CN101818136A - Method for producing cellulase by solid fermentation - Google Patents
Method for producing cellulase by solid fermentation Download PDFInfo
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- CN101818136A CN101818136A CN 201010145820 CN201010145820A CN101818136A CN 101818136 A CN101818136 A CN 101818136A CN 201010145820 CN201010145820 CN 201010145820 CN 201010145820 A CN201010145820 A CN 201010145820A CN 101818136 A CN101818136 A CN 101818136A
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Abstract
The invention discloses a method for producing cellulase by solid fermentation, which comprises the following steps of: culturing Trichoderma koningii strains at the temperature of between 28 and 33 DEG C for 16 to 48 hours, and then putting the strains into a seed tank to perform culture through ventilation and stirring at the temperature of between 28 and 33 DEG C for 16 to 40 hours; extracting 5 to 10 weight percent of culture strains, inoculating the culture strains to a disinfected solid fermentation culture medium, and after mixing uniformly, adding water to adjust the mixture until the moisture content of the disinfected mixture is between 65 and 70 percent; performing fermentation for 72 to 120 hours under the condition of keeping ambient temperature of between 26 and 30 DEG C and ambient humidity of between 65 and 70 percent; putting the maturely cultured solid materials into a stirring tank, immersing the maturely cultured solid materials by using clean water which is disinfected and cooled to room temperature in an amount 1 to 2 times the mass of the solid materials for 16 to 24 hours, and simultaneously, stirring the mixture at a speed of between 40 and 50 revolutions per minute to prepare liquid cellulase liquor after filtration. Compared with the activity of the cellulase produced by the conventional solid fermentation technology, the activity of the cellulase produced according to the method is improved by over 2 times, and simultaneously, the method can shorten the fermentation period, improve the production efficiency and reduce the production cost.
Description
Technical field
The present invention relates to the production method of enzyme, particularly relate to a kind of solid fermenting producing method of high active cellulase.
Background technology
Cellulase is that the difference according to each enzyme function is divided into three major types by the enzyme of the complexity that multiple lytic enzyme is formed: the C1 enzyme, be endoglucanase again, and act on the insoluble fibrin surface, random hydrolysis β-1,4-glycosidic link; The Cx enzyme claims the dextran excision enzyme again, acts on Mierocrystalline cellulose linear molecule end, hydrolysis β-1,4-glycosidic link; BG enzyme (being the CB enzyme again) can be hydrolyzed into glucose molecule with cellobiose, procellose.Utilize cellulase cellulose degradation can be become fermentable sugars, and then produce alcohol, single cell protein, organic acid, glycerine, acetone and other important chemical raw material by fermentation.Cellulase also can be used for cellulosic restricted degraded, is prepared into functional food additives, as straw powder, food fibre and functional oligose etc.At present, the utilization of Mierocrystalline cellulose and hemicellulose is international and domestic research hot subject, its objective is and seeks to develop renewable resources abundant, the most cheap on the earth---the approach of vegetable fibre.In addition, cellulase also be widely used in feed, food, brewage, industry such as weaving, papermaking, oil, Chinese patent medicine processing, beverage processing.Therefore, cellulase has boundless application prospect.Yet; the production status ubiquity enzyme activity of China's cellulase is low; survival rate is not high; easily dye assorted bacterium, fermentation period is long, the quality of production is unstable, the cost height; can not large-scale production etc. problem; cause the cellulase shortage, market value is high, is the bottleneck that the restriction cellulose raw material utilizes.
Summary of the invention
The object of the present invention is to provide a kind of method of solid fermentation production of cellulose enzyme, the cellulase survival rate height that utilizes this method to produce, component is complete, and fermentation period is short, the enzyme activity height, good product quality, production cost is low, can realize suitability for industrialized production.
