CN102002467A - Bacillus cereus DA3 strain and preparation method and application thereof - Google Patents
Bacillus cereus DA3 strain and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to a bacillus cereus DA3 strain and a preparation method and application thereof. The strain has the capacity of producing pectinase and hemicellulase and has flax retting activity. The provided strain and a culture system can be directly used for flax retting and have the advantages that: the retting period is short, the fiber dispersion rate is 100 percent, the retting efficiency is 95 percent, and the strain is not easily polluted, and is low in treatment cost, high in fiber quality, mild in retting condition, high in pollution resistance, good in heat resistance, free of environmental pollution and the like. Compared with the prior art, the invention has the characteristics of simple technology, suitability for large-scale industrial production and the like, and has potential theoretical and practical significance for exploring flax retting of the bacillus cereus under the alkaline condition.
Description
Technical field
The invention belongs to bacillus cereus strain and Application Areas thereof, particularly a kind of bacillus cereus DA3 bacterial strain and preparation method and application.
Background technology
Flax (flax) is ancient phloem fiber crop and oil crops.Flax originates from the Near East, Mediterranean Sea bank.The Neolithic Age before more than 5000 years, the Switzerland lake perch people and ancient egypt people, linum usitatissimum is also with its fibrous woven dress material, and " mummy " of Egyptian various places also is to use the linen clad.Oil is called flax again with type flax.Flax has cultivation history in 1000 at least in China.Fibrous type flax is to introduce from Japan in 1906.China mainly is distributed in Heilungkiang and Jilin two provinces.Nice and cool, the moistening weather of flax happiness.Flax fiber has characteristics such as pulling force is strong, soft, fineness is good, conduction is weak, the suction aproll is fast, rate of expansion is big, but high-count yarn spun is made senior dress material.
Flax series product are subjected to liking of consumers in general always.Especially in some developed countries, flax product belongs to expensive goods, is the symbol of fashion and identity, is subjected to consumers in general's favor with the characteristic of its natural green environmental protection.Along with improving constantly of our national living standards of the people, the unique concept that people also more and more have a preference for flax and brought.
At present, generally adopt methods such as traditional flax warm water retting, rain and dew retted fibre and natural water retted fibre in the production, all utilize natural microbial and enzyme thereof that the colloids such as pectin, hemicellulose and xylogen of flax are degraded, bacterial strain mixes, be difficult to effectively control, (warm water retting is about 4d to cause usually time length; The rain and dew retted fibre is 15-20d; Natural water retted fibre 5-7d), comprehensive long flax rate is low, quality is unstable (bundle strength is low) and environmental pollution heavily waits problems, is seriously restricting greatly developing of flax industry.The biological fast degumming technology of flax is the inexorable trend of China's flax retting technical development, since nineteen seventies, the farsighted scientific worker of China's linen textile battle line has carried out unremitting effort with the brainstrust of biotechnology, and obtained certain achievement, especially enter after the nineties, have breakthrough at the research of polygalacturonase and hemicellulase and the flax microorganism dominant microflora technical elements that comes unstuck.Crudefiber crop institute of the Chinese Academy of Agricultural Sciences obtains a strain flax retting bacterium (bacillus cereus) by a large amount of tests, through the experiment draw, come unstuck finish after, pectin, hemicellulose and delignification rate are respectively 82.1%17.0% and 11.4%.Akin etc. have also obtained three kinds of bacterial classifications by screening, and test finds that they can both remove colloid, can partly the degrade lignified cell wall at center of wherein a kind of bacterium.Other two kinds of bacterium fiber finer cell wall of when coming unstuck, also having degraded, but because making to a series of requirements of bacterial strain, suitability for industrialized production all fails practice at present.So far, although done a large amount of research to carrying out flax retting with biological method both at home and abroad, simple enzymatic degumming can not obtain gratifying result, does not also reach the requirement of spinning, and is laboratory lab scale research.Great number cost and complicated technology have restricted the industrial application of biotechnology in the flax retting field, and the full biological degumming technology of flax comprehensively, successfully is applied to produce also has long road to walk.
Summary of the invention
Technical problem to be solved by this invention provides a kind of bacillus cereus DA3 bacterial strain and preparation method and application, the invention provides bacterial strain and culture systems and can be directly used in flax retting, have the cycle of coming unstuck weak point, the fiber dispersion rate reaches 100%, and the efficient of coming unstuck reaches 95%; Compared with prior art, the present invention has characteristics such as simple, the suitable large-scale industrial production of technology, carries out flax retting for the exploration bacillus cereus under alkaline condition and has potential theory and practice meaning.
