CN101497844B - Method for preparing cigarette flavor by microbial fermentation of coffee - Google Patents
Method for preparing cigarette flavor by microbial fermentation of coffee Download PDFInfo
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- CN101497844B CN101497844B CN 200810046834 CN200810046834A CN101497844B CN 101497844 B CN101497844 B CN 101497844B CN 200810046834 CN200810046834 CN 200810046834 CN 200810046834 A CN200810046834 A CN 200810046834A CN 101497844 B CN101497844 B CN 101497844B
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- coffee
- tobacco
- liquid
- perfume
- tobacco leaf
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- 125000000999 tert-butyl group Chemical group [H]C([H])([H])C(*)(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- TUNFSRHWOTWDNC-HKGQFRNVSA-N tetradecanoic acid Chemical compound CCCCCCCCCCCCC[14C](O)=O TUNFSRHWOTWDNC-HKGQFRNVSA-N 0.000 description 1
- HNBDQABBWNOTRU-UHFFFAOYSA-N thalline Chemical compound C1=CC=[Tl]C=C1 HNBDQABBWNOTRU-UHFFFAOYSA-N 0.000 description 1
- OLTHARGIAFTREU-UHFFFAOYSA-N triacontane Natural products CCCCCCCCCCCCCCCCCCCCC(C)CCCCCCCC OLTHARGIAFTREU-UHFFFAOYSA-N 0.000 description 1
- RYYVLZVUVIJVGH-UHFFFAOYSA-N trimethylxanthine Natural products CN1C(=O)N(C)C(=O)C2=C1N=CN2C RYYVLZVUVIJVGH-UHFFFAOYSA-N 0.000 description 1
- 229940005605 valeric acid Drugs 0.000 description 1
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Abstract
The invention discloses a method for preparing tobacco perfume by adopting organism fermented coffee, and relates to a preparation method of tobacco perfume, which belongs to the technical field of tobacco. The method comprises the following steps: (1) preparing tobacco leaf synthetic medium; (2) selecting the organism producing perfume; (3) performing liquid fermentation or solid fermentation to coffee by using selected burley tobacco leaf surface dominant bacteria and obtaining the fermented coffee tobacco perfume. The method has simple process and low cost, and the obtained coffee tobacco perfume can be directly used as the perfume of cigarettes. The coffee tobacco perfume has special fragrant characteristic of acid fragrance, thereby lowering the stimulation, richening the smoke gas hiding the mixed gas, leading the smoke gas to be soft and improving the mouthfeel when being added into the cigarettes.
Description
Technical field
The present invention relates to the preparation method of tobacco aromatics using, belong to technical field of tobacco, specifically is the method for prepared using microbial fermentation technology production characteristic tobacco aromatics using with coffee.
Background technology
Flavoring and casing is the key link in the cigarette process, is to improve the deciding factor of product style and improve one of effective means of cigarette flavor quality.Tobacco aromatics using mainly is divided into 2 kinds by the difference of spices production method at present, the natural perfume and the employing chemical reaction synthetic spices that promptly adopt various separation means directly to extract from tobacco or other plant.These two kinds of production methods all exist its limitation separately at present.For certain plant perfume, its aroma component and feature are constant, and we are difficult to make plant produce the spices with another kind of local flavor according to the needs to fragrance, and the exploitation of plant extract spices is owing to the limited limitation that exists of available plant resources.For the chemical industry synthetic perfume, except that fragrance was single, owing to be subjected to the restriction of synthetic method and condition, a lot of good aroma components still can not synthetic, or cost is too high.
