CN101948788A - Screening method for degumming strain and degumming strain screened by same - Google Patents
Screening method for degumming strain and degumming strain screened by same Download PDFInfo
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- CN101948788A CN101948788A CN 201010274633 CN201010274633A CN101948788A CN 101948788 A CN101948788 A CN 101948788A CN 201010274633 CN201010274633 CN 201010274633 CN 201010274633 A CN201010274633 A CN 201010274633A CN 101948788 A CN101948788 A CN 101948788A
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Abstract
The invention relates to the microbial field, and in particular discloses a screening method for a degumming strain and the degumming strain screened by the same. The degumming strain is preserved at China Center for Type Culture Collection with a preservation number of CCTCC No. M 2010174. The degumming strain is screened and separated from rotten vegetable piles, has the advantages of short culture period, high degumming speed and high degumming rate and can be applied to the field of biological degumming . The screening method for the degumming strain screens by taking ramie juice as a unique organic nutrient component, so that the strain for decomposing the pectin component of the ramie can be quickly and repeatedly obtained without damaging the cellulose component of the ramie; and the strain has high degumming rate and industrial application.
Description
Technical field
The present invention relates to microorganism field, relate to a kind of screening method of degumming strain in particular, also relate to a kind of degumming strain that utilizes this method to filter out.
Background technology
Ramie, call white leaf ramie, perennial perennial root herbaceous plant, be China distinctive be the farm crop of main application with the weaving, its ultimate fibre is long, intensity is big, moisture absorption and diffusing wet fast, heat-conductive characteristic is good, and the back pure white mercerising that has that comes unstuck can purely spin, also can with blending such as cotton, silk, hair, chemical fibre, be important textile fibres crop.
Contain a large amount of impurity in the ramie, wherein based on the polysaccharide gelatinoid, require before the spinning most colloids in the bast are removed, and the ultimate fibre of ramie is separated from each other, this process is called comes unstuck.Traditional degumming technology adopts chemical degumming law, i.e. the Degumming method of caustic soda boiling, and this method is seriously polluted, energy consumption is high, fiber is had damage, no longer adapts to the requirement that modern industry is produced.
Along with the science and technology and modern textile industry development, a kind of new degumming technology has appearred, i.e. biological degumming.Biological degumming mainly is that the polygalacturonase that utilizes microorganisms, hemicellulase etc. decompose materials such as pectin in the plant phloem fiber, hemicellulose, but does not damage cellulosic structure, has the reaction conditions gentleness, saves water and energy, pollutes advantages such as few.Biological degumming of ramie generally comprises that enzyme comes unstuck and the bacterium dual mode that comes unstuck.It is that the enzyme that directly utilizes degummase preparation or degumming strain to produce acts on the ramie that enzyme comes unstuck, and utilizes the biological activity of enzyme, the colloidal complex of degradation of ramie fiber external parcel, thus fiber separation is come out; It is being inoculated on the raw ramie through the degumming strain that screens that bacterium is come unstuck, with the colloid on the ramie is nutrition source, allow degumming bacterium on raw ramie, breed in a large number, in the degumming bacterium reproductive process, secrete degummase and decompose colloid, make macromole such as high molecular weight pectin and hemicellulose resolve into small-molecule substance and soluble in water.But no matter which kind of biological degumming mode is all closely related with degumming bacterium, the characteristic of degumming bacterium directly affects the variation and the final degumming effect of the process of coming unstuck, and therefore in biological degumming, the screening of bacterial strain is most important link.
The report of the screening method of research degumming bacterium is more at present, mainly be two kinds of methods, first kind is to adopt pectin preparation substratum (to see that Wu Li is gorgeous etc., the separation of flax retting bacterium, screening and evaluation, Yunnan University's journal (natural science edition), 2007,29 (4): 419-423), this method can obtain the bacterial strain of energy decompose pectin quickly, but because do not have only pectin in the colloid of crudefiber crop, also have materials such as hemicellulose, therefore this method screening scope that can limit to degumming bacterium causes some degumming bacterium not obtain.Second method mainly is to adopt crudefiber crop directly to screen, and for example screens degumming strain with sisal leaves and (sees Chen Qing etc., the screening of sisal hemp bio-pulping degumming strain and evaluation and Preliminary Applications, Transactions of the Chinese Society of Agricultural Engineering, 2008,24 (6): 277-281); Employing with the ramie powder be the screening of carrying out degumming bacterium of the plate culture medium of sole carbon source (Chen Yangdong etc., ramie microbial degumming bacterial strain screening and degumming effect are estimated textile journal, 2010,31 (5): 69-72); (see Peng Yuan moral etc., the seed selection of quickly hemp degumming bacterial strain and the Performance Testing of coming unstuck, textile journal, 2007,28 (12): 65-68) with big straw screening degumming strain.It is all kinds of bacterial strains of nutrition that this method can screen with fiber crops, the bacterial strain that has comprised compositions such as energy decompose pectin, hemicellulose and Mierocrystalline cellulose, but the quality of cellulolytic bacterial strain meeting major injury bast-fibre in crudefiber crop is come unstuck, therefore the bacterial strain that is obtained by this method also not all is applicable to biological degumming.
