CN104403980A - Complex bacterial preparation and method for preparing cellulose by using complex bacterial preparation - Google Patents
Complex bacterial preparation and method for preparing cellulose by using complex bacterial preparation Download PDFInfo
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- CN104403980A CN104403980A CN201410759440.5A CN201410759440A CN104403980A CN 104403980 A CN104403980 A CN 104403980A CN 201410759440 A CN201410759440 A CN 201410759440A CN 104403980 A CN104403980 A CN 104403980A
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- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
- C12N9/2437—Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)
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Abstract
The invention relates to a complex bacterial preparation and a method for preparing cellulose by using the complex bacterial preparation. The complex bacterial preparation is a fermentation mixed solution which is prepared by fermentation culture of slow fiber clostridium, xylan degrading bacteroid, cytophaga and coprinus cinereus. The complex bacterial preparation breaks the limit of low activity of bacteria-producing extracellular enzyme and low decomposition rate of fungus; the agricultural wastes which are straws are used as fermentation substrates; a high-addition-value bio-enzyme preparation can be prepared by a solid-state fermentation process; the method of the preparing cellulose by using the complex bacterial preparation is simple and liable to implement and low in cost, and is capable of changing waste into wealth; the cellulose complex enzyme has a high capability of degrading cellulose substrates; a key technology is provided for decomposing the straws to produce cellulose enzyme; the method has wide application prospects in the field of straws fermentation feeds and feed enzyme preparations.
Description
Technical field
The present invention relates to a kind of method of composite fungus agent and cellulase-producing processed thereof, belong to agricultural fermentation technical field.
Background technology
Along with the fast development of livestock industry, how to improve the nutritive value of roughage and increase the focus that its ruminal digestion rate has become whole world research.In recent years, add the extensive concern that cellulase preparation causes people in ensiling or daily ration, but by the impact of various factors, as the reaction conditions etc. of the production cost of enzyme, kind and addition, enzyme, it is promoted the use of and remains in some restrictions.
For the production bacterial strain of cellulase, research in the past focuses mostly on purifying bacterium, but with natural lignocellulose materials such as stalks for substrate time, the field planting of purifying bacterial strain is slow, and it is low to produce enzyme efficiency.Recent study finds the amount that during the fermentation microbial co culture can be increased cellulase, and screening structure compound system degrades to lignocellulose and production of cellulose enzyme becomes study hotspot.
In addition, existing zymin production technique comprises liquid state fermentation (SubmergedFermentation, SmF) and solid state fermentation (Solid-state Fermentation, SSF).But the degraded difficulty that liquid state fermentation exists ligno-cellulosic materials is large, and transform the defect that cost is high, therefore Produced by Solid-state Fermentation enzyme becomes the technique means with application prospect.
But the method for existing production of cellulose enzyme exists efficiency is low, cost is high defect, be far from meeting actual production demand, the fermentation substrate therefore need to find a kind of efficient zymogenic bacteria, producing enzymatic process and cheapness.
Summary of the invention
The present invention first object is to provide a kind of composite fungus agent, take stalk as substrate, solid state fermentation is carried out to it and produces lignocellulolyticenzymes, the vigor of gained cellulase is high, improve and produce enzyme efficiency, reduce the production cost of enzyme, too increase the utilization ratio of stalk simultaneously, for the utilization of the agricultural wastes such as stalk and the development and application of unconventional water resources provide new way.
To achieve these goals, the present invention adopts following technical scheme:
A kind of composite fungus agent is inoculated in substratum respectively by slow fiber clostridium, Bacteroides xylanolyticus, Cytophaga, dust cover ghost toadstool, the fermentation mixed solution be mixed to get by fermented liquid after fermentation culture; In wherein said fermentation mixed solution, the number of colony forming units of slow fiber clostridium, Bacteroides xylanolyticus, Cytophaga, dust cover ghost toadstool is than being 20-25:15-20:15-20:40-50, and number of colony forming units is 0.8 × 10
6-1.2 × 10
6cFU.
