CN105733962A - Trichoderma aureoviride HLMM-04 strain and method for preparing tobacco flavor - Google Patents
Trichoderma aureoviride HLMM-04 strain and method for preparing tobacco flavor Download PDFInfo
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- CN105733962A CN105733962A CN201610239982.9A CN201610239982A CN105733962A CN 105733962 A CN105733962 A CN 105733962A CN 201610239982 A CN201610239982 A CN 201610239982A CN 105733962 A CN105733962 A CN 105733962A
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- fermentation
- hlmm
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- trichoderma aureoviride
- tobacco aromatics
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
- C12N1/145—Fungal isolates
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
- C12R2001/885—Trichoderma
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- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
- C11B9/00—Essential oils; Perfumes
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
The invention discloses a trichoderma aureoviride HLMM-04 strain and a method for preparing a tobacco flavor.The strain is trichoderma aureoviride HLMM-04 CGMCC No.10839, and has the capacity of producing cellulase and promoting decomposition of cellulase into oligosaccharide or monosaccharide.The strain is subjected to activation, fermentation, filtering and dialysis and prepared into cellulase crude enzyme liquid which is used for fermenting a mixture of loquat, loquat leaves, lilies, hawthorn fruit and boat-fruited scaphium seeds, and then the mixture is subjected to extraction, filtering and vacuum concentration to be prepared into the tobacco flavor.According to the most appropriate enzyme production conditions of the HLMM-04 strain, the culture medium initial pH is 6.5, culture temperature is 28 DEG C, and culture time is 5 d.A flavoring result shows that the prepared flavor has the effects of reducing irritation and improving the comfort degree of the throat, and has remaining taste.
Description
Technical field
The invention belongs to fermentable and application thereof, be specifically related to a strain trichoderma aureoviride new strains.
Meanwhile, the invention still further relates to utilize this bacterial strain preparation to have the tobacco aromatics using improving throat's comfortableness effect
Method.
Background technology
Flavoring and casing is the key link in cigarette process, is effectively to promote cigarette smoking quality, forms volume
One of effective means of the peculiar style of cigarette.The tobacco aromatics using added in China Nicotiana tabacum L. at present is to use to pass mostly
Gained is allocated in the extraction of system method again, although production operation is simple, but there is also fragrance dullness, improves
The shortcomings such as cigarette smoking DeGrain.Utilize microorganism that perfume material carries out fermentation to increase tradition and carry
Take the content of multiple aroma substance in spice, promote fragrance rich, and then it is abundant, high to produce fragrance
The Medicated cigarette of quality, meets consumer's requirement to cigarette quality.
Trichoderma (Trichoderma spp.) is distributed widely in nature, residual rotten wood, seed, plant
In body, fertilizer, soil and air especially the most.Trichoderma growing nutrient requires simple, fast growth,
Highly beneficial for industrialized production.Trichoderma aureoviride (Trichoderma aureoviride) is trichoderma
One, as a kind of important bio-control factors, has broad spectrum activity to the antagonism of pathogenic, makes more
Property, moreover it is possible to producing multiple enzyme decomposes the plant and animal residues in soil, increases soil fertility.To Huang
The report of green trichoderma focuses mostly at aspects such as soil improvement, Biological control and bio-feritlizers.In prior art
There is not yet and trichoderma aureoviride is used for fermentation prepares the report of tobacco aromatics using.
Summary of the invention
Present invention aims to the deficiencies in the prior art, it is provided that a strain trichoderma aureoviride new strains, should
Bacterial strain has cellulase-producing, and then promotes that cellulose decomposition becomes the ability of oligosaccharide or monosaccharide.
The present invention also aims to provide a kind of method utilizing this strain fermentation to prepare tobacco aromatics using.
The purpose of the present invention is achieved by the following technical programs.
Except as otherwise noted, percent of the present invention is mass percent.
A kind of trichoderma aureoviride (Trichoderma aureoviride) HLMM-04 bacterial strain, CGMCC No.
10839, it has cellulase-producing, and then promotes that cellulose decomposition becomes the ability of oligosaccharide or monosaccharide.
