CN102978127B - Tobacco leaf extract and cocoa extract composite fermentation perfume preparation method and applications of tobacco leaf extract and cocoa extract composite fermentation perfume in recombinant tobacco leaf - Google Patents
Tobacco leaf extract and cocoa extract composite fermentation perfume preparation method and applications of tobacco leaf extract and cocoa extract composite fermentation perfume in recombinant tobacco leaf Download PDFInfo
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- 238000002360 preparation method Methods 0.000 title claims abstract description 15
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Abstract
The present invention discloses a tobacco leaf extract and cocoa extract composite fermentation perfume preparation method and applications of a tobacco leaf extract and cocoa extract composite fermentation perfume in recombinant tobacco leaf. According to the present invention, a name of the strain is Raoultella planticola VP4-4, a preservation number is CCTCC M 2012005, the total 16SrRNA sequence length is 1408 bp, and similarity between the 16SrRNA sequence and Raoultella planticola is 99.563%. According to the method, the Raoultella planticola VP4-4 is adopted, and bacteria seed activation, seed culture liquid preparation, fermentation culture and fermentation solution transformation are performed to obtain the tobacco leaf extract and cocoa extract composite fermentation concrete. With the present invention, the process is simple, the raw materials are the cigarette product origin tobacco leaves and cocoa perfumes capable of applying in cigarettes, and the method has an industrial application prospect.
Description
Technical field
The present invention relates to recombination tobacco leaf additive technology field, specifically refer to a kind of tobacco leaf, the preparation method of cocoa extract complex ferment spices and the application in recombination tobacco leaf thereof.
Background technology
Recombination tobacco leaf claims again reconstituted tobacco or homogeneous tobacco leaf, mainly adds tackiness agent and other additives etc. to form by offal, fragment, offal or discarded tobacco leaf.Domestic existing paper process thin slice production technique is by the water-soluble substances water extraction in tobacco leaf and offal, and after solid-liquid separation, solid part is made paper substrate after slurrying again, and liquid portion concentrated rear returning is coated onto on paper substrate.This production technique is a raw-material physics regrouping process to a great extent, and the recombination tobacco leaf obtaining by the method is with little as tobacco leaf, the offal chemical composition difference of raw material.Cause like this recombination tobacco leaf to have that assorted gas is heavy, pungency is large and mouthfeel is uncomfortable etc. inhales taste defect, affected result of use and the addition of recombination tobacco leaf in cigarette product.Domestic common way is to adopt reinforced way to improve part weak point, but this traditional chemical process is very limited to the improvement degree of restructuring quality of tobacco, is difficult to solve some profound problems of recombination tobacco leaf quality.Cigarette generally adopts traditional method to make with more tobacco, natural plant kind extract, adopting monomer tobacco leaf or composite leaf group is that raw material obtains by water, ethanol-extracted or molecular distillation, after interpolation, the improvement of cigarette is had to certain limitation, improvement degree is limited, still the many defects of irritant large, assorted gas.
Cocoa beans (raw beans) moisture content 5.58%, fat 50.29%, nitrogenous substances 14.19%, Theobromine 1.55%, other non-nitrogen materials 13.91%, starch 8.77%, robust fibre 4.93%, contains phosphatase 24 0.4%, potassium 31.28%, magnesium oxide 16.22% in its ash content.In cocoa beans, also contain nervous center excitatory substance and the tannin such as caffeine, tannin and chocolate color have much relations.Wherein Theobromine, caffeine can stimulate pallium, eliminates sleepiness, strengthens sense of touch and the power to think and capable of regulating heart body, has again the effects such as expansion renal blood vessels, diuresis.
By the prepared leaf tobacco extract medicinal extract of traditional refluxing extraction, there is the intrinsic special natural style of tobacco, fragrance is pure and stay length, has prepared opium paste perfume (or spice), plays supplement cigarette perfume (or spice), the effect that strengthens mouthfeel and improve quality; Cocoa extract medicinal extract, has pure natural cocoa aroma, enriches cigarette perfume (or spice); But meanwhile, also there is the pungency of increasing, increase residual counter productive.
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, with tobacco leaf, cocoa extract is that raw material decomposes, 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 tobacco or the aroma component for the additive of tobacco, restructuring tobacco leaf cigarette gas is carried out to characteristic strengthening, improve mouthfeel.Adopt microbial fermentation technology to produce natural tobacco bioflavours, natural green is environmentally friendly, and technological process is simple to operation, does not produce poisonous byproduct, pollution-free etc.
