CN102392050B - Endophyte bio-transformed raisin extract and preparation and application in flavoring thereof - Google Patents
Endophyte bio-transformed raisin extract and preparation and application in flavoring thereof Download PDFInfo
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- CN102392050B CN102392050B CN 201110342923 CN201110342923A CN102392050B CN 102392050 B CN102392050 B CN 102392050B CN 201110342923 CN201110342923 CN 201110342923 CN 201110342923 A CN201110342923 A CN 201110342923A CN 102392050 B CN102392050 B CN 102392050B
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Abstract
The present invention belongs to the technical field of perfume for tobacco, and particularly relates to endophyte bio-transformed raisin extract and preparation and application in flavoring thereof. The method is implemented as follows: (1) separating and purifying endophyte; (2) culturing endophyte; (3) extracting and refining raisin; and (4) bio-transforming raisin extract with the endophyte to obtain bio-transformed raisin extract. The method of the present invention has simple technological process and low cost, and the obtained bio-transformed raisin extract can be directly used for the flavoring and casing of cigarettes. The perfume of bio-transformed raisin extract has a unique fresh and sweet smell, and can lower stimulation after being added to cigarettes. The perfume of bio-transformed raisin extract has a strong fresh and sweet smell as well as a strong toasting smell.
Description
Technical field
The present invention relates to biological preparation tobacco aromatics using technical field, specifically a kind of endophyte of plant bio-transformation raisin extract and preparation thereof and the application method in tobacco flavoring.
Background technology
The manufacture method of tobacco aromatics using has two at present, extracts natural perfume from tobacco or other plant, perhaps adopts traditional chemical industry method synthetic perfume.Yet, all there is its limitation separately in these two kinds of methods, for certain plant perfume, its aroma component and feature are constant, 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 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.People put into more sight the alternative resource of exploitation natural perfume product aspect when continuing original production technology, biotechnology is produced one of the most promising method of natural perfume product exactly.
Endophyte of plant is a microbe groups that diversity is very abundant.Being distributed in does not have in the outside plant tissue that infects, and with the host plant coevolution, can produce the meta-bolites of rich and varied biologically active.Utilize plant endogenesis epiphyte, research and develop natural perfume, very wide prospect will be arranged.
Raisin are nutritious, and its water content has only 15-25%, are rich in sugar, amino acid, vitamins and other nutritious components, are described as " jewel of world's fruit ".Raisin are that new fresh grape is being made under airing after a while under certain temperature and the humidity, it is the process of a spontaneous fermentation, compare with new fresh grape, give off a strong fragrance, mouthfeel is sweet, is subjected to the inspiration of raisin spontaneous fermentation, and the inventor utilizes endophyte of plant that raisin are carried out bio-transformation, in the hope of raisin are further fermented, enrich the fragrance of raisin.
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 synergy of the interior complicated metabolism 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 components of tobacco.In the prior art, utilize endophyte of plant that the method that raisin extract carries out bio-transformation production characteristic tobacco aromatics using is not appeared in the newspapers.
Summary of the invention
The invention provides a kind of endophyte of plant bio-transformation raisin extract and preparation thereof and the application method in tobacco flavoring.The endophyte of plant bio-transformation raisin extract that the present invention makes has unique fresh and sweet note feature, is applied in the tobacco flavoring, has fruit fragrant and sweet in the flue gas, and the fine and smooth sense of flue gas increases, and stimulates and residual minimizing, and pleasant impression is comfortable.
