CN106834131A - Fungi and its application of a kind of degrading tobacco stem pectin and xylan - Google Patents
Fungi and its application of a kind of degrading tobacco stem pectin and xylan Download PDFInfo
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- CN106834131A CN106834131A CN201611052682.6A CN201611052682A CN106834131A CN 106834131 A CN106834131 A CN 106834131A CN 201611052682 A CN201611052682 A CN 201611052682A CN 106834131 A CN106834131 A CN 106834131A
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- pectin
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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
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- A—HUMAN NECESSITIES
- A24—TOBACCO; CIGARS; CIGARETTES; SIMULATED SMOKING DEVICES; SMOKERS' REQUISITES
- A24B—MANUFACTURE OR PREPARATION OF TOBACCO FOR SMOKING OR CHEWING; TOBACCO; SNUFF
- A24B15/00—Chemical features or treatment of tobacco; Tobacco substitutes, e.g. in liquid form
- A24B15/18—Treatment of tobacco products or tobacco substitutes
- A24B15/20—Biochemical treatment
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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
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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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2477—Hemicellulases not provided in a preceding group
- C12N9/248—Xylanases
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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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/88—Lyases (4.)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y402/00—Carbon-oxygen lyases (4.2)
- C12Y402/02—Carbon-oxygen lyases (4.2) acting on polysaccharides (4.2.2)
- C12Y402/02002—Pectate lyase (4.2.2.2)
Abstract
The invention belongs to microbial strains technical field, and in particular to the fungi of a kind of degrading tobacco stem pectin and xylan, and its application in Field of Tobacco is further disclosed.The fungi of degrading tobacco stem pectin of the present invention and xylan, its Classification And Nomenclature is coronoid process dissipate capsule bacterium Eurotium cristatum 502, has been preserved in Wuhan University's China typical culture collection center, and deposit number is CCTCC M2016204.Bacterial strain of the present invention can secrete various biology enzymes such as zytase, pectase and amylase, the materials such as the xylan and pectin that can be degraded in stem, xylan etc. is converted into monose, so as to reduce the xylon gas of stem, the quality and availability of stem can be significantly improved.
Description
Technical field
The invention belongs to microbial strains technical field, and in particular to a kind of degrading tobacco stem pectin and xylan it is true
Bacterium, and its application in Field of Tobacco is further disclosed.
Background technology
Tobacco is Solanaceae Nicotiana plant, and offal is the thick and stiff vein of tobacco leaf, accounts for leaf weight 25%-30%.Offal is passed through
The filament of the one fixed width formulated after chopping operation, the processing for cigarette is used.
It is well known that stem plays an important role in cigarette composition, it is one of Main Means of lowering harm and decreasing coking：One side
Face, stem can reduce the content of the harmful components such as coke tar in cigarette, nicotine and CO, so as to reduce the harm of cigarette；On the other hand, obstruct
Silk can improve the Filling power of pipe tobacco, single case consumption silk amount of cigarette be reduced, so as to reduce production cost.But, due in stem
Content is more and fragrance matter content is little for cell wall substance (pectin, cellulose, hemicellulose and lignin etc.) so that sense organ
Quality haves the shortcomings that big excitant, miscellaneous gas weight, aroma quality are poor, and be there is also with cut tobacco in terms of form and color and luster compared with
Big difference.Research shows that the main component of tobacco stem shred cell membrane is pectin and hemicellulose, and pectin can make stem burned
Xylon gas are produced in journey, the quality of stem is had a strong impact on；And xylan is the main component of hemicellulose in plant cell, plant is accounted for
The 35% of thing dry cell weight, be in nature in addition to cellulose the most abundant polysaccharide of content, xylan causes stem in burning
During produce xylon gas, have a strong impact on the quality of stem.Therefore, stem is more used in Low-end Cigarettes, in top grade
Do not used in cigarette or consumption is little, the mixture proportion in cigarette also than relatively low, causes there are a large amount of offals to discard every year.
