CN102392050A - 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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- CN102392050A CN102392050A CN2011103429231A CN201110342923A CN102392050A CN 102392050 A CN102392050 A CN 102392050A CN 2011103429231 A CN2011103429231 A CN 2011103429231A CN 201110342923 A CN201110342923 A CN 201110342923A CN 102392050 A CN102392050 A CN 102392050A
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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 method of manufacture of tobacco aromatics using has two at present, from tobacco or other plant, extracts natural perfume, 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 characteristic 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 receive the restriction of compound method and condition, a lot of good aroma components still can not synthetic, or cost is too high.People put into the alternative resource of exploitation natural perfume product aspect with more sight when continuing original production technology, biotechnology is produced one of the most promising method of natural perfume product exactly.
Endophyte of plant is the very abundant microbe groups of a variety.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 cut 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 new fresh grapes under airing after a while, processing under certain temperature and the humidity, are the processes of a spontaneous fermentation, compare with new fresh grape; Give off a strong fragrance; Mouthfeel is sweet, receives 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 mikrobe can produce huge high active enzyme system in breeding; Like polysaccharide hydrolysis enzyme, protease, Mierocrystalline cellulose enzyme, esterification enzyme, redox enzymes and lyases etc.; In enzymatic action, chemical action and microbe body under the complicated metabolic synergy; Just can make effects such as substrate decomposes, degraded, oxidation, reduction, polymerization, coupling, conversion; Form complicated low molecular compound, comprising various perfume compounds, like alcohols, aldehydes, ketone, acids, ester class, phenols, furans, 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 present invention provides a kind of endophyte of plant bio-transformation raisin extract and preparation and the application method in tobacco flavoring.The endophyte of plant bio-transformation raisin extract that the present invention processes has unique fresh and sweet note characteristic, 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; At rotating speed is shake-flask culture under the 150r/min, obtains the activatory seed liquor, and described endophyte of plant is 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 filtrating.To filtrate at temperature 45-55 ℃, vacuum tightness is not less than that to be concentrated into solvent evaporation under the condition of 0.95Mpa half the, 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 a seed liquor: raisin extract=1: 50-100, place 30-37 ℃ constant temperature oscillator, and rotating speed is that 150r/min cultivates 24-28h down;
(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 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 effect that the variation of odor component comes comparison endophyte bio-transformation raisin extract in the product in research process, and raisin extract is used dichloromethane extraction, with the organic phase concentrating under reduced pressure; Advance GC-MS; Analyze aroma component wherein, and the DDMP after the discovery bio-transformation in the raisin (2,3-dihydro-3; 5-dihydroxyl-6-methyl-4H-pyrone) and 5 hydroxymethyl furfural content increase is all 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 directly be used for cigarette flavoring.The raisin tobacco aromatics using of bio-transformation has unique fresh and sweet note characteristic, and being added on to have in the cigarette to reduce stimulates, abundant and full flue gas, and increasing has fruit fragrant and sweet, the fine and smooth sense increase of flue gas, pleasant impression is comfortable.
Endophyte of plant of the present invention on October 10th, 2011 Wuhan University in Chinese Wuhan City, Hubei Province Chinese typical culture collection center carried out preservation.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 NSC 94600 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 explain the present invention rather than to further qualification of the present invention.
Embodiment 1
(1) separation of NSC 94600 endophyte and purifying
After scraping off NSC 94600 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, use 75% ethanol disinfection 1min again, aseptic water washing 2-3 time with the 2.5% chlorine bleach liquor 10min that sterilizes; 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, carry out separation and purification repeatedly according to the plate streaking partition method and cultivate, 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 mikrobe can disperse one by one; After cultivating, can obtain single bacterium colony at planar surface.
(2) NSC 94600 endophyte morphology research
NSC 94600 endophyte to behind the purifying has 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 a effect, and therefore whole mycelia demonstrates 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) NSC 94600 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; Produced 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
, the 500mL round-bottomed flask of return line, stirrer adds 50g raisin and 300mL 70% ethanol in being housed; 80 ℃ of following reflux 3h; Extract 2 times, filter merging filtrate; 50 ℃ of following vacuum tightness is not less than 0.95Mpa, and to be evaporated to solvent evaporation half the, 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 HP 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 cultivate behind the 25h seed liquor, 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 a seed liquor: raisin extract=1: 80, the 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 through the raisin extract of gas chromatography-mass spectrum after to raisin extract and bio-transformation, and its result sees table 1.
Odor 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 NSC 94600 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.Can find out that 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 obvious increase is all arranged, 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 odor component increases to some extent, furfural and acetate 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 a seed liquor: raisin extract=1: 50-100, place 30-37 ℃ constant temperature oscillator, and rotating speed is that 150r/min cultivates 24-28h down; 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
The 100mLPDB 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 30-37 ℃ the constant incubator and make actication of culture; At rotating speed is shake-flask culture under the 150r/min, obtains the activatory seed liquor, and described endophyte of plant is 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, extracts 1-2 time, is cooled to room temperature; Filter, collect filtrating; To filtrate at temperature 45-55 ℃, vacuum tightness is not less than under the condition of 0.95Mpa and concentrates, and it is half the to be concentrated into solvent evaporation, raisin extract, 115 ℃ of sterilization 15-20min down 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 a seed liquor: raisin extract=1: 50-100, place 30-37 ℃ constant temperature oscillator, and rotating speed is that 150r/min cultivates 24-28h down;
(4) raisin extract after will cultivating uses Plate Filtration, filters bacterial classification wherein, obtains the raisin extract of bio-transformation.
2. an endophyte of plant bio-transformation raisin extract is characterized in that, is prepared from the described method of claim 1.
3. the application method of the described endophyte of plant bio-transformation of claim 2 raisin extract is characterized in that, as tobacco aromatics using, adds in the cigarette.
4. an endophyte of plant is characterized in that, is penicillium spp (Penicillium sp.) EFCI-24.
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CN103146485A (en) * | 2013-03-04 | 2013-06-12 | 湖北中烟工业有限责任公司 | Method for preparing fruit composite extract for fermented cigarette |
CN105713842A (en) * | 2016-04-18 | 2016-06-29 | 云南中烟工业有限责任公司 | Method for fermentation preparation of jasmine flower perfume for cigarettes with Trichoderma aureoviride |
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Publication number | Priority date | Publication date | Assignee | Title |
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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 |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103146485A (en) * | 2013-03-04 | 2013-06-12 | 湖北中烟工业有限责任公司 | Method for preparing fruit composite extract for fermented cigarette |
CN105713842A (en) * | 2016-04-18 | 2016-06-29 | 云南中烟工业有限责任公司 | Method for fermentation preparation of jasmine flower perfume for cigarettes with Trichoderma aureoviride |
CN105713842B (en) * | 2016-04-18 | 2019-04-05 | 云南中烟工业有限责任公司 | A method of cigarette perfume of jasmines is prepared using trichoderma aureoviride fermentation |
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