CN104232492A - Tobacco-derived cellulase-producing microbe strain and application thereof - Google Patents
Tobacco-derived cellulase-producing microbe strain and application thereof Download PDFInfo
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- CN104232492A CN104232492A CN201410434948.8A CN201410434948A CN104232492A CN 104232492 A CN104232492 A CN 104232492A CN 201410434948 A CN201410434948 A CN 201410434948A CN 104232492 A CN104232492 A CN 104232492A
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Abstract
The invention relates to a tobacco-derived cellulase-producing microbe strain and application thereof in tobacco. The invention is characterized in that the microbe strain is a Trichoderma harzianum TC10-13 strain which is collected by China General Microbiological Culture Collection Center on December 17th, 2013, and the collection number is CGMCC No.8587. The ITS sequence of the strain is the main characteristic reference for identifying the strain. The application method comprises the following steps: by using wheat straw powder or cabo powder as a carbon source and a yeast extract as a nitrogen source, carrying out liquid fermentation to obtain a crude enzyme solution, and proportionally spraying the fermentation crude enzyme solution onto the tobacco shred, sheet tobacco and cabo to obviously lower the cellulose content in tobacco. The cellulose degradation strain is convenient to apply and can enhance the inherent quality of the tobacco.
Description
Technical field
The present invention relates to and a kind ofly derive from the cellulase-producing microorganism strains of tobacco and the application in tobacco thereof, belong to fermentable and Application Areas thereof.
Background technology
Mierocrystalline cellulose is a kind of polysaccharide, and its molecular formula is (C
6h
10o
5) n.In tobacco, cell wall substance is made up of materials such as Mierocrystalline cellulose, hemicellulose, xylogen, these materials play a part to maintain tobacco cell wall construction, account for 26% ~ 35% of tobacco dry matter weight, wherein, 13% ~ 18%(Yan Ke that Mierocrystalline cellulose accounts for dry-matter gross weight is beautiful, Yan Hongyang, Li Xingbo, Deng the comparative analysis of .2005. Cell Wall Substances in Flue-cured Tobacco Leaf. tobacco science and technology, 2005(10): 6-11).
The incendivity of Mierocrystalline cellulose to tobacco leaf has certain promoter action, makes tobacco leaf hold firepower and strengthens.But its too high levels is then unfavorable for the lifting of cigarette quality, content of cellulose and tobacco aroma matter, perfume quantity, flue gas concentration extremely significantly negative correlation, with pleasant impression, mix and be so incensed that a point negative correlation, the tobacco leaf pungency that content of cellulose is high is large, assorted gas weight, and fragrance can not appear (Yan Keyu, Wang Jianmin, Qu Jianbo, etc. the correlation analysis [J] of 2001. Henan flue cured tobacco judging parameter and main physical and chemical index. tobacco science and technology, (10): 5-9).Existing research is thought, cell walls class material can produce irritating heat-decomposing compound, Mierocrystalline cellulose is the important as precursors thing of catechol in flue gas, be 7% ~ 12%(Carmella, S.G., S.S. Hecht to the minimum contribution degree of catechol in main flume, Tc Tso, D. Hoffmann. 1984. Roles of tobacco cellulose, sugars, and chlorogenic acid as precursors to catechol in cigarette smoke [J]
.journal of Agricultural and Food Chemistry,
32(2): 267-273); The materials such as Mierocrystalline cellulose are also the precursor substance (Mcgrath generating PAHs under tobacco cold condition simultaneously, T.E., W.G. Chan, M.R. Hajaligol. 2003. Low temperature mechanism for the formation of polycyclic aromatic hydrocarbons from the pyrolysis of cellulose [J]
.journal of analytical and applied pyrolysis,
66(1-2): 51-70).Reducing cellulosic content in tobacco and not only can improve sucking quality, also having certain promoter action to sucking security.In degrading tobacco, content of cellulose mainly takes two kinds of methods at present: enzymolysis process and microbial method.Disclose in Chinese patent literature CN101912149A and a kind ofly apply steam explosion and combine with enzyme treatment technique and improve the method for tobacco stalk quality, wetting offal is first carried out steam explosion by this invention, add lignoenzyme and cellulase wherein again, reduce Mierocrystalline cellulose to the impact of offal cigarette quality, but the method cost is higher, and there is certain requirement to equipment.
