CN103497941A - Method for preparing cellulase through trichoderma viride high-efficiency fermentation - Google Patents
Method for preparing cellulase through trichoderma viride high-efficiency fermentation Download PDFInfo
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Abstract
The invention discloses a method for preparing cellulase through trichoderma viride high-efficiency fermentation. The key points of the method are that: trichoderma viride is inoculated on a Czapek medium and is activated; a single colony is selected and is inoculated in a liquid activation culture medium and is subjected to amplification culture, such that a seed liquid is obtained. The seed liquid is inoculated into a liquid enzyme-production culture medium according to a volume ratio of 1:10-20; 5-20g/L of bamboo powder with a specification of 40-60 meshes is adopted as a fermentation substrate; liquid fermentation is carried out for 72-120h, such that cellulase is prepared. The method is stable and highly efficient. The method is especially suitable for producing cellulase through trichoderma viride high-efficiency fermentation. According to the invention, the entire set of liquid fermentation process is obtained through optimization aiming at trichoderma viride physiological characteristics, such that cellulase with high enzyme activity can be obtained. Also, the bamboo raw material is used as a resource and as a cellulase induction substrate, and the biological enzyme preparation with high added value is prepared. The invention provides an innovative idea for utilizing bamboo resource with high value. With the method, Bamboo area farmers' economic income can be effectively improved. The invention has important significance.
Description
Technical field
The present invention relates to a kind of method for preparing cellulase, particularly a kind of high-efficiency fermenting viride prepares the method for cellulase, belongs to microorganism culturing technical field in biological chemistry.
Background technology
Cellulase be one group can the hydrocellulose molecular chain glucoside bond make it change into the general name of the polycomponent enzyme of glucose, be several enzyme complexs with different enzyme activities, mainly comprise circumscribed cellulase, endo cellulase, beta-glucosidase etc.Mierocrystalline cellulose needs the common appearance of these enzymes synergy, co-catalysis just can be completely degraded.Utilizing cellulase is food, the energy, chemical materials by cellulose conversion, and the many Environmental and resource issues that solve facing mankind are had to great realistic meaning.
The current obstruction mankind extensively efficiently utilize the bottleneck of cellulase, and the one, lack the High-Cellulase-Yielding bacterium, the 2nd, enzymatic production technique is still treated further optimization.At present more for the production of the microbial strains of cellulase is filamentous fungus, and wherein, the bacterial classification that enzyme activity is stronger is comparatively typical case of Trichoderma, Aspergillus and Penicillium, particularly viride and nearly edge bacterial strain thereof, is to generally acknowledge cellulase production bacterium preferably.In addition, ruminating animal relies on the rumen microorganism digestible cellulose, and as rumen bacteria and Clostridium thermocellum etc., but output and activity are lower.Both at home and abroad the preparation method of cellulase mainly contains two kinds of solid fermentation method and solution fermentations.The solid fermentation apparatus less investment, but floor space greatly, is easily dyed miscellaneous bacteria, production stability is poor, and also the difficult grasp of technology, the production that is difficult to magnify, the most enterprises of China still adopt solid fermentation process to prepare cellulase at present; Liquid fermenting has that production stability is high, technology is easily grasped, be convenient to the advantages such as suitability for industrialized production, but because cellulase is inducible enzyme, adopt corn stalk, wheat straw, straw etc. as inductor, effect is not very good, therefore enterprise is numerous and confused adopts expensive timber as raw material, greatly increase enterprise's production cost, restricted the promotion and application of cellulase production technology.
