CN1374398A - Prepn and use of enzyme prepn for use in hemp degluing industrial process - Google Patents
Prepn and use of enzyme prepn for use in hemp degluing industrial process Download PDFInfo
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- CN1374398A CN1374398A CN 02110152 CN02110152A CN1374398A CN 1374398 A CN1374398 A CN 1374398A CN 02110152 CN02110152 CN 02110152 CN 02110152 A CN02110152 A CN 02110152A CN 1374398 A CN1374398 A CN 1374398A
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Abstract
The present invention relates to the field of microbial engineering technology. The enzyme preparation is prepared through the process including slant culture and storing, shaking culture, seeding tank culture and fermenting tank production of high enzyme activity bacteria strain. Bacillus subtilis of No.13 strain and preservation number CCTCC No.M200038 is selected. The hemp degumming process includes the fermentation of enzyme liquid filtering residue, diluting bacteria liquid to 3-6 times, regulating the pH value of fermented enzyme liquid in degluing tank to 7.5-9.0, hemp material pre-treatment, degumming with degumming liquid for 2-10 hr. The process of the present invention has no environmental pollution, and can produce hemp top with length, fineness and breaking strength superior to those of chemical process.
Description
(1) technical field
The present invention relates to a kind of preparation method and application thereof of the zymin of hemp degluing industrial efficiently, belong to the microbial engineering field.
(2) background technology
Hemp (Cannabis stative) is the annual woody plant of Cannabaceae Cannabis, is one of the most ancient in the world fibre crops.The major ingredient of its bast is Mierocrystalline cellulose, hemicellulose, pectin, xylogen and crude protein etc.Not only colloid composition complexity, and content height, particularly content of lignin are about 5～8 times of ramie, 2～3 times of flax.The generally long 7mm～50mm of hemp fibre, wide 14 μ m～17 μ m.Utilize modern comfort to carry out flax spinning is world-famous puzzle always.
For making big hemp spinning yarn become possibility, can only use its process of fiber, this colloid appropriateness that just should remove between fibrous bundle is decomposed the fibrous bundle of hemp, and it is too many to come unstuck again.Adopt enzymatic degumming that the combined action of several enzymes must be arranged, and can effectively control the degree of enzymic hydrolysis and improve carding technology, make the length of hemp fibre, the requirement that fineness meets textile technology.Mentioned with the No.13 bacterial strain in No. 001127440.9 patent application that Shandong University proposes on September calendar year 2001 26 and carried out the production of hemp degumming enzyme, but the concrete steps of not coming unstuck.
" transplanting " china grass degumming process always over surplus the hemp degumming ten year, its defective is: environmental pollution and degumming " living, ripe " are irregular, and row yielding is on the low side.Enzymatic degumming technology can make it obtain essence and improve.Once there was the microflora that utilizes on natural air microorganism species or the raw ramie to come unstuck in the prior art, but owing to bacterial classification mixes, the degumming effect instability, usually time reaches a couple of days, is difficult to industrialization.Patent 1089669 provides the enzymolysis method for degumming of hemp technology, and its content is the anaerobic bacteria flora growth that the control certain condition adheres to by hempen original ramie, to reach the purpose to hemp degumming.Clearly, this is still the natural retted fibre under the manual control condition, and effect is difficult to stablize, and industrial application is very difficult.
(3) summary of the invention
The present invention is directed to the deficiencies in the prior art, a kind of preparation method and application thereof of the zymin of hemp degluing industrial efficiently are provided.
The present invention selects subtilis No.13 bacterial strain for use, its biological nature: thalline is shaft-like, Gram-positive (G
+), the statospore near-end is given birth to, and sporocyst is less than or equal to thalline, spore ellipse, aerobic, hydrogen peroxide enzyme positive, to energy fermentation and acids such as glucose, fructose, sucrose, lactose, semi-lactosi are not produced acid, can hydrolyzed starch, well-grown in containing the meat soup of 7%NaCl.Identify that according to uncle's outstanding Bacteria Identification handbook (the 8th edition 1984 Science Presses) and pertinent literature this bacterium is subtilis No.13 (Bacillus subtilis).