At the problems referred to above, China has filtered out the bacterial strain that some are applicable to biological degumming at present, as a kind of degumming bacterium B.Subtilis A2-5 that announces among the Chinese patent CN85104285; A kind of bacterial strain B.Subtilis No.13 that produces phloem fibre degumming enzyme that Chinese patent CN1371990 announces; A kind of combined bacterial-chemical degumming technology that Chinese patent CN85103481 announces, this technology utilizes mutagenic strain subtilis T66 to carry out biological degumming.But, among the Chinese patent CN85104285 strain culturing cycle longer, need about 24h; Coming unstuck among the Chinese patent CN1371990 is consuming time long, and wherein the I and II seed culture time is 8-14h, produces enzymic fermentation time 12-18h, and carrying out former retting again after degummase liquid is suitably handled needs 2-6h; And the degumming rate among the Chinese patent CN85103481 only is about 69%.From foregoing as can be known, the domestic degumming strain that screens at present is more, but exist mostly because of the cultivation and fermentation time is long and cause long shortcoming of production cycle, and part bacterial strain degumming rate is lower, the speed of coming unstuck is slower, has restricted biological degumming application aborning.
Summary of the invention
In view of this, the object of the present invention is to provide a kind of screening purification process of degumming strain, this method is that unique organotrophy composition screens with ramie juice, can obtain to decompose the bacterial strain of ramie colloid composition fast.
Another object of the present invention is to provide a kind of degumming strain, and this strain culturing cycle is short, the speed of coming unstuck is fast.
For achieving the above object, the invention provides a kind of screening purification process of degumming strain, may further comprise the steps:
Step 1, the sample that will include pectin bacillus, genus bacillus or enterobacteria are inoculated into constant-temperature shaking culture in the enrichment medium, described enrichment medium comprises 3-10% ramie, 0.5-1.5% peptone, 0.1-0.5% extractum carnis, 0.3-1.0%NaCl, and all the other compositions are water;
Step 2, the culture after step 1 cultivated are coated on constant temperature culture on the primary dcreening operation substratum, and the bacterium colony that will produce the hydrolysis circle on the primary dcreening operation substratum carries out purifying, and described primary dcreening operation substratum comprises 5-10% ramie, 0.01-0.08%K
2HPO
4, 0.03-0.1%NH
4Cl and 1.5-2.5% agar, all the other compositions are water;
Step 3, the inoculation behind step 2 purifying is measured the bacterial strain degumming rate to sieving substratum constant-temperature shaking culture 15-24h again, degumming rate is the purpose bacterial strain that filters out greater than 80% bacterial strain, and the described substratum that sieves again comprises 8-15g raw ramie, 0.01-0.08%K
2HPO
4And 0.03-0.1%NH
4Cl, all the other compositions are water.
When sieving again, experimental group and control group are set, experimental group is that step 3 is described; Control group promptly sieves in the substratum not inoculating strain again, measures the gum content of control group and experimental group according to the method for surveying gum content among the standard GB 5889-86, is designated as G respectively
0And G
1, degumming rate is Gr.Gr=1-G by formula
1/ G
0Calculate degumming rate.
Wherein, the described cultivation of step 1 is for cultivating 24-48h in 35 ℃, the constant temperature shaking table of rotating speed 170rpm.
Wherein, the described cultivation of step 2 is for to cultivate 24-48h in 35 ℃ of constant temperature shaking tables.
Wherein, the described cultivation of step 3 is for cultivating in 35 ℃, the constant temperature shaking table of rotating speed 180rpm.
Preferably, described enrichment medium comprises 5% ramie, 1% peptone, 0.3% extractum carnis, 0.5%NaCl, and all the other compositions are water.
The preparation of enrichment medium: the 5g raw ramie is cut into segment long about 1cm, adding water heats on electric furnace, after waiting to seethe with excitement, use little fiery infusion instead, ramie deliquescing in cup is after solution becomes pale brown look, ramie is pulled out, add 0.3g extractum carnis, 1g peptone, 0.5g NaCl then, solution is settled to 100ml, prepares enrichment medium in a large number all with reference to this ratio.
Preferably, described primary dcreening operation substratum comprises 8% ramie juice, 0.03%K
2HPO
4, 0.05%NH
4Cl and 2% agar, all the other compositions are water.
The preparation of primary dcreening operation substratum: the 8g raw ramie is cut into segment long about 1cm, adds water and on electric furnace, heat, after waiting to seethe with excitement, use little fiery infusion instead, ramie deliquescing in cup, solution after becoming pale brown look is pulled ramie out, adds 0.03g K then
2HPO
4, 0.05gNH
4Cl, 2g agar, solution is settled to 100ml, prepares the primary dcreening operation substratum in a large number all with reference to this ratio.
Preferably, the described substratum that sieves again comprises 10g raw ramie, 0.03%K
2HPO
4And 0.05%NH
4Cl, all the other compositions are water.
Sieve the preparation of substratum again: add 0.03g K
2HPO
4, 0.05gNH
4Cl, solution is settled to 100ml, adds the 10g raw ramie then, and a large amount of preparations are sieved substratum again all with reference to this ratio.