A kind of preparation method of endophyte of plant bio-transformation raisin extract may further comprise the steps:
(1) cultivation of endophyte of plant
The 100mLPDB substratum is loaded in the 250mL triangular flask, choose an amount of endophyte of plant mycelium inoculation in triangular flask with transfering loop, be positioned over and cultivate 24-28h in 30-37 ℃ the constant incubator and make actication of culture, be shake-flask culture under the 150r/min at rotating speed, the seed liquor that obtains activating, described endophyte of plant are Penicillium (Penicillium sp.) EFCI-24; Deposit number is CCTCC NO:M2011342;
(2) preparation of raisin extract
The raisin of choosing, cleaning are put into round-bottomed flask, and by raisin: concentration of volume percent is that the weight ratio of alcohol=1 of 65-75%: 5.5-6.5 is mixed, and extracts 2.5-3.5h 75-85 ℃ of reflux, extract 1-2 time, be cooled to room temperature, filter, collect filtrate.With filtrate at temperature 45-55 ℃, vacuum tightness be not less than be concentrated under the condition of 0.95Mpa solvent evaporation half, get raisin extract, 115 ℃ of sterilization 15-20min down;
(3) bio-transformation raisin extract
Cultured endophyte of plant is inoculated in the raisin extract after the sterilization, and the inoculation volume ratio is seed liquor: raisin extract=1: 50-100, place 30-37 ℃ constant temperature oscillator, and rotating speed is to cultivate 24-28h under the 150r/min;
(4) raisin extract after will cultivating uses Plate Filtration, filters bacterial classification wherein, obtains the raisin extract of bio-transformation.
A kind of endophyte of plant bio-transformation raisin extract is to be prepared from by above-mentioned method.
Above-mentioned endophyte of plant bio-transformation raisin extract can be used as tobacco aromatics using, adds in the cigarette.
The contriver finds to utilize the variation of volatile component in the product to come the effect of comparison endophyte bio-transformation raisin extract in research process, the raisin extract dichloromethane extraction, with the organic phase concentrating under reduced pressure, advance GC-MS, analyze aroma component wherein, DDMP (2, the 3-dihydro-3 after the discovery bio-transformation in the raisin, 5-dihydroxyl-6-methyl-4H-pyrone) and 5 hydroxymethyl furfural content increase is arranged, the raisin extract after the bio-transformation is fresh and sweet fragrant increasing.
Method technological process of the present invention is simple, with low cost, and it is reinforced that the raisin extract tobacco aromatics using of gained bio-transformation can be directly used in cigarette flavoring.The raisin tobacco aromatics using of bio-transformation has unique fresh and sweet note feature, and being added on to have in the cigarette to reduce stimulates, abundant and full flue gas, and increasing has fruit fragrant and sweet, and the fine and smooth sense of flue gas increases, and pleasant impression is comfortable.
Endophyte of plant of the present invention has carried out preservation on October 10th, 2011 at the Chinese typical culture collection center of Chinese Wuhan City, Hubei Province Wuhan University.Deposit number is CCTCC NO:M2011342; Called after Penicillium (Penicillium sp.) EFCI-24.
Description of drawings
Fig. 1 is for separating the morphology picture of camptothecine endophyte under opticmicroscope that obtains among the present invention.
Embodiment
Below in conjunction with specific embodiment the present invention is further described.Following examples are intended to illustrate the present invention rather than limitation of the invention further.
Embodiment 1
(1) separation of camptothecine endophyte and purifying
After scraping off camptothecine bark exterior skin with aseptic cutter, clean with aseptic water washing, be cut into the fritter of 1cm * 1cm, place 75% ethanol disinfection 1min, then with the 2.5% chlorine bleach liquor 10min that sterilizes, use 75% ethanol disinfection 1min again, aseptic water washing 2-3 time, cut off the edge, insert on the PDA plate culture medium.Treat the bark surface grow mycelia after its tip portion of picking move on the fresh PDA substratum, carrying out separation and purification repeatedly according to the plate streaking partition method cultivates, pick a little bark with transfering loop with aseptic technique and show the mycelia that grows, rule continuously at aseptic planar surface, hyphal cell quantity will reduce along with the increase of line number of times, and progressively spread out, and microorganism can disperse one by one, after cultivating, can obtain single bacterium colony at planar surface.