The technical scheme of usefulness that the present invention takes may further comprise the steps:
(1) bacterial classification is chosen: pick out koning trichoderma as producing bacterial strain in cellulase producing bacteria, preserve standby on the inclined-plane;
(2) spawn culture: the koning trichoderma bacterial strain of selecting is put into culturing bottle under 28～33 ℃ temperature condition, cultivated 16～48 hours, put under 28～33 ℃ temperature condition, ventilate stir culture 16～40 hours of seeding tank then;
(3) fermentative processing: get 5～10% cultivation bacterial classification by weight percentage, insert in the disinfectant solid fermentation substratum, constituting of this substratum: straw powder 60～70%, wheat bran 20～30%, ammonium sulfate 4～5%, yeast powder 3～4%, corn steep liquor 1～2% adds water and is adjusted to sterilization back water content and is weight percentage 65～70% behind the mixing; At the natural supply sterile air, keeping envrionment temperature is 26～30 ℃, the condition bottom fermentation of ambient moisture 65～70% 72～120 hours; Cultivate sophisticated solid material and drop into stirred pot, soak with the clean water that disappeared poison and be cooled to room temperature of cultivating 1～2 times of ripe solid material quality, speed with 40～50rpm stirs simultaneously, soaks after 16～24 hours, and the elimination solid residue promptly makes the cellulase solution of liquid; Be to improve yield, once more the filtrate that had obtained with having disappeared poison and be cooled to solid residue of clean water washing of room temperature, and having filtered once more can with filter concentrating filtrate mixing of obtaining for the first time, obtain finished product, filter residue is as solid feed.
Described stalk is paddy rice stalk, cornstalk, straw or straw.
The beneficial effect that the present invention has is:
Production of cellulose enzyme according to the method described above, adopt the koning trichoderma bacterial strain of high cellulase-producing, straw powder, wheat bran, yeast powder, sulfate of ammoniac, corn steep liquor are as the fermentation base-material, can guarantee that the mould-growth breeding is fast, superior product quality, the cellulase activity that vigor is produced than existing solid-fermented technique improves more than 2 times, can shorten cultivation, fermentation period simultaneously, enhance productivity, reduce production costs.This method also has advantages such as production technique is simple, easy and simple to handle, technology is grasped easily, raw material sources extensive, environmentally safe, the scale production that is fit to suit.
Embodiment
The production method of high active cellulase of the present invention is: pick out earlier the koning trichoderma bacterial strain as producing bacterial strain in cellulase producing bacteria, so that enhance productivity, reduce production costs, the bacterial strain of selecting is placed on the inclined-plane, bacterial strain that then will select inserts in the triangle culturing bottle and cultivated under 28～33 ℃ temperature condition 16～48 hours, puts into ventilate stir culture 16～40 hours of seeding tank; Get 5～10% cultivation bacterial classification more by weight percentage, insert in the disinfectant solid fermentation substratum, constituting of this substratum: straw powder 60～70%, wheat bran 20～30%, ammonium sulfate 4～5%, yeast powder 3～4%, corn steep liquor 1～2% adds water and is adjusted to sterilization back water content between 65～70% behind the mixing; At the natural supply sterile air, keep 26～30 ℃ of envrionment temperatures, the condition bottom fermentation of ambient moisture about 65～70% got final product in 72～120 hours.Select for use straw powder, wheat bran, yeast powder, sulfate of ammoniac, corn steep liquor etc. as the fermentation base-material, help the growth and breeding of koning trichoderma, can shorten the production cycle.During the fermentation according to mould-growth fermentation situation, envrionment temperature is controlled between 26～30 ℃, mould-growth is fast, growing way good, envrionment temperature then may command is a bit low relatively, otherwise envrionment temperature can suitably be controlled a bit high, fermented 72～120 hours, and looked the mould-growth situation and reach optimum regime, can stop fermentation.Cultivate sophisticated solid material and drop into stirred pot, soak with the clean water that disappeared poison and be cooled to room temperature of cultivating 1～2 times of ripe solid material quality, speed with 40～50rpm slowly stirs simultaneously, soak after 16～24 hours, the elimination solid residue promptly makes the high active cellulase liquid of liquid; Be to improve yield, the filtrate that can be have once more obtained with having disappeared poison and be cooled to solid residue of clean water washing of room temperature, and having filtered once more can with filter concentrating filtrate mixing of obtaining for the first time, obtain finished product, filter residue can be used as solid feed.