Offal as tobacco material one kind, be the accessory substance of tobacco industry, be that current tobacco productive corporation cannot be complete
The low-quality raw materials of digestion at present to make Disposal more.Used as tobacco leaf production big country, China is even more and there are about few hundred thousand tonnes of offal every year
Discarded object is thrown aside waste, both causes environmental pollution, while being also the waste to natural resources.But meanwhile, offal is again a kind of
Include the natural resources of various effective natural components rich in various chemical compositions, there are some researches show the composition contained in offal
Species is basically identical with tobacco leaf ingredient, more have research can be with extraction nicotine, solanesol, nicotinic acid, pectin, vegetable protein from offal
Etc. active ingredient and it is used, also becomes the important channel that comprehensive tobacco stems are utilized.
In China, offal is currently used primarily in and is prepared into stem and reconstituted tobacoo, then again mixes different substances together stem and thin slice
To Ye Zuzhong, for the production of cigarette.But with the continuous propulsion that lowering harm and decreasing coking works, the requirement to tobacco stem shred quality is got over
Come higher, therefore, for the utilization of stem, the quality for how improving stem has great importance.
In recent years, in order to improve the quality of stem, many scientific and technical personnel are from technology, chemical method and biological method
Stem is studied etc. different angles.Wherein, biological method is because reaction condition is gentle, it is low to react single-minded and cost
Feature and receive much concern, as improve stem quality research important research direction.The offal that can effectively degrade is screened from nature
The microorganism of cell wall substance, the ferment treatment offal produced using microorganism, macromolecular substances therein of degrading can be in certain journey
Offal mouthfeel is improved on degree, with broad prospect of application.
The content of the invention
Therefore, the technical problems to be solved by the invention are to provide a kind of eurotium cristatum strain, to solve existing skill
Tobacco stem shred second-rate problem in art.
In order to solve the above technical problems, the fungi of degrading tobacco stem pectin of the present invention and xylan, its classification
Coronoid process dissipate capsule bacterium Eurotium cristatum 502 are named as, have been preserved in Wuhan University's China typical culture collection
The heart, deposit number is CCTCC M2016204.
The invention also discloses a kind of method for cultivating the fungi, it includes being suitable for producing by the coronoid process dissipate capsule bacterium
Bacterial strain seed culture is carried out under conditions of raw ascospore, and in the case where the condition of culture of pectase and zytase expression is suitable for
The step of carrying out fermented and cultured.
The pectase and the condition of culture of zytase expression of being suitable for includes：In the culture containing 40-60g/L stems
In base, and 28-32 DEG C of the temperature is controlled to carry out fermented and cultured.
The invention also discloses the enzyme encoded by the fungi of the degrading tobacco stem pectin and xylan, the enzyme includes
Pectase and zytase.
The invention also discloses a kind of method for preparing the enzyme, including according to coronoid process dissipate capsule bacterium described in methods described culture
The step of, and after gained zymotic fluid is filtered into thalline, gained filtrate is crude enzyme liquid.
It is used to improve the purposes of tobacco stem shred quality the invention also discloses described enzyme.
Fungi the invention also discloses described degrading tobacco stem pectin and xylan is used to improve tobacco stems silk quality
The purposes of amount.
The invention also discloses a kind of method for improving tobacco stem shred quality, comprise the following steps：
(1) described coronoid process dissipate capsule bacterium Eurotium cristatum 502 are carried out into fermented and cultured according to methods described,
And obtain crude enzyme liquid；
(2) crude enzyme liquid for accounting for tobacco stem shred weight 0.01-0.5wt% is taken, the tobacco is uniformly sprayed onto after dilution
In stem, the water content of the stem is controlled to reach 30-50%, and fermented at a temperature of 30-40 DEG C, you can.
In the step (2), the dilution step of the crude enzyme liquid be using sterile distilled water or 0.1-0.2mol/L,
The citric acid-sodium citrate buffer solution of pH5.0-5.5 is diluted.
The invention also discloses a kind of Cigarette processing method, including the tobacco stem shred quality is improved according to methods described
Step.
Scheme of the present invention filters out one plant of advantage eurotium cristatum strain, the bacterium from the six fort tea that is produced from Guangxi Wuzhou
Strain can secrete various biology enzymes such as zytase, pectase and amylase, xylan and pectin in the stem that can degrade etc.
Material, makes xylan etc. be converted into monose, so as to reduce the xylon gas of stem.