At present, publication number is that the Chinese invention patent of CN100394860C discloses a kind of extract to the inhibited cocoa beans of carcinogenesis and cocoa pod shells, this extract is by with 50% acetone extraction cocoa beans, then washes with 40% ethanol, then carries out fractional separation acquisition with 60% ethanol.Tobacco leaf, cocoa are prepared and can be reduced stimulation, the tobacco leaf that enriches cigarette perfume (or spice), cocoa extract complex ferment spices and the application in recombination tobacco leaf thereof by microbial fermentation technology after extracting, have not been reported.
Summary of the invention
The present invention is directed to that current recombination tobacco leaf exists that assorted gas is heavy, pungency is large, flue gas is weak and mouthfeel is uncomfortable etc. inhales taste defect, a kind of tobacco leaf, the preparation method of cocoa extract complex ferment spices and the application in recombination tobacco leaf thereof are provided.What the present invention made generates cocoa extract complex ferment spices by planting raw Raoul bacterium bio-transformation, and recombination tobacco leaf flue gas matter is promoted, and increases cocoa breath and baking fragrance, and reducing flue gas stimulates, and enriches the tobacco leaf cocoa extract complex ferment spices of cigarette perfume (or spice),
The invention provides one kind and plant raw Raoul bacterium, this bacterial strain is called Raoultella planticola VP4-4, be preserved in Wuhan University's Chinese Typical Representative culture collection center on January 11st, 2012, preserving number is CCTCC M 2012005, the biological property of planting raw Raoul bacterium Raoultella planticola VP4-4 CCTCC M 2012005 is: Gram-negative bacteria, can produce acid, milk liquefy and generate indoles produce, lysine decarboxylase test be positive; Ornithine decarboxylase experiment, H2S produce, gelatine liquefication is negative; Can be with monose, polysaccharide, ester class and amino acid are sole carbon source growth,
Described Molecular Identification of planting raw Raoul bacterium Raoultella planticola VP4-4 is characterized as: its 16SrRNA sequence total length 1408bp, the similarity of 16S rRNA sequence and Raoultella planticola reaches 99.563%.
The preparation method who the invention provides a kind of tobacco leaf, cocoa extract complex ferment spices, it comprises the following steps:
1) tobacco leaf powder is broken to 80 ~ 100 orders, then offal, cocoa powder 1: 0.5 in mass ratio ~ 2 are mixed, it is 1: 30 ~ 50 water that mixture adds mass ratio, after refluxing extraction 1 ~ 3h, filter, repeat 3 times refluxing extraction filtrate, merging filtrate, obtain tobacco leaf, cocoa mixed extract, and sterilizing;
2) actication of culture: will plant raw Raoul bacterium Raoultella planticola VP4-4 CCTCC M 2012005 and be inoculated on slant medium, and at 25 ~ 38 DEG C, obtain activated spawn after standing cultivation 24 ~ 72h, and be placed in refrigerator;
3) prepare seed culture fluid: by step 2) in the slant culture of gained, be inoculated into seed culture medium, under 25 ~ 38 DEG C of conditions, 100 ~ 150r/min shaking culture 24h ~ 72h, transfers 1 ~ 3 time continuously, makes seed culture fluid;
4) fermentation: seed culture fluid 10 ~ 20% is inoculated into above-mentioned steps 1 by volume) in tobacco leaf, the cocoa mixed extract of sterilizing, the tinning amount of postvaccinal filtrate is 30 ~ 40%, pH5 ~ 8,32 ~ 38 DEG C of temperature, rotating speed 100 ~ 150r/min, lucifuge condition bottom fermentation 3 ~ 5 days, does not obtain fermented liquid;
5) by fermented liquid and 95% ethanol 1: 3 by volume ~ 6, precipitation is spent the night, and the centrifugal 10 ~ 20min of 4000 ~ 8000r/min is evaporated to relative density and is 1.1054 ~ 1.2241 medicinal extract after filtration, obtain tobacco leaf, cocoa extract complex ferment medicinal extract.
As preferred version, described step 2) in, described slant medium is: maltose: 120mL, glucose: 20g/L, agar: 15g/L.
As preferred version, in described step 3), described seed liquor substratum consists of: fructose: 50g/L, yeast powder: 10g/L, sodium-chlor: 5g/L.