Coffee generally will be handled before becoming commodity by fermentation, and coffee aroma by fermentation and local flavor are not inferior.The fermentation of coffee berry generally takes method of piling to implement, the fermenting process of coffee berry is quite complicated Biochemical processes, by the multiple yeast in the pulp, bacterium and mould participate in microbial activities, the reductase enzyme of while pulp itself, saccharase, maltin, amylase, the proteolytic enzyme fellowship fermenting process.The cotyledon of the coffee drupe of Chu Liing partly separates by fermentation, and pigment cell breaks, and the content of trimethyl-xanthine and tannin descends; Pectin content increases, and protein digestion becomes the nitrogenous substances of solubility.Produced ethanol, acetate, lactic acid, flavour precursors, little peptide, the reducing sugar of free amino acid and degraded.Through storing transportation and removal of contamination, baking process next, baking can be removed smell substance further with the coffee berry drying, forms final local flavor and color and luster.Most importantly produced the distinctive fragrance of coffee by the Maillard reaction in the process of baking, baking has simultaneously also changed the polysaccharide in the coffee berry, protein, thus the interaction between polyphenol and Maillard product has produced insoluble substance.Remove part coffee fat and obtain the coffee cake, the coffee cake is ground obtain coffee powder again.The strong fragrance that coffee powder has is a kind of good aroma component.
There are a large amount of microfloras in the flue-cured tobacco blade face of this external not ageing and preaging, utilize the bacterium in the tobacco in breeding, can produce huge high active enzyme system, as the polysaccharide hydrolysis enzyme, protease, the Mierocrystalline cellulose enzyme, the esterification enzyme, redox enzymes and lyases etc., in enzymatic action, under the interior complicated metabolic synergy of chemical action and microbe, with coffee is that raw material decomposes, degraded, oxidation, reduction, polymerization, coupling, effects such as conversion, form complicated low molecular compound, comprising various perfume compounds, as alcohols, aldehydes, ketone, acids, the ester class, phenols, furans, the pyrazine class, pyridines and terpenes etc., these aroma substances also are the aroma component of tobacco undoubtedly.But in the prior art, utilize coffee powder not appear in the newspapers for raw material adopts the method for microbial fermentation technology production characteristic tobacco aromatics using.
Summary of the invention
At above-mentioned deficiency of the prior art, it is the method for prepared using microbial fermentation technology production characteristic tobacco aromatics using with coffee that the technical problem to be solved in the present invention provides a kind of.Method technological process of the present invention is simple, with low cost, and it is reinforced to be directly used in cigarette flavoring.
Technical scheme of the present invention is a kind of method that adopts microbial fermentation of coffee to prepare tobacco aromatics using, and described method is carried out according to following step:
(1) preparation of tobacco leaf complex medium:
The burley tobaccos in 1 year to 2 years of natural ageing and water by 1: 5 mixed post-heating to 100 ℃, and were kept 1-2 hour, filter and obtain burley tobaccos water extract; With burley tobaccos water extract and microbial solid substratum mixed, make the tobacco leaf complex medium by 3: 7;
(2) produce fragrant Screening of Bioflocculant-producing Bacteria:
Take from the burley tobacco leaf 5-10g in 1 year to 2 years of right ageing, join in the sterile distilled water, concussion 30-60min, therefrom get 100-1000 μ l liquid, be added drop-wise on the tobacco leaf complex medium that has prepared, evenly be coated with out, with aerobic or anaerobic condition, separate or after dilution was coated with the flat band method purifying, the burley tobacco leaf surface dominant bacteria that obtains was the fragrant microorganism of product of screening with plate streaking;
(3) the burley tobacco leaf surface dominant bacteria with screening carries out liquid fermenting or solid fermentation to coffee, obtains coffee tobacco perfume.
Microbial solid substratum described in the above step is beef extract-peptone, PDA substratum or minimum medium.
The burley tobacco leaf surface dominant bacteria that is screened is genus bacillus, gemma clostridium, pseudomonas, aspergillus tubigensis, Penicillium notatum, yeast, and bacterial classification can be bacterium song or bacterium liquid form.