(2) camptothecine endophyte morphology research
Camptothecine endophyte behind the purifying has been carried out morphological observation with opticmicroscope, and microscopically is observed the mycelia multi-branched, and barrier film is arranged, and hyphal diameter is 2.6-3.5 μ m has a similar contraction at the barrier film place effect, and therefore whole mycelia presents the rhizoma nelumbinis shape.Conidium grows from hyphal cell, and branch is arranged, and the conidiophore diameter is about 2.4-2.7 μ m, the tool tabula, and does not expand on the top, and broom shape branch (Fig. 1) is arranged.Spore shape is circular or ellipse, bunchiness, rough, and size is 1.2-1.4 μ m.
(3) camptothecine endophyte physiological characteristic and evaluation
The 100mLPDB substratum is loaded in the 250mL triangular flask, choose an amount of endophyte of plant mycelium inoculation in triangular flask with transfering loop, be positioned over cultivate 24-28h in 30-37 ℃ the constant incubator after, can produce white hypha, white hypha crawls to grow afterwards, produce blackish green Powdered spore on the 2nd day at the center of bacterium colony, produce strumae, edge adularescent aerial hyphae to the bosom of the 3rd day bacterium colony; The back side bluish yellow look of substratum.
Grow in the time of liquid culture fast, 3d has just reached the logarithmic phase of growth, stops shaking table then, has blackish green lawn to be covered with the surface behind the 6d.Bacterium ball white, bacterium liquid is dark green.Through consulting document, tentatively can be accredited as Penicillium.
(4) raisin extract
In being housed, the 500mL round-bottomed flask of return line, stirrer adds 50g raisin and 300mL 70% ethanol, 80 ℃ of following reflux 3h, extract 2 times, filter, merging filtrate, 50 ℃ of following vacuum tightness be not less than 0.95Mpa be evaporated to solvent evaporation half, get the raisin extract about 200g.
(5) sterilization of raisin extract
With the above-mentioned raisin extract that obtains 115 ℃ of 15-20min that sterilize down in high pressure steam formula Autoclave.
(6) bio-transformation raisin extract
The 100mLPDB substratum is loaded in the 250mL triangular flask, choose an amount of endophyte of plant mycelium inoculation in triangular flask with transfering loop, be positioned in 35 ℃ the constant incubator, the 150r/min shaking table gets seed liquor after cultivating 25h, cultured endophyte seed liquor is inoculated in the raisin extract after the sterilization, placing rotating speed is that the following 35 ℃ constant incubator of 150r/min transforms 25h, the inoculation volume ratio is seed liquor: raisin extract=1: 80, bacterial classification in the raisin extract that bio-transformation is good uses Plate Filtration, obtains the raisin extract tobacco aromatics using of bio-transformation.
The product that the contriver produces method of the present invention detects, and detects by the raisin extract of gas chromatography-mass spectrum after to raisin extract and bio-transformation, and it the results are shown in Table 1.
Volatile component GC-MS data relatively before and after the bio-transformation of table 1 raisin extract
Data in the table 1 are to obtain with the raisin tobacco aromatics using detection of gas chromatography-mass spectrum to bio-transformation.
The camptothecine endogenetic fungus is smelt the fragrant fragrance of the raisin extract of bio-transformation of finding and is compared with raisin extract the extract microbial transformation, has increased delicate fragrance.Main volatile composition in the raisin extract is DDMP (2,3-dihydro-3,5-dihydroxyl-6-methyl-4H-pyrone) and 5 hydroxymethyl furfural.That 5 hydroxymethyl furfural has is sweet, colored, flue-cured tobacco is fragrant, and cigarette perfume (or spice) is had important effect, and DDMP (2,3-dihydro-3,5-dihydroxyl-6-methyl-4H-pyrans-4-ketone) is the precursor of voitol, has caramel fragrance, and cigarette perfume (or spice) is had important effect.As can be seen from Table 1, in the raisin extract through this endophyte conversion, DDMP (2,3-dihydro-3,5-dihydroxyl-6-methyl-4H-pyrone) and 5 hydroxymethyl furfural all be significantly increased DDMP (2,3-dihydro-3,5-dihydroxyl-6-methyl-4H-pyrone) content has increased about 5 times, and the content of 5 hydroxymethyl furfural has also increased much.And contained volatile component increases to some extent, furfural and acetic acid etc. have occurred.