A kind of bacillus cereus DA3 bacterial strain of the present invention (CGMCC No.3838) is characterized in that: this bacterial strain has the polygalacturonase of producing and hemicellulase ability, has the flax retting activity.
The preparation method of a kind of bacillus cereus DA3 bacterial strain of the present invention comprises:
(1) from the rotten sea grass of marine site, Zhoushan, East Sea sampling, earlier sea grass and enrichment medium are left standstill cultivation 30 days in ratio room temperature in enrichment medium of 1g: 20ml, get the 0.1ml enrichment medium then and coat isolation medium, 37 ℃ leave standstill and cultivated 1～4 day, obtain from the wild-type dominant strain that comes unstuck;
Wherein, the enrichment culture based formulas is: flax powder 5g, sterilized water 200ml, pH nature; The separation and Culture based formulas is: flax powder 5g, (NH
4)
2SO
45g, K
2HPO
41g, MgSO
40.5g, KCl 0.5g, FeSO
40.01, agar 20g, water 1000mL, with NaOH with pH regulator to 9;
(2) with the inoculation that obtains in the step (1) in the pectin nutritional medium, cultivate 72h for 28 ℃, carry disease germs after the formation, add 0.2% Congo red aqueous solution dyeing 4h, remove the Congo red solution of surface residue, it is Congo red to add a small amount of distillation pond thin laminar surface, to the promptly visible significantly magneta colour hydrolysis circle of light, magneta colour hydrolysis circle person occurs in periphery of bacterial colonies and be microbes of pectin discomposing (measure transparent circle diameter and colony diameter around the bacterium on the pectin agar plate, the hydrolysis circle generally has higher pectinase activity with the big person of ratio of colony diameter);
Microbes of pectin discomposing is inoculated in the hemicellulose nutritional medium then, cultivate 72h for 28 ℃, get final product water breakthrough Xie Quan (measure hydrolysis circle and lawn size, and calculate the ratio of the two size, the hydrolysis circle generally has higher hemicellulose enzyme activity with the big person of ratio of colony diameter);
(3) from above-mentioned substratum, select bacterial strain by the transparent circle method with product polygalacturonase and hemicellulase ability.Pectin nutritional medium in the described step (2), its component comprises:
Pectin 2g
NANO3 3g
K2HPO4 0.5g
MgSO4 1g
Agar 20g
Water 1000mL
The pH nature, 7.0-7.2
The hemicellulose nutritional medium, its component comprises:
Hemicellulose (self-control) 20g
NH4NO3 2g
K2HPO4 2g
MgSO4 0.2g
Yeast extract paste 5g
Agar 20g
Water 1000mL
pH 7.2。
The application of a kind of bacillus cereus DA3 bacterial strain of the present invention in flax retting comprises:
(1) bacillus cereus DA3 bacterial strain is stored in beef-protein medium, and 37 ℃ of 200rpm cultivate 24h, add frozen damping fluid;
(2) in the cooled 50mL nutrient broth medium of sterilization, insert bacterium one ring of above-mentioned slant culture 24h, shake-flask culture 24h under rotating speed 200r/min, 40 ℃ of conditions of temperature;
(3) culture medium inoculated behind the 50ml shake-flask culture is gone into flax fermention medium 5L, shake-flask culture 24h obtains zymocyte liquid under rotating speed 200r/min, 40 ℃ of conditions of temperature; Wherein, flax fermentative medium formula is: flax raw ramie 5g, KCl0.05g, dipotassium hydrogen phosphate 0.1g, MgSO
40.05g, FeSO
40.001g, (NH
4)
2SO
40.5g moisturizing is to 100ml constant volume (the 5L fermention medium is equal to increases each amounts of components);
In 4 ℃, 8000r/min is centrifugal with zymocyte liquid, gets supernatant liquor and is gained enzyme liquid, through the DNS colorimetric method for determining, the polygalacturonase enzyme activity is 242.96U/mL in the enzyme liquid then, and xylanase activity power is 354.67U/ml, it is at pH6.5-9.0, and enzyme below 50 ℃ is lived more stable;
(4) flax straw after will sterilizing mixes by bath raio with step 3 gained zymocyte liquid at 1: 20, at 40 ℃, comes unstuck respectively in the 200rpm shaking table 1～4 day, usually time is then removed come unglued liquid, boils 5min in boiling water bath,, stop to come unstuck for several times to remove the bacterium on flax with the tap water flushing.
