CN106191209A - A kind of method detecting Starch in Tobacco degrading microorganism - Google Patents
A kind of method detecting Starch in Tobacco degrading microorganism Download PDFInfo
- Publication number
- CN106191209A CN106191209A CN201610505577.7A CN201610505577A CN106191209A CN 106191209 A CN106191209 A CN 106191209A CN 201610505577 A CN201610505577 A CN 201610505577A CN 106191209 A CN106191209 A CN 106191209A
- Authority
- CN
- China
- Prior art keywords
- starch
- tobacco
- sample
- nicotiana tabacum
- liquid
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
- C12Q1/40—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving amylase
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/914—Hydrolases (3)
- G01N2333/924—Hydrolases (3) acting on glycosyl compounds (3.2)
- G01N2333/926—Hydrolases (3) acting on glycosyl compounds (3.2) acting on alpha -1, 4-glucosidic bonds, e.g. hyaluronidase, invertase, amylase
- G01N2333/928—Hydrolases (3) acting on glycosyl compounds (3.2) acting on alpha -1, 4-glucosidic bonds, e.g. hyaluronidase, invertase, amylase acting on alpha -1, 4-glucosidic bonds, e.g. hyaluronidase, invertase, amylase
Abstract
The present invention relates to a kind of method detecting Starch in Tobacco degrading microorganism, concrete steps include sample collecting, sample treatment, dilute sample, cell separation, ultrasonic Treatment, the demarcation of starch degradation microorganism and the step such as spike and microscope observation.The method can apply to flue-cured tobacco different cultivars, different parts, the Nicotiana tabacum L. of differing maturity class, obtain the information of Nicotiana tabacum L. starch degradation flora under state in position, reduce labor intensity significantly, cost is relatively low, operating process is easy, and operator need not special training, to the place sampled without special requirement, experimental result is accurate, can be quickly provided to the data that technical staff is correlated with.Simultaneously, the method also contributes to technical staff and understands fully concrete tobacco fermentation flavouring mechanism, in order to preferably Guiding Practice, explore and improve Nicotiana tabacum L. process technological measure and condition, widen innovation tobacco fermentation technical research thinking, actively develop associated frontier research.
Description
Technical field
The present invention relates to a kind of microorganism detection method, a kind of side detecting Starch in Tobacco degrading microorganism
Method.
Background technology
Flue-cured tobacco is one of mainstay industry of China, occupies critical role in the development of China Nicotiana tabacum L..Due to Nicotiana tabacum L.
Reason in cultivation, baking and ageing process, generally there is the problems such as starch residual quantity is higher in current domestic flue-cured tobacco.Starch is
The primary product of tobacco leaf photosynthetic effect, starch is a kind of storage form of Nicotiana tabacum L. matter and energy, in chloroplast, photosynthate
In addition to meeting the matter and energy needed for cell own metabolism activity, unnecessary part is essentially converted to starch and stores
Come, needed for cigarette strain vital movement when, starch degradable again and be converted into other form saccharide supply various demands.Nicotiana tabacum L.
The content of starch difference at three positions, upper, middle and lower is relatively big, and Nicotiana tabacum L. is in growth course, and its content of starch is stepped up, with blade
Aging and substantially reduce.The content of starch of Mature Tobacco Leaves is generally about 25%, and the content of starch of cured tobacco leaf is at 2%-
8%, when content of starch is more than 5%, then it is assumed that unfavorable to the quality of Nicotiana tabacum L., because firing in cigarette with the saccharide of starch form existence
Burning velocity and combustion completion can be affected during suction, produce during burning and stick with paste burnt flavour, make tobacco flavor degenerate, so content of starch
Number decide the interior quality of Nicotiana tabacum L. and suck the quality of taste, and the height of the sugared content that Starch Conversion is formed, direct shadow
Ringing the amount of starch of residual in Nicotiana tabacum L., therefore starch degradation degree is the important indicator that quality of tobacco is good and bad.
Baking and ageing process are that in Nicotiana tabacum L., macromole biologic artifact Degradation and Transformation improves for the most fragrant odor level
The significant process of little molecular biosciences compound.Along with the Chinese-style cigarette raising to feed quality requirements, it is necessary to baking and
The enzymatic activity of various macromole biologic artifact of degrading in ageing process is studied, in order to creates an enabling environment, promotes various
The degraded of macromole biologic artifact.Starch is a kind of important nitrogen-containing compound in Nicotiana tabacum L., meeting in baking and ageing process
Decompose, convert.Appropriate degradation Nicotiana tabacum L. starch has become one of key improving tobacco leaf usability.The degraded of Starch in Tobacco
It is under the conditions of uniform temperature, humidity and moisture content in leaves content, by the hydrolysis of relevant starch degradation bacterial secretory hydrolytic enzyme
Complete.In tobacco flue-curing and ageing process, existing means include the starch utilizing additional amylase to reduce in Nicotiana tabacum L.
Content, or utilize a large amount of microorganisms that tobacco leaf surface adheres to, on Nicotiana tabacum L., during growth and breeding, secretion discharges multiple
The hydrolysis occurred during sleep is played facilitation by enzyme.The starch degradation antibacterial of tobacco leaf surface attachment, except
Acted on baking and the ageing process of Nicotiana tabacum L. by the vital movement of self, participate in the life of Nicotiana tabacum L. also by its amylase secreted
Reason biochemical reaction, promotes the degraded of Starch in Tobacco, and the diastatic effect of starch degradation bacterial secretory of tobacco leaf surface is that it dries
Bake and an important factor in order in ageing process, by amylase and the synergism of related chemical species, one can be induced
Series improves the biochemical reaction of tobacco leaf chemical composition harmony and judging parameter, promotes the life of the biomacromolecules such as Starch in Tobacco
Thing converts, and coordinates the inherent composition of Nicotiana tabacum L., promotes decomposition and the conversion of the internal organic substance of Nicotiana tabacum L., adjusts various chemistry in Nicotiana tabacum L.
The ratio of composition, improves formation and the accumulation of fragrance component in Nicotiana tabacum L., thus improves the sucking quality of Nicotiana tabacum L..
