CN104962542B - A kind of method of sporosarcina production pectase and mannase - Google Patents
A kind of method of sporosarcina production pectase and mannase Download PDFInfo
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- CN104962542B CN104962542B CN201510409007.3A CN201510409007A CN104962542B CN 104962542 B CN104962542 B CN 104962542B CN 201510409007 A CN201510409007 A CN 201510409007A CN 104962542 B CN104962542 B CN 104962542B
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Abstract
A kind of method of sporosarcina production pectase and mannase is related to a kind of method of bacterium production pectase and mannase.The present invention provides a kind of method for producing pectase and mannase using bacterium, has expanded the microorganism fungus kind source of production pectase and β mannases.Method：Sporosarcina bacterium HDYM 26 is inoculated in konjaku flour culture medium, 22~26h of shaken cultivation under conditions of 36~37 DEG C, 130~150r/min obtains zymotic fluid, extracted to zymotic fluid, obtains pectase and mannase.This method is easy, of low cost.The zymotic fluid is short for the flax degumming time, colloid removal is thorough, residual gum content is low, and flax fiber yield is high, intensity is high.The present invention is for producing pectase and mannase.
Description
Technical field
The present invention relates to a kind of methods that bacterium produces pectase and mannase.
Background technology
Pectase refers to the enzyme for decomposing plant main component-pectic substance.Pectase is distributed widely in higher plant and micro-
In biology, according to the difference of its substrate specificity, and it can be divided into three classes.Wherein two classes (pectinesterase and polygalacturonase) are deposited
It is in higher plant and microorganism, also a kind of (pectin lyase) is present in microorganism, especially certain infection plants
In pathogenic microorganisms.
'beta '-mannase, English name β-mannanase are to hydrolyze the inscribe water that Isosorbide-5-Nitrae-β-D- mannopyranoses are main chain
Solve enzyme.'beta '-mannase substrate spectrum is wide in range, including glucomannan, mannosan and galactomannans.β-sweet dew
Dextranase has the juice clarification in extensive industrial use, such as the association with pulp bleaching of paper-making industry, grocery trade.'beta '-mannase will
Polysaccharide is degraded to oligosaccharide, is conducive to enteron aisle probiotic microorganisms and grows, increasingly noticeable in recent years.Microorganism 'beta '-mannase
Source is wide, action condition is mild, is easy to purify, and is the hot spot of 'beta '-mannase theory and practice research.Compared with fungi, carefully
Bacterium, which produces 'beta '-mannase, has the superiority such as fermentation period is short, nutritional requirement is simple.Excavate bacterium money abundant in nature
The novel bacteria of high yield 'beta '-mannase is found in source, is obtained more 'beta '-mannases with short cycle, low cost, is always
The research hotspot of microbiological art.
Invention content
The present invention provides a kind of method for producing pectase and mannase using bacterium, has expanded production pectase and β-is sweet
Reveal the microorganism fungus kind source of dextranase.
The method of sporosarcina production pectase and mannase of the present invention, carries out according to the following steps：
Sporosarcina bacterium (Sporosarcina sp.) HDYM-26 is inoculated in konjaku flour culture medium,
36~37 DEG C, 22~26h of shaken cultivation under conditions of 130~150r/min, obtain zymotic fluid, are extracted to zymotic fluid, obtained
Obtain pectase and mannase；Wherein, konjaku flour culture medium by 19~21g konjaku flours, 4~6g sodium nitrate and 1000mL originally
Water is made.
The cell concentration of the zymotic fluid of acquisition is (0.8~1.2) × 108A/mL.
The konjaku flour culture medium is made of 20g konjaku flours, 5g sodium nitrate and 1000mL tap water.
The pH value of the konjaku flour culture medium is 7.0~8.0.
The Sporosarcina ferment product of above-mentioned acquisition can be used for flax degumming, with 1:20 volume ratio is added to
In retting liquid, the retted fibre 72h at 20~30 DEG C.