This method adopts the koning trichoderma bacterial strain of high cellulase-producing as producing bacterial strain, in low sugar culture-medium, can grow rapid and enzymic synthesis is not checked, and utilize straw powder, wheat bran, yeast powder, sulfate of ammoniac, corn steep liquor etc. to carry out solid fermentation production of cellulose zymin.The cellulose components that makes is complete, and enzyme is lived and cost ratio height, and technology is easily grasped, fermentation period short, constant product quality, and nontoxic pollution-free can reach the standard as foodstuff additive and fodder additives that China Ministry of Health announces for 2003 No. 4 fully.This cellulase can be applicable to feed, weaving, oil, garden stuff processing, paper-making industry, Chinese patent medicine processing, particularly biomass ethanol and the field such as produces.Isolated solid residue contained nutritive substances such as a certain amount of cellulase and tropina after fermentation obtained zymin, can directly be used as the feed of high nutritive value.
Embodiment 1:
Below again with 1m
3The incubator test is done with detailed explanation the present invention for embodiment.
Picking out earlier the koning trichoderma bacterial strain in cellulase producing bacteria is placed on the inclined-plane as producing bacterial strain, bacterial strain that then will select inserts in the triangle culturing bottle and cultivated 16 hours under 30 ℃ temperature condition, puts into ventilate stir culture 35 hours of the 25L seeding tank that the 20L nutrient solution is housed; All insert in the disinfectant 280kg solid fermentation substratum the constituting of this substratum: first water rice straw powder 60%, wheat bran 30%, ammonium sulfate 4%, yeast powder 4%, corn steep liquor 2% mixes, and adds water behind the mixing and is adjusted to sterilization back water content and is weight percentage 70%.At the natural supply sterile air, keep 26～30 ℃ of envrionment temperatures, the condition bottom fermentation of ambient moisture about 65～70% got final product in 72 hours.Cultivate sophisticated solid material and drop into 2m
3Stirred pot soaks with the clean water that disappeared poison and be cooled to room temperature of 280kg, and the speed with 40～50rpm slowly stirs simultaneously, soaks after 16 hours, and the elimination solid residue promptly makes the high active cellulase liquid of liquid; Be to improve yield, once more with disappeared poison and be cooled to solid residue of clean water washing of room temperature of 280kg, filter once more the filtrate that obtains can with filter concentrating filtrate mixing of obtaining for the first time, obtain finished product, filter residue can be used as solid feed.
Embodiment 2:
Below again with 10m
3The incubator test is done with detailed explanation the present invention for embodiment.
Picking out earlier the koning trichoderma bacterial strain in cellulase producing bacteria is placed on the inclined-plane as producing bacterial strain, bacterial strain that then will select inserts in the triangle culturing bottle and cultivated 36 hours under 30 ℃ temperature condition, is equipped with in the 250L seeding tank of 200L nutrient solution to cultivate 40 hours; All insert in 2.8 tons of solid fermentation substratum of disinfectant the constituting of this substratum: earlier with cornstalk powder 66%, wheat bran 25%, ammonium sulfate 4.5%, yeast powder 3.5%, corn steep liquor 1% mixes, and adds water behind the mixing and is adjusted to sterilization back water content and is weight percentage 67%.At the natural supply sterile air, keep 26～28 ℃ of envrionment temperatures, the condition bottom fermentation of ambient moisture about 65～70% got final product in 100 hours.Cultivate sophisticated solid material and drop into 12m
3Stirred pot soaks with 4.5 tons of clean waters that disappeared poison and be cooled to room temperature, and the speed with 40～50rpm slowly stirs simultaneously, soaks after 20 hours, and the elimination solid residue promptly makes the high active cellulase liquid of liquid; Be to improve yield, once more with 4.5 tons of poison and be cooled to solid residue of clean water washing of room temperature of having disappeared, filter once more the filtrate that obtains can with concentrating filtrate mixing that filtration for the first time obtains, obtain finished product, filter residue can be used as solid feed.
Embodiment 3:
Below again with 50m
3The incubator test is done with detailed explanation the present invention for embodiment.