It is a kind of according to the application of claim 2 gained tobacco leaf, cocoa extract complex ferment spices that the present invention also provides, described tobacco leaf cocoa extract complex ferment spices adds in recombination tobacco leaf as natural baccy additive, and adding the mass ratio of tobacco leaf in recombination tobacco leaf, cocoa extract complex ferment spices is 0.1 ~ 0.3%.Recombination tobacco leaf flue gas matter is promoted, increase cocoa breath and baking fragrance, reducing flue gas stimulates, and enriches cigarette perfume (or spice), has improved recombination tobacco leaf and has stimulated large, the uncomfortable suction taste defect of mouthfeel.
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, with tobacco leaf, cocoa composite extract is that raw material decomposes, 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.
Advantage of the present invention is:
1, the present invention, taking natural tobacco leaf, cocoa composite extract as sole carbon source, nitrogenous source and substrate, easily allows microbial enzyme system be used to complex ferment and produces perfume, and its tobacco leaf cocoa extract complex ferment spices has the harmless advantage of natural environmental-protective.
2, adopt plant raw Raoul bacterium Raoultella planticola VP4-4 CCTCC M 2012005 come fermentative production make recombination tobacco leaf flue gas matter promote, increase cocoa breath and baking fragrance, reducing flue gas stimulates, enrich tobacco leaf, the cocoa extract complex ferment spices of cigarette perfume (or spice), improve recombination tobacco leaf and stimulated large, the uncomfortable suction taste defect of mouthfeel, can use with other essence spice for cigarette mixing preparation.
3, the inventive method technological process is simple, and raw material is cigarette product origin tobacco leaf and can be applied to the cacao flavor in cigarette, has industrialization prospect of the application.
Embodiment
Enumerate a part of embodiment below Relational Questions of the present invention is described in further detail, be necessary to point out that at this following specific embodiment just further illustrates of the present invention, does 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 preparation method that microorganism of the present invention is planted raw Raoul bacterium Raoultella planticolaVP4-4 CCTCC M 2012005 bio-transformations production tobacco leaves, cocoa extract complex ferment spices is as follows:
1) tobacco leaf powder is broken to 80 orders, then offal, cocoa powder are mixed in mass ratio at 1: 2, it is the water of 1: 30 that mixture adds mass ratio, after refluxing extraction 3h, filter, repeat 3 times refluxing extraction filtrate, merging filtrate, obtain tobacco leaf, cocoa mixed extract, and sterilizing;
2) actication of culture: will plant raw Raoul bacterium Raoultella planticola VP4-4 CCTCC M 2012005 and be inoculated on slant medium, and at 25 DEG C, obtain activated spawn after standing cultivation 24h, and be placed in 4 DEG C of refrigerators;
Described slant medium is: maltose: 120mL, glucose: 20g/L, agar: 15g/L.
3) prepare seed culture fluid: by step 2) in the slant culture of gained, be inoculated into seed culture medium, under 38 DEG C of conditions, 100r/min shaking culture 72h, transfers 3 times continuously, makes seed culture fluid;
Described seed liquor substratum consists of: fructose: 50g/L, yeast powder: 10g/L, sodium-chlor: 5g/L.
4) fermentation: seed culture fluid 20% is inoculated into above-mentioned steps 1 by volume) in tobacco leaf, the cocoa mixed extract of sterilizing, the tinning amount of postvaccinal filtrate is 40%, pH5,32 DEG C of temperature, rotating speed 100r/min, lucifuge condition bottom fermentation 5 days, does not obtain fermented liquid;
5) by fermented liquid and 95% ethanol 1: 3 by volume, precipitation was spent the night, and the centrifugal 20min of 4000r/min is evaporated to relative density and is 1.1054 medicinal extract after filtration, obtain tobacco leaf, cocoa extract complex ferment medicinal extract.
Embodiment 2
The preparation method that microorganism of the present invention is planted raw Raoul bacterium Raoultella planticolaVP4-4 CCTCC M 2012005 bio-transformations production tobacco leaves, cocoa extract complex ferment spices is as follows:
1) tobacco leaf powder is broken to 100 orders, then offal, cocoa powder are mixed in mass ratio at 1: 0.5, it is the water of 1: 50 that mixture adds mass ratio, after refluxing extraction 1h, filter, repeat 3 times refluxing extraction filtrate, merging filtrate, obtain tobacco leaf, cocoa mixed extract, and sterilizing;
2) actication of culture: will plant raw Raoul bacterium Raoultella planticola VP4-4 CCTCC M 2012005 and be inoculated on slant medium, and at 38 DEG C, obtain activated spawn after standing cultivation 72h, and be placed in 4 DEG C of refrigerators;
Described slant medium is: maltose: 120mL, glucose: 20g/L, agar: 15g/L.