Prepare the cellulase field at organism of fermentation, Chinese patent (ZL200810023480.8) " a kind of trichoderma reesei cultivation method that improves yield of cellulase " produces in enzyme process and regulates ventilation at Trichodermareesei, make to produce enzyme main phase dissolved oxygen concentration 20～30%, be beneficial to thalli growth, the filter paper enzyme activity of fixedly ventilation volume production of the cellulase enzyme of output improves 30%, produces enzyme time shorten 14%; Chinese patent (ZL200510009000.9) " high active cellulase and manufacture method thereof " is usingd basidiomycetes CGMCCNo.1294 as producing bacterial classification, sterilizing access triangular flask liquid spawn, carry out liquid submerged fermentation, after filtration, concentrated and spraying drying is made the cellulase powder, measure producing enzyme activity is 40000u/g, and process stability good, be difficult for microbiological contamination, be convenient to large-scale industrial production; The microorganism that Chinese patent (ZL200610002014.2) " method of producing low temperature cellulase using microbe fermentation " will produce cellulase is domestication by low temperature step by step, make its well-grown in low temperature environment, through enlarged culturing, enzymatic production, according to difference, need and the use object, by further concentrated, separation and purification of crude enzyme liquid, prepare the cellulase preparation of different activities, purity and formulation; United States Patent (USP) (US61110732) " Hosts and fermentation processes for cellulase production " will be through the filamentous fungus of gene modification as cultivating bacterial classification, using glucose, glycerol or the mixture of the two of the hemicellulose source alcohol sugar of 25～100wt% and 0～75wt% as carbon source, and fermentation prepares the prozyme that simultaneously contains cellulase and hemicellulose enzyme activity.United States Patent (USP) (US60920942) " Process for producing cellulase " is mould by long shoot wood, Trichoderma lignorum, aspergillus niger, wooden aspergillus are cultivated and zymotechnique by difference, prepares two kinds of products of cellulase and sophorolipid simultaneously.So far, yet there are no and utilize the mao bamboon raw material as inducing substrate, the related process technology that the high-efficiency fermenting viride prepares cellulase occurs.
Summary of the invention
The bacterial classification for preparing the cellulase technological process in order to solve the fermentation of current microorganism produces that enzyme efficiency is low, the solid fermentation poor stability, induce the practical problemss such as the substrate effect is undesirable, simultaneously recycling China's abundant, cheap bamboo resource, preparation high added value biological enzyme formulation, the object of the invention is to provide a kind of method that high-efficiency fermenting viride prepares cellulase.
For achieving the above object, technical scheme of the present invention is to adopt following steps:
1) not activated viride is dissolved with sterilized water in gnotobasis, adopt plate streak that bacterial classification is inoculated on czapek's solution, cultivate 72～168h in the constant incubator of 24～32 ℃, to growing single bacterium colony, obtain the viride through overactivation;
2) viride through overactivation step 1) obtained is picking list bacterium colony in gnotobasis, is inoculated in the liquid activation medium, in 24～32 ℃, the constant temperature oscillator of 180～260r/min, cultivates 48～72h, obtains the seed liquor of enlarged culturing;
3) by step 2) seed liquor of the enlarged culturing that obtains is inoculated in the liquid culture medium according to the inoculum size of volume ratio 1:10～20, according to 60% Intake Quantity, pack in fermentor tank, produce the enzyme cultivation and prepare cellulase, controlled fermentation liquid pH value 3～7,24～32 ℃ of leavening temperatures, air flow 1:0.4～1.2, fermentation time 72～120h, the fermented liquid obtained is centrifugal 10min in the refrigerated centrifuge of 4000r/min, adopt concentrated its concentration that makes of poly (ether sulfone) film of molecular weight cut-off 100～500k to promote 20 times, obtain cellulase.
Described liquid activation culture based formulas is: the PBS that glucose 10～20g/L, peptone 1～2g/L, Tween800.2～0.4g/L, ammonium sulfate 1～2g/L, potassium primary phosphate 1～3g/L, sal epsom 0.1～0.3g/L, calcium chloride 0.1～0.3g/L, urea 0.4～0.8g/L, the Mandels nutritive salt that accounts for liquid activation medium cumulative volume 10～20%, solvent are pH=3～7,120 ℃ of high pressure steam sterilization 20min.