Starch can be divided into amylose and amylopectin according to the difference of structure.Amylose is to be bonded by α-1,4-glucosides
The linear polysaccharide connect, typically constitutes from the 30% of starch composition；Amylopectin is by α-1,4-glycosidic bond and β-1,6-glucosides
The bonded polysaccharide with branch, is the key component of starch, accounts for 70% more than.α-amylase is starch water
Solve initial enzyme, can random degenerate amylose and the α-Isosorbide-5-Nitrae-glycosidic bond of amylopectin non-end, act on amylose
Product is maltose, when acting on amylopectin, can only hydrolyze the α-Isosorbide-5-Nitrae-glycosidic bond of peripheral carbochain, act on to α-1,6-glucosides
Limit dextrin is generated during key.Ripe fresh tobacco leaf of flue-cured tobacco starch-containing about 40%, in baking process, the degraded of Nicotiana tabacum L. starch is necessarily
Under temperature, humidity and Nicotiana tabacum L. Water Content Conditions, through α-amylase catalytic cleavage α-Isosorbide-5-Nitrae-or 1,6-glycosidic bond, generation reducing sugar etc.
Little molecule is carried out.
The expansion development of tobacco leaf production enterprise is played an important role by Nicotiana tabacum L. raw material.Due to kind, cultivate and modulate
Etc. reason, the lowest inferior Starch in Tobacco content of China's part Nicotiana tabacum L. is higher, have impact on the harmony of tobacco leaf chemical composition,
Reduce the sucking quality of Medicated cigarette.Appropriate degradation cured tobacco leaf content of starch become improve tobacco leaf usability key technology it
One, existing research shows, tobacco leaf surface is attached with numerous microorganisms, especially in the majority with antibacterial, and its type and quantity are directly closed
Being tied to the quality quality of Nicotiana tabacum L., the starch degradation antibacterial of tobacco leaf surface attachment synthesizes multiple amylases in its growth course,
These enzyme accelerate degraded and the conversion rate of Starch in Tobacco just.In baking of flue-cured tobacco, Nicotiana tabacum L. starch degradation degree is to cigarette
Leaf quality has important impact, in baking flue-cured tobacco starch non-degradable or degraded not exclusively can have a strong impact on the color of cured tobacco leaf, perfume (or spice),
The shallow lake of residual in taste, in Medicated cigarette, content of starch is too high when can affect suction burning velocity and combustion completion, particularly Nicotiana tabacum L.
Powder can produce when burning and sucking and be charred taste, zest and miscellaneous QI, and not shelf-stable.The too low meeting of content of reducing sugar that starch degradation produces
Destroy flue gas acid-base balance, nitrogen-containing compound increases, and not only makes that flue gas is coarse, produces and be charred taste, and can produce one when burning and sucking
The harmful gass such as nitrogen oxide (nitric oxide), phenols (phenols) and benzene class (benzenes).So, flue-cured tobacco in baking
Whether starch is fully degraded into a major criterion into evaluating Flue-cured tobacco Quality, it is considered that, the Starch Content of Tobacco modulated
Should be less than 3.0%, the most external sound tobacco content of starch is l%-2%, and domestic Nicotiana tabacum L. is 4%-6%, and cured tobacco leaf forms sediment
Powder residual quantity is too high has become one of key factor restricting the raising of domestic quality of tobacco.
In baking of flue-cured tobacco, starch degradation and further converted product thereof are closely related with scent of fire cured tobacco, and after baking, starch drops
Solve the reducing sugar produced cracking when suction and produce acid product, balance is produced by nicotine and nitrogen-containing compound in aspiration procedure
Raw alkaline flue gas has positive role, and it decides the fragrance style of former cigarette to a great extent.In tobacco flue-curing be aged
Cheng Zhong, due to diastatic catalytic action, the starch in Nicotiana tabacum L. is decomposed, synthesizes and converts.Starch degradation in Nicotiana tabacum L. is
Water-soluble sugar, the tobacco aroma quality after ferment treatment improves, and miscellaneous QI and zest alleviate, and flue gas sugariness increases, and pleasant impression improves,
Strength decreases, and Nicotiana tabacum L. oeverall quality is improved.Content of starch in Appropriate degradation cured tobacco leaf has become raising Nicotiana tabacum L.
One of critical process of quality.The conversion of Starch in Tobacco and decomposition depend on the starch fall of tobacco leaf surface attachment greatly
Solving the diastatic effect of bacterial secretory, diastatic effect grows from Nicotiana tabacum L., baking, until ageing exists the most all the time.By
In starch degradation bacterial secretory diastatic effect through tobacco flue-curing and ageing process all the time, the impact on quality of tobacco
Relatively big, the most more effectively utilize the amylase of starch degradation bacterial secretory to the residual starch degrading in Nicotiana tabacum L., monitor cigarette
Diastatic activity change rule in leaf growth, the course of processing, having become as lifting quality of tobacco further becomes tobacco business
The new challenge of research.The content of the bioconversion how promoting Starch in Tobacco and the starch controlled in Nicotiana tabacum L. is for flue-cured tobacco
Interior quality has important effect.
Tobacco bred, position, Maturity have important impact to content of starch and the fragrance taste thereof of Nicotiana tabacum L..Flue-cured tobacco is different
Kind, different parts, the Starch Content of Tobacco Changing Pattern of differing maturity class are the starch degrading bacteriums of tobacco leaf surface attachment
One of key factor of group's activity.Existing research shows that Starch in Tobacco enzyme and content of starch are in baking process each period
Activity change rule is different, and in baking process, Nicotiana tabacum L. amylase activity peak occurs when 24h, 36h and 72h of baking respectively,
Employing low temperature and low humidity turns yellow, and the curing environment of the fixation that heats up at a slow speed makes Nicotiana tabacum L. endo-amylase and starch phosphorylation enzymatic activity higher,
Be conducive to the degraded of Nicotiana tabacum L. starch.Although Nicotiana tabacum L. is subjected to destroy at cellularity after high-temperature baking, but at the ageing initial stage
It is also found that diastatic activity, has illustrated Nicotiana tabacum L. ageing process after baking remains partial starch enzyme in Nicotiana tabacum L., and
Amylase has preferable heat stability.