3) prepare seed culture fluid: by step 2) in the slant culture of gained, be inoculated into seed culture medium, under 25 DEG C of conditions, 150r/min shaking culture 24h, transfers 1 time continuously, makes seed culture fluid;
Described seed liquor substratum consists of: fructose: 50g/L, yeast powder: 10g/L, sodium-chlor: 5g/L.
4) fermentation: seed culture fluid 10% is inoculated into above-mentioned steps 1 by volume) in tobacco leaf, cocoa mixed extract, the tinning amount of postvaccinal filtrate is 30%, pH8,38 DEG C of temperature, rotating speed 150r/min, lucifuge condition bottom fermentation 5 days, does not obtain fermented liquid;
5) by fermented liquid and 95% ethanol 1: 6 by volume, precipitation was spent the night, and the centrifugal 10min of 8000r/min is evaporated to relative density and is 1.2241 medicinal extract after filtration, obtain tobacco leaf, cocoa extract complex ferment medicinal extract.
Embodiment 3
The preparation method that microorganism of the present invention is planted raw Raoul bacterium Raoultella planticolaVP4-4 CCTCC M 2012005 bio-transformations production tobacco leaves, cocoa extract complex ferment spices is as follows:
1) tobacco leaf powder is broken to 90 orders, then offal, cocoa powder are mixed in mass ratio at 1: 1, it is the water of 1: 40 that mixture adds mass ratio, after refluxing extraction 2h, filter, repeat 3 times refluxing extraction filtrate, merging filtrate, obtain tobacco leaf, cocoa mixed extract, and sterilizing;
2) actication of culture: will plant raw Raoul bacterium Raoultella planticola VP4-4 CCTCC M 2012005 and be inoculated on slant medium, and at 28 DEG C, obtain activated spawn after standing cultivation 72h, and be placed in 4 DEG C of refrigerators;
Described slant medium is: maltose: 120mL, glucose: 20g/L, agar: 15g/L.
3) prepare seed culture fluid: by step 2) in the slant culture of gained, be inoculated into seed culture medium, under 35 DEG C of conditions, 100r/min shaking culture 48h, transfers 2 times continuously, makes seed culture fluid;
Described seed liquor substratum consists of: fructose: 50g/L, yeast powder: 10g/L, sodium-chlor: 5g/L.
4) fermentation: seed culture fluid 20% is inoculated into above-mentioned steps 1 by volume) in tobacco leaf, cocoa mixed extract, the tinning amount of postvaccinal filtrate is 40%, pH6,32 DEG C of temperature, rotating speed 120r/min, lucifuge condition bottom fermentation 3 days, does not obtain fermented liquid;
5) by fermented liquid and 95% ethanol 1: 3 by volume, precipitation was spent the night, and the centrifugal 20min of 6000r/min is evaporated to relative density and is 1.1528 medicinal extract after filtration, obtain tobacco leaf, cocoa extract complex ferment medicinal extract.
Embodiment 4
The preparation method that microorganism of the present invention is planted raw Raoul bacterium Raoultella planticolaVP4-4 CCTCC M 2012005 bio-transformations production tobacco leaves, cocoa extract complex ferment spices is as follows:
1) tobacco leaf powder is broken to 100 orders, then offal, cocoa powder are mixed in mass ratio at 1: 0.5, it is the water of 1: 35 that mixture adds mass ratio, after refluxing extraction 3h, filter, repeat 3 times refluxing extraction filtrate, merging filtrate, obtain tobacco leaf, cocoa mixed extract, and sterilizing;
2) actication of culture: will plant raw Raoul bacterium Raoultella planticola VP4-4 CCTCC M 2012005 and be inoculated on slant medium, and at 35 DEG C, obtain activated spawn after standing cultivation 72h, and be placed in 4 DEG C of refrigerators;
Described slant medium is: maltose: 120mL, glucose: 20g/L, agar: 15g/L.
3) prepare seed culture fluid: by step 2) in the slant culture of gained, be inoculated into seed culture medium, under 32 DEG C of conditions, 100r/min shaking culture 72h, transfers 2 times continuously, makes seed culture fluid;
Described seed liquor substratum consists of: fructose: 50g/L, yeast powder: 10g/L, sodium-chlor: 5g/L.