Frozen damping fluid in the described step (1) is by potassium primary phosphate 0.0627g, dipotassium hydrogen phosphate 0.0177g, and Trisodium Citrate 0.0588g, bitter salt 0.02645g, glycerine 10ml adds water and is settled to 100ml and makes.
The application of a kind of bacillus cereus DA3 bacterial strain of the present invention in the linen thread and yarn pre-treatment comprises:
Step (1)～(3) are identical with claim 4;
(4) linen thread and yarn after will sterilizing mixes by bath raio with step 3 gained zymocyte liquid at 1: 20, at 40 ℃, comes unstuck respectively in the 200rpm shaking table 1～4 day, usually time is then removed come unglued liquid, boils 5min in boiling water bath,, stop to come unstuck for several times to remove the bacterium on flax with the tap water flushing; Wherein, linen thread and yarn is flax roving or second hards.
The application of a kind of bacillus cereus DA3 bacterial strain of the present invention in the sodolin kiering comprises:
Step (1)～(3) are identical with claim 4;
(4) sodolin after will sterilizing mixes by bath raio with step 3 gained zymocyte liquid at 1: 20, at 40 ℃, comes unstuck respectively in the 200rpm shaking table 1～4 day, usually time is then removed come unglued liquid, boils 5min in boiling water bath,, stop to come unstuck for several times to remove the bacterium on flax with the tap water flushing.
This bacterial strain is except that can being used for bast fiber degummings such as jute, hemp and fabric pretreatment thereof etc.
Bacillus cereus provided by the invention (Bacillus cereus) DA3 bacterial strain has been deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC) on May 17th, 2010, the address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, preserving number are CGMCC No.3838.According to 16sRDNA order-checking Blast and cluster analysis result, this bacterium and bacillus cereus similarity are 99%, and in conjunction with physiological and biochemical test result and microscopy analysis, naming this bacterial strain is bacillus cereus (Bacillus cereus) DA3 bacterial strain.
Bacillus cereus provided by the invention (Bacillus cereus) DA3 bacterial strain, feature is as follows: be ellipse or rod, cell dia＞1 μ m, the bacterium colony circle, projection, smooth surface is moistening, and neat in edge is the opaque shape of creamy white; Type of respiration is aerobic respiration, well-grown under aerobic conditions; Gram-positive, adnation flagellum, statospore ovalize (Fig. 1), hydrogen peroxide enzyme positive; In glucose, sucrose, wood-sugar fermentation test, can produce acid, but aerogenesis not; On citrate medium, produce alkali; This bacterial strain energy gelatin hydrolysate, starch and grease; The reduction litmus milk digests casein rapidly and grumeleuse is few; V-P measures positive; The indole reaction feminine gender; Methyl red is measured positive.
The DNA that extracts bacterial strain carries out pcr amplification, select bacterial 16 S rRNA gene universal primer for use, forward primer BSF8/20,5 '-AGAGTTTGATCCTGGCTAAG-3 ' (the E.coli correspondence position is 8～27), reverse primer BSR1541/20,5 '-AAGGAGGTGATCCAGCCG-3 ' (the E.coli correspondence position is 1541～1522); And then record bacterial strain 16S rRNA complete sequence, as follows:
AGAGTTTGATCCTGGCTCAGGACGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAGCGAACAGAAAAGGAGCTTGCTCCTTTGACGTTAGCGGCGGACGGGTGAGTAACACGTGGGCAACCTACCCTATAGTTTGGGATAACTCCGGGAAACCGGGGCTAATACCGAATAATCTCTTTTGCTTCATGGCAAGAGACTGAAAGACGGCATCTCGCTGTCGCTATAAGATGGGCCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACT?CCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGGCGAAAGCCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGTTTTCGGATCGTAAAACTCTGTTGTAAGGGAAGAACAAGTACAGTAGTAACTGGCTGTACCTTGACGGTACCTTATTAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGCGCGCGCAGGCGGTCCTTTAAGTCTGATGTGAAAGCCCACGGCTCAACCGTGGAGGGTCATTGGAAACTGGGGGACTTGAGTGCAGAAGAGGAAAGTGGAATTCCAAGTGTAGCGGTGAAATGCGTAGAGATTTGGAGGAACACCAGTGGCGAAGGCGACTTTCTGGTCTGTAACTGACGCTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCCGTTGACCACTGTAGAGATATAGTTTCCCCTTCGGGGGCAACGGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCATCATTTAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACGATACAAACGGTTGCCAACTCGCGAGAGGGAGCTAATCCGATAAAGTCGTTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGCCGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTTGGAGCCAGCCGCCGAAGGTGGGATAGATGATTGGGGTGAAGTCGTAACAAGGTAACC。
Carry out phylogeny cluster analysis (the 16S rRNA complete sequence of bacterial strain) with Molecular Evolutionary Genetics Analysis (version 3), obtain phyletic evolution tree shown in Figure 1.
