60 DEG C of water-bath boilings, timing extraction 2h after layering appears in middle separating tube place liquid level.After extraction finishes, in dichloromethane extraction liquid, add anhydrous sodium sulphate, spend the night, filter and be concentrated into 1mL, concentrated solution is used for carrying out GC/MS analysis.
(2) GC/MS analyzes: HP-5MS (m) capillary column of 50m × 0.25mm × 0.25 μ; Sample introduction temperature: 250 DEG C; Splitting ratio 20:1; Carrier gas: He, 1mL/min; Heating schedule: be raised to 250 DEG C (5min) with 5 DEG C/min from 50 DEG C (1min); Transmission line temperature: 250 DEG C; Ion source: EI source; Electron energy: 70eV; Sweep limit: 50～550amu; Use WILEY and MINILAB spectrum storehouse to retrieve.
(3) volatile component comparative analysis
What GC/MS carried out analysis of aroma components to fermentation type tobacco of the present invention with wheat bran medicinal extract and wheat bran extract medicinal extract the results are shown in Table 1, as can be seen from Table 1, fermentation type tobacco of the present invention identifies altogether 40 kinds of aroma components with wheat bran medicinal extract, wherein 5 kinds of ketones, 6 kinds of acids, 5 kinds of alcohols, 3 kinds of aldehydes, and alkane, alkene class, phenols, ester class, pyrans, pyridine, pyrazoles, thiocyanate-, to 21 kinds of aroma substances such as vinyl guaiacols.
Contrast wheat bran extract medicinal extract, fermentation type tobacco of the present invention is with having newly increased 27 kinds of aroma substances after the fermentation of wheat bran medicinal extract, and 9 kinds of aroma substances have disappeared.9 kinds of volatile aroma materials that disappear are mainly alkanes, phenol and phthalate, belong to the composition of unfavorable flue gas.In 27 kinds of aroma substances that increase: pyruvic aldehyde has the pungent smell of pungency and caramel sample sweet taste; That 2,3-dimethyl diketone has is sweet, cream, butter fragrance, has natural existence at drinks, milk preparation; Acetic acid is important organic acid, is pungency special aroma; 3-hydroxyl-2 butanone, percentage composition 40.935% relatively, for the important aroma component of wheat bran medicinal extract for fermentation type tobacco, is butter aroma after Macrodilution; 1,1-2 Ethoxyethane, is happy nut and fresh blue or green fruit fragrance, and natural product are present in rice wine, whisky, Rum etc., or in berry, vegetables and milk-product etc.; Isopropylformic acid has strong seemingly breathing out and lose cream smell and cheesy; Butyric acid has the cream and cheese fragrance and the whipped fat rancid flavor that are strong; 2,3-butanediol has the faint fruit perfume (or spice) of fragrance, natural being present in molasses fermented product; Furfural has burnt odor taste; 3-methyl-2-butene, 1% or the ethanolic soln of greater concn, there is berry, the red certain kind of berries, grape, raspberry, Rubus fruticocus and cherry sample fruital fragrance, there is the acid gas of spine, and with fermentation honey sample aromatic, be present in black currant, cocoa, nut etc., belong to natural equivalent spices; Vinyl guaiacol is to strong spice, cloves and fermentation like fragrance, has the breath of the peanut of stir-fry.
Relatively in the vicissitudinous aroma substance of percentage composition: phenylethyl alcohol has special rose fragrance, butyric acid-2-methyl-2-phenethyl ester has blue or green perfume, the fragrance of a flower, sweet fruital and wax sample fragrance.Above-mentioned fermentation type tobacco blend into one with all these fragrance of wheat bran medicinal extract and give flue gas coordination, mellow sweet fragrance, make flue gas soft, enrich fragrance, improve tobacco inhale taste.
Fermentation type tobacco of the present invention can successfully improve cigarette odor-absorbing with wheat bran medicinal extract, because microorganism can produce huge high active enzyme system in breeding, as polysaccharide hydrolysis enzyme, protease, esterification enzyme, redox enzymes and lyases etc., in enzymatic action, under the synergy of the interior complicated metabolism of chemical action and microbe, decompose taking wheat bran as raw material, degraded, oxidation, reduction, polymerization, coupling, the effects such as conversion, form complicated low molecular compound, comprising various perfume compounds, as alcohols, aldehydes, ketone, acids, lipid, phenols, pyrans class, pyridines and terpenes etc., these aroma substances well quality and suction abundant and that improved cigarette are hidden quality.
Table 1, wheat bran medicinal extract and the contrast of wheat bran extract medicinal extract volatile aroma composition for fermentation type tobacco
Remarks: phenylethyl propionate is interior mark, setting it is 6.833:5.928=1.153:1 at wheat bran extract medicinal extract with the relative percentage composition ratio in fermentation type tobacco wheat bran medicinal extract, is worth to come both aroma substances variations of comparison and the variation of percentage composition relatively thereof with this.
Enumerate embodiment below.Be necessary to point out at this, below specific embodiment just further illustrate of the present invention, do not represent limiting the scope of the invention.Some nonessential amendment and adjustment that other people make according to the present invention, still belong to protection scope of the present invention.