Nature have numerous can the microorganism of degraded cellulose, comprise fungi, bacterium and actinomycetes, wherein, Trichoderma (
trichoderma), Penicillium (
penicillium) be to the stronger two kinds of fungies of Mierocrystalline cellulose effect (Liu Shuxia, Wu Haiyan, Wu Haiwen, etc. 2008. black earth corn field soil cellulose decomposition fungal species qualifications and capacity of decomposition measure [J]. Maize Sciences, 16(3): 112-116).From tobacco, be separated the microorganism strains obtained have good adaptability to tobacco, its cellulase system composition also more easily utilizes tobacco natural cellulose.Show according to another research, trichoderma harziarum can by the cell walls of the material decomposition pathogenic bacterias such as eccrine fiber element enzyme, good preventive effect (Li Meiyun is had to Phytophthora nicotianae etc., Tan Lihua, Fang Dunhuang, Deng. the cultivation of 2006. trichoderma harziarums and suppression research [J] to Phytophthora nicotianae growth thereof. microbiology is circulated a notice of, 33(6): 79-83), there is good application prospect.At present, the application of this microorganism strains in tobacco there is not yet open report.
Summary of the invention
Object of the present invention provides a kind of cellulase-producing microorganism and the application method in tobacco thereof based on above-mentioned prior art situation just.
The object of the invention is to be achieved through the following technical solutions:
Cellulase producing strain trichoderma harziarum (
trichoderma harzianum) TC10-13 system applicant is separated from tobacco, the strain identified has eccrine fiber element enzyme ability fungal bacterial strain, this bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on December 17th, 2013, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, preserving number is: CGMCC No.8587.
With cellulase producing strain trichoderma harziarum (
trichoderma harzianum) TC10-13 fermentation liquor treatment pipe tobacco, sheet cigarette or stem, significantly can reduce content of cellulose, promote interior quality.
The present invention screens the function stem with degraded cellulose ability from tobacco, derives from tobacco and is applied to tobacco, and tobacco has better adaptability, significantly can reduce content of cellulose, and promotes tobacco interior quality.
Trichoderma harziarum (
trichoderma harzianum) TC10-13 bacterial strain grows rapidly, expand radially on PDA substratum, cultivate 3d and be namely paved with flat board, see Fig. 1, white mycelium fine hair shape, closely, edge presents light green, conidium oval.TC10-13 bacterial strain spore microscopic morphology is shown in Fig. 2.The ITS sequence of this bacterial strain is the principal character foundation of this bacterial strain of qualification, and the TC10-13 bacterial strain phylogenetic tree built according to ITS sequence is shown in Fig. 3.
The present invention utilizes trichoderma harziarum TC10-13 bacterial strain through liquid fermenting gained crude enzyme liquid process pipe tobacco, sheet cigarette or stem, thus degrading tobacco content of cellulose, promote cigarette quality,
Main contents of the present invention comprise:
Liquid fermenting: bacterial strain spore suspension is seeded in best culture medium, carries out liquid culture.
Extract crude enzyme liquid: utilize whizzer that fermented liquid is centrifugal, extract supernatant liquor as crude enzyme liquid.
Spray: crude enzyme liquid is evenly sprayed in certain proportion on pipe tobacco, sheet cigarette or stem, can significantly reduce baccy fiber cellulose content, promote cigarette quality.
Concrete culturing process is as follows:
Trichoderma harzianum strain TC10-13 is added sterilized water to make spore suspension (spore concentration is 10
5~ 10
6individual mL), the ratio with 1% ~ 15% is seeded in the 250 mL triangular flasks that 20 mL ~ 80 mL culture mediums are housed carries out liquid fermentation and culture.Culture temperature 20 DEG C ~ 33 DEG C, shaking speed 150 rpm ~ 250 rpm, fermentation time 48 h ~ 144 h.
The enzymatic production substratum that this bacterial strain is suitable for is composed as follows: contain in every 1000 mL distilled water: wheat stalk 5.0 g ~ 20.0 g, yeast extract 0.5 g ~ 2.5 g,
,mgSO
47H
2o 0.1 g ~ 0.3 g, Tween-80 1 mL ~ 8 mL.
Extract supernatant liquor as crude enzyme liquid, with the atomizer of band vacuum pump, crude enzyme liquid is atomized, evenly spray on pipe tobacco, sheet cigarette or stem, crude enzyme liquid addition is 5% ~ 20%(V/W), temperature of reaction 30 DEG C ~ 45 DEG C, reaction times 4h ~ 18 h, processes 10 min ~ 30 min after reaction terminates, cellulase is lost activity at 80 DEG C ~ 85 DEG C.Carry out content of cellulose and routine chemical components to the sample after process to measure, result show trichoderma harziarum (
trichoderma harzianum) TC10-13 bacterial strain crude enzyme liquid can significantly reduce baccy fiber cellulose content, promotes interior quality.