Under the culture condition that makes up, this strain culturing condition is extensive, good heat resistance, and growth and breeding is fast, produces polygalacturonase and xylanase activity height, thereby can effectively remove the colloid in flax bast, and enzyme is alive stable, bacterium, the equal nontoxicity of enzyme.This bacterial strain and culture systems can be directly used in flax retting.
Beneficial effect
(1) the invention provides bacterial strain and culture systems, simultaneously high yield polygalacturonase and hemicellulase can be directly used in flax retting after testing, have the cycle of coming unstuck weak point, the fiber dispersion rate reaches 100%, and is respond well to flax retting, the efficient of coming unstuck reaches 95%, bacterial classification is difficult for contaminated, and processing cost is low, and fiber quality is good, the mild condition of coming unstuck, contamination resistance is strong, and resistance toheat is good, advantages such as non-environmental-pollution;
(2) the present invention has characteristics such as simple, the suitable large-scale industrial production of technology.Utilize this system to carry out flax retting and have the usually time weak point, the flax fiber scatter coefficient reaches 100%, and degumming rate reaches more than 90%, and the degummed ramie quality is good.
Description of drawings
Fig. 1 is that bacterial strain 16S rRNA sequential system is grown tree;
Fig. 2 is capsule stain photo (an oily mirror);
Fig. 3 is an electromicroscopic photograph;
Fig. 4 is fermentation example photo.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after the content of having read the present invention's instruction, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Embodiment 1
The screening of flax retting bacterial classification obtains
(1) from the rotten sea grass of marine site, Zhoushan, East Sea sampling, earlier sea grass and enrichment medium are left standstill cultivation 30 days in ratio room temperature in enrichment medium of 1g: 20ml, get the 0.1ml enrichment medium then and coat isolation medium, 37 ℃ leave standstill and cultivated 1～4 day, obtain from the wild-type dominant strain that comes unstuck;
Wherein, the enrichment culture based formulas is: flax powder 5g, sterilized water 200ml, pH nature; The separation and Culture based formulas is: flax powder 5g, (NH
4)
2SO
45g, K
2HPO
41g, MgSO
40.5g, KCl 0.5g, FeSO
40.01, agar 20g, water 1000mL, with NaOH with pH regulator to 9;
(2) bacterial strain that obtains in the step (1) is chosen 4 rings and be inoculated into (culture dish diameter 10cm) in the pectin nutritional medium, cultivate 72h for 28 ℃, carry disease germs after the formation, add 0.2% Congo red aqueous solution dyeing 4h, remove the Congo red solution of surface residue, it is Congo red to add a small amount of distillation pond thin laminar surface, to the promptly visible significantly magneta colour hydrolysis circle of light, magneta colour hydrolysis circle person occurs in periphery of bacterial colonies and be microbes of pectin discomposing (measure transparent circle diameter and colony diameter around the bacterium on the pectin agar plate, the hydrolysis circle generally has higher pectinase activity with the big person of ratio of colony diameter);
Then microbes of pectin discomposing is chosen 4 rings and be inoculated into (culture dish diameter 10cm) in the hemicellulose nutritional medium, cultivate 72h for 28 ℃, can (measure hydrolysis circle and lawn size by water breakthrough Xie Quan, and calculate the ratio of the two size, the hydrolysis circle generally has higher hemicellulose enzyme activity with the big person of ratio of colony diameter);
The pectin nutritional medium, its component comprises:
Pectin 2g
NANO3 3g
K2HPO4 0.5g
MgSO4 1g
Agar 20g
Water 1000mL
The pH nature, 7.0-7.2.