Screening method of the present invention utilizes the main component of ramie juice for screening culture medium, ramie juice be raw ramie in boiling water during boiling ramie glue be shed in the aqueous solution and form, so the main component of ramie juice is all kinds of colloid materials in the ramie, cellulose not substantially.Therefore, utilizing with ramie juice is that unique organic substratum screens degumming strain, can overcome the problem of the plain and restriction degumming strain screening scope of institute's bacterial strain that sieve damage bast-fibre in the existing screening method, the efficient that the raising degumming strain screens.
The present invention also provides a kind of degumming strain, this bacterial strain utilizes screening method of the present invention screening and separating gained from the dish heap that rots, described degumming strain has been deposited in Chinese typical culture collection center on July 12nd, 2010, the address is that deposit number is CCTCCNO:M 2010174 in the Wuhan University of Lopa Nationality an ancient woman's ornament mountain, wuchang, wuhan.
Less than 4 or do not have bacterium colony greater than 10 o'clock and grow, the suitableeest growth pH value is 6.5-7.5 to degumming strain of the present invention in the medium pH value, and optimum growth temperature is 35 ℃-37 ℃, and this bacterial strain reproduction speed is fast, and 10-12h enters growth stationary phase.
Degumming strain hydrolyzed pectin of the present invention experiment is positive, and Mierocrystalline cellulose decomposes experiment and is negative, and shows that this bacterial strain can decompose pectin, but can not cause damage to Mierocrystalline cellulose; In the carbohydrate fermentation experiment, this bacterial strain can utilize Zulkovsky starch, glucose, lactose, sucrose and maltose to do the uniqueness carbon source for growth; In other Physiology and biochemistries detected, this bacterial strain catalase, VP measured and are positive, the energy hydrolyzed starch, and liquefy gelatin, urease-producing can not utilize propanedioic acid and Citrate trianion, does not produce fluorochrome, and the methyl red experiment is negative oxidable decomposition acetate.
The 16S rDNA sequence homology of the 16S rDNA sequence of degumming strain of the present invention and pectin bacillus shows that greater than 99% this bacterial strain belongs to Pectobacterium.Simultaneously, bacterial strain of the present invention is being inoculated into back 8h on the ramie, and degumming rate just reaches more than 90%.
Screening method of the present invention is that unique organotrophy composition screens with ramie juice, can obtain to decompose the bacterial strain of ramie colloid composition fast, does not damage the plain composition of ramee, and the degumming rate height of bacterial strain, has industrial applicability.In addition, degumming strain culture cycle of the present invention is short, and the speed of coming unstuck is fast, and the degumming rate height can be applied to the biological degumming field.
Biological preservation explanation
The classification name: pectin bacillus RJ6, Pectobacterium sp.RJ6. is deposited in Chinese typical culture collection center on July 12nd, 2010, and the address is Lopa Nationality an ancient woman's ornament mountain, a wuchang, wuhan Wuhan University, and deposit number is CCTCC NO:M 2010174.
Description of drawings
Fig. 1 shows degumming strain sem photograph of the present invention, and magnification is 8000 times.
Embodiment
The invention discloses a kind of screening method of degumming strain and the degumming strain that filters out thereof, those skilled in the art can use for reference this paper content, suitably improve processing parameter and realize.Special needs to be pointed out is that all similarly replace and change apparent to those skilled in the art, they all are regarded as being included in the present invention.Screening method of the present invention and degumming strain are described by preferred embodiment, the related personnel obviously can change or suitably change and combination methods and applications as herein described in not breaking away from content of the present invention, spirit and scope, realizes and use the technology of the present invention.
According to the present invention, a kind of screening method of degumming strain may further comprise the steps:
Step 1, the sample that will include pectin bacillus, genus bacillus or enterobacteria are inoculated into constant-temperature shaking culture in the enrichment medium, described enrichment medium comprises 3-10% ramie, 0.5-1.5% peptone, 0.1-0.5% extractum carnis, 0.3-1.0%NaCl, and all the other compositions are water;
Step 2, the culture after step 1 cultivated are coated on constant temperature culture on the primary dcreening operation substratum, and the bacterium colony that will produce the hydrolysis circle on the primary dcreening operation substratum carries out purifying, and described primary dcreening operation substratum comprises 5-10% ramie, 0.01-0.08%K
2HPO
4, 0.03-0.1%NH
4Cl and 1.5-2.5% agar, all the other compositions are water;
Step 3, the inoculation behind step 2 purifying is measured the bacterial strain degumming rate to sieving substratum constant-temperature shaking culture 15-24h again, degumming rate is the purpose bacterial strain that filters out greater than 80% bacterial strain, and the described substratum that sieves again comprises 8-15g raw ramie, 0.01-0.08%K
2HPO
4And 0.03-0.1%NH
4Cl, all the other compositions are water.
Wherein, the described cultivation of step 1 is for cultivating 24-48h in 35 ℃, the constant temperature shaking table of rotating speed 170rpm.
Wherein, the described cultivation of step 2 is for to cultivate 24-48h in 35 ℃ of constant temperature shaking tables.