Up to now, starch degradation antibacterial and the diastatic means of all research tobacco leaf surfaces attachment are all by pure training
Support and carry out, promote that the mode of starch degradation mainly makes various artificial enzyme's preparation by enzyme at present, utilize growth or produce
The starch degradation bacteria culture of the competitive advantages such as enzyme adds in baking and aging tobacco leaves, accelerates starch degradation process, increases cigarette
Leaf fragrance and improve quality of tobacco.First the antibacterial with higher starch enzymatic activity that separation screening obtains from flue-cured tobacco surface
Bacterial strain, then by being prepared as diastase after fermentation, is applied on junior tobacco leaf, degrades the starch in Nicotiana tabacum L.,
Make it be converted into monosaccharide and the oligosaccharide having facilitation to quality of tobacco, thus improve quality and the industrial applicability of Nicotiana tabacum L..
Existing research shows, to the product amylase bacterial isolates bacillus amyloliquefaciens (Bacillus being isolatable from flue-cured tobacco blade face
Amyloliquefaciens A1) carry out fermentation culture, extract crude enzyme liquid, detection enzymatic activity is also applied on junior tobacco leaf, knot
Fruit shows, its total sugar increment rate after processing with the produced diastase of strains A 1 is 12.78%, and reducing sugar increment rate is
12.03%, the enzyme preparation utilizing the bacterium producing multi enzyme preparation separated from tobacco leaf surface to carry out fermenting and producing can promote starch junior tobacco leaf
Degraded, improves content of reducing sugar.The starch degradation antibacterial that tobacco leaf surface exists can pass through own metabolism secreting amylase, promotes
Content of starch declines further, such as tobacco bacterial wilt pathogenic bacteria (Pseudomonas solanacearum), can produce decomposition and form sediment
The enzyme of powder, such as α-amylase.But no matter the artificial diastase using pure culture mode to obtain has the disadvantage that (1)
It is that external enzyme preparation is improved or added to tobacco fermentation technical conditions, owing to various places Nicotiana tabacum L. interior quality is different, it is impossible to
Form the most unified tobacco fermentation technology；(2) affected by factors such as temperature, pH, inhibitor due to the activity of enzyme, and not
Congener tobacco leaf chemical composition is incomplete same, and fermentation condition can not be suitable for the action condition of every kind of enzyme；(3) the most most
Use the business enzyme of commercialization, owing to the catalysis activity of enzyme by many regulations and controls (such as temperature, soda acid
Degree, enzyme and the concentration of substrate and the existence etc. of inhibitor), and the special of himself that have of tobacco fermentation and processing technique will
Asking, therefore the use of simple commercial enzyme may not be fully able to adapt to the needs of ferment treatment in all tobacco fermentation.
The starch degradation flora of tobacco leaf surface attachment is one of critical function flora in tobacco flue-curing and ageing process, due to
Lack the original position information of the starch degradation flora of the relevant participation tobacco fermentation of system, at present starch degradation flora is dried at Nicotiana tabacum L.
The roasting understanding with the effect in ageing process is mainly studied (i.e. by isolation of pure culture bacterial strain) by pure culture, but, Nicotiana tabacum L.
In fermentation flora, only minority (< 20%) can be by pure culture, and the microorganism obtained by pure culture research is raw
Reason Biochemical Information differs reflection microorganism surely in the effect in situ of complicated ecosystem and function.
Summary of the invention
The present invention provides a kind of method detecting Starch in Tobacco degrading microorganism, uses in position under (in situ) state
Detect starch degradation antibacterial and the diastatic vigor of they secretions of monitoring of tobacco leaf surface attachment, be i.e. to need not by pure
Culture of isolated bacterial strain, can be applicable to, under different Nicotiana tabacum L. kinds and fermentation condition, monitor, adjust and promote Nicotiana tabacum L. starch degradation
The vigor of antibacterial, explores a kind of technological measure that can control Nicotiana tabacum L. amylase activity further.Its principle is: with fluorescence
Organic macromolecule BODIPY be combined with starch, make the fluorescence in BODIPY be wrapped in the inside of conjugate, at fluorescence microscopy
Microscopic observation less than, be positioned at the α-amylase between Cytoplasm wall and can hydrolyze BODIPY fluorescence starch, discharge with fluorescence is short
Fragment hydrolyzate, these hydrolyzate are deposited on around the microorganism wall of relevant enzyme vigor, under fluorescence microscope
The single cell with specific alpha-amylase activity that be fluorescently labeled be can be observed.
Meanwhile, in order to get rid of the false positive in dyeing course, distinguish there is hydrolytic enzyme activities with there is no hydrolytic enzyme activities but
Can absorb the cell (error flag) of hydrolyzate, the present invention adds various electron transmission chain inhibitor to ensure in cultivation
The reliability of result.Its principle be microorganism species can hydrolyze or ferment add the labelled compound (shallow lake of BODIPY labelling
Powder), therefore all cells of the markd metabolite of absorption band the most all can be labeled, and add electron transmission chain inhibitor
The microorganism that can suppress absorb with the hydrolyzate of fluorescently-labeled starch, prevent owing to active absorption is with fluorescent labeling
The hydrolyzate of thing and the false positive that causes, it is ensured that viewed be entirely starch fall with fluorescently-labeled microorganism
Solve microorganism.
In order to obtain optimized incubation time, to ensure observe the activity of α-amylase to greatest extent and demarcate starch
Bacterium for degrading, the present invention has collected the tobacco sample (3h of different baking period；9h；24h；36h), different trainings is tested respectively
The time of supporting,.The present situation that viewed antibacterial maintains like, simply fluorescence intensity is otherwise varied.Process software I mage J divides
Analysis and contrast picture, thus obtain optimized incubation time.