4) fermentation: seed culture fluid 15% is inoculated into above-mentioned steps 1 by volume) in tobacco leaf, cocoa mixed extract, the tinning amount of postvaccinal filtrate is 35%, pH6,33 DEG C of temperature, rotating speed 150r/min, lucifuge condition bottom fermentation 5 days, does not obtain fermented liquid;
5) by fermented liquid and 95% ethanol 1: 4 by volume, precipitation was spent the night, and the centrifugal 10min of 5500r/min is evaporated to relative density and is 1.6584 medicinal extract after filtration, obtain tobacco leaf, cocoa extract complex ferment medicinal extract.
Embodiment 5
Recombination tobacco leaf amount 0.1%, 0.2%, 0.3% pressed by above-mentioned tobacco leaf cocoa extract complex ferment spices by contriver, adds to respectively on recombination tobacco leaf.Recombination tobacco leaf is packed in sealing bag, place 80 DEG C of constant temperature 30min in baking oven, suitable to recombination tobacco leaf moisture, roll into cigarette, put into climatic chamber 22 DEG C+1, relative humidity is 60%+2%, smoothly 48h, asks the expert group of smokeing panel test to be smoked panel test.Contrast adds the cigarette that the recombination tobacco leaf of same amount tobacco leaf cocoa composite extract rolls for spray.Repeatedly smoke panel test through the expert group of smokeing panel test, think: this fermenting tobacco leaf extract, while being 0.1%, 0.2% with recombination tobacco leaf amount ratio, having the recombination tobacco leaf of making flue gas matter promotes, increase cocoa breath and baking fragrance, reducing flue gas stimulates, and enriches the advantage of cigarette perfume (or spice), has improved recombination tobacco leaf and has stimulated large, the uncomfortable suction taste defect of mouthfeel.
Table 1. fermenting tobacco leaf extract adds the sensory evaluating smoking in recombination tobacco leaf to
Claims (4)
1. a preparation method for tobacco leaf, cocoa extract complex ferment spices, comprises the following steps:
1) tobacco leaf powder is broken to 80～100 orders, then offal, cocoa powder 1: 0.5 in mass ratio～2 are mixed, it is 1: 30～50 water that mixture adds mass ratio, after refluxing extraction 1～3h, filter, repeat 3 times refluxing extraction filtrate, merging filtrate, obtain tobacco leaf, cocoa mixed extract, and sterilizing;
2) actication of culture: will plant raw Raoul bacterium Raoultella planticola VP4-4 CCTCC M 2012005 and be inoculated on slant medium, at 25～38 DEG C, after leaving standstill cultivation 24～72h, obtain activated spawn, be placed in refrigerator, wherein, plant raw Raoul bacterium Raoultella planticola VP4-4, preserving number is CCTCC M 2012005;
3) prepare seed culture fluid: by step 2) in the slant culture of gained, be inoculated into seed culture medium, under 25～38 DEG C of conditions, 100～150r/min shaking culture 24h～72h, transfers 1～3 time continuously, makes seed culture fluid;
4) fermentation: seed culture fluid 10～20% is inoculated into above-mentioned steps 1 by volume) in tobacco leaf, the cocoa mixed extract of sterilizing, the tinning amount of postvaccinal filtrate is 30～40%, pH5～8,32～38 DEG C of temperature, rotating speed 100～150r/min, lucifuge condition bottom fermentation 3～5 days, does not obtain fermented liquid;
5) by fermented liquid and 95% ethanol 1: 3 by volume～6, precipitation is spent the night, and the centrifugal 10～20min of 4000～8000r/min is evaporated to relative density and is 1.1054～1.2241 medicinal extract after filtration, obtain tobacco leaf, cocoa extract complex ferment medicinal extract.
2. the preparation method of a kind of tobacco leaf according to claim 1, cocoa extract complex ferment spices, is characterized in that: described step 2) in, described slant medium is: wort 120mL/L, glucose: 20g/L, agar: 15g/L.
3. the preparation method of a kind of tobacco leaf according to claim 1, cocoa extract complex ferment spices, is characterized in that: in described step 3), described seed culture medium consists of: fructose: 50g/L, yeast powder: 10g/L, sodium-chlor: 5g/L.
4. the application of the tobacco leaf that method makes according to claim 1, cocoa extract complex ferment spices, it is characterized in that: described tobacco leaf cocoa extract complex ferment spices adds in recombination tobacco leaf as natural baccy additive, and adding the mass ratio of tobacco leaf in recombination tobacco leaf, cocoa extract complex ferment spices is 0.1～0.3%.
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