The method utilizing described bacterial strain to prepare tobacco aromatics using, comprises the following steps:
(1), after trichoderma aureoviride HLMM-04CGMCC No.10839 bacterial strain being activated twice, it is inoculated in dress
Have in the triangular flask of 200mL PDA fluid medium, at 20～35 DEG C, 180rpm bottom fermentation cultivate 1～
7d；Described PDA fluid medium: Rhizoma Solani tuber osi 200g, glucose 20g, mend distilled water to 1L,
PH5.5～8.5,115 DEG C of autoclaving 15min；
(2) filtering fermentation liquor removes thalline, takes supernatant and is slowly added to ammonium sulphate to 80% saturation, and 4
Standing overnight at DEG C, centrifugal collecting precipitate is also dissolved in 2mL buffer, then carry out with buffer
Analysis, obtains crude enzyme liquid；Described buffer is: 0.02mol/L Na2HPO4-NaH2PO4, pH 7.3；
(3) Folium Eriobotryae, Folium Eriobotryae, Bulbus Lilii, Fructus Crataegi, Semen Sterculiae Lychnophorae 2:1:1:1:2 in mass ratio is mixed,
Decontamination is cleaned, and adds the water of double weight, mix homogeneously, adjust pH to 6.0 after crushing；Add yellowish green wood
Mould HLMM-04 crude enzyme liquid, fermentation, crude enzyme liquid addition is the 5%～20% of plant material quality, fermentation
Temperature is 30 DEG C, fermentation time 2～5h；Reflux, extract, 3h, filter cleaner, by supernatant concentrating under reduced pressure,
Obtain required tobacco aromatics using.
Further, for preventing spice from going bad, by 5% interpolation propylene glycol of tobacco aromatics using weight.
In step (1), optimum condition of enzyme production is initial pH 6.5, and cultivation temperature 28 DEG C, during cultivation
Between 5d.
Relative to prior art, the invention have the advantages that
(1) Folium Eriobotryae, Folium Eriobotryae, Bulbus Lilii, Fructus Crataegi, Semen Sterculiae Lychnophorae are carried out multiple by the present invention by proper proportion
Joining, exploitation has the tobacco aromatics using improving throat's comfortableness effect, and having minimizing throat stimulates, and promotes the production of body fluid,
The effect of comfortable pleasant impression.
(2) first trichoderma aureoviride is applied to tobacco aromatics using preparation field.Use trichoderma aureoviride
HLMM-04 bacterial strain fermentation liquor processes Folium Eriobotryae, Folium Eriobotryae, Bulbus Lilii, Fructus Crataegi, Semen Sterculiae Lychnophorae compound, with
Non-fermented product extract is compared, and miscellaneous QI has reduced, and fragrance is more mellow and fuller, and the improvement to throat's comfortableness is made
With increase.
(3) present invention is with low cost, simple to operate, it is simple to realize large-scale industrial production, application
Have good prospects.
The explanation of preservation biomaterial
The bacterial strain of the present invention, on May 22nd, 2015, is deposited in Chinese microorganism strain preservation pipe
Reason committee's common micro-organisms center (CGMCG)；This centre address: city of the BeiJing, China Chaoyang District North Star
West Road 1 institute 3, Institute of Microorganism, Academia Sinica.The named trichoderma aureoviride of this strain classification
(Trichoderma aureoviride) HLMM-04, preserving number is CGMCC No.10839.
Detailed description of the invention
Below by embodiment, the present invention is described in further detail, but embodiment is not to this
The restriction of bright technical scheme.
Embodiment 1
1, the suitable condition of enzyme production of trichoderma aureoviride HLMM-04 bacterial strain
The 1.1 initial pH of the culture medium impact on HLMM-04 strain enzyme-producing
The pH of fluid medium is transferred to 5.5,6.0,6.5,7.0,7.5,8.0,8.5 respectively, 28
DEG C, shaken cultivation, after 3 days, measures cellulase activity, shows that optimum initial pH is under 120rpm
6.5。
The impact on HLMM-04 strain enzyme-producing of 1.2 cultivation temperature
Postvaccinal liquid fermentation medium is respectively placed in 20 DEG C, 25 DEG C, 28 DEG C, 32 DEG C, at 35 DEG C,
After 120r/min shaken cultivation 3d, measure cellulase activity, show that optimum cultivation temperature is 28 DEG C.