The hemicellulose nutritional medium, its component comprises:
Hemicellulose (self-control) 20g
NH4NO3 2g
K2HPO4 2g
MgSO4 0.2g
Yeast extract paste 5g
Agar 20g
(3) from above-mentioned substratum, select bacterial strain by the transparent circle method with product polygalacturonase and hemicellulase ability.
Embodiment 2
In order to make this bacterial strain can keep stable high yield characteristics, the slant preservation substratum uses beef-protein medium, culture medium raw material and preparation method are: extractum carnis 0.5g, peptone 1g, sodium-chlor 0.5g (pH7.4-7.6), add water to the 100ml constant volume, 37 ℃ of 200rpm overnight incubation are added frozen damping fluid 100ml; Wherein, frozen damping fluid is by potassium primary phosphate 0.0627g, dipotassium hydrogen phosphate 0.0177g, and Trisodium Citrate 0.0588g, bitter salt 0.02645g, glycerine 10ml adds water and is settled to 100ml and makes; With nutrient broth medium (sodium-chlor 0.5g adds water to the 100ml constant volume for extractum carnis 0.3g, peptone 1g, pH7.4-7.6) be sub-packed in the 250mL container, 50mL in each container, seal wrapping after, in 121 ℃ the sterilization 20min; Bacterium one ring of slant culture 24h, shake-flask culture 24h under rotating speed 200r/min, 40 ℃ of conditions of temperature are inserted in the cooling back.
Fermention medium used in the present invention is flax raw ramie 5g, KCl 0.05g, dipotassium hydrogen phosphate 0.1g, MgSO
40.05g, FeSO
40.001g, (NH
4)
2SO
40.5g; Fermention medium 5L is gone in inoculation behind the 50ml shake-flask culture, and shake-flask culture 24h obtains zymocyte liquid under rotating speed 200r/min, 40 ℃ of conditions of temperature; Again through high speed freezing centrifuge at 4 ℃, centrifugal bacterium liquid under the 8000r/min condition is got supernatant liquor and is gained degummase liquid.
The enzyme activity determination of degummase liquid
In order to measure the effective constituent of compound degummase liquid, polygalacturonase and xylanase activity have been measured respectively.Wherein adopt DNS colorimetric method for determining polygalacturonase to live, the enzyme activity determination substrate is respectively 0.25% pectin, 1% xylan (all preparing with the phosphoric acid buffer memory liquid of pH8).
Activity determination method is as follows:
(1) get the 25ml colorimetric cylinder, add substrate 1.8ml earlier, the fermented liquid supernatant liquid 0.2ml behind the 8000rpm that learns from else's experience again, 4 ℃ of centrifugal 15min, jumping a queue shakes up, and 50 ℃ of water bath heat preservation 30min take out;
(2) add 1.5mlDNS colour developing liquid and shaking up, put the 5min that develops the color in the boiling water immediately, take out the flowing water cooling.Adding distil water is diluted to scale, shakes up;
(3) the enzyme liquid of crossing with inactivation treatment compares, and usefulness UNICO VU2100 type spectrophotometer under 520nm and 540nm wavelength, is measured pectinase activity, zytase respectively.
The polygalacturonase enzyme activity is 240.62U/mL in the enzyme liquid, and xylanase activity power is 300.58U/ml, and it is at pH6.5-9.0, and enzyme below 50 ℃ is lived more stable.
Embodiment 3
Flax retting technology is as follows:
The 5g flax straw is inserted the 250ml triangular flask, be sub-packed at 1: 20 in the triangular flask by bath raio with flax straw and zymocyte liquid, liquid amount is 100ml, at 40 ℃, comes unstuck respectively in the 200rpm shaking table 1 day, 2 days and 4 days; Usually time is then removed come unglued liquid, boils 5min in boiling water bath,, stops to come unstuck for several times to remove the bacterium on flax with the tap water flushing.
Find that by the flax of coming unstuck is observed flax fiber has obvious dispersion after 1 day, fermented liquid darkens and becomes muddy; After coming unstuck 2 days originally flax of bunchy be dispersed into fully fibrous, the obvious retrogradation of fermented liquid; The flax retting rate reaches more than 90% after coming unstuck 4 days.