Wherein, the described cultivation of step 3 is for cultivating in 35 ℃, the constant temperature shaking table of rotating speed 180rpm.
Preferably, described enrichment medium comprises 5% ramie, 1% peptone, 0.3% extractum carnis, 0.5%NaCl, and all the other compositions are water.
The preparation of enrichment medium: the 5g raw ramie is cut into segment long about 1cm, adding water heats on electric furnace, after waiting to seethe with excitement, use little fiery infusion instead, ramie deliquescing in cup is after solution becomes pale brown look, ramie is pulled out, add 0.3g extractum carnis, 1g peptone, 0.5g NaCl then, solution is settled to 100ml, prepares enrichment medium in a large number all with reference to this ratio.
Preferably, described primary dcreening operation substratum comprises 8% ramie juice, 0.03%K
2HPO
4, 0.05%NH
4Cl and 2% agar, all the other compositions are water.
The preparation of primary dcreening operation substratum: the 8g raw ramie is cut into segment long about 1cm, adds water and on electric furnace, heat, after waiting to seethe with excitement, use little fiery infusion instead, ramie deliquescing in cup, solution after becoming pale brown look is pulled ramie out, adds 0.03g K then
2HPO
4, 0.05gNH
4Cl, 2g agar, solution is settled to 100ml, prepares the primary dcreening operation substratum in a large number all with reference to this ratio.
Preferably, the described substratum that sieves again comprises 10g raw ramie, 0.03%K
2HPO
4And 0.05%NH
4Cl, all the other compositions are water.
Sieve the preparation of substratum again: add 0.03g K
2HPO
4, 0.05gNH
4Cl, solution is settled to 100ml, adds the 10g raw ramie then, and a large amount of preparations are sieved substratum again all with reference to this ratio.
Adopt screening method of the present invention can from the sample that includes pectin bacillus, enterobacteria or genus bacillus, filter out the bacterial strain that decomposes ramie colloid composition, bacterial strain is being inoculated into back 8-24h on the ramie, degumming rate shows that all greater than 80% present method has industrial applicability.
The present invention also provides a kind of degumming strain, and this bacterial strain utilizes screening method of the present invention screening and separating gained from the dish heap that rots.
Degumming strain of the present invention in the medium pH value less than 4 or do not have bacterium colony greater than 10 o'clock and grow, the suitableeest growth pH value is 6.5-7.5, and culture temperature has a small amount of bacterium colony to produce when being 50 ℃, and optimum growth temperature is 35 ℃-37 ℃, this bacterial strain reproduction speed is fast, and 10-12h enters the stationary phase of growing.
Degumming strain hydrolyzed pectin of the present invention experiment is positive, and Mierocrystalline cellulose decomposes experiment and is negative, and shows that this bacterial strain can decompose pectin, but can not cause damage to Mierocrystalline cellulose; In the carbohydrate fermentation experiment, this bacterial strain can utilize Zulkovsky starch, glucose, lactose, sucrose and maltose to do the uniqueness carbon source for growth; In other Physiology and biochemistries detected, this bacterial strain catalase, VP measured and are positive, the energy hydrolyzed starch, and liquefy gelatin, urease-producing can not utilize propanedioic acid and Citrate trianion, does not produce fluorochrome, and the methyl red experiment is negative oxidable decomposition acetate.
16S rDNA is that " generally acknowledging ruler " identified in present division bacteria, and method in common is that the 16S rDNA sequence of bacterial classification is compared with all sequences among the database Genbank, and general similarity is higher than can think a genus more than 98%.The 16S rDNA sequence homology of the 16S rDNA sequence of degumming strain of the present invention and pectin bacillus shows that greater than 99% this bacterial strain belongs to Pectobacterium.Simultaneously, bacterial strain of the present invention is being inoculated into back 8h on the ramie, and degumming rate reaches 91%, shows that degumming strain of the present invention has the very high efficient of coming unstuck.。
Below in conjunction with embodiment, further set forth the present invention.
Embodiment 1: utilize screening method of the present invention to screen degumming strain of the present invention
(1) enrichment culture:
Rotten dish sample (comprising multiple rotten vegetables such as Chinese cabbage, Radix Dauci Sativae, the tomato) adding of 1g is equipped with in the 200ml triangular flask of 20ml sterilized water, adds sterile glass beads, 20min vibrates in the 150rpm shaking table.Draw the solution after the 10ml vibration, add in the 100ml enrichment medium, place 35 ℃, the constant temperature shaking table of rotating speed 170rpm to cultivate 48h.Enrichment medium: 5% ramie, 1% peptone, 0.3% extractum carnis, 0.5%NaCl, all the other compositions are water.
The preparation of enrichment medium: the 5g raw ramie is cut into segment long about 1cm, adding water heats on electric furnace, after waiting to seethe with excitement, use little fiery infusion instead, ramie deliquescing in cup is after solution becomes pale brown look, ramie is pulled out, add 0.3g extractum carnis, 1g peptone, 0.5g NaCl then, solution is settled to 100ml, prepares enrichment medium in a large number all with reference to this ratio.