The impact on HLMM-04 strain enzyme-producing of 1.3 incubation times
The fluid medium (pH 6.5) inoculated is placed in climatic chamber, 28 DEG C, 120r/min
Shaken cultivation, every 24h measures primary fiber element enzyme and lives, and cultivates and reaches to cellulase activity when the 5th day
Big value.
2, trichoderma aureoviride HLMM-04 crude enzyme liquid preparation method
According to above optimum results, after trichoderma aureoviride HLMM-04 bacterial strain is activated twice, it is inoculated in dress
Having in the triangular flask of 200mL PDA fluid medium (pH 6.5), at 28 DEG C, 180rpm issues
Ferment cultivates 5d.Filter and remove thalline.Take supernatant and be slowly added to ammonium sulphate to 80% saturation, at 4 DEG C
Standing overnight, centrifugal collecting precipitate is also dissolved in 2mL buffer (0.02mol/L
Na2HPO4-NaH2PO4Buffer, pH 7.3) in, dialyse with same buffer, obtain
Crude enzyme liquid.
3, the method that trichoderma aureoviride HLMM-04 strain fermentation prepares tobacco aromatics using
Folium Eriobotryae, Folium Eriobotryae, Bulbus Lilii, Fructus Crataegi, Semen Sterculiae Lychnophorae 2:1:1:1:2 in mass ratio is mixed, decontamination
Clean, add the water of double weight, mix homogeneously after crushing, adjust pH to 6.0.Add trichoderma aureoviride
HLMM-04 crude enzyme liquid, fermentation.Crude enzyme liquid addition be vegetable material (Folium Eriobotryae, Folium Eriobotryae, Bulbus Lilii,
Fructus Crataegi, Semen Sterculiae Lychnophorae mixture) quality 15%, fermentation temperature is 30 DEG C, fermentation time 3h.Backflow
Extract 3h, filter cleaner, by supernatant concentrating under reduced pressure, obtain fermentation tobacco aromatics using.
For preventing from going bad, by 5% interpolation propylene glycol of fermentation spice weight.
4, product feature and physicochemical property
Gained fermentation tobacco aromatics using is rufous stream shape mastic, does not clarifies.Product physicochemical property is shown in Table 1.
The physical and chemical index of table 1 trichoderma aureoviride HLMM-04 fermentation spice
5, perfuming cigarette experiment
Take fermentation tobacco aromatics using 0.1g, after taking sterilized water dilution 50 times, be uniformly sprayed at the non-perfuming of 50g
Ye Zuzhong, loads in clean sealing bag, after placing 2h at room temperature, by tobacco shred in 120 DEG C of baking ovens
Dry, make moisture reach 11%-13%.Tobacco shred and comparison after processing are put in climatic chamber,
Balancing 24h under the conditions of temperature 22 DEG C, humidity 60%, roll into Medicated cigarette by hand, by smokeing panel test, expert forms
The group of smokeing panel test carry out sensory evaluating smoking.With the mixed extraction spice that do not ferments for comparison.Perfuming result shows,
Trichoderma aureoviride HLMM-04 fermentation spice is obvious with control area, and miscellaneous QI reduces, and throat's comfortableness increases,
Pleasant impression improves.Evaluation result is shown in Table 2.
Table 2 ferments spice smoking result
Embodiment 2
Repeat embodiment 1, have following difference: crude enzyme liquid addition is the 5% of Yunnan Fructus Canarii albi quality, send out
Ferment time 2h.
1, product feature and physicochemical property
Gained fermentation tobacco aromatics using is rufous stream shape mastic, does not clarifies.Product physicochemical property is shown in Table 3.
The physical and chemical index of table 3 trichoderma aureoviride HLMM-04 fermentation spice
2, perfuming cigarette experiment
Perfuming result shows, trichoderma aureoviride HLMM-04 fermentation spice is obvious with control area, and fragrance is rich
Richness increases, and throat's comfortableness increases, and pleasant impression improves.Evaluation result is shown in Table 4.