Embodiment 4
Flax retting technology is as follows:
Linen thread and yarn (flax roving or second hards) after the 5g sterilization is inserted the 250ml triangular flask, be sub-packed in the triangular flask with flax straw and zymocyte liquid at 1: 20 by bath raio, liquid amount is 100ml, at 40 ℃, comes unstuck respectively in the 200rpm shaking table 1 day, 2 days and 4 days; Usually time is then removed come unglued liquid, boils 5min in boiling water bath,, stops to come unstuck for several times to remove the bacterium on flax with the tap water flushing.
Find that by the flax of coming unstuck is observed flax fiber has obvious dispersion after 1 day, fermented liquid darkens and becomes muddy; After coming unstuck 2 days originally flax of bunchy be dispersed into fully fibrous, the obvious retrogradation of fermented liquid; The flax retting rate reaches more than 90% after coming unstuck 4 days.
Embodiment 5
Flax retting technology is as follows:
Sodolin after the 5g sterilization is inserted the 250ml triangular flask, be sub-packed at 1: 20 in the triangular flask by bath raio with flax straw and zymocyte liquid, liquid amount is 100ml, at 40 ℃, comes unstuck respectively in the 200rpm shaking table 1 day, 2 days and 4 days; Usually time is then removed come unglued liquid, boils 5min in boiling water bath,, stops to come unstuck for several times to remove the bacterium on flax with the tap water flushing.
Find that by the flax of coming unstuck is observed sodolin whiteness, pliability are significantly improved after 1 day, prodding and itching feeling significantly lowers.
Claims (9)
1. a bacillus cereus DA3 bacterial strain (CGMCC No.3838) is characterized in that: this bacterial strain has the polygalacturonase of producing and hemicellulase ability, has the flax retting activity, and its 16S rRNA sequence is as follows:
AGAGTTTGATCCTGGCTCAGGACGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAGCGAACAGAAAAGGAGCTTGCTCCTTTGACGTTAGCGGCGGACGGGTGAGTAACACGTGGGCAACCTACCCTATAGTTTGGGATAACTCCGGGAAACCGGGGCTAATACCGAATAATCTCTTTTGCTTCATGGCAAGAGACTGAAAGACGGCATCTCGCTGTCGCTATAAGATGGGCCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGGCGAAAGCCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGTTTTCGGATCGTAAAACTCTGTTGTAAGGGAAGAACAAGTACAGTAGTAACTGGCTGTACCTTGACGGTACCTTATTAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGCGCGCGCAGGCGGTCCTTTAAGTCTGATGTGAAAGCCCACGGCTCAACCGTGGAGGGTCATTGGAAACTGGGGGACTTGAGTGCAGAAGAGGAAAGTGGAATTCCAAGTGTAGCGGTGAAATGCGTAGAGATTTGGAGGAACACCAGTGGCGAAGGCGACTTTCTGGTCTGTAACTGACGCTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCCGTTGACCACTGTAGAGATATAGTTTCCCCTTCGGGGGCAACGGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCATCATTTAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACGATACAAACGGTTGCCAACTCGCGAGAGGGAGCTAATCCGATAAAGTCGTTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGCCGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTTGGAGCCAGCCGCCGAAGGTGGGATAGATGATTGGGGTGAAGTCGTAACAAGGTAACC。
2. the preparation method of a bacillus cereus DA3 bacterial strain comprises:
(1) from the rotten sea grass of marine site, Zhoushan, East Sea sampling, earlier sea grass and enrichment medium are left standstill cultivation 30 days in ratio room temperature in enrichment medium of 1g: 20ml, get the 0.1ml enrichment medium then and coat isolation medium, 37 ℃ leave standstill and cultivated 1～4 day, obtain from the wild-type dominant strain that comes unstuck;
Wherein, the enrichment culture based formulas is: flax powder 5g, sterilized water 200ml, pH nature; The separation and Culture based formulas is: flax powder 5g, (NH
4)
2SO
45g, K
2HPO
41g, MgSO
40.5g, KCl 0.5g, FeSO
40.01, agar 20g, water 1000mL, with NaOH with pH regulator to 9;
(2) with the inoculation that obtains in the step (1) in the pectin nutritional medium, cultivate 72h for 28 ℃, carry disease germs after the formation, add 0.2% Congo red aqueous solution dyeing 4h, remove the Congo red solution of surface residue, it is Congo red to add a small amount of distillation pond thin laminar surface, to the promptly visible significantly magneta colour hydrolysis circle of light, magneta colour hydrolysis circle person occurs in periphery of bacterial colonies and is microbes of pectin discomposing; Then microbes of pectin discomposing is inoculated in the hemicellulose nutritional medium, cultivates 72h for 28 ℃, get final product water breakthrough Xie Quan;
(3) from above-mentioned substratum, select bacterial strain by the transparent circle method with product polygalacturonase and hemicellulase ability.