(2) primary dcreening operation:
With the dilution of the culture after the enrichment culture, being coated on ramie juice is on unique organic primary dcreening operation culture medium flat plate, cultivates 48h in 35 ℃ of constant incubators, will produce the result of the bacterium colony of hydrolysis circle as primary dcreening operation on the primary dcreening operation substratum.Primary dcreening operation substratum: ramie 8%, K
2HPO
40.03%, NH
4Cl 0.05%, agar 2%, all the other components are water.
The preparation of primary dcreening operation substratum: the 8g raw ramie is cut into segment long about 1cm, adds water and on electric furnace, heat, after waiting to seethe with excitement, use little fiery infusion instead, ramie deliquescing in cup, solution after becoming pale brown look is pulled ramie out, adds 0.03g K then
2HPO
4, 0.05gNH
4Cl, 2g agar, solution is settled to 100ml, prepares the primary dcreening operation substratum in a large number all with reference to this ratio.
(3) purifying:
With the bacterial strain that primary dcreening operation comes out, on the broth culture flat board, rule, place 35 ℃ of constant incubators to cultivate 48h, repeat streak culturely more than three times, examine under a microscope the single thalline of form as continuous several times and then think bacterial strain purifying.
(4) multiple sieve:
The bacterial strain of purifying is carried out China grass degumming, multiple sieve inoculation of medium bacterial strain after sterilization, shaking culture in 35 ℃, rotating speed 180rpm constant temperature shaking table, cultivate the degumming rate that 24h measures bacterial strain, degumming rate is the purpose bacterial strain that filters out greater than 80% bacterial strain, from degumming rate greater than the picking degumming rate is the highest 80% the bacterial strain a strain purpose bacterial strain as optimum.Sieve substratum again: raw ramie 10g, K
2HPO
40.03%, NH
4Cl 0.05%, and all the other components are water.
Sieve the preparation of substratum again: add 0.03g K
2HPO
4, 0.05gNH
4Cl, solution is settled to 100ml, adds the 10g raw ramie then, and a large amount of preparations are sieved substratum again all with reference to this ratio.
Embodiment 2: degumming strain 16S rDNA of the present invention sequential analysis
With the bacterial strain called after RJ6 that filters out among the embodiment 1, deliver to Chinese typical culture collection center and carry out preservation, deliver to the order-checking of Shanghai biotechnology company limited simultaneously, used sequenator is ABI-PRISM3730, sequencing reagent is BigDyeterminator v3.1, and gained 16S rDNA sequence is shown in SEQ ID NO:1.
The 16S rDNA sequence of RJ6 and all sequences in the GenBank database are compared, the 16S rDNA sequence homology of result's demonstration and pectin bacillus is greater than 99%, 16S rDNA is that " generally acknowledging ruler " identified in present division bacteria, general similarity is higher than can think a genus more than 98%, so degumming strain RJ6 of the present invention belongs to Pectobacterium.
Described degumming strain RJ6 has been deposited in Chinese typical culture collection center on July 12nd, 2010, the address is in the Wuhan University of Lopa Nationality an ancient woman's ornament mountain, wuchang, wuhan, deposit number is CCTCC NO:M2010174, classification name: pectin bacillus, Pectobacterium sp..
Embodiment 3: degumming strain morphological specificity of the present invention
With degumming strain RJ6 of the present invention on the organism agar plate under different pH values and culture temperature constant temperature culture 24-48h, the result show RJ6 in the medium pH value less than 4 or do not have bacterium colony greater than 10 o'clock and grow, the suitableeest growth pH value is 6.5-7.5, culture temperature has a small amount of bacterium colony to produce when being 50 ℃, optimum growth temperature is 35 ℃-37 ℃.Under optimum pH and culture temperature, this bacterial strain reproduction speed is fast, and 10-12h enters the stationary phase of growing.
Get degumming strain RJ6 bacterium sample of the present invention and carry out observing RJ6 cell growthhabit feature with scanning electron microscope after the respective handling, the result is referring to Fig. 1.The RJ6 morphological specificity is: the grand shape projection of bacterium colony circle, and oyster white or light yellow, opaque, smooth surface, moistening glossy, neat in edge, colony diameter reaches 3-5mm behind the cultivation 36h.The cell rod-short, the blunt circle in two ends, size is (0.4-0.5 μ m * 1.1-1.8 μ m), Gram-negative, amphitrichous, no gemma, not chromogenesis.
Embodiment 4: degumming strain physiological and biochemical property of the present invention
According to related contents such as " common bacteria system identification handbook " degumming strain RJ6 of the present invention is carried out the Physiology and biochemistry related assays, the result is referring to table 1.