Wherein, described slow fiber clostridium (Clostridium lentocellum), its deposit number is ATCC 49066.
Described Bacteroides xylanolyticus (Bacteroides xylanisolvens), its deposit number is DSM 18836.
Described phagocyte bacterium (Cytophaga sp.), its deposit number is ATCC13125.
Described dust cover ghost toadstool (Coprinus cinereus), its deposit number is CGMCCNO.4051.
Described substratum is Mandels substratum, and its formula is as follows:
Add wheat stalk in the medium as unique carbon source, length is 2-3cm, and addition is 1% of prior culture media total mass simultaneously.
Described fermentation culture conditions is: 33-37 DEG C of static gas wave refrigerator 7d.
Above-mentioned composite fungus agent also can be made into this area and commonly uses solid dosage, and described solid composite fungus agent is by 0.8 × 10 by above-mentioned fermentation mixed solution and carrier
6-1.2 × 10
6cFU:(1-10) g proportions and obtaining; Preferably, gained fermentation mixed solution and carrier are by 1 × 10
6cFU:5g proportions.
Described carrier is medical stone and/or straw powder.
Another object of the present invention is to provide a kind of method of cost effective cellulase-producing, is take stalk as substrate, is inoculated into by composite fungus agent in substratum and carries out solid state fermentation cultivation, obtain cellulose complex enzyme.Described cellulose complex enzyme comprises cellulase, hemicellulase and beta-glucosidase.
Described method concrete steps are as follows:
1) crushed stalk is mixed with substratum, 115-120 DEG C of sterilizing 15-30min, cooling;
2) above-mentioned composite fungus agent being inoculated into step 1) in gained system, every day stirs once, cultivates 7-10 days for 30-35 DEG C, by tunning vacuum-drying, obtains solid fabric element prozyme.
Wherein, described stalk is paddy rice, wheat, corn or other ligno-cellulosic materials, length < 1mm.
Described culture medium prescription is (g/l):
Described stalk and substratum blending ratio are 5-10:15-20 (g/ml), preferred 10:15 (g/ml).
Described composite fungus agent addition is that in every kg broth, number of colony forming units is 0.16 × 10
6-0.24 × 10
6cFU.
It is low that composite fungus agent of the present invention breaks through Production by Bacteria enzymatic activities, the limitation that fungi rate of decomposition is slow, with agricultural wastes-stalk for fermentation substrate, adopt solid-state fermentation process, preparation high added value biological enzyme formulation, simple and easy to do, with low cost and can turn waste into wealth, gained cellulose complex enzyme is comparatively strong to the degradation property of cellulosic substrate, for decomposing straw cellulase-producing provides gordian technique, will have broad application prospects at straw fermented feed and fodder enzyme preparation field.
Embodiment
Following examples for illustration of the present invention, but are not used for limiting the scope of the invention.
Embodiment 1 one kinds of composite fungus agents I
Obtain by the following method:
Be inoculated in Mandels substratum respectively by slow fiber clostridium, xylan bacterioide, phagocyte bacterium, dust cover ghost toadstool, 35 DEG C of static gas wave refrigerator 7d, fermentation culture product is respectively A, B, C, D, mixes and obtains composite microbial system culture.Wherein, in CFU, slow fiber clostridium: Bacteroides xylanolyticus: Cytophaga: the ratio of dust cover ghost toadstool is 25:20:15:40.
The composition of described Mandels substratum is:
And adding wheat stalk in the medium as unique carbon source, 2cm is long, and addition is 1% of prior culture media total mass.
Embodiment 2 one kinds of composite fungus agents II
Adopt the method preparation of embodiment 1, difference is, in composite microbial system culture, the number of colony forming units of slow fiber clostridium, Bacteroides xylanolyticus, Cytophaga, dust cover ghost toadstool is than being 25:15:15:45.