Embodiment 1
The present invention utilizes the preparation method of microorganism mulberry enterobacteria (Enterobacter mori) VL4-3CCTCC NO:M2012020 bio-transformation fermentation type tobacco wheat bran medicinal extract as follows:
1) by wheat bran and water in mass ratio 1:8 refluxing extraction after 3 hours, filter, get filtrate, 115 DEG C of sterilizings 25 minutes;
2) slant culture: mulberry enterobacteria CCTCC NO:M2012020 is inoculated on potato dextrose agar slant medium, under 25 DEG C, pH9 condition, leaves standstill to cultivate and be placed on for 24 hours in 4 DEG C of refrigerators;
Slant medium is: potato immersion liquid 200mL/L, glucose 20g/L, agar 13g/L;
3) prepare seed culture fluid: by above-mentioned steps 2) in the slant culture of gained, be inoculated into seed culture medium, at 25 DEG C, shaking culture 12h under rotating speed 150rpm/min condition, transfers continuously after 4 times and makes seed culture fluid;
Seed liquor substratum consists of: maltose 50g/L, peptone 10g/L, sodium-chlor 5g/L;
4) fermentation: by step 3) in the seed culture fluid of gained 5% be inoculated into above-mentioned steps 1 by volume) in wheat bran filtrate in form fermented liquid, the tinning amount of above-mentioned fermented liquid is 40%, at pH6, and 35 DEG C of temperature, rotating speed 100rpm/min, not lucifuge condition bottom fermentation 48 hours;
5) by step 4) in fermented liquid and the 95% ethanol 1:4 by volume of gained, precipitation spend the night after suction filtration, except desolventizing, making brown medicinal extract is fermentation type tobacco wheat bran medicinal extract by concentrated filtrate decompression, density is 1.2450.
Embodiment 2
The preparation method of microorganism mulberry enterobacteria (Enterobacter mori) VL4-3CCTCC NO:M2012020 bio-transformation fermentation type tobacco wheat bran medicinal extract that utilizes of the present invention is as follows:
1) by wheat bran and water in mass ratio 1:30 refluxing extraction after 2 hours, filter, get filtrate, 115 DEG C of sterilizings 25 minutes;
2) slant culture: mulberry enterobacteria CCTCC NO:M2012020 is inoculated on potato dextrose agar slant medium, under 30 DEG C, pH7 condition, leaves standstill to cultivate and be placed on for 36 hours in 4 DEG C of refrigerators;
Slant medium is: potato immersion liquid 200mL/L, glucose 20g/L, agar 13g/L;
3) prepare seed culture fluid: by above-mentioned steps 2) in the slant culture of gained, be inoculated into seed culture medium, at 30 DEG C, shaking culture 24h under rotating speed 120rpm/min condition, transfers continuously after 3 times and makes seed culture fluid;
Described seed liquor substratum consists of: maltose 50g/L, peptone 10g/L, sodium-chlor 5g/L;
4) fermentation: by upper step 3) in the seed culture fluid of gained 10% be inoculated into above-mentioned steps 1 by volume) in wheat bran filtrate in form fermented liquid, the tinning amount of above-mentioned fermented liquid is 30%, at pH8,30 DEG C of temperature, rotating speed 150rpm/min, not lucifuge condition bottom fermentation 36 hours;
5) by step 4) in fermented liquid and the 95% ethanol 1:3 by volume of gained, precipitation spend the night after suction filtration, except desolventizing, making brown medicinal extract is fermentation type tobacco wheat bran medicinal extract by concentrated filtrate decompression, density is 1.2710.
Embodiment 3
The present invention utilizes the preparation method of microorganism mulberry enterobacteria (Enterobacter mori) VL4-3CCTCC NO:M2012020 bio-transformation fermentation type tobacco wheat bran medicinal extract as follows:
1) by wheat bran and water in mass ratio 1:50 refluxing extraction after 1 hour, filter, get filtrate, 115 DEG C of sterilizings 25 minutes;
2) slant culture: mulberry enterobacteria CCTCC NO:M2012020 is inoculated on potato dextrose agar slant medium, under 35 DEG C, pH5 condition, leaves standstill to cultivate and be placed on for 72 hours in 4 DEG C of refrigerators;
Slant medium is: potato immersion liquid 200mL/L, glucose 20g/L, agar 13g/L;
3) prepare seed culture fluid: by above-mentioned steps 2) in the slant culture of gained, be inoculated into seed culture medium, at 35 DEG C, shaking culture 36h under rotating speed 100rpm/min condition, transfers continuously after 2 times and makes seed culture fluid;
Described seed liquor substratum consists of: maltose 50g/L, peptone 10g/L, sodium-chlor 5g/L
4) fermentation: by step 3) in the seed culture fluid of gained 20% be inoculated into above-mentioned steps 1 by volume) in wheat bran filtrate in form fermented liquid, the tinning amount of above-mentioned fermented liquid is 40%, at pH10, and 25 DEG C of temperature, rotating speed 200rpm/min, not lucifuge condition bottom fermentation 24 hours;
5) by step 4) in fermented liquid and the 95% ethanol 1:3 by volume of gained, precipitation spend the night after suction filtration, except desolventizing, making brown medicinal extract is fermentation type tobacco wheat bran medicinal extract by concentrated filtrate decompression, density is 1.3123.