Table 1 degumming strain RJ6 of the present invention Physiology and biochemistry detects
| Feature | The result | Feature | The result |
| V-P detects | + | Fluorochrome detects | - |
| The starch hydrolysis | + | The methyl red experiment | - |
| The acetate oxidation | - | Glucose utilization | + |
| The catalase experiment | + | Lactose utilization | + |
| Malonate utilization | + | Sucrose utilizes | + |
| Citrate trianion utilizes | + | Gelatine liquefication | + |
| The hydrolyzed pectin experiment | + | Maltose utilizes | + |
| Mierocrystalline cellulose decomposes experiment | - | The urase experiment | - |
The result shows that degumming strain RJ6 hydrolyzed pectin experiment of the present invention is positive, and Mierocrystalline cellulose decomposes experiment and is negative, and shows that this bacterial strain can decompose pectin, but can not cause damage to Mierocrystalline cellulose; In the carbohydrate fermentation experiment, this bacterial strain can utilize Zulkovsky starch, glucose, lactose, sucrose and maltose to do the uniqueness carbon source for growth; In other Physiology and biochemistries detected, this bacterial strain catalase, VP measured and are positive, the energy hydrolyzed starch, and liquefy gelatin, urease-producing can not utilize propanedioic acid and Citrate trianion, does not produce fluorochrome, and the methyl red experiment is negative oxidable decomposition acetate.
Embodiment 5: the degumming rate of degumming strain of the present invention detects
In experimental group, degumming strain RJ6 of the present invention is inoculated into is equipped with in the 60ml broth culture triangular flask, shaking culture 8h in 35 ℃, the constant temperature shaking table of rotating speed 170rpm, getting the access of 15ml bacterium liquid is equipped with in the triangular flask of 300ml tap water and 30g raw ramie, in 35 ℃, the constant temperature shaking table of rotating speed 170rpm, behind the shaking culture 8h, detect degumming rate.Control group is same treatment but inoculating strain RJ6 not.
According to the method mensuration control group of surveying gum content among the standard GB 5889-86 and the gum content of experimental group, be designated as G respectively
0And G
1, degumming rate is Gr.Gr=1-G by formula
1/ G
0Calculate degumming rate.Detecting the RJ6 degumming rate according to above method is 91%, wherein G
0Be 21.67%, G
1Be 1.95%.
The result shows that bacterial strain RJ6 of the present invention is being inoculated into back 8h on the ramie, and degumming rate just reaches more than 90%, show degumming strain RJ6 culture cycle of the present invention short (constant temperature vibration 8h promptly can be used for coming unstuck), the speed of coming unstuck is fast, and the degumming rate height can be applied to the biological degumming field.
Embodiment 6: screening method screening enterobacteria of the present invention
1, enterobacteria screening
(1) enrichment culture:
The sample adding that 1g is included enteroaerogen and enterobacter agglomerans is equipped with in the 200ml triangular flask of 20ml sterilized water, adds sterile glass beads, and 10min vibrates in the 150rpm shaking table.Draw the solution after the 10ml vibration, add in the 100ml enrichment medium, place 35 ℃, the constant temperature shaking table of rotating speed 170rpm to cultivate 24h.Enrichment medium: 3% ramie, 0.5% peptone, 0.1% extractum carnis, 0.3%NaCl, all the other compositions are water.
The preparation of enrichment medium: the 3g raw ramie is cut into segment long about 1cm, adding water heats on electric furnace, after waiting to seethe with excitement, use little fiery infusion instead, ramie deliquescing in cup is after solution becomes pale brown look, ramie is pulled out, add 0.1g extractum carnis, 0.5g peptone, 0.3g NaCl then, solution is settled to 100ml, prepares enrichment medium in a large number all with reference to this ratio.
(2) primary dcreening operation:
With the dilution of the culture after the enrichment culture, being coated on ramie juice is on unique organic primary dcreening operation culture medium flat plate, cultivates 24h in 35 ℃ of constant incubators, will produce the result of the bacterium colony of hydrolysis circle as primary dcreening operation on the primary dcreening operation substratum.Primary dcreening operation substratum: ramie 5%, K
2HPO
40.01%, NH
4Cl 0.03%, agar 1.5%, all the other components are water.
The preparation of primary dcreening operation substratum: the 5g raw ramie is cut into segment long about 1cm, adds water and on electric furnace, heat, after waiting to seethe with excitement, use little fiery infusion instead, ramie deliquescing in cup, solution after becoming pale brown look is pulled ramie out, adds 0.01g K then
2HPO
4, 0.03gNH
4Cl, 1.5g agar, solution is settled to 100ml, prepares the primary dcreening operation substratum in a large number all with reference to this ratio.
(3) purifying:
With the bacterial strain that primary dcreening operation comes out, on the broth culture flat board, rule, place 35 ℃ of constant incubators to cultivate 24h, repeat streak culturely more than three times, examine under a microscope the single thalline of form as continuous several times and then think bacterial strain purifying.
(4) multiple sieve:
The bacterial strain of purifying is carried out China grass degumming, multiple sieve inoculation of medium bacterial strain after sterilization, shaking culture in 35 ℃, rotating speed 180rpm constant temperature shaking table, cultivate the degumming rate that 24h measures bacterial strain, degumming rate is the purpose bacterial strain that filters out greater than 80% bacterial strain, from degumming rate greater than the picking degumming rate is the highest 80% the bacterial strain a strain purpose bacterial strain as optimum.Sieve substratum again: raw ramie 8g, K
2HPO
40.01%, NH
4Cl 0.03%, and all the other components are water.