Burley tobacco leaf surface dominant bacteria with screening carries out liquid fermenting to coffee, carries out according to the following steps:
1. actication of culture: get burley tobacco leaf surface dominant bacteria, be inoculated on the ready slant medium, be positioned over and cultivate 24～48h in 30～37 ℃ the constant incubator and make actication of culture, again with the activatory bacterial classification inoculation in the LB liquid nutrient medium that adds sugared source, in 30～37 ℃ of constant temperature culture 24～48h, obtain the activatory seed liquor;
2. the pre-treatment of raw material: choose coffee powder that exsiccant pulverized as raw material, with water: coffee powder=1: mixed (50-100), in above-mentioned mixed solution, add and account for the sugared source of above-mentioned mixed solution 1-5% and the inorganic salt of 0.1-2%, at 115 ℃ of 15～20min that sterilize down, be the good coffee liquid nutrient medium of sterilization;
3. liquid fermenting: will activate good seed liquor and be inoculated in the good coffee liquid nutrient medium of sterilization, the inoculation volume ratio is a seed liquor: coffee liquid nutrient medium=1: (50～100) after mixing, place 30～37 ℃ constant incubator, the 24～48h that ferments;
4. fermented liquid is concentrated and refining: with the coffee fermented liquid that ferments, sheet frame membrane filtration after the bacterial classification deactivation, concentrating under reduced pressure or lyophilize are condensed into paste again, according to 1: the mass ratio of (1～4) mixes paste mutually with extraction solvent, paste is dissolved fully, after refiltering, filtrate decompression is concentrated into the solvent of removing 80-95%, gained liquid paste substance is a coffee tobacco perfume.Therefore, also abbreviate this spices as coffee medicinal extract sometimes in the present specification.
With the fragrant microorganism of the product that screens coffee being carried out solid fermentation carries out according to the following steps:
1. actication of culture: the burley tobacco leaf surface dominant bacteria of getting purifying, be inoculated on the slant medium, be positioned over and cultivate 24～48h in 20～37 ℃ the constant incubator and make actication of culture, again with the activatory bacterial classification inoculation in the liquid nutrient medium that adds sugared source, in 20～37 ℃ of constant temperature culture 24～48h, with medium centrifugal reject supernatant liquor, the precipitation thalline with physiological saline dilution concussion evenly is the activated seed liquid that obtains;
2. the pre-treatment of raw material: choose coffee powder that exsiccant pulverized as raw material,, obtain aseptic coffee meal at 115 ℃ of sterilization 15～20min down;
3. solid fermentation: activated seed liquid is inoculated in the aseptic coffee meal according to 1: 1～10 mass ratio, stirs, make meal solid and seed liquor thorough mixing even, be positioned in 28～35 ℃ the incubator and fermented 2～7 days;
4. extract and make with extra care: the above-mentioned coffee meal that ferments, in fermentation solid and extracting solution mass ratio 1: the ratio of (1～5) adds an amount of extracting solution, be heated to 90-100 ℃ and refluxing extraction 2～3 times, each 1～3h that extracts, after the extracting solution collection and filtering, filtrate decompression is condensed into medicinal extract; According to 1: the mass ratio of (1～4) mixes paste mutually with extraction solvent, and paste is dissolved fully, and after further filtering, concentrating under reduced pressure is removed unnecessary solvent, and gained liquid paste substance is coffee tobacco perfume.Therefore, also abbreviate this spices as coffee medicinal extract sometimes in this paper application documents.
The 1. middle described slant medium of actication of culture of step is beef extract-peptone, PDA substratum and minimum medium, and substratum can be solid or liquid form.
Step 4. in fermented liquid to concentrate extracting solution, the extraction solvent described in 4. extracting and make with extra care with refining and step be water or ethanol, or both composite solutions.
The sugared source of adding in the described coffee fermenting process is one or more the mixture in sucrose, glucose, the fructose.
The step 2. described inorganic salt of pre-treatment of middle raw material is KH
2PO
4, (NH
4)
2SO
4, MgSO
4, or NaCl.
Method technological process of the present invention is simple, with low cost, and it is reinforced that the gained coffee tobacco perfume can be directly used in cigarette flavoring.Coffee tobacco perfume has unique fragrant note feature of acid, and being added on to have in the cigarette to reduce stimulates, and abundant and full flue gas is covered assorted gas, makes the effect that flue gas is soft and improve mouthfeel.