By method known to those skilled in the art, gather pedotheque in the suburbs, Jinan, select to separate, carry out just, sieve again through shake flask fermentation, then through ultraviolet ray and processing such as lithium chloride complex mutation and protoplastis fusion, and according to degumming effect and enzymic activity height binomial standard, screen, obtain the bacterial strain of strain laboratory numbering No.13, on November 20th, 2000 this bacterium is submitted to Chinese typical culture collection, preserving number is CCTCC NO.M200038.
The preparation method of hemp degluing industrial zymin comprises slant culture and preservation, shake-flask culture, seed tank culture and the fermentor tank production of live high-enzyme kind.
The slant culture of live high-enzyme kind and preservation condition: potato is cultivated inclined-plane or beef peptone agar slant.PH7.2～7.4,100Pa sterilization 20～30 minutes is placed to the inclined-plane, with this bacterial classification of transfering loop streak inoculation, cultivates 24～48 hours, and preserves standby for 30 ℃～34 ℃.
Potato is cultivated the substratum on inclined-plane: 100 parts of weight percent 10～20% murphy juices, and 1.0～2.0 parts of sucrose, 0.2～0.4 part of pectin, 0.03～0.1 part of yeast extract paste, 1.5～1.8 parts in agar is weight part, down together.
The substratum of beef peptone agar slant: 0.3 part of extractum carnis, 0.5 part of peptone, 0.5 part of NaCl, 1.8～2.0 parts in agar, 100 parts in water.
The substratum of shake-flask culture: 3.0～5.0 parts in (1) wheat bran, 1.0～2.0 parts of Semen Maydis powder, (NH
4)
2SO
40.5～1.5 parts, K
2HPO
40.05～0.1 part, MgSO
47H
20.1～0.2 part of O, 0.02～0.05 part of yeast extract paste.Perhaps (2) extractum carnis is 0.3 part, 0.5 part of peptone, 0.5 part of NaCl, 0.5～1.0 part of sucrose, 100 parts in water.Preceding PH7.5～8.5 disappear.30～34 ℃ of culture condition, 180～220 rev/mins, 48～72 hours time.
After cultivating end, measure the enzyme activity of polygalacturonase (is substrate with the Sigma polygalacturonic acid), zytase (is substrate with the Sigma xylan), mannase (is substrate with the Sigma locust bean gum) by the SomogyiShi method.Enzyme activity unit is defined as under condition determination, discharges the i.e. enzyme activity unit of the required enzyme amount of 1 μ mol product by the substrate per minute, represents with μ/ml.
Seed tank culture can be divided I and II, and the substratum of producing with fermentor tank (three grades) is identical.Substratum: 3.0～5.0 parts in wheat bran, 1.0～2.0 parts of Semen Maydis powder, 0.5～1.5 part of soybean cake powder, (NH
4)
2SO
40.5～1.0 parts, K
2HPO
40.05～0.10 part, MgSO
47H
20.10～0.20 part of O, preceding PH7.5～8.5 disappear.I and II seed and enzymatic production control condition: tank pressure: 0.03Mpa, temperature: 32 ℃～34 ℃, air flow (volume of air m
3/ fermentating liquid volume m
3Minute) 1: 0.3～0.5, incubation time I and II seed is 8～14 hours, 12～18 hours fermentor tank time.
Fermentation production process is noted mensuration and record jar temperature, PH, a bacterium growth situation, is had or not assorted bacterium, enzyme situation of growth alive and the variation of carbon nitrogen metabolism etc.
The application of hemp degluing industrial zymin comprises and uses the said products that hemp is carried out enzymatic degumming.Hemp degumming common process step is raw ramie → cut root → bundle handle → softening → dress cage → precook → come unstuck → scutching → wash → dewater → shake up → oil → dewater → shake up → dry → softening → heap storage health, and it is as follows that the present invention carries out the enzymatic degumming concrete steps to hemp:
(1) the fermenting enzyme liquid of fermentative production is through Plate Filtration or centrifugal slagging-off, and 3～6 times of dilute with water enzyme liquid are put into glue kettle, and the PH that dilutes enzyme liquid is transferred to 7.5～9.0, and is stand-by as come unglued liquid.
(2) the raw ramie pre-treatment can be carried out routinely, cuts the 158～162mm of root place, pricks, and softening adopts to be done soft 1～2 time, and 80～100 ℃ of water loggings 1～1.5 hour prepare to come unstuck.