In tobacco, content of cellulose measuring method is as follows:
Tobacco sample is dried and is clayed into power, cross 40 mesh sieves, accurately take 0.25 g offal and be placed in 100 mL Erlenmeyer flasks, add 25 mL 5% acetums, concussion extraction 30 min, centrifugal, abandoning supernatant, adds distilled water centrifugal 2 ~ 3 times, is transferred to by filter residue in 100 mL Erlenmeyer flasks, add 0.25 g cellulase, add the citrate buffer solution of 60 mL pH4.7 again, shake 24 h at 37 DEG C, then sample is cooled to room temperature, filter and be settled to 100 mL, utilizing the glucose content in Flow Analyzer mensuration solution.
Content of cellulose (mg/g)=
W=0.25
（1-ω）
Sample dry weight (g)=0.25
(1-sample water ratio (%))
x:glucose content, unit is mg/mL.
V: sample volume is 100 mL.
W: tobacco sample dry biomass, unit is g.
ω: tobacco sample water ratio, unit is %.
Accompanying drawing explanation
Fig. 1 is trichoderma harzianum strain TC10-13 colonial morphology
Fig. 2 be trichoderma harzianum strain TC10-13 spore microscopic morphology observe (
);
Fig. 3 is trichoderma harzianum strain TC10-13 phylogenetic tree.
Embodiment
The present invention is described further below in conjunction with specific examples,
Case study on implementation 1
Trichoderma harzianum strain TC10-13 is added sterilized water to make spore suspension (spore concentration is 10
6individual/mL),
Ratio with 7% is seeded in the 250 mL triangular flasks that 50 mL culture mediums are housed carries out fermentation culture.Culture temperature 28 DEG C, shaking speed 200 rpm, incubation time 96 h.Through test determination, gained crude enzyme liquid cellulase activity is 37.83 U/mL.
Culture medium consists of (g/L): wheat stalk powder 15.0, yeast extract 4.0, MgSO
47H
2o 0.3 g, Tween-80 2.0 mL, initial pH 5.0.
Test tobacco leaf is from Yunnan mountain of papers, and grade is C3F, adopts filament cutter to be prepared into pipe tobacco, by moisture in cut tobacco regulation and control to 12% ~ 13%.
Crude enzyme liquid respectively with 5% and 15%(V/W) ratio uniform spray on pipe tobacco, with the pipe tobacco not spraying crude enzyme liquid and spray deactivation crude enzyme liquid pipe tobacco in contrast.After mixing, be put in 40 DEG C of baking ovens and react 10 h, process 30 min under reaction end is placed on 80 DEG C of conditions, make cellulase inactivation.Sample is dried, respectively working sample water ratio, content of cellulose and chemical composition.
Sample chemical composition the results are shown in Table 1, and content of cellulose the results are shown in Table 2.
As can be seen from Table 1, after crude enzyme liquid process, pipe tobacco total sugar content, reducing sugar content, potassium content, sugared alkali increase than all to some extent, and total alkaloid content, total nitrogen content, pH value and nitrogen base are more not remarkable than change.Potassium is one of important indicator of flue-cured tobacco, and after process, the raising of potassium content improves cigarette quality to a certain extent.
Table 1 is Physicochemical composition result everywhere
| ? | Total alkaloid (%) | Total reducing sugar (%) | Reducing sugar (%) | Total nitrogen (%) | Potassium (%) | PH value | Sugar alkali ratio | Nitrogen base ratio |
| CK | 3.33 | 22.31 | 20.37 | 2.51 | 2.03 | 4.95 | 6.12 | 0.75 |
| 5% deactivation | 3.39 | 23.19 | 21.25 | 2.41 | 2.27 | 4.89 | 6.27 | 0.71 |
| 5% | 3.34 | 22.81 | 20.68 | 2.48 | 2.23 | 4.91 | 6.19 | 0.74 |
| 15% deactivation | 3.44 | 23.57 | 21.53 | 2.47 | 2.46 | 4.86 | 6.26 | 0.72 |
| 15% | 3.41 | 24.96 | 22.40 | 2.52 | 2.22 | 4.90 | 6.57 | 0.74 |
As can be seen from Table 2, after crude enzyme liquid process, content of cellulose has decline in various degree.The pipe tobacco content of cellulose of 5% crude enzyme liquid process declines 12.60%, the pipe tobacco content of cellulose of 15% crude enzyme liquid process declines 16.53%, content of cellulose reduces along with the increase of crude enzyme liquid addition, and visible, bacterial strain trichoderma harziarum TC10-13 has good degradation effect to content of cellulose.
Table 2 is process content of cellulose measurement result respectively
| ? | CK | 5% addition | 15% addition |
| Content of cellulose (mg/g) | 176.0 | 153.8 | 146.9 |
Claims (7)
1. derive from a cellulase-producing microorganism strains for tobacco, it is characterized in that: described cellulase-producing microorganism strains be trichoderma harziarum (
trichoderma harzianum) TC10-13 bacterial strain, this bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on December 17th, 2013, and preserving number is: CGMCC No.8587.