Description of drawings
Accompanying drawing 1 is volatility and half volatile aroma component total ion current figure in the coffee tobacco perfume of the present invention.
Data in this accompanying drawing 1 detect coffee tobacco perfume with gas chromatography-mass spectrum and liquid chromatography-mass spectrography and obtain.
Embodiment
Below in conjunction with embodiment relevant technologies problem of the present invention is described in further detail.
Embodiment 1: the mode with liquid state fermentation prepares coffee tobacco perfume.
1. actication of culture: get the gemma clostridium, be inoculated on the beef-protein medium inclined-plane, be positioned in the constant incubator, under anaerobic cultivate 36h for 30 ℃, inoculate in the basic liquid nutrient medium of deep layer, cultivate 24h in 30 ℃, obtain the activatory seed liquor.
2. the pre-treatment of raw material: choose coffee powder (coffee powder is commercially available product, and is as follows) that the 100g exsiccant pulverized as raw material, add sucrose and the 10g (NH of 100g
4)
2SO
4, 2gMgSO
4, 5g KH
2PO
3, add the 4000ml pure water, be mixed with liquid fermentation liquid, at 115 ℃ of 20min that sterilize down.
3. liquid fermenting: will activate good seed liquor 40ml and be inoculated in the good coffee liquid nutrient medium of sterilization, thorough mixing evenly after, place 37 ℃ constant incubator sealing and fermenting 48h.
4. fermented liquid is concentrated and refining: with the coffee fermented liquid that ferments, after the bacterial classification deactivation behind the sheet frame membrane filtration, concentrating under reduced pressure or lyophilize are condensed into paste, according to 1: 4 mass ratio paste is mixed mutually with 95% ethanol, paste is dissolved fully, after further filtering, concentrating under reduced pressure is removed unnecessary solvent just can obtain coffee tobacco perfume.
Embodiment 2: the mode with solid state fermentation prepares coffee tobacco perfume.
1. actication of culture: get yeast, be inoculated in the constant incubator that is positioned over 28 ℃ on the solid PDA substratum vamp and cultivate 48h, in the PDA liquid nutrient medium that is inoculated in,, obtain the activatory seed liquor in 28 ℃ of constant temperature culture 48h.
2. the pre-treatment of raw material: choose coffee powder that the 1000g exsiccant pulverized as raw material,, obtain aseptic coffee meal at 115 ℃ of sterilization 20min down.
3. solid fermentation: the seed liquor 100ml that handles well is inoculated in the above-mentioned aseptic coffee meal, stirs, make meal solid and seed liquor thorough mixing even, the coffee powder that inoculation is good is positioned in 28 ℃ the incubator and fermented 7 days.
4. extract and make with extra care: with the coffee meal that ferments, the ratio than 1: 3 adds an amount of pure water in fermentation solid and quality, and reflux extraction 3 times extracts 3h at every turn, and after the extraction liquid collection was further filtered, concentrating under reduced pressure became medicinal extract; According to 1: 3 mass ratio paste is mixed mutually with 95% ethanol, paste is dissolved fully, after further filtering, concentrating under reduced pressure is removed the solvent of 80-95%, just can obtain coffee tobacco perfume.
Method of the present invention is screened microorganism from tobacco leaf surface, and this is because the contriver recognizes after after deliberation, has a large amount of microfloras on the flue-cured tobacco blade face of not ageing and preaging.But kind of microorganism and quantity are different, and this is different relevant with flue-cured tobacco cultivars/place of production/time grade and alcoholization condition.Flue-cured tobacco blade face microbial host will comprise bacterium/mould and actinomycetes, and other has a small amount of yeast.Wherein bacterium has comparative advantage, and reaches 90% ~ 99%, and actinomycetes, mould and yeast ratio are very little.Bacterium in the tobacco mainly is a genus bacillus, the gemma clostridium, and pseudomonas and staphylococcus: actinomycetes mainly are streptomycetes; Mould mainly is aspergillus tubigensis and Penicillium notatum.Wherein genus bacillus and gemma clostridium account for about 90% of tobacco leaf surface microbe species.The contriver thinks, because microorganism can produce huge high active enzyme system in breeding, as the polysaccharide hydrolysis enzyme, protease, the Mierocrystalline cellulose enzyme, the esterification enzyme, redox enzymes and lyases etc., in enzymatic action, under the interior complicated metabolic synergy of chemical action and microbe, substrate is decomposed, degraded, oxidation, reduction, polymerization, coupling, effects such as conversion, form complicated low molecular compound, comprising various perfume compounds, as alcohols, aldehydes, ketone, acids, the ester class, phenols, furans, the pyrazine class, pyridines and terpenes etc., these aroma substances also are the aroma component of tobacco undoubtedly.