For reaching above-mentioned purpose, the invention provides a kind of method detecting Starch in Tobacco degrading microorganism, including with
Lower step:
(1) sample collecting: wear sterile gloves and use the baking of disposable sterile collection bag random collecting or be aged
Tobacco sample in journey, and be stored under 18-40 degree constant temperature in 20min, carry out sample treatment；
(2) sample treatment: use concentration 70% alcohol disinfecting shears, aseptically, sterilized tobacco sample use
Shears is cut into some fritters, and the unwanted tissues such as stem and vein of carefully removing Nicotiana tabacum L.；
(3) dilute sample: under aseptic condition, weighs fresh broken leaf sample, and joining pH4.0-9.0 is sterilizing 1 × PBS
Buffer, its consumption of buffer is 10-30 times of broken leaf sample quality, uniform with broken leaf sample mix；
(4) cell separation: the mixing liquid that step (3) obtains is poured into the most respectively blender stirring and smashes, every time
1-5 minute, pour the homogenate of blender top gear after the liquid mixing after then smashing three times into, filter, filtrate is centrifuged and goes
Except supernatant, being resuspended in by the precipitate removing supernatant equipped with pH4.0-9.0 is the centrifugal of sterilizing 1 × PBS
Guan Zhong, obtains cell suspending liquid；
(5) ultrasonic Treatment: being put into by centrifuge tube in ultrasonic instrument fixing, centrifuge tube is not when guaranteeing ultrasonic Treatment
On the premise of moving, stand-by with taking out after frequency processing 50-140 minute of 10000-80000Hz；At the place waiting next step
During reason, the centrifuge tube that will be equipped with cell suspending liquid is stored in the refrigerator of 4 DEG C；
(6) demarcation of starch degradation microorganism and spike: process by centrifugation and remove in the cell suspending liquid of supernatant, depend on
1 × Tris-HCl buffer of secondary addition 6-15mmol/L, pH 5.1-9.0 sterilizing and the boron fluoride two pyroles fluorescence of sterilizing
The green fluorescence starch dye liquor of dyestuff BODIPY labelling, forms mixing liquid, and immediately mixing liquid is transferred to aseptic width
In end serum bottle, add the electron transmission chain inhibitor of sterilizing, lid is built, by the tight lucifuge of Aluminium Foil Package；Room temperature shake 30-
120min, speed is maintained at 100-500rpm and cultivates；The reactant liquor that under gnotobasis, absorption 10-50 μ L cultivation obtains is uniform
Coat on gelatin coating microscope slide, and lucifuge air-dries in gnotobasis；Described electron transmission chain inhibitor is 1-10mmol/
L Hydrazoic acid,sodium salt, 1-10mmol/L 1080 (Monsanto) and the mixed solution of 1-10mmol/L iodo-acetamide sodium composition；
(7) microscope observe: dried microscope slide is placed on fluorescence microscopy endoscope objective lens platform, 100 × object lens under enter
Row is observed and takes pictures, and excites block to observe by green fluorescence, and starch degradation antibacterial presents green fluorescence and is starch degradation antibacterial.
Preferably, the gelatin coating microscope slide described in step (7) processes as follows:
(1) take 7 carry sheet glass shelf, microscope slide is filled in carry sheet glass shelf；In 3.5L distilled water, add 4-5 drip washing
Agent, puts into carry sheet glass shelf, boils 10min；Taking out carry sheet glass shelf, dry in the air 3min；With distilled water, detergent is rinsed well, glass will be carried
The sheet state of being kept upright is dried；
(2) in 3.5L distilled water, add ten sulfate dihydrate potassium chromium and the gelatin of 0.44g of 2.5g, be heated to 60-80 DEG C,
Carry sheet glass shelf, constant temperature 10min is put into after gelatin dissolves substantially；Take out carry sheet glass shelf, microscope slide is tiltedly stood on test tube rack limit
It is drying to obtain.
Preferably, step (4) is described to be poured the liquid of mixing domestic stirrer stirring the most respectively into and smashes, and each 3
Minute, the mixing liquid after then smashing three times is transferred in aseptic BioRad homogenate bag, in BioRad homogenizer 3 grades homogenate
3min, is filtered by the mistake filter grid of homogenate bag side, and by filtrate collection in aseptic 50ml centrifuge tube, 5000g is centrifuged 8min
Remove supernatant, and precipitate is put in 2ml centrifuge tube, be resuspended in buffering equipped with the sterilizing 1 × PBS of pH4.0-9.0
In liquid.
Preferably, the centrifugal treating specifically 4500g-5000g described in step (4) is centrifuged 5-10min.
Preferably, the centrifugal treating specifically 4500g-5000g described in step (6) is centrifuged 8-12min.
Preferably, the volume of the 1 × Tris-HCl buffer described in step (6) and the Cell suspension volumes phase taken
With；Volume and 1 × Tris-HCl buffer volume of the green fluorescence dye liquor of BODIPY labelling used are identical；Electron transport chain
Volume and 1 × Tris-HCl buffer volume of inhibitor are identical.
Spike and demarcation Nicotiana tabacum L. starch in the mixed microorganism group that the present invention is complicated from tobacco flue-curing and ageing process
Degradation flora, uses molecular microbial ecology ways and means i.e. BODIPY amylase (amylase) dyeing to demarcate and chase after
Nicotiana tabacum L. starch degrading bacterium under track home state.Comparing with former culture-dependent method, the inventive method can directly be demarcated and supervise
Tobacco Control leaf starch degradation flora is on quality of tobacco and on the degree of biochemical process of Nicotiana tabacum L. degraded and the impact of speed, significantly
Reducing labor intensity, cost is relatively low, and operating process is easy, and operator need not special training, to sampling position Wu Te
Different requirement, experimental result is accurate, can be quickly provided to the data that diagnostic personnel is relevant.
Use molecular microbial ecology ways and means i.e. BODIPY α-amylase (α-amylase) dyeing exempting to cultivate
Demarcate and follow the trail of starch degradation flora and their activity of tobacco leaf surface attachment under home state, not only for detection cigarette
The activity change rule of leaf starch degradation flora has great importance, moreover it is possible to further appreciate that Nicotiana tabacum L. starch degradation flora is at cigarette
Effect in leaf baking and ageing process and function, under acquisition home state, the Nomenclature Composition and Structure of Complexes of Nicotiana tabacum L. starch degradation flora is former
Position information, understands starch degradation flora and the Starch Content of Tobacco of different cultivars, different parts, differing maturity class is changed rule
The response of rule.By applying the method in tobacco fermentation or the course of processing, can control and promote Nicotiana tabacum L. starch degrading bacterium
The activity of group, makes up Nicotiana tabacum L. and because of growth or modulates the improper deficiency caused, thus reach to improve the purpose of Nicotiana tabacum L. smoking quality, with
Time also can be greatly shortened sweat, cost-effective, significantly promote economic benefit.
The described method of the present invention can apply to flue-cured tobacco different cultivars, different parts, the Nicotiana tabacum L. of differing maturity class,
Obtain the information of Nicotiana tabacum L. starch degradation flora under state in position, moreover it is possible to be quickly provided to the data that technical staff is correlated with, understand fully
Concrete tobacco fermentation flavouring mechanism, can preferably Guiding Practice, explore and improve Nicotiana tabacum L. process technological measure and condition,
Widen innovation tobacco fermentation technical research thinking, actively develop associated frontier research.