In addition, the present invention is inoculated into cultured endophyte of plant in the raisin extract after the sterilization, and the inoculation volume ratio is seed liquor: raisin extract=1: 50-100, place 30-37 ℃ constant temperature oscillator, and rotating speed is to cultivate 24-28h under the 150r/min; All obtained effect preferably.
The contriver estimates in cigarette the raisin extract that utilizes endophyte of plant bio-transformation front and back, has compared the perfuming effect of the raisin extract of bio-transformation front and back, sees Table 2.
The perfuming smoking result of raisin extract in cigarette before and after table 2 bio-transformation
Claims (4)
1. the preparation method of an endophyte of plant bio-transformation raisin extract is characterized in that: may further comprise the steps:
(1) cultivation of endophyte of plant
100mL PDB substratum is loaded in the 250mL triangular flask, choose the endophyte of plant mycelium inoculation in triangular flask with transfering loop, be positioned over and cultivate 24-28h in the constant incubator of 30-37 ° of C and make actication of culture, be shake-flask culture under the 150r/min at rotating speed, the seed liquor that obtains activating, described endophyte of plant are Penicillium notatum (Penicillium sp.) EFCI-24; Deposit number is CCTCC NO:M2011342;
(2) preparation of raisin extract
The raisin of choosing, cleaning are put into round-bottomed flask, and by raisin: concentration of volume percent is that the weight ratio of alcohol=1:5.5-6.5 of 65-75% is mixed, and extracts 2.5-3.5h 75-85 ° of C reflux, extracts 1-2 time, is cooled to room temperature; Filter, collect filtrate; At temperature 45-55 ° C, vacuum tightness is not less than under the condition of 0.95Mpa and concentrates with filtrate, be concentrated into solvent evaporation half, get raisin extract, 15-20min sterilizes under 115 ° of C in high-pressure steam sterilizing pan;
(3) bio-transformation raisin extract
Cultured endophyte of plant is inoculated in the raisin extract after the sterilization, and the inoculation volume ratio is seed liquor: raisin extract=1:50-100, places the constant temperature oscillator of 30-37 ° of C, and rotating speed is to cultivate 24-28h under the 150r/min;
(4) raisin extract after will cultivating uses Plate Filtration, filters bacterial classification wherein, obtains the raisin extract of bio-transformation.
2. the raisin extract of the bio-transformation of the described method of claim 1 preparation.
3. the application method of the raisin extract of the bio-transformation of the described method preparation of claim 1 is characterized in that, as tobacco aromatics using, adds in the cigarette.
4. an endophyte of plant is characterized in that, is Penicillium notatum (Penicillium sp.) EFCI-24; Deposit number is CCTCC NO:M2011342.
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| CN102090709A (en) * | 2010-12-22 | 2011-06-15 | 湖南中烟工业有限责任公司 | Tobacco perfume prepared by using tea polysaccharid as sugar source of Maillard reaction and preparation method |
| CN102100397A (en) * | 2010-12-22 | 2011-06-22 | 湖南中烟工业有限责任公司 | Flavoring essence for tobacco and preparation method thereof |
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| CN102090709A (en) * | 2010-12-22 | 2011-06-15 | 湖南中烟工业有限责任公司 | Tobacco perfume prepared by using tea polysaccharid as sugar source of Maillard reaction and preparation method |
| CN102100397A (en) * | 2010-12-22 | 2011-06-22 | 湖南中烟工业有限责任公司 | Flavoring essence for tobacco and preparation method thereof |
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