(3) come unglued liquid and raw ramie bath raio (10～20): 1 (volume L: weight KG), PH soldier 7.5～9.0, and the temperature of coming unstuck is controlled at 40 ℃～50 ℃, and it is static or dynamically that degummase liquid keeps, usually time 2～10 hours.Degummase liquid is because enzyme loss alive is not quite reusable.
Come unstuck fiber crops through washing fiber crops machine washing fiber crops, and subsequent handling is carried out routinely.
The hemp ramie stripes mean length 90～95mm (chemical method 86mm) that makes, average fineness 19dtex～20dtex (chemical method 14.29dtex), breaking tenacity 3.5～3.7g/D (chemical method is 3.12g/D).All can enough spin the pure hemp yarn of 8～24Nm and 32～80Nm hemp blending fiber crops without any handling.Compare with chemical method in addition, the present invention also has environmentally safe, improves advantages such as workman's labor condition.
(4) embodiment
Embodiment 1: the preparation method of hemp degluing industrial zymin
Degummase liquid is pressed the three grade fermemtation explained hereafter: first class seed pot 50L, secondary seed jar 500L, fermentor tank 10M
3
CCTCC No.M200038 bacterium slant strains is inoculated and is shaken in the bottle product enzyme substratum.Its preparation method is: 300ml shakes bottled product enzyme substratum 50ml and puts 32 ℃ ± 1 ℃, and 200 rev/mins of shaking tables were cultivated 72 hours.
Potato is cultivated the substratum on inclined-plane: 100 parts of weight percent 20% murphy juices, 1.5 parts of sucrose, 0.3 part of pectin, 0.05 part of yeast extract paste, 1.6 parts in agar.Be weight part, down together.
The substratum of shake-flask culture: 3.0 parts in (1) wheat bran, 1.0 parts of Semen Maydis powder, (NH
4)
2SO
41.0 part, K
2HPO
40.1 part, MgSO
47H
20.1 part of O, 0.03 part of yeast extract paste.Preceding PH8.0 disappears.
Jar is with producing the enzyme substratum: 5.0 parts in wheat bran, 2.0 parts of Semen Maydis powder, 1.0 parts of soybean cake powder, (NH
4)
2SO
41.0 part, K
2HPO
40.05 part, MgSO
47H
20.10 part of O, preceding PH8.0 disappears.Tinning volume 65%.Control condition: tank pressure: 0.03Mpa, temperature: 33 ℃ ± 1 ℃, air flow (volume of air m
3/ fermentating liquid volume m
3Minute) 1: 0.4, incubation time I and II seed is 10 hours, 16 hours fermentor tank time.
Put a jar standard: fermented liquid PH5.5, enzyme live and no longer rise pectinase activity 1000 μ/ml, Xylanase activity 60 μ/ml, mannosans enzyme activity 50 μ/ml.
Embodiment 2: the preparation method of hemp degluing industrial zymin.
As described in embodiment 1, different is:
The substratum on inclined-plane is the substratum of beef peptone agar slant: 0.3 part of extractum carnis, 0.5 part of peptone, NaCl0.5 part, 2.0 parts in agar, 100 parts in water.
The substratum of shake-flask culture: 0.3 part of extractum carnis, 0.5 part of peptone, 0.5 part of NaCl, 0.8 part of sucrose, 100 parts in water.
Degummase liquid is pressed first class seed pot and fermentor tank explained hereafter: first class seed pot 50L, fermentor tank 10M
3
Embodiment 3: the hemp degumming step is as follows: (1) after 5 times of water dilutions, transfers PH8.5 as embodiment 1 described degummase liquid.(2) hemp cuts root 150mm, pricks, adopts and does soft 2 times, soaks 1 hour in 90 ℃, the water of PH8.(3) (weight: volume), 40 ℃ of water bath heat preservations came unstuck PH8 6 hours to the bath raio of hemp and dilution back enzyme liquid by 1: 18.Every cage hemp dress 320Kg is divided into three cages, three crowdes of meter 960kg that come unstuck.
After taking out washing, dehydration is shaken up, and oils, and de-oiling is shaken up, oven dry, softening, heap storage health.Press chemical Degumming ramie stripes analysis test method test result: mean length 93mm, average fineness 19.23dtex, breaking tenacity 3.6g/D.