Accompanying drawing explanation
Accompanying drawing 1 is the method flow diagram of detection Starch in Tobacco degrading microorganism；
Accompanying drawing 2 is tobacco leaf surface microbiologic population Green fluorescence starch stained positive starch degradation microorganism microphotograph
Example, is intended that the starch degradation antibacterial of tobacco leaf surface attachment after BODIPY DQ starch dyes, refers at B at A in figure
Bright is the plant tissue after Nicotiana tabacum L. is broken.
Detailed description of the invention
Below by way of specific instantiation, embodiments of the present invention being described, those skilled in the art can be by this specification
Disclosed content understands other advantages and effect of the present invention easily.The present invention can also be by the most different concrete realities
The mode of executing is carried out or applies, the every details in this specification can also based on different viewpoints and application, without departing from
Various modification or change is carried out under the spirit of the present invention.
Before further describing the specific embodiment of the invention, it should be appreciated that protection scope of the present invention is not limited to down
State specific specific embodiments；It is also understood that the term used in the embodiment of the present invention is specific concrete in order to describe
Embodiment rather than in order to limit the scope of the invention；In description of the invention and claims, unless in literary composition
Additionally explicitly pointing out, singulative " ", " one " and " this " include plural form.
When embodiment provides numerical range, it should be appreciated that unless the present invention is otherwise noted, two ends of each numerical range
Between point and two end points, any one numerical value all can be selected for.Unless otherwise defined, in the present invention use all technology and
The same meaning that scientific terminology and those skilled in the art of the present technique are generally understood that.Except in embodiment use concrete grammar, equipment,
Outside material, according to those skilled in the art's grasp to prior art and the record of the present invention, it is also possible to use and this
Any method, equipment and the material of the prior art that the method described in inventive embodiments, equipment, material are similar or equivalent comes real
The existing present invention.
Unless otherwise indicated, the experimental technique that disclosed in this invention, detection method, preparation method all use this technology to lead
The routine techniques of molecular biology, biochemistry, technique of analytical chemistry and association area that territory is conventional.
Embodiment one: a kind of method detecting Starch in Tobacco degrading microorganism, step is as follows:
Sample collection: wear sterile gloves, use disposable sterile collection bag random collecting 1kg baking or ageing process
In tobacco sample, and be stored under 37 DEG C of constant temperatures and send laboratory to carry out sample treatment in 20min；
Sample treatment: at laboratory first with concentration 70% alcohol disinfecting shears, under aseptic condition, by tobacco sample with disappearing
The shears that poison is crossed is cut into 1-4cm2Fritter, and the unwanted tissues such as stem and vein of carefully removing Nicotiana tabacum L.；
Dilute sample: under aseptic condition, weighs the fresh broken leaf sample of 30g with aseptic 50ml beaker, adds 300ml sample
Product reagent treatment A is uniform with broken leaf sample mix；
Cell separation: the liquid of mixing is poured into the most respectively the stirring of Philip (PHILIPS) domestic stirrer and smashes,
Each 3 minutes, the mixing liquid after then smashing three times was transferred in aseptic BioRad homogenate bag, in BioRad homogenizer 3
Shelves homogenate 3min, by homogenate bag side mistakes filter grid filtration, by filtrate collection in aseptic 50ml centrifuge tube, 5000g from
Heart 8min；Remove supernatant, and precipitate is put in 2ml centrifuge tube, be resuspended in 2ml sample processing reagent A；
Ultrasonic Treatment: the centrifuge tube that will be equipped with mixing liquid is fixed, and is then placed in ultrasonic instrument, is guaranteeing super
On the premise of during sonicated, centrifuge tube will not move, take out after 60 minutes stand-by by the frequency processing of 53000Hz.Under waiting
In the experimentation of one step, cell suspending liquid can be stored in the refrigerator of 4 DEG C；
The demarcation of starch degradation microorganism and spike: take 400 μ L cell suspending liquid (obtaining from previous step) and be transferred to nothing
In the 2mL eppendof centrifuge tube of bacterium, centrifugal (4500g) 10 minutes, remove supernatant, add 400 μ L and demarcate tracer reagent
B1,200 μ L demarcate tracer reagent B2, and are transferred in serum bottle at the bottom of aseptic 10ml width by mixing liquid immediately, add electricity
Sub-transfer chain inhibitor C, builds lid, and the tight lucifuge of rapid Aluminium Foil Package；Room temperature shake 45min, speed is maintained at
220rpm；Draw 10 μ L reactant liquors under gnotobasis to be spread evenly across on gelatin coating microscope slide, and lucifuge in gnotobasis
Air-dry；
Microscope is observed: dried microscope slide is placed in fluorescence microscope, and (Lycra DM6000B is equipped with Lycra DFC500
Digital camera) on object lens platform, 100 × object lens under carry out observing and taking pictures exciting block to observe by green fluorescence, starch drops
Solve antibacterial and present green fluorescence.
Wherein, described gelatin smear processes according to the following steps:
Take 7 carry sheet glass shelf, microscope slide is filled in carry sheet glass shelf；In 3.5L distilled water, add 4-5 drip detergent,
Put into carry sheet glass shelf, boil 10min；Taking out carry sheet glass shelf, dry in the air 3min；With distilled water, detergent is rinsed well, by microscope slide
The state of being kept upright is dried；
In 3.5L distilled water, add ten sulfate dihydrate potassium chromium and the gelatin of 0.44g of 2.5g, be heated to about 70 DEG C, treat
Gelatin puts into carry sheet glass shelf, constant temperature 10min after substantially dissolving；Take out carry sheet glass shelf, microscope slide is tiltedly stood on test tube rack limit dry
Dry and get final product.
Wherein, described sample processing reagent A is sterilizing 1 × PBS, pH=6.0.
Wherein, described demarcation tracer reagent B1, B2 be respectively sterilizing Tris-HCl buffer (10mmol/L, pH 7.8) and
The green fluorescence starch dye of sterilizing BODIPY (boron fluoride two pyroles fluorescent dye, boron-dipyrromethene) labelling
Liquid.
Wherein, described electron transport chain inhibitor C be total amount be the sterilizing 3mmol/L Hydrazoic acid,sodium salt of 0.6ml, 2mmol/L
1080 (Monsanto), 4mmol/L iodo-acetamide sodium mixed solution.