Embodiment 3 one kinds of composite fungus agents III
Adopt the method preparation of embodiment 1, difference is, in composite microbial system culture, the number of colony forming units of slow fiber clostridium, Bacteroides xylanolyticus, Cytophaga, dust cover ghost toadstool is than being 20:15:15:50.
Embodiment 4 one kinds of solid type composite fungus agents
Embodiment 1 gained composite fungus agent and medical stone are pressed 1.0 × 10
6cFU:5g absorption carrier, to obtain final product.
Embodiment 5 one kinds of solid type composite fungus agents
Embodiment 2 gained composite fungus agent and medical stone are pressed 1.0 × 10
6cFU:1g absorption carrier, to obtain final product.
Embodiment 6 one kinds of solid type composite fungus agents
Embodiment 3 gained composite fungus agent and medical stone are pressed 1.0 × 10
6cFU:10g absorption carrier, to obtain final product.
Embodiment 7 one kinds utilizes composite fungus agent system to produce the method for cellulose complex enzyme
Comprise the steps:
1) rice straw is crushed to length <1mm, mixes in 10g:15ml ratio with substratum, 118 DEG C of sterilizing 20min, cooling;
2) embodiment 1 gained composite fungus agent I is inoculated into step 1) in gained system, every day stirs once, cultivate 7-10 days for 30-35 DEG C, by the 45 DEG C of vacuum-dryings of gained tunning to the weight content of moisture below 8.0%, obtain solid fabric element prozyme.
Wherein, described paddy rice can be replaced wheat, corn or other ligno-cellulosic materials.
Described culture medium prescription is (g/l):
Described stalk and substratum blending ratio are 10:15 (g/ml).
Described composite fungus agent addition is that in every kg broth, number of colony forming units is 0.2 × 10
6cFU.
Adopt identical method, utilize embodiment 2 gained composite fungus agent I (control group) in embodiment 2 composite fungus agent II, embodiment 3 composite fungus agent III, CN101560488A to produce solid fabric element prozyme.
Experimental example 1 tunning detected result
In the solid fabric element prozyme of embodiment 1-3 composite fungus agent, control group output, add 100mL respectively, pH5.5 citrate buffer, shake 20min at 10 DEG C of shaking table 150rpm, the centrifugal 10min of 8000rpm, get supernatant and measure cellulase activity.
According to the condition determination of GB/T 23881-2009 feeding cellulase activity, live for substrate measures filter paper enzyme activity, xylanase activity and beta-glucosidase with filter paper, 1% xylan and 0.5% saligenin respectively.At 37 DEG C, pH5.5, under the condition of reaction 60min, per minute degraded substrate discharges the enzyme amount needed for 1 μm of ol glucose, is defined as a Ge Meihuo unit, represents with IU.
Detected result shows:
The enzyme of composite fungus agent I tunning is lived: cellulase activity reaches 6.42IU/g, and xylanase activity reaches 23.22IU/g, and endoglucanase (CMCase) alive can reach 88.96IU/g.
The enzyme of composite fungus agent II tunning is lived: cellulase activity reaches 5.11IU/g, and xylanase activity reaches 20.51IU/g, and endoglucanase (CMCase) alive can reach 70.32IU/g.。
The enzyme of composite fungus agent III tunning is lived: cellulase activity reaches 6.29IU/g, and xylanase activity reaches 21.54IU/g, and endoglucanase (CMCase) alive can reach 78.36IU/g.
In CN101560488A, the enzyme of embodiment 2 composite fungus agent I tunning is lived: cellulase activity reaches 4.43IU/g; Xylanase activity reaches 11.46IU/g, and endoglucanase (CMCase) alive reaches 30.65IU/g.
As can be seen here, adopting composite fungus agent of the present invention, is that substrate carries out solid state fermentation with stalk, and in gained tunning, the enzyme of cellulose complex enzyme is lived and significantly improved.