Beneficial effects of the present invention：
The Sporosarcina ferment product ingredient that the present invention obtains is few, there was only konjaku flour in original culture medium
And sodium nitrate.With the progress of fermentation, konjaku flour is degraded to small molecule reduced sugar, can be utilized by bacterium；Sodium in sodium nitrate
Ion and nitrate anion can be utilized by bacterium.Therefore, the zymotic fluid to environment almost without pollution.Konjaku flour and sodium nitrate are
Technical grade, therefore the manufacturing cost of Sporosarcina ferment product of the present invention is cheap.
Sporosarcina bacterium (Sporosarcina sp.) HDYM-26 in the present invention is that the abnormal type of change energy is aerobic
Bacterium is isolated from flax warm water retting liquid, has good biocompatibility with retted fibre system, therefore be highly suitable for flax and macerate
The scouring processes of fiber crops.
Sporosarcina bacterium (Sporosarcina sp.) HDYM-26 is in konjaku flour fermentation medium in the present invention
In, since substrate for induction acts on, pectase and mannase can be generated.Therefore, which sends out in konjaku flour fermentation medium
In zymotic fluid obtained by ferment, containing pectase and mannase, the pectic substance in flax straw colloid of degrading respectively and half fiber
Dimension element, i.e., remove two kinds of colloids, have the advantages that the degumming period is short, degumming is thorough comprehensively.Bacterium in the bacterium seed liquor, can
To continue growth and breeding in retting liquid, more degummases are secreted, further shorten the degumming period, colloid of thoroughly degrading.And
And the bacterium does not generate cellulase, does not destroy the quality of flax fiber.
Specific implementation mode
Technical solution of the present invention is not limited to act specific implementation mode set forth below, further includes between each specific implementation mode
Arbitrary combination.
Specific implementation mode one：Present embodiment sporosarcina produce pectase and mannase method, by with
Lower step carries out：
Sporosarcina bacterium (Sporosarcina sp.) HDYM-26 is inoculated in konjaku flour culture medium,
36~37 DEG C, 22~26h of shaken cultivation under conditions of 130~150r/min, obtain zymotic fluid, are extracted to zymotic fluid, obtained
Obtain pectase and mannase.
The method of present embodiment is easy, of low cost.The zymotic fluid is short for the flax degumming time, colloid removal is thorough
Bottom, residual gum content are low, and flax fiber yield is high, intensity is high.
It is Sporosarcina bacterium that present embodiment, which is used to prepare flax degumming with the bacterial strain of bacterium seed liquor,
(Sporosarcina sp.) HDYM-26, " function bacterium bio-diversity grinds in flax retting liquid of Heilongjiang Province different regions
Study carefully " disclosure record, it is now preserved in microorganism key lab of colleges and universities of province of Heilongjiang University.
Specific implementation mode two：The present embodiment is different from the first embodiment in that：The konjaku flour culture medium by
19~21g konjaku flours, 4~6g sodium nitrate and 1000mL tap water are made.It is other same as the specific embodiment one.
Specific implementation mode three：The present embodiment is different from the first embodiment in that：The konjaku flour culture medium by
20g konjaku flours, 5g sodium nitrate and 1000mL tap water are made.It is other same as the specific embodiment one.
Specific implementation mode four：Present embodiment is unlike specific implementation mode two or three：The konjaku flour culture
The pH value of base is 7.0~8.0.It is other identical as specific implementation mode two or three.
Specific implementation mode five：Present embodiment is unlike specific implementation mode two or three：The konjaku flour culture
The pH value of base is 7.5.It is other identical as specific implementation mode two or three.
Specific implementation mode six：Unlike one of present embodiment and specific implementation mode one to five：37 DEG C,
Shaken cultivation is for 24 hours under conditions of 140r/min.It is other identical as one of specific implementation mode one to five.
Specific implementation mode seven：Unlike one of present embodiment and specific implementation mode one to six：The fermentation of acquisition
The cell concentration of liquid is (0.8~1.2) × 108A/mL.It is other identical as one of specific implementation mode one to six.