Described liquid culture medium formula is: glucose 4～6g/L, 40 to 60 order mao bamboon powder 5～20g/L, Tween800.2～0.6g/L, ammonium sulfate 1～3g/L, potassium primary phosphate 3～5g/L, sal epsom 0.3～0.5g/L, calcium chloride 0.3～0.5g/L, urea 0.8～1.2g/L, account for the Mandels trace element of liquid culture medium cumulative volume 0.1～0.3%, the PBS that solvent is pH=3～7,120 ℃ of high pressure steam sterilization 20min.
With background technology, compare, the beneficial effect that the present invention has is:
The present invention introduces viride in fermentation prepares the microbial strains of cellulase, for the physiological property optimization of viride, obtains a set of efficient liquid zymotechnique, can obtain the cellulase of high enzymatic activity under this technique; The induce substrate of recycling mao bamboon raw material as cellulase proposed simultaneously, preparation high added value biological enzyme formulation, open one's minds for one that is the higher value application bamboo resource, meet the national development circular economy policy, and can conscientiously improve China bamboo district peasant's income.
The accompanying drawing explanation
Fig. 1 is that embodiment 3 activates the viride photo that grows single bacterium colony through czapek's solution;
Fig. 2 is the processing condition according to embodiment 3, and the CMC enzyme of the cellulase prepared in the different fermentations time period is lived.
Embodiment
Below in conjunction with specific embodiment, the invention will be further described.
Embodiment 1:
1) viride that will be not activated dissolves with sterilized water in gnotobasis, adopts plate streak that bacterial classification is inoculated on czapek's solution, cultivates 72h in the constant incubator of 24 ℃, to growing single bacterium colony, obtains the viride through overactivation;
2) viride through overactivation step 1) obtained is picking list bacterium colony in gnotobasis, is inoculated in the liquid activation medium, in 24 ℃, the constant temperature oscillator of 180r/min, cultivates 48h, obtains the seed liquor of enlarged culturing; (liquid activation culture based formulas: the PBS that glucose 10g/L, peptone 1g/L, Tween800.2g/L, ammonium sulfate 1g/L, potassium primary phosphate 1g/L, sal epsom 0.1g/L, calcium chloride 0.1g/L, urea 0.4g/L, the Mandels nutritive salt that accounts for liquid activation medium cumulative volume 10%, solvent are pH=3,120 ℃ of high pressure steam sterilization 20min)
3) by step 2) seed liquor of the enlarged culturing that obtains is inoculated in the liquid culture medium according to the inoculum size of volume ratio 1:10, according to 60% Intake Quantity, pack in fermentor tank, produce the enzyme cultivation and prepare cellulase, controlled fermentation liquid pH value 3,24 ℃ of leavening temperatures, air flow 1:0.4, fermentation time 72h, the fermented liquid obtained is centrifugal 10min in the refrigerated centrifuge of 4000r/min, adopt concentrated its concentration that makes of poly (ether sulfone) film of molecular weight cut-off 100k to promote 20 times, obtain cellulase (a).(liquid culture medium formula: glucose 4g/L, 40 to 60 order mao bamboon powder 5g/L, Tween800.2g/L, ammonium sulfate 1g/L, potassium primary phosphate 3g/L, sal epsom 0.3g/L, calcium chloride 0.3g/L, urea 0.8g/L, account for the Mandels trace element of liquid culture medium cumulative volume 0.1%, the PBS that solvent is pH=3,120 ℃ of high pressure steam sterilization 20min)
Embodiment 2:
1) viride that will be not activated dissolves with sterilized water in gnotobasis, adopts plate streak that bacterial classification is inoculated on czapek's solution, cultivates 108h in the constant incubator of 28 ℃, to growing single bacterium colony, obtains the viride through overactivation;
2) viride through overactivation step 1) obtained is picking list bacterium colony in gnotobasis, is inoculated in the liquid activation medium, in 28 ℃, the constant temperature oscillator of 220r/min, cultivates 60h, obtains the seed liquor of enlarged culturing; (liquid activation culture based formulas: the PBS that glucose 15g/L, peptone 1.5g/L, Tween800.3g/L, ammonium sulfate 1.5g/L, potassium primary phosphate 2g/L, sal epsom 0.2g/L, calcium chloride 0.2g/L, urea 0.6g/L, the Mandels nutritive salt that accounts for liquid activation medium cumulative volume 15%, solvent are pH=5,120 ℃ of high pressure steam sterilization 20min)
3) by step 2) seed liquor of the enlarged culturing that obtains is inoculated in the liquid culture medium according to the inoculum size of volume ratio 1:15, according to 60% Intake Quantity, pack in fermentor tank, produce the enzyme cultivation and prepare cellulase, controlled fermentation liquid pH value 5,28 ℃ of leavening temperatures, air flow 1:0.8, fermentation time 96h, the fermented liquid obtained is centrifugal 10min in the refrigerated centrifuge of 4000r/min, adopt concentrated its concentration that makes of poly (ether sulfone) film of molecular weight cut-off 300k to promote 20 times, obtain cellulase (b).(liquid culture medium formula: glucose 5g/L, 40 to 60 order mao bamboon powder 10g/L, Tween800.4g/L, ammonium sulfate 2g/L, potassium primary phosphate 4g/L, sal epsom 0.4g/L, calcium chloride 0.4g/L, urea 1.0g/L, account for the Mandels trace element of liquid culture medium cumulative volume 0.2%, the PBS that solvent is pH=5,120 ℃ of high pressure steam sterilization 20min)
Embodiment 3:
1) viride that will be not activated dissolves with sterilized water in gnotobasis, adopts plate streak that bacterial classification is inoculated on czapek's solution, cultivates 144h in the constant incubator of 30 ℃, to growing single bacterium colony, obtains the viride through overactivation;
2) viride through overactivation step 1) obtained is picking list bacterium colony in gnotobasis, is inoculated in the liquid activation medium, in 30 ℃, the constant temperature oscillator of 240r/min, cultivates 72h, obtains the seed liquor of enlarged culturing; (liquid activation culture based formulas: the PBS that glucose 20g/L, peptone 2g/L, Tween800.3g/L, ammonium sulfate 1.5g/L, potassium primary phosphate 2g/L, sal epsom 0.3g/L, calcium chloride 0.2g/L, urea 0.8g/L, the Mandels nutritive salt that accounts for liquid activation medium cumulative volume 15%, solvent are pH=5,120 ℃ of high pressure steam sterilization 20min)
3) by step 2) seed liquor of the enlarged culturing that obtains is inoculated in the liquid culture medium according to the inoculum size of volume ratio 1:15, according to 60% Intake Quantity, pack in fermentor tank, produce the enzyme cultivation and prepare cellulase, controlled fermentation liquid pH value 5,30 ℃ of leavening temperatures, air flow 1:1, fermentation time 108h, the fermented liquid obtained is centrifugal 10min in the refrigerated centrifuge of 4000r/min, adopt concentrated its concentration that makes of poly (ether sulfone) film of molecular weight cut-off 300k to promote 20 times, obtain cellulase (c).(liquid culture medium formula: glucose 6g/L, 40 to 60 order mao bamboon powder 15g/L, Tween800.4g/L, ammonium sulfate 1.5g/L, potassium primary phosphate 4g/L, sal epsom 0.5g/L, calcium chloride 0.4g/L, urea 1.2g/L, account for the Mandels trace element of liquid culture medium cumulative volume 0.15%, the PBS that solvent is pH=5,120 ℃ of high pressure steam sterilization 20min)