Experimental example 2 tunning is to the hydrolysis and saccharification of wheat stalk
Take the tunning of the cryodesiccated composite fungus agent I of 1g, mix with 100g wheat stalk (length 1-1.5cm), regulate water content to 60%, 30 DEG C of quiescent culture 48h, obtain enzymolysis product.Enzymolysis product is dried to constant weight in 60 DEG C, pulverizes and measure Mierocrystalline cellulose and hemicellulose level.Separately get and take 1g enzymolysis product, add 9mL deionized water, concussion 5min, gets the concentration (anthrone colorimetry) of soluble sugar in the centrifugal rear mensuration stalk of supernatant liquor.3 repetitions are established in experiment, and result is taken the mean.
The assay step of Mierocrystalline cellulose and hemicellulose in stalk:
1. the rice straw sample decomposing front and back is taken 1g (same sample arranges 3 repetitions) respectively, be contained in cellulose acetate filter logical in, be arranged in SOKURE glass extracting tube, with alcohol-ether (1:1, volume ratio) mixed solution process 24 hours, after drying, obtain residual solids, then use water circulation 2 hours, the weight claimed after drying is M1.
2. by the residue 0.65mol/L HCl circulation that 1. obtains 2 hours, after drying, the weight claimed is M2.
3. the residue 15M H will 2. obtained
2sO
4soak after 2 hours, with 0.42MH2SO4 circulation 5 hours, the weight claimed after drying was M3.
Calculation formula:
Weight=the M1-M2 of hemicellulose;
Cellulosic weight=M2-M3;
Hemicellulose rate of decomposition=(before decomposing the rear hemicellulose weight of hemicellulose weight-decomposition)/(after the front stalk weight-decomposition of decomposition stalk weight);
Cellulose decomposition rate=(before decomposing the rear cellulose of cellulose-decomposition)/(after the front stalk weight-decomposition of decomposition stalk weight).
Meanwhile, adopt above-mentioned same procedure to detect to the solid fabric element prozyme of embodiment 2 composite fungus agent I (control group) output in composite fungus agent II, composite fungus agent III and CN101560488A, its result is as follows:
Composite fungus agent I result: enzymolysis 48h, in stalk, hemicellulose and cellulose decomposition rate are respectively 17.25% and 6.17%, and soluble sugar concentration is increased to 110.2mg/g by 21.18mg/g.
The result of composite fungus agent II: enzymolysis 48h, in stalk, hemicellulose and cellulose decomposition rate are respectively 16.58% and 5.93%, and soluble sugar concentration is increased to 108.43mg/g by 21.18mg/g.
The result of composite fungus agent III: enzymolysis 48h, in stalk, hemicellulose and cellulose decomposition rate are respectively 17.64% and 6.09%, and soluble sugar concentration is increased to 109.59mg/g by 21.18mg/g.
The result of embodiment 2 composite fungus agent I in CN101560488: enzymolysis 48h, in stalk, hemicellulose and cellulose decomposition rate are respectively 10.64% and 3.09%, and soluble sugar concentration is increased to 67.82mg/g by 21.18mg/g.
Experiment proves, the cellulase that the present invention's composite fungus agent used is produced, there is enzyme activity high, to Mierocrystalline cellulose and hemicellulose rate of decomposition high, and the feature that conversion coefficient is high, and low production cost, technique is simple, for decomposition, saccharification cellulose Wood Adhesives from Biomass provide gordian technique for high-quality roughage, will have broad application prospects in raising stalk digestibility field.
Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, all belong to the scope of protection of present invention.
Claims (10)
1. a composite fungus agent, is characterized in that, is to be inoculated in substratum respectively by slow fiber clostridium, Bacteroides xylanolyticus, Cytophaga, dust cover ghost toadstool, the fermentation mixed solution be mixed to get by fermented liquid after fermentation culture;
In wherein said fermentation mixed solution, the number of colony forming units of slow fiber clostridium, Bacteroides xylanolyticus, Cytophaga, dust cover ghost toadstool is than being 20-25:15-20:15-20:40-50, and number of colony forming units is 0.8 × 10
6-1.2 × 10
6cFU;
Described fermentation culture conditions is: 33-37 DEG C of static gas wave refrigerator 7d.