The contriver also thinks, carry out Screening of Bioflocculant-producing Bacteria with the substratum that adds tobacco leaf water extract and can better simulate condition and the needed nutritive ingredient that microorganism grows in natural ageing process, better filter out the fragrant microorganism of the needed product of tobacco leaf surface accurately, the blindness that reduces to screen increases the success ratio of screening.
The product that the contriver produces method of the present invention detects: by gas chromatography-mass spectrum and liquid chromatography-mass spectrography the composition of fermentation cigarette with coffee flavour detected, its result asks for an interview table 1.
Table 1, the fermentation cigarette composition of coffee flavour
| Peak number | Retention time | Chemical name | Molecular formula | Massfraction |
| 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 | 4.71 5.50 6.27 6.63 8.75 8.97 9.86 10.21 10.76 11.15 11.31 11.42 12.20 12.92 13.38 13.75 14.13 14.50 14.67 14.77 15.56 16.28 16.58 17.04 17.37 17.56 | The butanols 1-butanol amylalcohol 1-Pentanol styrene Styrene 3-hydroxyl-sad Octanoic acid of 2-acetone 2-Butanone 3-hydroxy-acetic acid Acetic acid 3-furfural 3-Furfural propionic acid Propanoic acid isobutyric acid Isobutyric acid butyric acid Butanoic acid butyrolactone Butyrolactone 2-furfuryl alcohol 2-furanmethanol isovaleric acid Isovaleric acid valeric acid Valeric acid 4-methylvaleric acid Valerio acid 4-methyl caproic acid Hexanoic acid phenmethylol Benzyl alcohol benzyl carbinol Benzeneethanol enanthic acid Heptanoic acid phenyl ethyl butyrate Butanoic acid 2 1-(1H-pyrroles-2-yl) ethyl ketone Ketone methyl pyrrol-2-yl stearaldehyde n-Octadecanal n-nonanoic acid Nonanoic acid methyl hexadecanoate Palmitic acid; Methylester ethyl palmitate Palmitic acid, ethylester capric acid Decanoic acid | C 4H 10O C 5H 12O C 8H 8 C 4H 8O 2 C 2H 4O 2 C 5H 4O 2 C 3H 6O 2 C 4H 8O 2 C 4H 8O 2 C 4H 6O 2 C 5H 6O 2 C 5H 10O 2 C 5H 10O 2 C 5H 10O 2 C 6H 12O 2 C 7H 8O C 8H 10O C 7H 14O 2 C 12H 16O 2 C 6H 7NO C 8H 16O 2 C 18H 36O C 9H 18O 2 C 17H 34O 2 C 18H 36O 2 C 10H 20O 2 | 0.22％ 0.21％ 0.24％ 0.47％ 1.08％ 0.29％ 0.22％ 0.20％ 40.96％ 0.33％ 0.32％ 1.24％ 0.15％ 0.18％ 0.28％ 0.18％ 0.52％ 0.52％ 0.35％ 0.42％ 0.3％ 0.15％ 0.41％ 0.74％ 0.43％ 0.70％ |
| 27 28 29 30 31 32 33 33 34 35 36 37 38 39 40 41 42 43? | 17.90 18.63 18.86 19.05 19.42 19.51 19.83 20.05 20.55 21.81 23.42 25.75 26.47 27.81 31.02 31.83 33.09 35.39 | 2,4-DI-tert-butylphenol compounds Phenol, 2,4-di-tert-butyl 18.16 tetracosane Tetracosane methyl stearate Octadecanoic acid methyl ester soft-methyl ester Hexadecanoic acid methyl ester laurostearic acid n- |