Sieve the preparation of substratum again: add 0.01g K
2HPO
4, 0.03gNH
4Cl, solution is settled to 100ml, adds the 8g raw ramie then, and a large amount of preparations are sieved substratum again all with reference to this ratio.
2, sieving enterobacteria degumming rate and Physiology and biochemistry detects
Enterobacteria called after RJ4 with filtering out carries out the relevant physiological biochemistry detection, and is inoculated on the raw ramie according to the detection method of embodiment 5, behind the shaking culture 24h, detects degumming rate in 35 ℃, the constant temperature shaking table of rotating speed 170rpm.The result is referring to table 2.
Table 2RJ4 degumming rate and Physiology and biochemistry detect
The result shows that the enterobacteria RJ4 degumming rate that filters out is 80.24%, and the hydrolyzed pectin experiment is positive, and Mierocrystalline cellulose decomposes experiment and is negative, and shows this bacterial strain degumming rate height, does not damage cellulose components.
Embodiment 7: screening method screening genus bacillus of the present invention
1, genus bacillus screening
(1) enrichment culture:
The sample adding that 1g is included subtilis, bacillus polymyxa and bacillus licheniformis is equipped with in the 200ml triangular flask of 20ml sterilized water, adds sterile glass beads, and 15min vibrates in the 150rpm shaking table.Draw the solution after the 10ml vibration, add in the 100ml enrichment medium, place 35 ℃, the constant temperature shaking table of rotating speed 170rpm to cultivate 36h.Enrichment medium: 10% ramie, 1.5% peptone, 0.5% extractum carnis, 1.0%NaCl, all the other compositions are water.
The preparation of enrichment medium: the 10g raw ramie is cut into segment long about 1cm, adding water heats on electric furnace, after waiting to seethe with excitement, use little fiery infusion instead, ramie deliquescing in cup is after solution becomes pale brown look, ramie is pulled out, add 0.5g extractum carnis, 1.5g peptone, 1.0g NaCl then, solution is settled to 100ml, prepares enrichment medium in a large number all with reference to this ratio.
(2) primary dcreening operation:
With the dilution of the culture after the enrichment culture, being coated on ramie juice is on unique organic primary dcreening operation culture medium flat plate, cultivates 36h in 35 ℃ of constant incubators, will produce the result of the bacterium colony of hydrolysis circle as primary dcreening operation on the primary dcreening operation substratum.Primary dcreening operation substratum: ramie 10%, K
2HPO
40.08%, NH
4Cl 0.1%, agar 2.5%, all the other components are water.
The preparation of primary dcreening operation substratum: the 10g raw ramie is cut into segment long about 1cm, adds water and on electric furnace, heat, after waiting to seethe with excitement, use little fiery infusion instead, ramie deliquescing in cup, solution after becoming pale brown look is pulled ramie out, adds 0.08g K then
2HPO
4, 0.1gNH
4Cl, 2.5g agar, solution is settled to 100ml, prepares the primary dcreening operation substratum in a large number all with reference to this ratio.
(3) purifying:
With the bacterial strain that primary dcreening operation comes out, on the broth culture flat board, rule, place 35 ℃ of constant incubators to cultivate 36h, repeat streak culturely more than three times, examine under a microscope the single thalline of form as continuous several times and then think bacterial strain purifying.
(4) multiple sieve:
The bacterial strain of purifying is carried out China grass degumming, multiple sieve inoculation of medium bacterial strain after sterilization, shaking culture in 35 ℃, rotating speed 180rpm constant temperature shaking table, cultivate the degumming rate that 24h measures bacterial strain, degumming rate is the purpose bacterial strain that filters out greater than 80% bacterial strain, from degumming rate greater than the picking degumming rate is the highest 80% the bacterial strain a strain purpose bacterial strain as optimum.Sieve substratum again: raw ramie 15g, K
2HPO
40.08%, NH
4Cl 0.1%, and all the other components are water.
Sieve the preparation of substratum again: add 0.08g K
2HPO
4, 0.1gNH
4Cl, solution is settled to 100ml, adds the 15g raw ramie then, and a large amount of preparations are sieved substratum again all with reference to this ratio.
2, sieving genus bacillus degumming rate and Physiology and biochemistry detects
Genus bacillus called after RJ9 with filtering out carries out the relevant physiological biochemistry detection, and is inoculated on the raw ramie according to the detection method of embodiment 5, behind the shaking culture 24h, detects degumming rate in 35 ℃, the constant temperature shaking table of rotating speed 170rpm.The result is referring to table 2.
Table 2RJ9 degumming rate and Physiology and biochemistry detect
The result shows that the genus bacillus RJ9 degumming rate that filters out is 83.47%, and the hydrolyzed pectin experiment is positive, and Mierocrystalline cellulose decomposes experiment and is negative, and shows this bacterial strain degumming rate height, does not damage cellulose components.