Embodiment 4: as described in embodiment 3, different is:
Every cage hemp dress 160Kg, altogether in five batches, meter 800Kg comes unstuck.
Fiber crops grain in the enzymatic degumming ramie stripes, fault such as intensive are almost nil, become sliver to do, and strength irregularity is better, end breakage rate descends more than 10% when weaving, because the enzymatic degumming fiber separation is even, moderate, noil reduces, and the combing row yielding improves 7～8 percentage points than the product of chemical method.
Claims (8)
1. the preparation method of a hemp degluing industrial zymin, comprise slant culture and preservation, shake-flask culture, seed tank culture and the fermentor tank production of live high-enzyme kind, it is characterized in that, select subtilis No.13 bacterial strain for use, preserving number is CCTCC NO.M200038, its biological nature: thalline is shaft-like, Gram-positive (G
+), the statospore near-end is given birth to, and sporocyst is less than or equal to thalline, spore ellipse, aerobic, hydrogen peroxide enzyme positive, to glucose, fructose, sucrose can fermentation and acid, lactose, semi-lactosi are not produced acid, can hydrolyzed starch, well-grown in containing the meat soup of 7%NaCl.
2. the preparation method of hemp degluing industrial zymin as claimed in claim 1, it is characterized in that, the slant culture of live high-enzyme kind and preservation condition: potato is cultivated inclined-plane or beef peptone agar slant, PH7.2～7.4,100Pa sterilization 20～30 minutes is placed to the inclined-plane, with this bacterial classification of transfering loop streak inoculation, cultivated 24～48 hours, and preserved standby for 30 ℃～34 ℃.
3. the preparation method of hemp degluing industrial zymin as claimed in claim 1, it is characterized in that, potato is cultivated the substratum on inclined-plane: 100 parts of weight percent 10～20% murphy juices, 1.0～2.0 parts of sucrose, 0.2～0.4 part of pectin, 0.03～0.1 part of yeast extract paste, 1.5～1.8 parts in agar is weight part.
4. the preparation method of hemp degluing industrial zymin as claimed in claim 1 is characterized in that, the substratum of beef extract-peptone agar slant: 0.3 part of extractum carnis, 0.5 part of peptone, 0.5 part of NaCl, 1.8～2.0 parts in agar, 100 parts in water is weight part.
5. the preparation method of hemp degluing industrial zymin as claimed in claim 1 is characterized in that, the substratum of shake-flask culture: 3.0～5.0 parts in (1) wheat bran, 1.0～2.0 parts of Semen Maydis powder, (NH
4)
2SO
40.5～1.5 parts, K
2HPO
40.05～0.1 part, MgSO
47H
20.1～0.2 part of O, 0.02～0.05 part of yeast extract paste; Perhaps (2) extractum carnis is 0.3 part, 0.5 part of peptone, 0.5 part of NaCl, 0.5～1.0 part of sucrose, 100 parts in water; Preceding PH7.5～8.5 disappear.30～34 ℃ of culture condition, 48～72 hours time, are weight part by 180～220 rev/mins.
6. the preparation method of hemp degluing industrial zymin as claimed in claim 1 is characterized in that seed tank culture is divided I and II, the substratum of producing with fermentor tank is identical, substratum: 3.0～5.0 parts in wheat bran, 1.0～2.0 parts of Semen Maydis powder, 0.5～1.5 part of soybean cake powder, (NH
4)
2SO
40.5～1.0 parts, K
2HPO
40.05～0.10 part, MgSO
47H
2O0.10～0.20 part, preceding PH7.5～8.5 disappear; I and II seed and enzymatic production control condition: tank pressure: 0.03Mpa, temperature: 32 ℃～34 ℃, air flow (volume of air m
3/ fermentating liquid volume m
3Minute) 1: 0.3～0.5, incubation time I and II seed is 8～14 hours, 12～18 hours fermentor tank time, is weight part.
7. the application of hemp degluing industrial zymin comprises that the product that uses claim 1～6 carries out enzymatic degumming to hemp.