Embodiment 4:
1) viride that will be not activated dissolves with sterilized water in gnotobasis, adopts plate streak that bacterial classification is inoculated on czapek's solution, cultivates 168h in the constant incubator of 32 ℃, to growing single bacterium colony, obtains the viride through overactivation;
2) viride through overactivation step 1) obtained is picking list bacterium colony in gnotobasis, is inoculated in the liquid activation medium, in 32 ℃, the constant temperature oscillator of 260r/min, cultivates 72h, obtains the seed liquor of enlarged culturing; (liquid activation culture based formulas: the PBS that glucose 20g/L, peptone 2g/L, Tween800.4g/L, ammonium sulfate 2g/L, potassium primary phosphate 3g/L, sal epsom 0.3g/L, calcium chloride 0.3g/L, urea 0.8g/L, the Mandels nutritive salt that accounts for liquid activation medium cumulative volume 20%, solvent are pH=7,120 ℃ of high pressure steam sterilization 20min)
3) by step 2) seed liquor of the enlarged culturing that obtains is inoculated in the liquid culture medium according to the inoculum size of volume ratio 1:20, according to 60% Intake Quantity, pack in fermentor tank, produce the enzyme cultivation and prepare cellulase, controlled fermentation liquid pH value 7,32 ℃ of leavening temperatures, air flow 1:1.2, fermentation time 120h, the fermented liquid obtained is centrifugal 10min in the refrigerated centrifuge of 4000r/min, adopt concentrated its concentration that makes of poly (ether sulfone) film of molecular weight cut-off 500k to promote 20 times, obtain cellulase (d).(liquid culture medium formula: glucose 6g/L, 40 to 60 order mao bamboon powder 20g/L, Tween800.6g/L, ammonium sulfate 3g/L, potassium primary phosphate 5g/L, sal epsom 0.5g/L, calcium chloride 0.5g/L, urea 1.2g/L, account for the Mandels trace element of liquid culture medium cumulative volume 0.3%, the PBS that solvent is pH=7,120 ℃ of high pressure steam sterilization 20min)
The CMC enzyme of four kinds of cellulases that mensuration embodiment 1,2,3,4 prepares is lived.Table 1 is the measurement result of being lived by four kinds of cellulase CMC enzymes of embodiment 1,2,3,4 preparations.Known by data in table 1, the CMC enzyme work of the cellulase (a) that adopts preparation method of the present invention to obtain, (b), (c), (d) is distributed in 2110～2640IU/mL, all there is higher cellulase activity, illustrate that the method for the present invention relates to meets the technology category that high-efficiency fermenting prepares cellulase.
As Fig. 1, activating through czapek's solution the viride photo that grows single bacterium colony from embodiment 3 can find out, it is the living green mycelia of gas, and mycelia is long, and and straight, side shoot is less, and at the contraction of the middle and later periods of growth and generation fibrillae of spores, spore is little, is oval.As Fig. 2, according to the processing condition of embodiment 3, in the fermenting process of 72～120h, the cellulase CMC enzyme work obtained is fallen after rising, and at 108h, reaches the highest enzyme 2640IU/mL alive, so the best of embodiment 3 product enzymic fermentation time is decided to be 108h.
Table 1
What more than enumerate is only specific embodiments of the invention.The invention is not restricted to above embodiment, many distortion can also be arranged.All distortion that those of ordinary skill in the art can directly derive or associate from content disclosed by the invention, all should think protection scope of the present invention.