Embodiment 4
Contriver by above-mentioned fermentation type tobacco with wheat bran medicinal extract in the ratio of pipe tobacco amount 0.05%, 0.1%, 0.5%, wheat bran extract medicinal extract in pipe tobacco amount ratio 0.05%, add in pipe tobacco and smoke panel test respectively, smoke panel test and carry out according to GB GB5606.4-1996.Smoking result is as shown in table 2 below.Repeatedly smoke panel test through the expert group of smokeing panel test, think: this cigarette wheat bran medicinal extract, while adding in pipe tobacco in the ratio of pipe tobacco amount 0.05%, 0.1%, have advantages of with flue gas coordinate, increase mellow sweet fragrance, make flue gas soft, enrich fragrance, stimulate little, assorted gas few, improve tobacco and inhale taste, can use with other essence spice for cigarette mixing preparation, improved wheat bran extract medicinal extract and added in pipe tobacco, inharmonious with flue gas, assorted gas is heavy, pungency is large, the critical defect of cannot cigarette using.
Table 2. fermentation type tobacco with wheat bran medicinal extract and wheat bran extract medicinal extract in the perfuming cigarette contrast of smokeing panel test
Claims (5)
1. the method for fermentation type tobacco wheat bran medicinal extract is prepared in a strain mulberry enterobacteria bio-transformation, it is characterized in that described method carries out according to following steps:
(1) refluxing extraction: dry wheat bran and water are mixed 1:8～50 in mass ratio, and refluxing extraction was filtered after 1～3 hour, got wheat bran filtrate, sterilizing 25 minutes at 115 DEG C;
(2) slant culture: mulberry enterobacteria CCTCC NO:M2012020 is inoculated on potato dextrose agar slant medium, under 25～35 DEG C, pH5～9 condition, leaves standstill to cultivate after 24～72 hours and obtain slant culture, be placed in 4 DEG C of refrigerators;
(3) prepare seed culture fluid: with the slant culture of gained in step (2), be inoculated into seed culture medium, at 25～35 DEG C, shaking culture 12～36h under rotating speed 100～150rpm/min condition, transfers continuously after 1～4 time and makes seed culture fluid;
(4) fermentation: the seed culture fluid of gained in step (3) is formed to fermented liquid in 2～20% wheat bran filtrates that are inoculated in step (1) by volume, the tinning amount of described fermented liquid is tank volume 10～40%, in pH6～10,25～35 DEG C of temperature, rotating speed 100～200rpm/min, not lucifuge condition bottom fermentation 24～72 hours;
(5) prepare medicinal extract: be 95% ethanol 1:3～4 by volume by fermented liquid and the weight percent of gained in step (4), precipitation spend the night after suction filtration, suction filtration liquid concentrating under reduced pressure is removed to desolventizing, gained tawny medicinal extract is fermentation type tobacco wheat bran medicinal extract, and described fermentation type tobacco is between 1.2100～1.3500 by the density of wheat bran medicinal extract.
2. the method for fermentation type tobacco wheat bran medicinal extract is prepared in a strain mulberry enterobacteria bio-transformation as claimed in claim 1, it is characterized in that: described wheat bran is Testa Tritici.
3. the method for fermentation type tobacco wheat bran medicinal extract is prepared in a strain mulberry enterobacteria bio-transformation as claimed in claim 1, it is characterized in that in described slant medium: potato immersion liquid 200mL/L, glucose 20g/L, agar 13g/L.
4. the method for fermentation type tobacco wheat bran medicinal extract is prepared in a strain mulberry enterobacteria bio-transformation as claimed in claim 1, it is characterized in that in described seed culture medium: maltose 50g/L, peptone 10g/L, sodium-chlor 5g/L.
5. the fermentation type tobacco that a strain mulberry enterobacteria bio-transformation as claimed in claim 1 prepares is the application in cigarette with wheat bran medicinal extract, it is characterized in that: in perfuming cigarette operation, add fermentation type tobacco wheat bran medicinal extract in the ratio of pipe tobacco gross weight 0.05%～0.1%.
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| CN105647644A (en) * | 2016-02-18 | 2016-06-08 | 湖北中烟工业有限责任公司 | Preparation method and application of flavor producing bacterial strain fermented honeysuckle flower flavor |
| CN106174681B (en) * | 2016-08-31 | 2017-10-17 | 湖北中烟工业有限责任公司 | A kind of method that biotechnology lifts reconstituted tobacco flavouring essence quality |
| CN109536393B (en) * | 2018-11-09 | 2021-05-18 | 湖北中烟工业有限责任公司 | Preparation method and application of Maryland tobacco leaf fermentation extract |
| CN113755388B (en) * | 2021-09-30 | 2023-03-14 | 福建省亚热带植物研究所 | Enterobacter mori LRZ01 strain for degrading cellulose and application thereof |
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