Improve the quality of tobacco stem shred raw material using the dominant strain for being screened of the invention, the technical process is simple,
Reaction condition is gentle；After treatment, the content of pectin reduces 8.2-50.3% after stem fermentation, and xylan contains in stem
Amount reduces 2.2-11.5%, and excitant is significantly reduced, and flue gas becomes alcohol and cigarette perfume increases, and significantly improves the sense organ matter of stem
Amount and availability, further increase the availability of stem.Meanwhile, the features such as the method has simple and practical, with low cost, can
Hope industrially popularization and application.
Specific embodiment
The bacterial strain screening of embodiment 1
The eurotium cristatum strain that pectase and zytase can be produced of the present invention, is six forts produced from Guangxi Wuzhou
It is isolated in tea, specifically include following steps：
(1) enriched medium separate microorganism bacterial strain from six fort tea is utilized, by the broken rear addition enrichment culture of six fort tea powders
Base culture is screened, and the enriched medium is：1g K2HPO4·3H2O, 0.5g MgSO4·7H2O, 3g NH4NO3, 32g
NaCl, 20g sucrose, water 1000mL, pH value 6.0；
(2) gained obtain microbial inoculum screened in plate isolation base through flat rubbing method, it is incubated after,
Select the larger bacterium colony of morphological differences to rule respectively purifying, until there is single bacterium colony, obtain bacterial strain, keep, it is standby；The flat board
Isolation medium is：1g K2HPO4·3H2O, 0.5g MgSO4·7H2O, 3g NH4NO3, 32g NaCl, 20g sucrose, 15g fine jades
Cosmetics, water 1000mL, pH value 6.0；
(3) the bacterial strain secondary screening Screening of Media that line is obtained can be produced the bacterial strain of pectase and zytase, institute
Stating secondary screening culture medium is：50g stems, 1000mL water, pH value 6.0, screening obtains one plant of performance preferably bacterial strain, is designated as coronoid process and dissipates
Capsule bacterium Eurotium cristatum 502, are deposited in China typical culture collection center on April 18th, 2016, its letter
Referred to as CCTCC, deposit number is CCTCC NO：M2016204.Preservation address：Luojiashan, Wuchang, Wuhan City, Hubei Province, postal service is compiled
Code 430072.
Coronoid process dissipate capsule bacterium Eurotium cristatum 502 obtained above are carried out into genetic stability investigation：By each
Bacterial strain was continuously transferred for 10 generations, and per generation accesses the fermentation medium carries out cultivation and fermentation (50g/L stems), and triangular flask is with eight layers
Gauze wrapped, condition of culture is 28 DEG C of fermentation temperature, and speed of agitator 150rpm is small in rotary shaker constant temperature shake flask fermentation 48
When, bacterial strain shows the fermenting property of stabilization.It can be seen that, the heritability of the bacterial strain is preferable.
The culture of the bacterial strain of embodiment 2
The incubation step of the bacterial strains of coronoid process dissipate capsule bacterium Eurotium cristatum 502 is described in the present embodiment：
(1) seed culture：Take 1g K2HPO4·3H2O, 0.5g MgSO4·7H2O, 3g NH4NO3, 32g NaCl, 20g sugarcanes
Sugar, 15g agar powders, the 1000mL that adds water prepares culture medium, and the strain of freezing is inoculated into training by pH value 6.0 in gnotobasis
Support on base, cultivated 7 days at 28 DEG C；
(2) Shaking culture：5g stems, plus distilled water 100mL preparation fermentation mediums, pH value 6.0 are taken, high steam goes out
Bacterium；Shaking flask liquid amount is 10-30v/v%, and shaking speed is 150rpm, is taken from seed culture flat board in gnotobasis oese
One ring spore, is accessed in fermentation medium, and 48h is cultivated at 28 DEG C, you can.
It is prepared by the crude enzyme liquid of embodiment 3
Obtained zymotic fluid in Example 2, bacterial strain is filtered with 8 layers of sterile gauze, obtains filtrate as crude enzyme liquid.
Gained crude enzyme liquid fragrant odour, detects, pectinase activity reaches 460U/mL, xylan through DNS (3,5- dinitrosalicylic acid) method
Enzyme activity reaches 328U/mL.