Claims (3)
1. a high-efficiency fermenting viride prepares the method for cellulase, it is characterized in that, comprises the following steps:
1) not activated viride (Trichoderma viviride) is dissolved with sterilized water in gnotobasis, adopt plate streak that bacterial classification is inoculated on czapek's solution (czapck), cultivate 72～168h in the constant incubator of 24～32 ℃, to growing single bacterium colony, obtain the viride through overactivation;
2) viride through overactivation step 1) obtained is picking list bacterium colony in gnotobasis, is inoculated in the liquid activation medium, in 24～32 ℃, the constant temperature oscillator of 180～260r/min, cultivates 48～72h, obtains the seed liquor of enlarged culturing;
3) by step 2) seed liquor of the enlarged culturing that obtains is inoculated in the liquid culture medium according to the inoculum size of volume ratio 1:10～20, according to 60% Intake Quantity, pack in fermentor tank, produce the enzyme cultivation and prepare cellulase, controlled fermentation liquid pH value 3～7,24～32 ℃ of leavening temperatures, air flow 1:0.4～1.2, fermentation time 72～120h, the fermented liquid obtained is centrifugal 10min in the refrigerated centrifuge of 4000r/min, adopt concentrated its concentration that makes of poly (ether sulfone) film of molecular weight cut-off 100～500k to promote 20 times, obtain cellulase.
2. a kind of high-efficiency fermenting viride according to claim 1 prepares the method for cellulase, it is characterized in that: described liquid activation culture based formulas is: the phosphoric acid buffer (PBS) that glucose 10～20g/L, peptone 1～2g/L, Tween800.2～0.4g/L, ammonium sulfate 1～2g/L, potassium primary phosphate 1～3g/L, sal epsom 0.1～0.3g/L, calcium chloride 0.1～0.3g/L, urea 0.4～0.8g/L, the Mandels nutritive salt that accounts for liquid activation medium cumulative volume 10～20%, solvent are pH=3～7,120 ℃ of high pressure steam sterilization 20min.
3. a kind of high-efficiency fermenting viride according to claim 1 prepares the method for cellulase, it is characterized in that: described liquid culture medium formula is: glucose 4～6g/L, 40 to 60 order mao bamboon powder 5～20g/L, Tween800.2～0.6g/L, ammonium sulfate 1～3g/L, potassium primary phosphate 3～5g/L, sal epsom 0.3～0.5g/L, calcium chloride 0.3～0.5g/L, urea 0.8～1.2g/L, account for the Mandels trace element of liquid culture medium cumulative volume 0.1～0.3%, the PBS that solvent is pH=3～7,120 ℃ of high pressure steam sterilization 20min.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104070573A (en) * | 2014-06-17 | 2014-10-01 | 福建农林大学 | Efficient and environment-friendly method for preventing bamboo from mildewing |
| CN105018447A (en) * | 2015-08-17 | 2015-11-04 | 湖南省核农学与航天育种研究所 | Method for high-yielding cellulose through trichoderma fungi |
| CN114081103A (en) * | 2021-11-25 | 2022-02-25 | 福建汇盛生物科技有限公司 | Preparation process and device of composite cellulase preparation |
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2013
- 2013-09-01 CN CN201310393491.6A patent/CN103497941B/en not_active Expired - Fee Related
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| 杨淑敏等: "利用X2射线衍射法测定竹材纤维素结晶度", 《东北林业大学学报》 * |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104070573A (en) * | 2014-06-17 | 2014-10-01 | 福建农林大学 | Efficient and environment-friendly method for preventing bamboo from mildewing |
| CN104070573B (en) * | 2014-06-17 | 2016-01-27 | 福建农林大学 | A kind of anti-mildew method of bamboo wood of high-efficiency environment friendly |
| CN105018447A (en) * | 2015-08-17 | 2015-11-04 | 湖南省核农学与航天育种研究所 | Method for high-yielding cellulose through trichoderma fungi |
| CN105018447B (en) * | 2015-08-17 | 2018-01-05 | 湖南省核农学与航天育种研究所 | A kind of method using fungus Trichoderma High Cellulase Production |
| CN114081103A (en) * | 2021-11-25 | 2022-02-25 | 福建汇盛生物科技有限公司 | Preparation process and device of composite cellulase preparation |
| CN114081103B (en) * | 2021-11-25 | 2024-02-09 | 福建汇盛生物科技有限公司 | Preparation process and device of composite cellulase preparation |
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