C 14H 22O C 24H 50 C 19H 38O 2 C 19H 36O 2 C 12H 24O 2 C 19H 34O 2 C 20H 36O 2 C 16H 22O 4 C 26H 54 C 14H 28O 2 C 28H 58 C 16H 30O 2 C 16H 30O 2 C 30H 62 C 34H 70 C 18H 36O 2 C 18H 34O 2 C 18H 32O 2? | 0.23％ 0.21％ 0.23％ 0.41％ 0.95％ 0.34％ 0.31％ 0.57％ 0.48％ 3.73％ 1.50％ 2.06％ 2.06％ 1.25％ 0.79％ 1.51％ 3.46％ 1.38％ |
The present invention utilizes the method for microbial fermentation that coffee is handled to prepare the coffee tobacco aromatics using.The contriver thinks that coffee is under action of microorganisms, and very big variation has taken place for volatility and half volatile material composition, mainly shows the organic acid of low carbon chain, alcohols, ester class and alkanes substance.And the coffee after the fermentation also preserved the original aroma component of coffee, and phenyl aldehyde, phenylacetic aldehyde, furfural, vinylbenzene, furans, higher fatty acid and ester class thereof are the essential substance that forms the crucial aroma component of characteristic coffee flavor.Phenyl aldehyde has pleasant almond perfume (or spice), nutty and fruit perfume (or spice); Phenylacetic aldehyde has the fragrance like jacinthe, Syringa oblata Lindl. sample, and rose nut inside information is arranged, and fragrance is strong, and lasting is very lasting.Acetate in the fermentation of coffee spices, propionic acid, butyric acid can relax pungent flue gas, cover assorted gas; The organic acid of a series of low carbon chain such as valeric acid, 4-methylvaleric acid, isovaleric acid, caproic acid, enanthic acid, sad, capric acid, n-nonanoic acid is favourable to the flue gas of cigarette, and they can reduce the alkalescence in the flue gas, thereby can reduce pungency makes jealous pure and mild; It is of poor quality and make it have light sweet soft suction flavor that senior lipid acid such as laurostearic acid, lauric acid, myristic acid, palmitic acid, 9.12-18 diolefinic acids, 7-gaidic acid can improve flue gas, and ethyl palmitate, Uniphat A60, methyl stearate, soft-methyl ester, 8, the ester class of senior lipid acid such as 11-octadecadienoic acid methyl esters, 9,12 octadecadienoic acid ethyl ester then can improve and improve cigarette fragrance; 5-methyl furan aldehyde, 3-furfural, 2-furfuryl alcohol be promoting or transferring cigarette perfume (or spice) in cigarette, modifies coffee, caramel, nut and hot fragrant breeze flavor; Phenylcarbinol, phenylethyl alcohol can improve the quality of tobacco and the fragrant flavor of inhaling of cigarette of cigarette, increase the suction flavor of skillful and Tianjin alcohol of cigarette fragrance.The test of smokeing panel test shows that the coffee of handling that undergoes microbial fermentation has good perfuming effect, has increased the fine and smooth degree and the soft sense of flue gas through perfuming cigarette, for the excitement of cigarette tangible reduction effect is arranged also.