2. composite fungus agent according to claim 1, is characterized in that, described slow fiber clostridium (Clostridium lentocellum), and its deposit number is ATCC 49066;
Described Bacteroides xylanolyticus (Bacteroides xylanisolvens), its deposit number is DSM 18836;
Described phagocyte bacterium (Cytophaga sp.), its deposit number is ATCC13125;
Described dust cover ghost toadstool (Coprinus cinereus), its deposit number is CGMCCNO.4051.
3. composite fungus agent according to claim 1, is characterized in that, described substratum is Mandels substratum, and its formula is as follows:
Add wheat stalk in the medium as unique carbon source, length is 2-3cm, and addition is 1% of prior culture media total mass simultaneously.
4. according to the arbitrary described composite fungus agent of claim 1-3, it is characterized in that, described composite fungus agent also can be made into solid dosage, and described solid composite fungus agent is by 0.8 × 10 by above-mentioned fermentation mixed solution and carrier
6-1.2 × 10
6cFU:(1-10) g proportions and obtaining; Preferred gained fermentation mixed solution and carrier are by 1 × 10
6cFU:5g proportions.
5. composite fungus agent according to claim 4, is characterized in that, described carrier is medical stone and/or straw powder.
6. a method for cellulase-producing processed, is characterized in that, comprises the steps:
1) crushed stalk is mixed with substratum, 115-120 DEG C of sterilizing 15-30min, cooling;
2) arbitrary for claim 1-5 described composite fungus agent being inoculated into step 1) in gained system, every day stirs once, cultivates 7-10 days for 30-35 DEG C, by tunning vacuum-drying, obtains solid fabric element prozyme.
7. method according to claim 6, is characterized in that, described stalk is paddy rice, wheat, corn or other ligno-cellulosic materials, length < 1mm.
8. method according to claim 6, is characterized in that, described culture medium prescription is (g/l):
9. method according to claim 6, is characterized in that, described stalk and substratum blending ratio are 5-10:15-20 (g/ml), is preferably 10:15 (g/ml).
10. method according to claim 6, is characterized in that, described composite fungus agent inoculum size is that in every kg broth, number of colony forming units is 0.16 × 10
6-0.24 × 10
6cFU.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107455548A (en) * | 2017-09-07 | 2017-12-12 | 中国林业科学研究院林产化学工业研究所 | A kind of method that edible mushroom solid state fermentation Soybean Meal prepares feeding level protein additive |
| CN113880612A (en) * | 2021-10-25 | 2022-01-04 | 中国农业大学烟台研究院 | Method for preparing seaweed biological organic liquid fertilizer, seaweed biological organic liquid fertilizer and application |
Citations (1)
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|---|---|---|---|---|
| CN101560488A (en) * | 2009-05-27 | 2009-10-21 | 中国农业大学 | Enzyme and microbial inoculum for decomposing lignocellulose |
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2014
- 2014-12-11 CN CN201410759440.5A patent/CN104403980A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101560488A (en) * | 2009-05-27 | 2009-10-21 | 中国农业大学 | Enzyme and microbial inoculum for decomposing lignocellulose |
Non-Patent Citations (3)
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| 刘长莉 等: "常温秸秆还田菌群的筛选及分解稻秆特性研究", 《中国农业科学》 * |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107455548A (en) * | 2017-09-07 | 2017-12-12 | 中国林业科学研究院林产化学工业研究所 | A kind of method that edible mushroom solid state fermentation Soybean Meal prepares feeding level protein additive |
| CN113880612A (en) * | 2021-10-25 | 2022-01-04 | 中国农业大学烟台研究院 | Method for preparing seaweed biological organic liquid fertilizer, seaweed biological organic liquid fertilizer and application |
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