Embodiment two: a kind of method detecting Starch in Tobacco degrading microorganism, step is as follows:
Sample collection: wear sterile gloves, use disposable sterile collection bag random collecting 1kg baking or ageing process
In tobacco sample, and be stored under 30 DEG C of constant temperatures and send laboratory to carry out sample treatment in 20min；
Sample treatment: at laboratory first with concentration 70% alcohol disinfecting shears, under aseptic condition, by tobacco sample with disappearing
The shears that poison is crossed is cut into 1-4cm2Fritter, and the unwanted tissues such as stem and vein of carefully removing Nicotiana tabacum L.；
Dilute sample: under aseptic condition, weighs the fresh broken leaf sample of 30g with aseptic 50ml beaker, adds 600ml sample
Product reagent treatment A is uniform with broken leaf sample mix；
Cell separation: the liquid of mixing is poured into the most respectively the stirring of Philip (PHILIPS) domestic stirrer and smashes,
Each 4 minutes, the mixing liquid after then smashing three times was transferred in aseptic BioRad homogenate bag, in BioRad homogenizer 3
Shelves homogenate 3min, by homogenate bag side mistakes filter grid filtration, by filtrate collection in aseptic 50ml centrifuge tube, 4500g from
Heart 10min；Remove supernatant, and precipitate is put in 2ml centrifuge tube, be resuspended in 2ml sample processing reagent A；
Ultrasonic Treatment: the centrifuge tube that will be equipped with mixing liquid is fixed, and is then placed in ultrasonic instrument, is guaranteeing super
On the premise of during sonicated, centrifuge tube will not move, take out after 50 minutes stand-by by the frequency processing of 73000Hz.Under waiting
In the experimentation of one step, cell suspending liquid can be stored in the refrigerator of 4 DEG C；
The demarcation of starch degradation microorganism and spike: take 400 μ L cell suspending liquid (obtaining from previous step) and be transferred to nothing
In the 2mL eppendof centrifuge tube of bacterium, centrifugal (5000g) 8 minutes, remove supernatant, add 400 μ L demarcate tracer reagent B1,
200 μ L demarcate tracer reagent B2, and are transferred in serum bottle at the bottom of aseptic 10ml width by mixing liquid immediately, add electronics and pass
Pass chain inhibitor C, lid is built, and the tight lucifuge of rapid Aluminium Foil Package；Room temperature shake 60min, speed is maintained at 280rpm；Nothing
Draw 10 μ L reactant liquors under collarium border to be spread evenly across on gelatin coating microscope slide, and lucifuge air-dries in gnotobasis；
Microscope is observed: dried microscope slide is placed in fluorescence microscope, and (Lycra DM6000B is equipped with Lycra DFC500
Digital camera) on object lens platform, 100 × object lens under carry out observing and taking pictures exciting block to observe by green fluorescence, starch drops
Solve antibacterial and present green fluorescence.
Wherein, described gelatin smear processes according to the following steps:
Take 7 carry sheet glass shelf, microscope slide is filled in carry sheet glass shelf；In 3.5L distilled water, add 4-5 drip detergent,
Put into carry sheet glass shelf, boil 10min；Taking out carry sheet glass shelf, dry in the air 3min；With distilled water, detergent is rinsed well, by microscope slide
The state of being kept upright is dried；
In 3.5L distilled water, add ten sulfate dihydrate potassium chromium and the gelatin of 0.44g of 2.5g, be heated to about 60 DEG C, treat
Gelatin puts into carry sheet glass shelf, constant temperature 10min after substantially dissolving；Take out carry sheet glass shelf, microscope slide is tiltedly stood on test tube rack limit dry
Dry and get final product.
Wherein, described sample processing reagent A is sterilizing 1 × PBS, pH=9.0.
Wherein, described demarcation tracer reagent B1, B2 be respectively sterilizing Tris-HCl buffer (10mmol/L, pH 7.8) and
The green fluorescence starch dye of sterilizing BODIPY (boron fluoride two pyroles fluorescent dye, boron-dipyrromethene) labelling
Liquid.
Wherein, described electron transport chain inhibitor C be total amount be the sterilizing 3mmol/L Hydrazoic acid,sodium salt of 0.6ml, 2mmol/L
1080 (Monsanto), 4mmol/L iodo-acetamide sodium mixed solution.
Embodiment three: a kind of method detecting Starch in Tobacco degrading microorganism, step is as follows:
Sample collection: wear sterile gloves, use disposable sterile collection bag random collecting 1kg baking or ageing process
In tobacco sample, and be stored under 25 DEG C of constant temperatures and send laboratory to carry out sample treatment in 20min；
Sample treatment: at laboratory first with concentration 70% alcohol disinfecting shears, under aseptic condition, by tobacco sample with disappearing
The shears that poison is crossed is cut into 1-4cm2Fritter, and the unwanted tissues such as stem and vein of carefully removing Nicotiana tabacum L.；
Dilute sample: under aseptic condition, weighs the fresh broken leaf sample of 50g with aseptic 50ml beaker, adds 1000ml
Sample processing reagent A, uniform with broken leaf sample mix；
Cell separation: the liquid of mixing is poured into the most respectively the stirring of Philip (PHILIPS) domestic stirrer and smashes,
Each 5 minutes, the mixing liquid after then smashing three times was transferred in aseptic BioRad homogenate bag, in BioRad homogenizer 3
Shelves homogenate 5min, by homogenate bag side mistakes filter grid filtration, by filtrate collection in aseptic 50ml centrifuge tube, 5000g from
Heart 10min；Remove supernatant, and precipitate is put in 2ml centrifuge tube, be resuspended in 2ml sample processing reagent A；
Ultrasonic Treatment: the centrifuge tube that will be equipped with mixing liquid is fixed, and is then placed in ultrasonic instrument, is guaranteeing super
On the premise of during sonicated, centrifuge tube will not move, take out after 120 minutes stand-by by the frequency processing of 80000Hz.Waiting
In next step experimentation, cell suspending liquid can be stored in the refrigerator of 4 DEG C；
The demarcation of starch degradation microorganism and spike: take 400 μ L cell suspending liquid (obtaining from previous step) and be transferred to nothing
In the 2mL eppendof centrifuge tube of bacterium, centrifugal (5000g) 12 minutes, remove supernatant, add 400 μ L and demarcate tracer reagent
B1,200 μ L demarcate tracer reagent B2, and are transferred in serum bottle at the bottom of aseptic 10ml width by mixing liquid immediately, add electricity
Sub-transfer chain inhibitor C, builds lid, and the tight lucifuge of rapid Aluminium Foil Package；Room temperature shake 90min, speed is maintained at
300rpm；Draw 10 μ L reactant liquors under gnotobasis to be spread evenly across on gelatin coating microscope slide, and lucifuge in gnotobasis
Air-dry；
Microscope is observed: dried microscope slide is placed in fluorescence microscope, and (Lycra DM6000B is equipped with Lycra DFC500
Digital camera) on object lens platform, 100 × object lens under carry out observing and taking pictures exciting block to observe by green fluorescence, starch drops
Solve antibacterial and present green fluorescence.