Embodiment 4
15g stems are weighed, the μ g of crude enzyme liquid 1.5 prepared by another Example 3 use 3.9mL distilled water dilutings, load micro spray
Day with fog spray is uniformly sprayed onto on stem, the water content of stem is reached 30%, and stem then is put into 30 DEG C of incubator fermentations
24h。
Through detection after fermentation, the content of pectin reduces 6.2% in stem, and the content of xylan is reduced in stem
3.6%.The xylon gas of stem are reduced, excitant reduction, and flue gas becomes alcohol and cigarette perfume increases, and aesthetic quality is obviously improved.
Embodiment 5
15g stems are weighed, the μ g of crude enzyme liquid 7.5 prepared by another Example 3 use 7mL distilled water dilutings, load ulv spraying
Device spray is uniformly sprayed onto on stem, the water content of stem is reached 40%, and stem then is put into 30 DEG C of incubator fermentation 36h.
By detection after fermentation, the content of pectin reduces 12.3% in stem, and the content of xylan is reduced in stem
5.4%.The xylon gas of stem are reduced, excitant reduction, and flue gas becomes alcohol and cigarette perfume increases, and aesthetic quality is obviously improved.
Embodiment 6
Weigh 15g stems, the μ g of crude enzyme liquid 7.5 prepared by another Example 3, with the citric acid-lemon of 0.1mol/L pH5.2
Lemon acid sodium buffer solution 7mL dilutions, make the water content of stem reach 40%, load micro sprayer spray and are uniformly sprayed onto on stem,
Then stem is put into 35 DEG C of incubator fermentation 24h.
Through detection after fermentation, the content of pectin reduces 16.9% in stem, and the content of xylan is reduced in stem
7.2%.The xylon gas of stem are reduced, excitant reduction, and flue gas becomes alcohol and cigarette perfume increases, and aesthetic quality is obviously improved.
Embodiment 7
Weigh 15g stems, the μ g of crude enzyme liquid 15 prepared by another Example 3, with the citric acid-lemon of 0.2mol/L pH5.2
Sour sodium buffer solution 11.4mL dilutions, make the water content of stem reach 50%, load micro sprayer spray and are uniformly sprayed onto on stem,
Then stem is put into 35 DEG C of incubator fermentation 48h.
Through detection after fermentation, the content of pectin reduces 28.3% in stem, and the content of xylan is reduced in stem
10.3%.The xylon gas of stem are reduced, excitant reduction, and flue gas becomes alcohol and cigarette perfume increases, and aesthetic quality is obviously improved.
Embodiment 8
Weigh 15g stems, the μ g of crude enzyme liquid 4.5 prepared by another Example 3, with the citric acid-lemon of 0.1mol/L pH5.2
Lemon acid sodium buffer solution 11.4mL dilutions, load micro sprayer spray and are uniformly sprayed onto on stem, reach the water content of stem
50%, stem is then put into 40 DEG C of incubator fermentation 12h.
Through detection after fermentation, the content of pectin reduces 10.3% in stem, and the content of xylan is reduced in stem
3.9%.The xylon gas of stem are reduced, excitant reduction, and flue gas becomes alcohol and cigarette perfume increases, and aesthetic quality is obviously improved.
Embodiment 9
Weigh 15g stems, the μ g of crude enzyme liquid 75 prepared by another Example 3, with the citric acid-lemon of 0.2mol/L pH5.2
Sour sodium buffer solution 11.4mL dilutions, load micro sprayer spray and are uniformly sprayed onto on stem, the water content of stem is reached 50%,
Then stem is put into 35 DEG C of incubator fermentation 48h.
Through detection after fermentation, the content of pectin reduces 38.5% in stem, and the content of xylan is reduced in stem
12.8%.The xylon gas of stem are reduced, excitant reduction, and flue gas becomes alcohol and cigarette perfume increases, and aesthetic quality is obviously improved.
Obviously, above-described embodiment is only intended to clearly illustrate example, and not to the restriction of implementation method.It is right
For those of ordinary skill in the art, can also make on the basis of the above description other multi-forms change or
Change.There is no need and unable to be exhaustive to all of implementation method.And the obvious change thus extended out or
Among changing still in the protection domain of the invention.