Following tests is carried out for verification beneficial effects of the present invention：
Contrast test 1：
One, tri- groups of A, B, C are respectively set, A groups are by Sporosarcina bacterium (Sporosarcina sp.) HDYM-26
It is inoculated in konjaku flour culture medium 1, shaken cultivation for 24 hours, obtains zymotic fluid under conditions of 37 DEG C, 140r/min；Zymotic fluid
Cell concentration is (0.8~1.2) × 108A/mL；Konjaku flour culture medium 1 by 20g konjaku flours, 5g sodium nitrate and 1000mL originally
Water is made, and the pH value of konjaku flour culture medium is 7.5.
Sporosarcina bacterium (Sporosarcina sp.) HDYM-26 is inoculated in konjaku flour culture medium 2 by B groups
In, shaken cultivation for 24 hours, obtains zymotic fluid under conditions of 37 DEG C, 140r/min；The cell concentration of zymotic fluid is (0.8~1.2)
×108A/mL；Konjaku flour culture medium 2 is made of 20g konjaku flours, 5g ammonium nitrate, 5g sodium chloride and 1000mL tap water, konjaku
Powder Medium's PH Value is 7.
Sporosarcina bacterium (Sporosarcina sp.) HDYM-26 is inoculated in konjaku flour culture medium 3 by C groups
In, shaken cultivation for 24 hours, obtains zymotic fluid under conditions of 37 DEG C, 140r/min；The cell concentration of zymotic fluid is (0.8~1.2)
×108A/mL；Konjaku flour culture medium 3 is made of 20g konjaku flours, 2g sodium chloride and 1000mL tap water, konjaku flour medium pH
Value is 7.
Two, pectinase activity, mannosan enzyme activity and cellulase in the zymotic fluid of tri- groups of acquisitions of A, B, C are detected respectively
Vigor；The flax degumming that A groups obtain is 650U/mL with pectinase activity in bacterium seed liquor, and mannosan enzyme activity is
3800U/mL, cellulase activity ＜ 5U/mL；The flax degumming that B groups obtain is 400U/ with pectinase activity in bacterium seed liquor
ML, mannosan enzyme activity are 2600U/mL, cellulase activity ＜ 5U/mL；The flax degumming bacterium seed liquor that C groups obtain
Middle pectinase activity is 320U/mL, and mannosan enzyme activity is 2200U/mL, cellulase activity ＜ 5U/mL.
Three, dust soil processing first is carried out to flax straw, i.e., flax straw is impregnated to about 1h in tap water, taken out
After be placed at room temperature for a few hours and dry.The above-mentioned processed flax straws of 600g are taken, are divided into three parts, every part of 200g.By three parts of Asias
Numb original stem is smooth, it is closely knit be laid in three electric-heated thermostatic water baths, tap water is added, numb water ratio is 1kg：20L.First
The flax degumming bacterium seed liquor of 1L A groups is added in a water-bath, the flax degumming of 1L B groups is added in second water-bath
With bacterium seed liquor, the flax degumming bacterium seed liquor of 1L C groups, progress retted fibre experiment are added in third water-bath.
Four, three groups of retted fibre test periods are 72h.
Five, flax straw is taken out after retted fibre, the ventilation for being positioned over Yanguan Pass abundance is dried, then by scapus health 48h.
Upper machine scutching obtains flax fiber.
Flax fiber intensity, fiber yield and residual gum content are measured, the results are shown in Table 2.
2 flax fiber intensity of table, fiber yield and residual gum content
By the above test result it is found that only (certainly by 20g konjaku flours, 5g sodium nitrate and 1000mL with konjaku flour culture medium 1
Water is made, and the pH value of konjaku flour culture medium is to contain pectase and mannosan in the bacterium seed liquor that 7.5) culture obtains
Enzyme, the bacterium seed liquor obtained with the culture of konjaku flour culture medium 1 are used for the best results of flax degumming.
Contrast test 2：
One, Sporosarcina bacterium (Sporosarcina sp.) HDYM-26 is inoculated in konjaku flour culture medium,
For 24 hours, the culture solution of acquisition is flax degumming bacterium seed liquor to shaken cultivation under conditions of 37 DEG C, 140r/min；Seed
The cell concentration of liquid is (0.8~1.2) × 108A/mL；
Konjaku flour culture medium is made of 20g konjaku flours, 5g sodium nitrate and 1000mL tap water, the pH value of konjaku flour culture medium
It is 7.5.