8. the application of hemp degluing industrial zymin as claimed in claim 7, comprise step: raw ramie → cut root → bundle handle → softening → dress cage → precook → come unstuck → scutching → wash → dewater → shake up → oil → dewater → shake up → dry → softening → heap storage health is characterized in that concrete operations are as follows:
(1) the fermenting enzyme liquid of fermentative production is through Plate Filtration or centrifugal slagging-off, and 3～6 times of dilute with water enzyme liquid are put into glue kettle, and the PH that dilutes enzyme liquid is transferred to 7.5～9.0, and is stand-by as come unglued liquid;
(2) the raw ramie pre-treatment can be carried out routinely, cuts numbly from the 150～160mm of root place, pricks, and softening adopts to be done soft 1～2 time, and 80～100 ℃ of water loggings 1～1.5 hour prepare to come unstuck;
(3) come unglued liquid and raw ramie bath raio (10～20): 1 (volume L: weight KG), PH7.5～9.0, the temperature of coming unstuck is controlled at 40 ℃～50 ℃, and it is static or dynamic that degummase liquid keeps, usually time 2～10 hours.
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|---|---|---|---|
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| CN1188513C CN1188513C (en) | 2005-02-09 |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101187066B (en) * | 2007-09-27 | 2010-06-09 | 江苏紫荆花纺织科技股份有限公司 | Method for degelatinizing jute applying composite enzyme |
| CN101838856A (en) * | 2010-05-14 | 2010-09-22 | 华中科技大学 | Online ramie biological degumming method |
| CN101575817B (en) * | 2009-06-15 | 2011-05-04 | 大连工业大学 | Method for degumming mulberry bast fiber microbe in solid state |
| CN104250856A (en) * | 2014-07-14 | 2014-12-31 | 东华大学 | Preparation method of hibiscus cannabinus fiber by using lysine bacillus DY2 strain for retting |
| CN104630908A (en) * | 2015-02-15 | 2015-05-20 | 东华大学 | Method for preparing flax fiber by combined process of mucor circinelloides DK1 strain and hydrogen peroxide |
| CN104630910A (en) * | 2015-02-15 | 2015-05-20 | 东华大学 | Method for preparing flax fiber by combined process of candida tropicalis DK2 strain and hydrogen peroxide |
| CN104630909A (en) * | 2015-02-15 | 2015-05-20 | 东华大学 | Method for preparing flax fiber by ultrasonic combination of mucor circinelloides DK1 strain and hydrogen peroxide |
| 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 |
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| CN101713148B (en) * | 2009-05-13 | 2011-11-02 | 上海龙之杰企业发展有限公司 | Process for pre-treating biological enzymes of ramie yarn and bast fiber cloth |
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101187066B (en) * | 2007-09-27 | 2010-06-09 | 江苏紫荆花纺织科技股份有限公司 | Method for degelatinizing jute applying composite enzyme |
| CN101575817B (en) * | 2009-06-15 | 2011-05-04 | 大连工业大学 | Method for degumming mulberry bast fiber microbe in solid state |
| CN101838856A (en) * | 2010-05-14 | 2010-09-22 | 华中科技大学 | Online ramie biological degumming method |
| CN104250856A (en) * | 2014-07-14 | 2014-12-31 | 东华大学 | Preparation method of hibiscus cannabinus fiber by using lysine bacillus DY2 strain for retting |
| CN104250856B (en) * | 2014-07-14 | 2016-06-08 | 东华大学 | A kind of method utilizing Methionin genus bacillus DY2 bacterial strain retted fibre to prepare bastose |
| CN104630908A (en) * | 2015-02-15 | 2015-05-20 | 东华大学 | Method for preparing flax fiber by combined process of mucor circinelloides DK1 strain and hydrogen peroxide |
| CN104630910A (en) * | 2015-02-15 | 2015-05-20 | 东华大学 | Method for preparing flax fiber by combined process of candida tropicalis DK2 strain and hydrogen peroxide |
| CN104630909A (en) * | 2015-02-15 | 2015-05-20 | 东华大学 | Method for preparing flax fiber by ultrasonic combination of mucor circinelloides DK1 strain and hydrogen peroxide |
| CN104630909B (en) * | 2015-02-15 | 2016-08-17 | 东华大学 | A kind of volume branch Mucor DK1 bacterial strain and the ultrasonic combined method preparing flaxen fiber of hydrogen peroxide |
| 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 |
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