Claims (10)
1. a kind of fungi of degrading tobacco stem pectin and xylan, its Classification And Nomenclature is coronoid process dissipate capsule bacterium Eurotium
Cristatum 502, has been preserved in Wuhan University's China typical culture collection center, and deposit number is CCTCC
M2016204。
2. it is a kind of cultivate claim 1 described in fungi method, it is characterised in that it includes dissipating coronoid process described in claim 1
Capsule bacterium carries out bacterial strain seed culture under conditions of being suitable for producing ascospore, and is being suitable for pectase and zytase table
The step of fermented and cultured being carried out under the condition of culture for reaching.
3. it is according to claim 2 culture fungi method, it is characterised in that it is described to be suitable for pectase and zytase
The condition of culture of expression includes：In the culture medium containing 40-60g/L stems, and 28-32 DEG C of the temperature is controlled to carry out fermentation training
Support.
4. the enzyme for being encoded as the fungi of degrading tobacco stem pectin and xylan described in claim 1, the enzyme includes pectase
And zytase.
5. a kind of method for preparing enzyme described in claim 4, it is characterised in that including being trained according to Claims 2 or 3 methods described
The step of supporting the coronoid process dissipate capsule bacterium, and after gained zymotic fluid is filtered into thalline, gained filtrate is crude enzyme liquid.
6. the enzyme described in claim 4 is used to improve the purposes of tobacco stem shred quality.
7. degrading tobacco stem pectin and the fungi of xylan described in claim 1 is used to improve the use of tobacco stem shred quality
On the way.
8. a kind of method for improving tobacco stem shred quality, it is characterised in that comprise the following steps：
(1) according to claim 5 methods described, described coronoid process dissipate capsule bacterium Eurotium cristatum 502 are fermented
Culture, and obtain crude enzyme liquid；
(2) crude enzyme liquid for accounting for tobacco stem shred weight 0.01-0.5wt% is taken, is uniformly sprayed onto after dilution described tobacco stem shred
In, control the water content of the stem to reach 30-50%, and fermented at a temperature of 30-40 DEG C, you can.
9. the method for improving tobacco stem shred quality according to claim 8, it is characterised in that in the step (2), it is described thick
The dilution step of enzyme liquid is the citric acid-sodium citrate buffer solution using sterile distilled water or 0.1-0.2mol/L, pH5.0-5.5
It is diluted.
10. a kind of Cigarette processing method, it is characterised in that including improving the tobacco stems according to the methods described of claim 8 or 9
The step of yarn quality.
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| CN201611052682.6A CN106834131B (en) | 2016-11-24 | 2016-11-24 | Fungus for degrading pectin and xylan in tobacco stems and application of fungus |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106676013A (en) * | 2016-11-24 | 2017-05-17 | 广西中烟工业有限责任公司 | Method for improving quality of redrying tobacco leaves by utilizing fermentation of eurotium cristatum tea leaves |
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| CN103110180A (en) * | 2012-12-24 | 2013-05-22 | 贵州大学 | Early-flue-cured tobacco quality improving method with eurotium cristatum |
| CN103275876A (en) * | 2013-05-23 | 2013-09-04 | 湖南农业大学 | Tea source eurotium cristatum strain and application thereof |
| CN106676013A (en) * | 2016-11-24 | 2017-05-17 | 广西中烟工业有限责任公司 | Method for improving quality of redrying tobacco leaves by utilizing fermentation of eurotium cristatum tea leaves |
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2016
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| CN103110180A (en) * | 2012-12-24 | 2013-05-22 | 贵州大学 | Early-flue-cured tobacco quality improving method with eurotium cristatum |
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| CN106676013A (en) * | 2016-11-24 | 2017-05-17 | 广西中烟工业有限责任公司 | Method for improving quality of redrying tobacco leaves by utilizing fermentation of eurotium cristatum tea leaves |
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| CN106676013A (en) * | 2016-11-24 | 2017-05-17 | 广西中烟工业有限责任公司 | Method for improving quality of redrying tobacco leaves by utilizing fermentation of eurotium cristatum tea leaves |
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