The flax degumming of acquisition is 650U/mL with pectinase activity in bacterium seed liquor, and mannosan enzyme activity is
3800U/mL, cellulase activity ＜ 5U/mL.
Two, dust soil processing first is carried out to flax straw, i.e., flax straw is impregnated to about 1h in tap water, taken out
After be placed at room temperature for a few hours and dry.The above-mentioned processed flax straws of 600g are taken, are divided into three parts, every part of 200g.By three parts of Asias
Numb original stem is smooth, it is closely knit be laid in three electric-heated thermostatic water baths, tap water is added, numb water ratio is 1kg：20L.First
1L flax degummings bacterium seed liquor is added in a water-bath, 1L tap water, third water-bath is added in second water-bath
Middle pure enzyme mixation (the pectinase activity 650U/mL, mannosan enzyme activity 3800U/ that 1L pectases and mannase is added
ML), retted fibre experiment is carried out.
Three, tap water retted fibre test period is 96h, remaining two groups of retted fibre test period is 72h.
Four, flax straw is taken out after retted fibre, the ventilation for being positioned over Yanguan Pass abundance is dried, then by scapus health 48h.
Upper machine scutching obtains flax fiber.
Flax fiber intensity, fiber yield and residual gum content are measured, the results are shown in Table 1.
1 flax fiber intensity of table, fiber yield and residual gum content
Sporosarcina ferment product is added directly into retting liquid in this test method, operation letter
Just；The growth optimum temperature range of Sporosarcina bacterium (Sporosarcina sp.) HDYM-26 is wide in range in this experiment,
Within the scope of 20~30 DEG C can vigorous growth, be not necessarily to additional heating cost；Usually time only needs 72h, than conventional natural retted fibre
Time (96-120h) significantly shortens；The flax fiber residual gum content of acquisition is low, fiber yield is high, fibre strength is high.
This experiment Sporosarcina ferment product manufacturing cost is cheap, thin using this experiment Sporosarcina
The cost of fermented liquid is only 20%~25% using pure enzyme preparation.Component of enzyme system is various in the zymotic fluid, contains fruit simultaneously
Glue enzyme and mannase can remove the colloid complex component in flax straw comprehensively, therefore residual gum content is lower.
Claims (6)
1. a kind of method of sporosarcina production pectase and mannase, it is characterised in that this method according to the following steps into
Row：
Sporosarcina bacterium (Sporosarcina sp.) HDYM-26 is inoculated in konjaku flour culture medium, 36~
37 DEG C, 22~26h of shaken cultivation under conditions of 130~150r/min, obtain zymotic fluid, are extracted to zymotic fluid, obtain fruit
Glue enzyme and mannase；Wherein, konjaku flour culture medium is by 19~21g konjaku flours, 4~6g sodium nitrate and 1000mL tap water systems
At.
2. the method for a kind of sporosarcina production pectase and mannase according to claim 1, feature exist
It is made of 20g konjaku flours, 5g sodium nitrate and 1000mL tap water in the konjaku flour culture medium.
3. the method for a kind of sporosarcina production pectase and mannase according to claim 1 or 2, feature
It is that the pH value of the konjaku flour culture medium is 7.0~8.0.
4. the method for a kind of sporosarcina production pectase and mannase according to claim 1 or 2, feature
It is that the pH value of the konjaku flour culture medium is 7.5.
5. the method for a kind of sporosarcina production pectase and mannase according to claim 3, feature exist
In shaken cultivation is for 24 hours under conditions of 37 DEG C, 140r/min.
6. the method for a kind of sporosarcina production pectase and mannase according to claim 5, feature exist
In acquisition zymotic fluid cell concentration be (0.8~1.2) × 108A/mL.
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| "沤麻系统中一株甘露聚糖酶产生菌产酶条件的优化";平文祥 等;《黑龙江大学自然科学学报》;20081231;第25卷(第6期);第783-789页 * |
| "黑龙江省不同地区亚麻沤麻液中功能菌生物多样性的研究";冯永刚;《中国优秀硕士学位论文全文数据库（电子期刊）农业科技辑》;20061115(第11期);正文第47、68页 * |
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