3. the preparation method of a kind of bacillus cereus DA3 bacterial strain according to claim 2 is characterized in that: the pectin nutritional medium in the described step (2), and its component comprises:
Pectin 2g
NANO3 3g
K2HPO4 0.5g
MgSO4 1g
Agar 20g
Water 1000mL
The pH nature, 7.0-7.2
The hemicellulose nutritional medium, its component comprises:
Hemicellulose (self-control) 20g
NH4NO3 2g
K2HPO4 2g
MgSO4 0.2g
Yeast extract paste 5g
Agar 20g
Water 1000mL
pH 7.2。
4. the application of a bacillus cereus DA3 bacterial strain in flax retting comprises:
(1) bacillus cereus DA3 bacterial strain is stored in beef-protein medium, and 37 ℃ of 200rpm cultivate 24h, add frozen damping fluid;
(2) in the cooled 50 mL nutrient broth mediums of sterilization, insert bacterium one ring of above-mentioned slant culture 24 h, shake-flask culture 24 h under rotating speed 200 r/min, 40 ℃ of conditions of temperature;
(3) culture medium inoculated behind the 50ml shake-flask culture is gone into flax fermention medium 5L, shake-flask culture 24 h obtain zymocyte liquid under rotating speed 200 r/min, 40 ℃ of conditions of temperature; Wherein, flax fermentative medium formula is: flax raw ramie 5g, KCl0.05g, dipotassium hydrogen phosphate 0.1g, MgSO
40.05g, FeSO
40.001g, (NH
4)
2SO
40.5g moisturizing is to the 100ml constant volume;
(4) flax straw after will sterilizing mixes by bath raio with step 3 gained zymocyte liquid at 1: 20, at 40 ℃, comes unstuck respectively in the 200rpm shaking table 1～4 day, usually time is then removed come unglued liquid, boils 5min in boiling water bath,, stop to come unstuck for several times to remove the bacterium on flax with the tap water flushing.
5. the application of a kind of bacillus cereus DA3 bacterial strain according to claim 4 in flax retting, it is characterized in that: the frozen damping fluid in the described step (1) is by potassium primary phosphate 0.0627g, dipotassium hydrogen phosphate 0.0177g, Trisodium Citrate 0.0588g, bitter salt 0.02645g, glycerine 10ml adds water and is settled to 100ml and makes.
6. the application of a kind of bacillus cereus DA3 bacterial strain according to claim 4 in flax retting, it is characterized in that: with step (3) gained zymocyte liquid in 4 ℃, 8000 r/min are centrifugal, get supernatant liquor and be gained enzyme liquid, through the DNS colorimetric method for determining, the polygalacturonase enzyme activity is 242.96U/mL in the enzyme liquid then, and xylanase activity power is 354.67U/ml, it is at pH6.5-9.0, and enzyme below 50 ℃ is lived more stable.
7. the application of a bacillus cereus DA3 bacterial strain in the linen thread and yarn pre-treatment comprises:
Step (1)～(3) are identical with claim 4;
(4) linen thread and yarn after will sterilizing mixes by bath raio with step 3 gained zymocyte liquid at 1: 20, at 40 ℃, comes unstuck respectively in the 200rpm shaking table 1～4 day, usually time is then removed come unglued liquid, boils 5min in boiling water bath,, stop to come unstuck for several times to remove the bacterium on flax with the tap water flushing.
8. the application of a kind of bacillus cereus DA3 bacterial strain according to claim 7 in the linen thread and yarn pre-treatment is characterized in that: described linen thread and yarn is flax roving or second hards.
9. the application of a bacillus cereus DA3 bacterial strain in the sodolin kiering comprises:
Step (1)～(3) are identical with claim 4;
(4) sodolin after will sterilizing mixes by bath raio with step 3 gained zymocyte liquid at 1: 20, at 40 ℃, comes unstuck respectively in the 200rpm shaking table 1～4 day, usually time is then removed come unglued liquid, boils 5min in boiling water bath,, stop to come unstuck for several times to remove the bacterium on flax with the tap water flushing.
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