Wherein, described gelatin smear processes according to the following steps:
Take 7 carry sheet glass shelf, microscope slide is filled in carry sheet glass shelf；In 3.5L distilled water, add 4-5 drip detergent,
Put into carry sheet glass shelf, boil 10min；Taking out carry sheet glass shelf, dry in the air 3min；With distilled water, detergent is rinsed well, by microscope slide
The state of being kept upright is dried；
In 3.5L distilled water, add ten sulfate dihydrate potassium chromium and the gelatin of 0.44g of 2.5g, be heated to about 80 DEG C, treat
Gelatin puts into carry sheet glass shelf, constant temperature 10min after substantially dissolving；Take out carry sheet glass shelf, microscope slide is tiltedly stood on test tube rack limit dry
Dry and get final product.
Wherein, described sample processing reagent A is sterilizing 1 × PBS, pH=5.0.
Wherein, described demarcation tracer reagent B1, B2 be respectively sterilizing Tris-HCl buffer (10mmol/L, pH 7.8) and
The green fluorescence starch dye of sterilizing BODIPY (boron fluoride two pyroles fluorescent dye, boron-dipyrromethene) labelling
Liquid.
Wherein, described electron transport chain inhibitor C be total amount be the sterilizing 3mmol/L Hydrazoic acid,sodium salt of 0.6ml, 2mmol/L
1080 (Monsanto), 4mmol/L iodo-acetamide sodium mixed solution.
Below it is only the concrete exemplary applications of the present invention, protection scope of the present invention is not constituted any limitation.All employings
The technical scheme that equivalents or equivalence are replaced and formed, within the scope of all falling within rights protection of the present invention.
Claims (6)
1. the method detecting Starch in Tobacco degrading microorganism, it is characterised in that: comprise the following steps:
(1) sample collecting: wear sterile gloves and use in the baking of disposable sterile collection bag random collecting or ageing process
Tobacco sample, and be stored under 18-40 degree constant temperature in 20min, carry out sample treatment；
(2) sample treatment: with concentration 70% alcohol disinfecting shears, aseptically, by the tobacco sample shears sterilized
It is cut into some fritters, and the unwanted tissues such as stem and vein of carefully removing Nicotiana tabacum L.；
(3) dilute sample: under aseptic condition, weighs fresh broken leaf sample, joins pH4.0-9.0 sterilizing 1 × PBS
In, buffer consumption is 10-30 times of broken leaf sample quality, uniform with broken leaf sample mix；
(4) cell separation: the mixing liquid that step (3) obtains being poured into blender stirring the most respectively and smashes, each 1-5 divides
Clock, pours the homogenate of blender top gear into, filters, filtrate be centrifuged and remove supernatant after the liquid mixing after then smashing three times
Liquid, is resuspended in the precipitate removing supernatant equipped with in the centrifuge tube that pH4.0-9.0 is sterilizing 1 × PBS, obtains
To cell suspending liquid；
(5) ultrasonic Treatment: being put into by centrifuge tube in ultrasonic instrument fixing, centrifuge tube will not move when guaranteeing ultrasonic Treatment
On the premise of Dong, stand-by with taking out after frequency processing 50-140 minute of 10000-80000Hz；Waiting next step process
Cheng Zhong, the centrifuge tube that will be equipped with cell suspending liquid is stored in the refrigerator of 4 DEG C；
(6) demarcation of starch degradation microorganism and spike: process by centrifugation and remove in the cell suspending liquid of supernatant, add successively
Enter 1 × Tris-HCl buffer and the sterilizing boron fluoride two pyroles fluorescent dye of 6-15mmol/L, pH 5.1-9.0 sterilizing
The green fluorescence starch dye liquor of BODIPY labelling, forms mixing liquid, and immediately mixing liquid is transferred to aseptic blood of the wide end
In clear bottle, add sterilizing electron transmission chain inhibitor, lid is built, by the tight lucifuge of Aluminium Foil Package；Room temperature shake 30-120min,
Speed is maintained at 100-500rpm and cultivates；The reactant liquor that under gnotobasis, absorption 10-50 μ L cultivation obtains is spread evenly across
On gelatin coating microscope slide, and lucifuge air-dries in gnotobasis；Described electron transmission chain inhibitor is 1-10mmol/L nitrine
Change sodium, 1-10mmol/L 1080 (Monsanto) and the mixed solution of 1-10mmol/L iodo-acetamide sodium composition；
(7) microscope observe: dried microscope slide is placed on fluorescence microscopy endoscope objective lens platform, 100 × object lens under, by green
Color fluorescence excitation block is observed, and presents green fluorescence and is starch degradation antibacterial.
The method detecting Starch in Tobacco degrading microorganism the most according to claim 1, it is characterised in that: described gelatin coating
Microscope slide processes as follows:
(1) take 7 carry sheet glass shelf, microscope slide is filled in carry sheet glass shelf；In 3.5L distilled water, add 4-5 drip detergent,
Put into carry sheet glass shelf, boil 10min；Taking out carry sheet glass shelf, dry in the air 3min；With distilled water, detergent is rinsed well, by microscope slide
The state of being kept upright is dried；
(2) in 3.5L distilled water, add ten sulfate dihydrate potassium chromium and the gelatin of 0.44g of 2.5g, be heated to 60-80 DEG C, treat bright
Carry sheet glass shelf, constant temperature 10min is put into after this dissolving of gum base；Take out carry sheet glass shelf, microscope slide is tiltedly stood on and is dried on test tube rack limit
Obtain.