Table 4 ferments spice smoking result
Embodiment 3
Repeat embodiment 1, have following difference: crude enzyme liquid addition is the 20% of Yunnan Fructus Canarii albi quality, send out
Ferment time 5h.
1, product feature and physicochemical property
Gained fermentation tobacco aromatics using is rufous stream shape mastic, does not clarifies.Product physicochemical property is shown in Table 5.
The physical and chemical index of table 5 trichoderma aureoviride HLMM-04 fermentation spice
2, perfuming cigarette experiment
Perfuming result shows, trichoderma aureoviride HLMM-04 fermentation spice is obvious with control area, and flue gas is assisted
Adjusting, throat's comfortableness increases, and stimulates and reduces, and pleasant impression improves.Evaluation result is shown in Table 6.
Table 6 ferments spice smoking result
Claims (4)
1. trichoderma aureoviride (Trichoderma aureoviride) HLMM-04 bacterial strain, CGMCC
No.10839, it has cellulase-producing, and then promotes that cellulose decomposition becomes the ability of oligosaccharide or monosaccharide.
2. the method utilizing bacterial strain described in claim 1 to prepare tobacco aromatics using, comprises the following steps:
(1), after trichoderma aureoviride HLMM-04 CGMCC No.10839 bacterial strain being activated twice, it is inoculated in dress
Have in the triangular flask of 200mL PDA fluid medium, at 20～35 DEG C, 180rpm bottom fermentation cultivate 1～
7d；Described PDA fluid medium: Rhizoma Solani tuber osi 200g, glucose 20g, mend distilled water to 1L,
PH5.5～8.5,115 DEG C of autoclaving 15min；
(2) filtering fermentation liquor removes thalline, takes supernatant and is slowly added to ammonium sulphate to 80% saturation, and 4
Standing overnight at DEG C, centrifugal collecting precipitate is also dissolved in 2mL buffer, then carry out with buffer
Analysis, obtains crude enzyme liquid；Described buffer is: 0.02mol/L Na2HPO4-NaH2PO4, pH 7.3；
(3) Folium Eriobotryae, Folium Eriobotryae, Bulbus Lilii, Fructus Crataegi, Semen Sterculiae Lychnophorae 2:1:1:1:2 in mass ratio is mixed,
Decontamination is cleaned, and adds the water of double weight, mix homogeneously, adjust pH to 6.0 after crushing；Add yellowish green wood
Mould HLMM-04 crude enzyme liquid, fermentation, crude enzyme liquid addition is the 5%～20% of plant material quality, fermentation
Temperature is 30 DEG C, fermentation time 2～5h；Reflux, extract, 3h, filter cleaner, by supernatant concentrating under reduced pressure,
Obtain required tobacco aromatics using.
The method preparing tobacco aromatics using the most according to claim 2, it is characterised in that: for preventing perfume (or spice)
Material is rotten, by 5% interpolation propylene glycol of tobacco aromatics using weight.
The method preparing tobacco aromatics using the most according to claim 2, it is characterised in that: step (1)
In optimum fermentation culture conditions be initial pH 6.5, cultivation temperature 28 DEG C, incubation time 5d.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108753839A (en) * | 2018-05-25 | 2018-11-06 | 颐中（青岛）实业有限公司 | A kind of preparation method for the filter material of cigarette that there is drop evil to protect lung function |
| CN109097404A (en) * | 2018-08-23 | 2018-12-28 | 江西中烟工业有限责任公司 | A kind of preparation and its application of the dry composite fermentation extract of loquat |
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Cited By (2)
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| CN108753839A (en) * | 2018-05-25 | 2018-11-06 | 颐中（青岛）实业有限公司 | A kind of preparation method for the filter material of cigarette that there is drop evil to protect lung function |
| CN109097404A (en) * | 2018-08-23 | 2018-12-28 | 江西中烟工业有限责任公司 | A kind of preparation and its application of the dry composite fermentation extract of loquat |
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| CN105733962B (en) | 2019-05-17 |
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