2. cellulase-producing microorganism strains according to claim 1, it is characterized in that: this bacterial strain grows rapidly on PDA substratum, expand radially, cultivate 3 d and be namely paved with flat board, white mycelium fine hair shape, closely, edge presents light green, conidium oval is the principal character foundation of this bacterial strain of qualification by the ITS sequence of this bacterial strain of specific amplification gained.
3. utilize the cellulase-producing microorganism strains described in claim 1 to prepare a method for Mierocrystalline cellulose crude enzyme liquid, it is characterized in that: trichoderma harzianum strain TC10-13 being added sterilized water, to make spore concentration be 10
5~ 10
6the spore suspension of individual mL, ratio with 1% ~ 15% is seeded in the 250 mL triangular flasks that 20 mL ~ 80 mL culture mediums are housed carries out liquid fermentation and culture, culture temperature 20 DEG C ~ 33 DEG C, shaking speed 150 rpm ~ 250 rpm, fermentation time 48 h ~ 144 h, utilize whizzer that fermented liquid is centrifugal, extract supernatant liquor as crude enzyme liquid.
4. cellulase-producing microorganism strains according to claim 3 prepares the method for Mierocrystalline cellulose crude enzyme liquid, it is characterized in that: described culture medium composition is as follows: contain in every 1000 mL distilled water: wheat stalk 5.0 g ~ 20.0 g, yeast extract 0.5 g ~ 2.5 g, MgSO
47H
2o 0.1 g ~ 0.3 g, Tween-80 1mL ~ 8 mL, pH4.5 ~ 6.0.
5. the application of a cellulase-producing trichoderma harziarum TC10-13 bacterial strain according to claim 1 in tobacco, it is characterized in that: by the crude enzyme liquid process tobacco of TC10-13 bacterial strain through liquid fermenting gained, can obviously reduce content of cellulose in tobacco, after process, tobacco interior quality gets a promotion.
6. the application of cellulase-producing trichoderma harziarum TC10-13 bacterial strain according to claim 5 in tobacco, is characterized in that: described tobacco is pipe tobacco, sheet cigarette or stem.
7. the application of cellulase-producing trichoderma harziarum TC10-13 bacterial strain in tobacco according to claim 5 or 6, it is characterized in that: with trichoderma harzianum strain TC10-13 when liquid fermenting gained crude enzyme liquid process tobacco, crude enzyme liquid addition is 5% ~ 20%(V/W), temperature of reaction 30 DEG C ~ 45 DEG C, reaction times 4 h ~ 18 h, at 80 DEG C ~ 85 DEG C, process 10 min ~ 30 min after reaction terminates, cellulase is lost activity.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104911110A (en) * | 2015-05-31 | 2015-09-16 | 中国烟草总公司郑州烟草研究院 | Functional bacterial strain capable of degrading cellulose and application of functional bacterial strain in tobacco |
| CN106754494A (en) * | 2016-12-09 | 2017-05-31 | 湖北工业大学 | A kind of cellulase-producing microbial strains and its application in tobacco |
| CN114410483A (en) * | 2022-01-27 | 2022-04-29 | 山东农业大学 | Trichoderma harzianum and application thereof in degradation of waste orchard branches |
| CN115851545A (en) * | 2022-12-21 | 2023-03-28 | 福建中烟工业有限责任公司 | Korean bacillus and culture medium for improving activity of enzyme produced by same |
-
2014
- 2014-08-30 CN CN201410434948.8A patent/CN104232492A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104911110A (en) * | 2015-05-31 | 2015-09-16 | 中国烟草总公司郑州烟草研究院 | Functional bacterial strain capable of degrading cellulose and application of functional bacterial strain in tobacco |
| CN104911110B (en) * | 2015-05-31 | 2018-07-24 | 中国烟草总公司郑州烟草研究院 | It is a kind of can degraded cellulose function stem and its application in tobacco |
| CN106754494A (en) * | 2016-12-09 | 2017-05-31 | 湖北工业大学 | A kind of cellulase-producing microbial strains and its application in tobacco |
| CN106754494B (en) * | 2016-12-09 | 2019-10-18 | 湖北工业大学 | A kind of cellulase-producing microbial strains and its application in tobacco |
| CN114410483A (en) * | 2022-01-27 | 2022-04-29 | 山东农业大学 | Trichoderma harzianum and application thereof in degradation of waste orchard branches |
| CN115851545A (en) * | 2022-12-21 | 2023-03-28 | 福建中烟工业有限责任公司 | Korean bacillus and culture medium for improving activity of enzyme produced by same |
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Application publication date: 20141224 |