The perfuming cigarette smoking result of table 2, coffee fermentation concentrated extract
| Cigarette leaf group | The perfuming amount | Smoking result | |
| Fermentation of coffee medicinal extract not | Fermentation of coffee medicinal extract | ||
| Mixed type leaf group | 0.2% | Oral cavity sense is better than blank sample slightly, is mainly reflected in moisture feeling and residual aspect, and fragrance matter is coarse, and preceding half what embody is burnt fragrant and sweet, and later half has assorted gas to occur, and returns sweet sense behind the oral cavity, and dry sensation is bigger. | It is more obvious that the exquisiteness of flue gas and level and smooth sense improve, and flue gas has moisture feeling and slight sweet sense, and the pungency of going into larynx also obviously alleviates, and cigarette perfume (or spice) is more clear, and is roasting fragrant and sweet, do not have obviously residually at whole lingual surface, and puckery sense is arranged slightly. |
| Imperial leaf group is risen in nine divisions of China in remote antiquity | 0.2% | Cigarette is fragrant similar with blank sample, and the fragrant saturating property sent out of cigarette is better, and the sense of the fragrant inlet of cigarette burr is many, the residual initiation convergence sense of lingual surface, and there is dry sensation in the oral cavity, influences anti-absorption, and major defect is not ideal enough on the clean level of comfort. | The flue gas exquisiteness, soft, fragrant embodiment is very natural, stream is long, and aftertaste is arranged slightly, mainly is roasting fragrant and sweet and very harmony of flue gas, the level and smooth sense of flue gas embodies to some extent, and flue gas brings in the blank sample goes into laryngoxerosis and excitement does not all embody, and lingual surface has slight residual. |
Above smoking result shows: the bacterial classification that method of the present invention screens on the one hand can utilize outer sugaring source, by self a series of pathways metabolism, compounds such as the methyl esters of formation lower fatty acid and lower fatty acid and ethyl ester, lower fatty acid has the effect of covering intrinsic assorted gas of tobacco leaf and abirritate and fragrance matter is had some improvement, compounds such as the methyl esters of lower fatty acid and ethyl ester have fruit perfume (or spice) or aroma, fat is fragrant, wax is fragrant, wherein much be the important component of flue gas essence, play an important role for enriching flue gas.It is that coffee itself is had the longer chain fatty acid of certain negative interaction and lipid acid, ester class, heterocyclic compound, the alcohols that Ester is degraded into low carbon chain thereof, carbonyl compound etc. and the closely-related material of fragrance to flue gas that the bacterial classification that screens on the other hand utilizes the various enzymes that produce in its cell.With protein degradation is amino acid, coordinates the irritating smell that too much produces because of protein.Prove that thus method of the present invention is very successful.
Claims (5)
1. method that adopts microbial fermentation of coffee to prepare tobacco aromatics using is characterized in that:
Described method is carried out according to following step:
(1) preparation of tobacco leaf complex medium:
The burley tobaccos in 1 year to 2 years of natural ageing and water by 1: 5 mixed post-heating to 100 ℃, and were kept 1-2 hour, filter and obtain burley tobaccos water extract; With burley tobaccos water extract and microbial solid substratum mixed, make the tobacco leaf complex medium by 3: 7;
(2) produce fragrant Screening of Bioflocculant-producing Bacteria:
Take from the burley tobacco leaf 5-10g in 1 year to 2 years of right ageing, join in the sterile distilled water, concussion 30-60min, therefrom get 100-1000 μ l liquid, be added drop-wise on the tobacco leaf complex medium that has prepared, evenly be coated with out, with aerobic or anaerobic condition, separate or after dilution was coated with the flat band method purifying, the burley tobacco leaf surface dominant bacteria that obtains was the fragrant microorganism of product of screening with plate streaking;
The burley tobacco leaf surface dominant bacteria that is screened is genus bacillus, gemma clostridium, pseudomonas, aspergillus tubigensis, Penicillium notatum, yeast, and bacterial classification is bacterium song or bacterium liquid form;
(3) the burley tobacco leaf surface dominant bacteria with screening carries out liquid fermenting or solid fermentation to coffee, obtains coffee tobacco perfume;