The method detecting Starch in Tobacco degrading microorganism the most according to claim 1, it is characterised in that: step (4) is described
The liquid of mixing is poured into domestic stirrer stirring the most respectively smash, each 3 minutes, the mixing after then smashing three times
Liquid is transferred in aseptic BioRad homogenate bag, is homogenized 3min BioRad homogenizer 3 grades, by being homogenized the mistake filter grid of bag side
Filtering, by filtrate collection in aseptic 50ml centrifuge tube, 5000g is centrifuged 8min and removes supernatant, and precipitate is put into 2ml
In centrifuge tube, it is resuspended in equipped with in the sterilizing 1 × PBS of pH4.0-9.0.
The method detecting Starch in Tobacco degrading microorganism the most according to claim 1, it is characterised in that: step (4) is described
Centrifugal treating specifically 4500g-5000g be centrifuged 5-10min.
The method detecting Starch in Tobacco degrading microorganism the most according to claim 1, it is characterised in that: step (6) is described
Centrifugal treating specifically 4500g-5000g be centrifuged 8-12min.
The method detecting Starch in Tobacco degrading microorganism the most according to claim 1, it is characterised in that: step (6) is used
The volume of 1 × Tris-HCl buffer identical with the Cell suspension volumes taken；The green of BODIPY labelling used is glimmering
Volume and 1 × Tris-HCl buffer volume of light dye liquor are identical；The volume of electron transmission chain inhibitor used and 1 ×
Tris-HCl buffer volume is identical.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610505577.7A CN106191209B (en) | 2016-06-30 | 2016-06-30 | A method of detection Starch in Tobacco degrading microorganism |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610505577.7A CN106191209B (en) | 2016-06-30 | 2016-06-30 | A method of detection Starch in Tobacco degrading microorganism |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106191209A true CN106191209A (en) | 2016-12-07 |
| CN106191209B CN106191209B (en) | 2019-10-18 |
Family
ID=57462846
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610505577.7A Active CN106191209B (en) | 2016-06-30 | 2016-06-30 | A method of detection Starch in Tobacco degrading microorganism |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106191209B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108130360A (en) * | 2017-12-14 | 2018-06-08 | 孔云虹 | A kind of method of quick detection broiler chicken caecum starch degradation bacterium |
| CN109401975A (en) * | 2018-11-28 | 2019-03-01 | 云南中烟工业有限责任公司 | A method of collecting ageing tobacco leaf surface microorganism |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102559548A (en) * | 2011-12-22 | 2012-07-11 | 红塔烟草(集团)有限责任公司 | Diastase high-producing strain and method for improving quality of low-grade tobacco through same |
-
2016
- 2016-06-30 CN CN201610505577.7A patent/CN106191209B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102559548A (en) * | 2011-12-22 | 2012-07-11 | 红塔烟草(集团)有限责任公司 | Diastase high-producing strain and method for improving quality of low-grade tobacco through same |
Non-Patent Citations (2)
| Title |
|---|
| YUN XIA ET AL.: "In situ identiication and quantication of starch-hydrolyzing bacteria attached to barley and corn grain in the rumen of cows fed barley-based diets", 《FEMS MICROBIOLOGY ECOLOGY》 * |
| 陈瑞铭主编: "《动物组织培养技术及其应用》", 30 September 1998 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108130360A (en) * | 2017-12-14 | 2018-06-08 | 孔云虹 | A kind of method of quick detection broiler chicken caecum starch degradation bacterium |
| CN109401975A (en) * | 2018-11-28 | 2019-03-01 | 云南中烟工业有限责任公司 | A method of collecting ageing tobacco leaf surface microorganism |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106191209B (en) | 2019-10-18 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CA2910287C (en) | Sporoderm-breaking method for ganoderma spore powder and products obtained by the same | |
| CN104351901B (en) | A kind of probiotics fermention chlorella drink and preparation method thereof | |
| CN102217786B (en) | Method for preparing tobacco stem cellulose through microbial solid fermentation process | |
| CN104560891A (en) | Screening method of pseudomonas solanacearum bacteriophage resource | |
| CN103952317B (en) | Esterified red yeast strain and its production technology | |
| CN106191209B (en) | A method of detection Starch in Tobacco degrading microorganism | |
| CN107150390A (en) | A kind of sofening treatment method of wicker | |
| CN103243013B (en) | Low temperature gelatinization and saccharification and alcoholic fermentation technology and process for mature vinegar | |
| CN103315058B (en) | Application of leuconostoc mesenteroides strain | |
| CN101849613A (en) | Biological detoxification technology for rape-seed meal | |
| CN105104986B (en) | A kind of preparation method of bitter buckwheat red yeast rice | |
| CN108486002A (en) | The Siraitia grosvenorii endophyte bacterial strain of one plant of extracellular polysaccharide and its produce exocellular polysaccharide method and exocellular polysaccharide application | |
| CN107586727A (en) | A kind of fermentation process of ganoderma lucidum mycelium | |
| CN108102983B (en) | Lactobacillus plantarum for high yield of amylase and application thereof | |
| Archibong et al. | Ethanol production from cassava wastes (pulp and peel) using alcohol tolerant yeast isolated from palm wine | |
| CN105505799A (en) | Fermentation method and application of trichoderma viride mutant strains | |
| CN108546719A (en) | Composite enzyme deodorant and its preparation method and application | |
| KR20150084660A (en) | Method for preparing fermented Gastrodia elata BL using plant originated lactic acid bacteria | |
| CN106497871A (en) | A kind of method for improving human amnion mesenchymal stem cell Osteoblast Differentiation efficiency and application | |
| CN101695338B (en) | Method for producing feed additive for reducing livestock cholesterol | |
| CN107496987A (en) | A kind of method for preparing self-bone grafting osteogenic formulation | |
| CN106676087B (en) | A kind of preparation method of proline restriction endonuclease | |
| CN110408546A (en) | A kind of Trichoderma viride solid fermentation process | |
| CN102115725B (en) | Preparation method of alga-lysing burden-alleviating compound biological agent | |
| JP5732274B2 (en) | Product obtained using moss and process for producing the same |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20190902 Address after: 650214 Kunming economic and Technological Development Zone, Yunnan Pu Road, No. 2 Applicant after: Kunming University Address before: 650118 Department of Life Science and Technology, Kunming University, No. 2 Puxin Road, Kunming Economic and Technological Development Zone, Yunnan Province Applicant before: Kong Yunhong |
|
| TA01 | Transfer of patent application right | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |