CN101671645B - Method for preparing beta-mannase and special strain thereof - Google Patents
Method for preparing beta-mannase and special strain thereof Download PDFInfo
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Abstract
The invention discloses a bacillus subtilis strain. The strain TT-68 is collected in General Microorganism Center of China Committee for Culture Collection of Microorganisms (short for CGMCC, address: Datun road, Zhaoyang district, Beijing, China) on Oct. 5, 2009, and the collection number is CGMCC No. 3340. Experiments prove that the zymogenic level is 6200 to 12000U/mL by adopting a method that the strain is zymogenic-fermented and cultured by a shake flask, which is greatly improved than the zymogenic level studied by China before. Expanding culture is carried out for 48 hours by 5L and 5T fermentation tank, and the enzyme activation can respectively reach 8500 to 20000U/mL and 11000 to 28000U/mL, which are highest level in relevant reports at present.
Description
Technical field
The present invention relates to a kind of method and special strain therefore thereof for preparing 'beta '-mannase.
Background technology
Mannosans is the important component of plant hemicellulose; by β-1; the wire polymer that the 4-D-seminose is formed by connecting; side chain at polysaccharide mainly contains the substituted radicals such as glucosyl group, ethanoyl and galactosyl; its source is abundant, has different types of mannosans to exist in Rhizoma amorphophalli powder, locust bean gum, artemisia glue, guar gum.β-Isosorbide-5-Nitrae-D-mannase (β-Isosorbide-5-Nitrae-D-mannan mannohydrolase; EC.3.2.1.78), (β-mannanase) is that a class can be hydrolyzed the mannooligo saccharide that contains β-Isosorbide-5-Nitrae-D-MANNOSE glycosidic bond and the inscribe lytic enzyme of mannocarolose, extensively is present in animal, plant and the microorganism to be called for short 'beta '-mannase.'beta '-mannase is the enzyme of hydrolysis mannosans most critical as a kind of hemicellulase, can be widely used in the industries such as food, papermaking, feed, weaving, printing and dyeing, has great application prospect and productive value.Wherein microbe-derived 'beta '-mannase has active high, the distinguishing features such as cost is low, steady sources, extraction convenience, has broad application prospects.
The research of 'beta '-mannase starts from late 1950s, and early stage research focuses on the screening of bacterium producing multi enzyme preparation, separation and purification of enzyme and zymologic property, and it is applied to the analysis of natural polysaecharides material sugar chain structure as toolenzyme.The eighties, the research of 'beta '-mannase began to using and the industrialization future development.So far, the research of production by biological beta-mannase enzymic fermentation mainly concentrates on black-koji mould (Aspergillus) and genus bacillus (Bacillus), and in the genus bacillus research more be subtilis (Bacillus subtilis), Bacillus licheniformis (Bacillus licheniformis) and Alkaliphilic bacillus (alkalophilic Bacillussp.), and the highest with the producing bacillus subtilis enzymic activity.Domestic related microorganism produces the report of 'beta '-mannase also take mesophilic microorganism as main: genus bacillus M-21 is take guar gum as carbon source, yeast extract paste, peptone are nitrogenous source, 32 ℃ of lower liquid fermentation and culture, it produces enzyme level is 1487U/mL (Zhou Fang, Mu Haijin, Jiang Xiaolu. genus bacillus M-21 produces 'beta '-mannase Fermentation Conditions [J]. food and fermentation industries, 2007,33:10-14); Thermophilic subtilis WY45 and WY34 are take Rhizoma amorphophalli powder as carbon source, yeast extract and Tryptones are nitrogenous source, liquid fermentation and culture under 50 ℃ of conditions, the level of producing 'beta '-mannase is respectively 2800U/mL (bavin duckweed duckweed Wei Yun, Jiang Zhengqiang, Li Lite, day bottom merit. subtilis WY45 produces 'beta '-mannase fermentation condition optimization [J]. China Agricultural University's journal, 2005,10 (3): 77-80) and 1105U/mL (Jiang, Z.Q., Wei, Y., Li, D.Y., Li, L.T., Chai, P.P.and Kusakabe, I.High-level production, purification and characterization of a thermostable β-mannanase from thenewly isolated Bacillus subtilis WY34[J] .Carbohydrate Polymers, 2006,66:88-96).Less about the patent application of natural microbial liquid fermenting production mannase both at home and abroad, and yield of enzyme is on the low side, wherein the patent No. is to disclose in 01144782.6 the patent a kind ofly to produce liquid fermenting with the aspergillus niger starting strain and produce acid mannase, and enzyme is lived and is 150U/mL.And the product enzyme activity in the foreign patent is also below 2000U/mL.The patent No. is to disclose a kind of engineering strain with the Pichia anomala expression mannase gene in 200810229197.0 the patent to carry out the fermentor tank liquid fermenting and produce mannase, and the work of fermentation 70-100h enzyme can reach 23320U/mL, but it is partially long to produce the enzyme cycle.The patent No. is to disclose a kind of employing Erwinia dissociant CXJZ95-198 fermentor tank liquid fermenting in 200510032497.6 the patent to produce mannase, and enzyme is lived and is 180U/mL.Above horizontal application is on the low side in industrial production mannosans enzyme level, and the production cycle is long and fermentation costs is higher.
Summary of the invention
The object of the present invention is to provide a kind of bacillus subtilis strain.This bacterial strain by fermentation can the High-efficient Production 'beta '-mannase.
Subtilis provided by the invention (Bacillus subtilis) TT-68 is preserved in Chinese microorganism strain preservation board of trustee reason person on October 15th, 2009 and understands common micro-organisms center (abbreviation CGMCC, the address is: Da Tun road, Chaoyang District, BeiJing, China city), preserving number is CGMCC № .3340.
Another object of the present invention is to provide a kind of method of 'beta '-mannase processed.
Method provided by the invention is with above-mentioned subtilis TT-68 fermentation, obtains 'beta '-mannase.
The fermention medium of above-mentioned fermentation usefulness comprises the carbon source of 30-50g/L, the nitrogenous source of 5-8g/L, the KH of 3-7g/L
2PO
4MgSO with 0.2-0.5g/L
47H
2O.
Above-mentioned carbon source can be at least a in konjaku powder, flower taro powder, konjak gum, locust bean gum and the guar gum; Above-mentioned nitrogenous source can be at least a in yeast extract, Tryptones, soy peptone, beef peptone, corn steep liquor, bean cake powder and the cottonseed meal.
In order further to improve the productive rate of enzyme, can in above-mentioned fermention medium, add amino acid and/or VITAMIN.
Above-mentioned amino acid can be ILE, DL-L-LEU and/or Valine; Said vitamin can be riboflavin, pyridoxine hydrochloride and/or xitix.
Above-mentioned fermention medium can be prepared as follows: konjaku powder 30-50g, bean cake powder 5-8g, KH
2PO
43-7g, MgSO
47H
2O 0.2-0.5g, Valine 2g, pyridoxine hydrochloride 0.5mg adds water to 1L, natural pH.
Further, above-mentioned fermention medium is to prepare as follows: konjaku powder 40-50g, bean cake powder 7g, KH
2PO
45g, MgSO
47H
2O 0.3g, Valine 2g, pyridoxine hydrochloride 0.5mg adds water to 1L, natural pH.
Above-mentioned fermentation is can be in shaking flask, in the 5L fermentor tank or carry out in 5 tons of fermentor tanks.
When above-mentioned fermentation was carried out in shaking flask, described fermentation condition was: temperature 40-55 ℃, rotating speed is 100-220rpm, and rotation radius is 12mm; When above-mentioned fermentation was carried out in the 5L fermentor tank, described fermentation condition was temperature 40-55 ℃, and air flow is 1-2vvm, and stir speed (S.S.) is 300-600rpm; When above-mentioned fermentation was carried out in 5 tons of fermentor tanks, described fermentation condition was temperature 40-55 ℃, and air flow is 1-1.5vvm, and stir speed (S.S.) is 150-300rpm.
Experimental results show that: adopt above-mentioned bacterial strains and aforesaid method, produce enzymic fermentation by shaking flask and cultivate, the product enzyme level is 6200-12000U/mL, improves a lot than the product enzyme level of before domestic research.Carry out 5L and 5 tons of fermentor tank enlarged culturing 48h, enzyme is lived can reach 8500-20000U/mL and 11000-28000U/mL respectively, is the highest level in the present relevant report.
Source of the present invention (konjaku powder, bean cake powder etc.) is extensive, and low price has reduced production cost.The method is produced 'beta '-mannase and is produced the enzyme level height, and fermentation period is short, and production cost is low, has preferably industrial applications prospect.
Description of drawings
Fig. 1 is different carbon sources are produced the beta-mannase enzymic activity on subtilis TT-68 shaking flask liquid fermenting impact.
Fig. 2 is different nitrogen sources is produced the beta-mannase enzymic activity on subtilis TT-68 shaking flask liquid fermenting impact.
Fig. 3 is amino acid produces the beta-mannase enzymic activity on subtilis TT-68 shaking flask liquid fermenting impact.
Fig. 4 is VITAMIN is produced the beta-mannase enzymic activity on subtilis TT-68 shaking flask liquid fermenting impact.
Embodiment
Main raw material and reagent: locust bean gum, guar gum are available from Switzerland SIGMA company; Yeast extract, Tryptones are available from OXOID company; Amino acid and VITAMIN are all available from the extensive and profound in meaning star biotechnology in Beijing limited liability company; Agar, soy peptone, beef peptone, bean cake powder, cottonseed meal and corn steep liquor are available from Beijing Kang Mingwei substratum technology limited liability company.KH
2PO
4, MgSO
47H
2O, NaCl, NaOH, phenol, sodium sulphite anhydrous 99.3 analytical pure are available from the Beijing Chemical Plant; Konjaku powder (product type 3A, operative norm NY/T494-2002, the milky white granules shape, granularity: the 60-100 order, viscosity: 〉=20000mpa.s, active substance content is greater than 90%.Annotate: common Rhizoma amorphophalli powder granularity is at the 0-65 order.), flower taro powder (product type 3A, operative norm NY/T494-2002, faint yellow particulate state, granularity 0-65 order, viscosity 〉=20000mpa.s, active substance content is greater than 90%.), konjak gum (product specification 03-TK, operative norm NY/T494-2002, white powder, granularity 0-65 order, viscosity 40000mpa.s, active substance content is greater than 90%.) available from the Qingjian River, Wuhan City konjak products company limited; 3,5-dinitrosalicylic acid is available from Shanghai sail preparation far away factory.
The invention will be further described below in conjunction with specific embodiment, but the present invention is not limited to following examples.
Among the following embodiment, if no special instructions, be ordinary method.
Among the following embodiment, described percentage composition is the quality percentage composition if no special instructions.
Subtilis TT-68 provided by the present invention screens from the soil rotted leaf and obtains.Subtilis TT-68 is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on October 15th, 2009 and (is called for short CGMCC, the address is: the Datun Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica), preservation registration number is CGMCC № .3340.
The bacterial characteristics of subtilis TT-68 provided by the present invention is: bacterium colony is creamy white, circle or subcircular, the edge is more neat, central uplift, dry fold, opaque, chromogenesis not, colony diameter 2-3mm, microscope inspection is rod-short, long 1.5-3.0 μ m, diameter 0.6-0.7 μ m produces gemma.Physiological and biochemical property is: Gram-positive, aerobic, chemoheterotrophic bacteria, and V-P reaction, catalase, glucose fermentation, nitrate reduction are positive, and clark and Lubsreaction is negative, its growth energy hydrolyzed starch, gelatin.
The screening of embodiment 1, shake-flask culture condition
One, the screening of carbon source in the fermention medium
1, actication of culture
The t bacteria T-68 of preservation is activated at dull and stereotyped activation medium.
Above-mentioned activation medium can be prepared in accordance with the following methods: locust bean gum 10g, yeast extract 6g, beef extract-peptone 4g, KH
2PO
45g, MgSO
47H
2O 0.3g, agar 15g adds water to 1L, natural pH.
2, seed liquor preparation
Fresh inoculation after the activation is cultivated in seed culture medium.
Above-mentioned seed culture medium can be prepared in accordance with the following methods: yeast extract 5g, and Tryptones 10g, NaCl 10g adds water to 1L, natural pH.
3, the shaking flask liquid fermenting is produced 'beta '-mannase
The 50mL fermention medium of in the 250mL triangular flask, packing into, access 1.5% (volume percent) seed liquor, on 50 ℃ of constant temperature air bath shaking tables with 180rpm shaking culture 4d, with the centrifugal 10min of fermented liquid 10000rpm after the fermentation, get supernatant as crude enzyme liquid, carry out the 'beta '-mannase enzyme activity determination.
Wherein, fermention medium is prepared in accordance with the following methods: carbon source 30g, yeast extract 6g, KH
2PO
45g, MgSO
47H
2O 0.3g adds water to 1L, natural pH.The kind of experimental basis carbon source is different, establishes 5 kinds of substratum, and its carbon source is respectively konjaku powder, flower taro powder, konjak gum, locust bean gum and guar gum.
Wherein, enzyme activity determination is that (3,5-dinitrosalicylic acid solution and reducing sugar solution are reduced into henna aminocompound after the heat to employing DNS method altogether, and within the specific limits, the shade of the amount of reducing sugar and red-brown thing is the certain proportion relation.Under the 540nm wavelength, measure the absorbance of red-brown material, just can obtain the content of reducing sugar in the sample.) carry out enzyme activity determination (Miller, G.L.Use of dinitrosalicylic acid reagentfor determination of reducing sugars[J] .Analytical Chemistry, 1959,31:426-428).Concrete grammar is as follows: at the locust bean gum substrate (50mM of 0.9mL 0.5%, the citrate buffer solution preparation of pH 6.0) adds the suitably enzyme liquid 0.1mL of dilution in, react 10min in 60 ℃ of water-baths, with DNS reagent (1%3,5-dinitrosalicylic acid, 1%NaOH, 0.2% phenol, use the distilled water preparation as storage liquid, add 0.05% sodium sulphite anhydrous 99.3 before using) measure the reducing sugar amount that produces, take D-MANNOSE as standard.Under the above-mentioned reaction conditions, it is 1 unit of enzyme activity (U) that per minute produces the needed enzyme amount of 1 μ mol D-MANNOSE.
Three repetitions are established in experiment.The impact that carbon source is produced the beta-mannase enzymic activity to TT-68 cultivates take konjaku powder as carbon source that to produce enzyme the highest as shown in Figure 1, can reach 2400U/mL.
Two, the screening of nitrogenous source in the fermention medium
1, actication of culture
The method of the actication of culture of step 2 and above-mentioned steps one is just the same.
2, seed liquor preparation
The seed liquor preparation of step 2 is just the same with the method for above-mentioned steps one.
3, the shaking flask liquid fermenting is produced 'beta '-mannase
The shaking flask liquid fermenting of step 2 is produced 'beta '-mannase and is that from the difference of step 1 fermention medium is different, and all the other steps are identical.
The fermention medium of this step is prepared in accordance with the following methods: konjaku powder 30g, nitrogenous source 6g, KH
2PO
45g, MgSO
47H
2O 0.3g adds water to 1L, natural pH.The kind of this experimental basis nitrogenous source is different, establishes 7 kinds of substratum, and its nitrogenous source is respectively yeast extract, Tryptones, soy peptone, beef peptone, bean cake powder, cottonseed meal and corn steep liquor.
Three repetitions are established in experiment.Carry out 'beta '-mannase enzyme activity determination and determining the protein quantity according to the method that step 1 provides.The impact that nitrogenous source produces the beta-mannase enzymic activity to TT-68 cultivates take bean cake powder as nitrogenous source that to produce enzyme the highest as shown in Figure 2, can reach 4800U/mL.
Three, amino acid whose screening in the fermention medium
1, actication of culture
The method of the actication of culture of step 3 and above-mentioned steps two is just the same.
2, seed liquor preparation
The seed liquor preparation of step 3 is just the same with the method for above-mentioned steps two.
3, the shaking flask liquid fermenting is produced 'beta '-mannase
The shaking flask liquid fermenting of step 3 is produced 'beta '-mannase and is that from the difference of step 2 fermention medium is different, and all the other steps are identical.
The fermention medium of this step is prepared in accordance with the following methods: konjaku powder 30g, bean cake powder 6g, KH
2PO
45g, MgSO
47H
2O 0.3g, amino acid 2g adds water to 1L, natural pH.The amino acid whose kind of this experimental basis is different, establishes 3 kinds of substratum, and its amino acid is respectively ILE, DL-L-LEU and Valine.
Three repetitions are established in experiment.Carry out 'beta '-mannase enzyme activity determination and determining the protein quantity according to the method that step 1 provides.Different aminoacids produces the impact of beta-mannase enzymic activity such as Fig. 3 to TT-68, and (Control is not for adding amino acid whose contrast, L-Isoleucine is ILE) shown in, ILE, DL-L-LEU, Valine improve a lot to producing enzyme in the amino acid, wherein the Valine effect is the most obvious, and enzyme work can reach 6200U/mL.
Four, the screening of VITAMIN in the fermention medium
1, actication of culture
The method of the actication of culture of step 4 and above-mentioned steps three is just the same.
2, seed liquor preparation
The seed liquor preparation of step 4 is just the same with the method for above-mentioned steps three.
3, the shaking flask liquid fermenting is produced 'beta '-mannase
The shaking flask liquid fermenting of step 4 is given birth to 'beta '-mannase and is that from the difference of step 3 fermention medium is different, and all the other steps are identical.
The fermention medium of this step is prepared in accordance with the following methods: konjaku powder 30g, bean cake powder 6g, KH
2PO
45g, MgSO
47H
2O 0.3g, VITAMIN 0.5mg adds water to 1L, natural pH.The kind of this experimental basis VITAMIN is different, establishes 3 kinds of substratum, and wherein VITAMIN is respectively riboflavin, pyridoxine hydrochloride and xitix.
Three repetitions are established in experiment.Carry out the 'beta '-mannase enzyme activity determination according to the method that step 1 provides.Different VITAMIN are produced the impact of beta-mannase enzymic activity shown in Fig. 4 (Control is the not contrast of vitaminize) to TT-68, riboflavin, pyridoxine hydrochloride, xitix improve a lot to producing enzyme in the VITAMIN, wherein the pyridoxine hydrochloride effect is best, and enzyme work can reach 6600U/mL.
Five, the screening of fermentation culture conditions
1, actication of culture
The method of the actication of culture of step 5 and above-mentioned steps four is just the same.
2, seed liquor preparation
The seed liquor preparation of step 5 is just the same with the method for above-mentioned steps four.
3, the shaking flask liquid fermenting is produced 'beta '-mannase
The difference that the shaking flask liquid fermenting of step 5 is produced 'beta '-mannase and step 4 is following 3 points, and all the other steps are identical:
1, fermention medium: konjaku powder (30,40 or 50g/L), bean cake powder (5,7 or 8g/L), KH
2PO
45g/L, MgSO
47H
2O 0.3g/L, Valine 2g/L, pyridoxine hydrochloride 0.5mg/L;
2, fermentation condition: inoculum size (1%, 2% or 3%), shaking flask rotating speed (100,160 or 220rpm) and leavening temperature (40,50 or 55 ℃);
3, fermentation time is 6 days.
Wherein, different konjaku powder concentration, different bean cake powder concentration, different inoculum sizes, different rotating speeds see Table 1 with 15 processing that different culture temperature is combined into.Carry out 'beta '-mannase enzyme activity determination and determining the protein quantity according to the method that step 1 provides.Three repetitions are established in experiment.It is as shown in table 1 to produce the enzyme situation, ferments 6 days, and thermophilic subtilis TT-68 shaking flask liquid fermenting produces the beta-mannase enzymic activity can reach 6200-12000U/mL.
Table 1 fermentation condition produces the impact of 'beta '-mannase on subtilis TT-68 shaking flask liquid fermenting
Embodiment 2, in the 5L fermentation cylinder for fermentation
1, actication of culture
The method of the step 1 of the actication of culture of embodiment 2 and embodiment 1 is just the same.
2, seed liquor preparation
The seed liquor preparation of embodiment 2 is just the same with the method for the step 1 of embodiment 1.
3, the shaking flask liquid fermenting is produced 'beta '-mannase
In 5L fermentor tank (substratum is 2.5L), access respectively 1%, 2% and 3% (volume percent) seed liquor, in the lower cultivation of differing temps (40,50 and 55 ℃), air flow is 1,1.5 and 2vvm, stir speed (S.S.) is 300,450 and 600rpm, fermentation period 48h, three repetitions are established in experiment.The processing setting of different inoculum sizes, temperature, air flow and stir speed (S.S.) sees Table 2.
Wherein, the fermention medium of present embodiment is prepared in accordance with the following methods: konjaku powder 40g, bean cake powder 7g, KH
2PO
45g, MgSO
47H
2O 0.3g, Valine 2g, pyridoxine hydrochloride 0.5mg adds water to 1L, natural pH.
Carry out 'beta '-mannase enzyme activity determination and determining the protein quantity according to the method that the step 1 of embodiment 1 provides.Different condition sees Table 2 to the impact that the TT-685L ferment tank produces the beta-mannase enzymic activity.Thermophilic subtilis TT-68 48h in the 5L fermentor tank produces the beta-mannase enzymic activity and can reach 8500-20000U/mL.
Table 2 different fermentations condition is produced the impact of 'beta '-mannase in the 5L fermentor tank on thermophilic subtilis TT-68
Embodiment 3,5 tons of fermentation cylinder for fermentation
1, actication of culture
The method of the step 1 of the actication of culture of embodiment 3 and embodiment 1 is just the same.
2, first order seed
Fresh inoculation after the activation is cultivated in seed culture medium, obtain the first order seed nutrient solution.Wherein seed culture medium is the same with the seed culture medium of the step 1 of embodiment 1.
3, secondary seed
The first order seed nutrient solution is inoculated in the secondary seed fermentor tank, and inoculum size is 1% (volume percent), and 50 ℃ of lower cultivations, air flow is 1.5vvm, and stirring velocity is 250rpm.
Above-mentioned secondary seed fermentation tank culture medium can be prepared in accordance with the following methods: konjaku powder 40g, bean cake powder 7g, KH
2PO
45g, MgSO
47H
2O 0.3g, Valine 2g, pyridoxine hydrochloride 0.5mg adds water to 1L, natural pH.
4, produce enzyme 5 tons of fermentation cylinder for fermentation
The secondary seed nutrient solution is inoculated in 5 tons of fermentor tanks (fermentation culture fiduciary point tank volume 75%), and inoculum size is 2%, cultivates in that differing temps (40,50 and 55 ℃) is lower, and air flow is 1.5vvm, and stir speed (S.S.) is 200rpm, incubation time 48h.The processing setting of differing temps and different rotating speeds sees Table 3.
The fermention medium of above-mentioned 5 tons of fermentor tanks can be prepared in accordance with the following methods: konjaku powder 40g, bean cake powder 7g, KH
2PO
45g, MgSO
47H
2O 0.3g, Valine 2g, pyridoxine hydrochloride 0.5mg adds water to 1L, natural pH.
Experiment repeats 3 times.Carry out 'beta '-mannase enzyme activity determination and determining the protein quantity according to the method that the step 1 of embodiment 1 provides.Culture temperature and stir speed (S.S.) are as shown in table 3 in the impact of 5 tons of fermentation cylinder for fermentation product beta-mannase enzymic activitys on TT-68, thermophilic subtilis TT-68 48h in 5 tons of fermentor tanks produces the beta-mannase enzymic activity and can reach 11000-28000U/mL, is the maximum of reporting at present.
Table 3 different fermentations condition is produced the impact of 'beta '-mannase in 5 tons of fermentor tanks on subtilis TT-68
Claims (9)
1. method for preparing 'beta '-mannase, be fermentative preservation number for the subtilis of CGMCCNo.3340 (Bacillus subtilis) TT-68, obtain 'beta '-mannase.
2. method according to claim 1, it is characterized in that: the fermention medium of described fermentation usefulness comprises the nitrogenous source of the carbon source of 30-50g/L, 5-8g/L, the KH of 3-7g/L
2PO
4MgSO with 0.2-0.5g/L
47H
2O.
3. method according to claim 2 is characterized in that: described carbon source is at least a in konjaku powder, flower taro powder, konjak gum, locust bean gum and the guar gum; Described nitrogenous source is at least a in yeast extract, Tryptones, soy peptone, beef peptone, corn steep liquor, bean cake powder and the cottonseed meal.
4. according to claim 2 or 3 described methods, it is characterized in that: described fermention medium also comprises amino acid and/or VITAMIN.
5. method according to claim 4, it is characterized in that: described amino acid is ILE, DL-L-LEU and/or Valine; Described VITAMIN is riboflavin, pyridoxine hydrochloride and/or xitix.
6. method according to claim 5, it is characterized in that: described fermention medium is to prepare as follows: konjaku powder 30-50g, bean cake powder 5-8g, KH
2PO
43-7g, MgSO
47H
2O 0.2-0.5g, Valine 2g, pyridoxine hydrochloride 0.5mg adds water to 1L.
7. method according to claim 6, it is characterized in that: described fermention medium is to prepare as follows: konjaku powder 40-50g, bean cake powder 7g, KH
2PO
45g, MgSO
47H
2O 0.3g, Valine 2g, pyridoxine hydrochloride 0.5mg adds water to 1L.
8. method according to claim 2 is characterized in that: described fermentation is in shaking flask, in the 5L fermentor tank or carries out in 5 tons of fermentor tanks.
9. method according to claim 8, it is characterized in that: described fermentation is carried out in shaking flask, and described fermentation condition is: temperature 40-55 ℃, rotating speed is 100-220rpm, and rotation radius is 12mm; Described fermentation is carried out in the 5L fermentor tank, and described fermentation condition is temperature 40-55 ℃, and air flow is 1-2vvm, and stir speed (S.S.) is 300-600rpm; Described fermentation is carried out in 5 tons of fermentor tanks, and described fermentation condition is temperature 40-55 ℃, and air flow is 1-1.5vvm, and stir speed (S.S.) is 150-300rpm.
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| CN102154247B (en) * | 2011-02-15 | 2013-07-10 | 华东理工大学 | Method for preparing beta-mannanase by using natural Bacillus subtilis |
| CN102191235B (en) * | 2011-04-18 | 2013-01-23 | 黑龙江大学 | Method for producing beta-mannanase through fermentation by utilizing konjac flour microorganisms |
| WO2013053074A1 (en) * | 2011-10-10 | 2013-04-18 | Huang Daiyong | Bacillus subtilis for high-efficiency expression of β-mannanase, enzyme product thereof and production method therefor |
| CN102373168A (en) * | 2011-10-10 | 2012-03-14 | 武汉东方天琪生物工程有限公司 | Bacillus subtilis capable of expressing beta-mannase at high efficiency, as well as enzyme product and production method thereof |
| CN107365756A (en) * | 2017-08-01 | 2017-11-21 | 天津大学 | A kind of method for preparing β mannases using the fermentation of mannosan type natural plant gum liquid hydrolyzate |
| CN108359700B (en) * | 2018-02-28 | 2021-08-17 | 亿利耐雀生物科技有限公司 | Method for preparing artemisia desertorum oligosaccharide through enzymolysis and application of artemisia desertorum oligosaccharide |
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| CN101492667A (en) * | 2008-01-21 | 2009-07-29 | 中国农业大学 | Method for producing mannase and special immobilized biomembrane thereof |
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