Burley tobacco leaf surface dominant bacteria with screening carries out liquid fermenting to coffee, carries out according to the following steps:
1. actication of culture: get burley tobacco leaf surface dominant bacteria, be inoculated on the ready slant medium, be positioned over and cultivate 24～48h in 30～37 ℃ the constant incubator and make actication of culture, again with the activatory bacterial classification inoculation in the LB liquid nutrient medium that adds sugared source, in 30～37 ℃ of constant temperature culture 24～48h, obtain the activatory seed liquor;
2. the pre-treatment of raw material: choose coffee powder that exsiccant pulverized as raw material, with water: coffee powder=1: mixed (50-100), in above-mentioned mixed solution, add and account for the sugared source of above-mentioned mixed solution 1-5% and the inorganic salt of 0.1-2%, at 115 ℃ of 15～20min that sterilize down, be the good coffee liquid nutrient medium of sterilization;
3. liquid fermenting: will activate good seed liquor and be inoculated in the good coffee liquid nutrient medium of sterilization, the inoculation volume ratio is a seed liquor: coffee liquid nutrient medium=1: (50～100) after mixing, place 30～37 ℃ constant incubator, the 24～48h that ferments;
4. fermented liquid is concentrated and refining: with the coffee fermented liquid that ferments, sheet frame membrane filtration after the bacterial classification deactivation, concentrating under reduced pressure or lyophilize are condensed into paste again, according to 1: the mass ratio of (1～4) mixes paste mutually with extraction solvent, paste is dissolved fully, after refiltering, filtrate decompression is concentrated into the solvent of removing 80-95%, gained liquid paste substance is a coffee tobacco perfume;
With the fragrant microorganism of the product that screens coffee being carried out solid fermentation carries out according to the following steps:
1. actication of culture: the burley tobacco leaf surface dominant bacteria of getting purifying, be inoculated on the slant medium, be positioned over and cultivate 24～48h in 20～37 ℃ the constant incubator and make actication of culture, again with the activatory bacterial classification inoculation in the liquid nutrient medium that adds sugared source, in 20～37 ℃ of constant temperature culture 24～48h, with medium centrifugal reject supernatant liquor, the precipitation thalline with physiological saline dilution concussion evenly is the activated seed liquid that obtains;
2. the pre-treatment of raw material: choose coffee powder that exsiccant pulverized as raw material,, obtain aseptic coffee meal at 115 ℃ of sterilization 15～20min down;
3. solid fermentation: activated seed liquid is inoculated in the aseptic coffee meal according to 1: 1～10 mass ratio, stirs, make meal solid and seed liquor thorough mixing even, be positioned in 28～35 ℃ the incubator and fermented 2～7 days;
4. extract and make with extra care: the above-mentioned coffee meal that ferments, in fermentation solid and extracting solution mass ratio 1: the ratio of (1～5) adds an amount of extracting solution, be heated to 90-100 ℃ and refluxing extraction 2～3 times, each 1～3h that extracts, after the extracting solution collection and filtering, filtrate decompression is condensed into medicinal extract; According to 1: the mass ratio of (1～4) mixes paste mutually with extraction solvent, and paste is dissolved fully, and after further filtering, concentrating under reduced pressure is removed the 80-95% solvent, and gained liquid paste substance is coffee tobacco perfume.
2. a kind of method that adopts microbial fermentation of coffee to prepare tobacco aromatics using according to claim 1 is characterized in that: described microbial solid substratum is beef extract-peptone, PDA substratum or minimum medium.
3. a kind of method that adopts microbial fermentation of coffee to prepare tobacco aromatics using according to claim 1, it is characterized in that: step 4. fermented liquid to concentrate extracting solution, the extraction solvent described in 4. extracting and make with extra care with refining and step be water or ethanol, or both composite solutions.
4. a kind of method that adopts microbial fermentation of coffee to prepare tobacco aromatics using according to claim 1 is characterized in that: the sugared source of adding in the described coffee fermenting process is one or more the mixture in sucrose, glucose, the fructose.
5. a kind of method that adopts microbial fermentation of coffee to prepare tobacco aromatics using according to claim 1 is characterized in that: the step 2. inorganic salt described in the pre-treatment of raw material is KH
2PO
4, (NH
4)
2SO
4, MgSO
4, or NaCl.
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