The above only is a preferred implementation of the present invention; should be pointed out that for those skilled in the art, under the prerequisite that does not break away from the principle of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (9)
1. the screening method of a degumming strain may further comprise the steps:
Step 1, the sample that will include pectin bacillus, genus bacillus or enterobacteria are inoculated in the enrichment medium and cultivate, described enrichment medium comprises 3-10% ramie, 0.5-1.5% peptone, 0.1-0.5% extractum carnis, 0.3-1.0%NaCl, and all the other compositions are water;
Step 2, the culture after step 1 cultivated are coated on the primary dcreening operation substratum and cultivate, and the bacterium colony that will produce the hydrolysis circle on the primary dcreening operation substratum carries out purifying, and described primary dcreening operation substratum comprises 5-10% ramie, 0.01-0.08%K
2HPO
4, 0.03-0.1%NH
4Cl and 1.5-2.5% agar, all the other compositions are water;
Step 3, the inoculation behind step 2 purifying is measured the bacterial strain degumming rate to sieving culture medium culturing 15-24h again, degumming rate is the purpose bacterial strain that filters out greater than 80% bacterial strain, and the described substratum that sieves again comprises 8-15g raw ramie, 0.01-0.08%K
2HPO
4And 0.03-0.1%NH
4Cl, all the other compositions are water.
2. according to the described screening method of claim 1, it is characterized in that described enrichment medium comprises 5% ramie, 1% peptone, 0.3% extractum carnis, 0.5%NaCl, all the other compositions are water.
3. according to the described screening method of claim 1, it is characterized in that described primary dcreening operation substratum comprises 8% ramie, 0.03%K
2HPO
4, 0.05%NH
4Cl and 2% agar, all the other compositions are water.
4. according to the described screening method of claim 1, it is characterized in that the described substratum that sieves again comprises 10g raw ramie, 0.03%K
2HPO
4And 0.05%NH
4Cl, all the other compositions are water.
5. according to the described screening method of claim 1, it is characterized in that the described cultivation of step 1 is for cultivating 24-48h in 35 ℃, the constant temperature shaking table of rotating speed 170rpm.
6. according to the described screening method of claim 1, it is characterized in that the described cultivation of step 2 is for to cultivate 24-48h in 35 ℃ of constant temperature shaking tables.
7. according to the described screening method of claim 1, it is characterized in that the described cultivation of step 3 is for cultivating in 35 ℃, the constant temperature shaking table of rotating speed 180rpm.
8. a degumming strain is characterized in that, deposit number is CCTCC NO:M2010174.
9. the application of the described degumming strain of claim 8 in biological degumming.
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| CN102863116A (en) * | 2012-07-12 | 2013-01-09 | 湖南润久科技有限公司 | Method and device for repeatedly utilizing ramie biological degumming waste water |
| CN103074247A (en) * | 2012-09-17 | 2013-05-01 | 贾平 | Composite flora and textile fiber preparation method by using the same |
| CN105463587A (en) * | 2015-12-31 | 2016-04-06 | 华中科技大学 | Biological degumming method for ramie |
| CN105567592A (en) * | 2016-01-05 | 2016-05-11 | 天津科技大学 | Bacillus subtilis capable of simultaneously producing pectase and hemicellulase, screening method and applications thereof |
| CN106367350A (en) * | 2016-09-29 | 2017-02-01 | 中国农业科学院麻类研究所 | Long-term preservation and activation method of efficient degumming strain of ramie |
| CN107904186A (en) * | 2017-10-11 | 2018-04-13 | 中国农业科学院麻类研究所 | Bacterial strain and its crudefiber crop degumming tech available for crudefiber crop microbial degumming |
| CN113005058A (en) * | 2021-02-25 | 2021-06-22 | 阿勒泰戈宝茶股份有限公司 | Apocynum degumming biological preparation and preparation method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102863116A (en) * | 2012-07-12 | 2013-01-09 | 湖南润久科技有限公司 | Method and device for repeatedly utilizing ramie biological degumming waste water |
| CN103074247A (en) * | 2012-09-17 | 2013-05-01 | 贾平 | Composite flora and textile fiber preparation method by using the same |
| CN103074247B (en) * | 2012-09-17 | 2014-08-27 | 北京天安生物科技有限公司 | Composite flora and textile fiber preparation method by using the same |
| CN105463587A (en) * | 2015-12-31 | 2016-04-06 | 华中科技大学 | Biological degumming method for ramie |
| CN105463587B (en) * | 2015-12-31 | 2017-06-16 | 华中科技大学 | A kind of method of biological degumming of ramie |
| CN105567592A (en) * | 2016-01-05 | 2016-05-11 | 天津科技大学 | Bacillus subtilis capable of simultaneously producing pectase and hemicellulase, screening method and applications thereof |
| CN106367350A (en) * | 2016-09-29 | 2017-02-01 | 中国农业科学院麻类研究所 | Long-term preservation and activation method of efficient degumming strain of ramie |
| CN107904186A (en) * | 2017-10-11 | 2018-04-13 | 中国农业科学院麻类研究所 | Bacterial strain and its crudefiber crop degumming tech available for crudefiber crop microbial degumming |
| CN113005058A (en) * | 2021-02-25 | 2021-06-22 | 阿勒泰戈宝茶股份有限公司 | Apocynum degumming biological preparation and preparation method thereof |
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