In cellulase producing bacteria, pick out earlier the koning trichoderma bacterial strain and be placed on the inclined-plane, the bacterial strain of selecting is inserted in the triangle culturing bottle under 30 ℃ temperature condition, cultivated 48 hours then, 0.8m is housed as producing bacterial strain
3The 1m of nutrient solution
3Cultivated 16 hours in the seeding tank; All insert in 13.2 tons of solid fermentation substratum of disinfectant the constituting of this substratum: earlier with straw powder 70%, wheat bran 20%, ammonium sulfate 5%, yeast powder 4%, corn steep liquor 1% mixes, and adds water behind the mixing and is adjusted to sterilization back water content and is weight percentage 65%.At the natural supply sterile air, keep 26～27 ℃ of envrionment temperatures, the condition bottom fermentation of ambient moisture about 65～70% got final product in 120 hours.Cultivate sophisticated solid material and drop into 60m
3Stirred pot soaks with 26.4 tons of clean waters that disappeared poison and be cooled to room temperature, and the speed with 40～50rpm slowly stirs simultaneously, soaks after 24 hours, and the elimination solid residue promptly makes the high active cellulase liquid of liquid; Be to improve yield, once more with 26.4 tons of poison and be cooled to solid residue of clean water washing of room temperature of having disappeared, filter once more the filtrate that obtains can with concentrating filtrate mixing that filtration for the first time obtains, obtain finished product, filter residue can be used as solid feed.
Claims (2)
1. the solid fermenting producing method of a cellulase, it is characterized in that: it may further comprise the steps:
(1) bacterial classification is chosen: pick out koning trichoderma as producing bacterial strain in cellulase producing bacteria, preserve standby on the inclined-plane;
(2) spawn culture: the koning trichoderma bacterial strain of selecting is put into culturing bottle under 28～33 ℃ temperature condition, cultivated 16～48 hours, put under 28～33 ℃ temperature condition, ventilate stir culture 16～40 hours of seeding tank then;
(3) fermentative processing: get 5～10% cultivation bacterial classification by weight percentage, insert in the disinfectant solid fermentation substratum, constituting of this substratum: straw powder 60～70%, wheat bran 20～30%, ammonium sulfate 4～5%, yeast powder 3～4%, corn steep liquor 1～2% adds water and is adjusted to sterilization back water content and is weight percentage 65～70% behind the mixing; At the natural supply sterile air, keeping envrionment temperature is 26～30 ℃, the condition bottom fermentation of ambient moisture 65～70% 72～120 hours; Cultivate sophisticated solid material and drop into stirred pot, soak with the clean water that disappeared poison and be cooled to room temperature of cultivating 1～2 times of ripe solid material quality, speed with 40～50rpm stirs simultaneously, soaks after 16～24 hours, and the elimination solid residue promptly makes the cellulase solution of liquid; Be to improve yield, once more the filtrate that had obtained with having disappeared poison and be cooled to solid residue of clean water washing of room temperature, and having filtered once more can with filter concentrating filtrate mixing of obtaining for the first time, obtain finished product, filter residue is as solid feed.
2. the solid fermenting producing method of a kind of cellulase according to claim 1, it is characterized in that: described stalk is paddy rice stalk, cornstalk, straw or straw.
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| CN 201010145820 CN101818136A (en) | 2010-04-13 | 2010-04-13 | Method for producing cellulase by solid fermentation |
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| CN 201010145820 CN101818136A (en) | 2010-04-13 | 2010-04-13 | Method for producing cellulase by solid fermentation |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103283953A (en) * | 2013-06-22 | 2013-09-11 | 河南歌瑞农牧股份有限公司 | Beef cattle health care type feed production method |
| CN104164463A (en) * | 2014-06-09 | 2014-11-26 | 泰安生力源生物工程有限公司 | Low-moisture raw material solid fermentation method for assimilating inorganic nitrogen of fiber byproducts |
| CN108299029A (en) * | 2018-03-01 | 2018-07-20 | 商丘职业技术学院 | A kind of maize straw quick rotting agent and preparation method thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101532003A (en) * | 2009-04-20 | 2009-09-16 | 杭州贝奥生物技术工程有限公司 | Production method of acidophilic cellulose |
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2010
- 2010-04-13 CN CN 201010145820 patent/CN101818136A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101532003A (en) * | 2009-04-20 | 2009-09-16 | 杭州贝奥生物技术工程有限公司 | Production method of acidophilic cellulose |
Non-Patent Citations (3)
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| 《宁夏大学学报（自然科学版）》 20070630 李爱华等 培养条件对康宁木霉产酶的影响 第173-175页 1-2 第28卷, 第2期 2 * |
| 《湖南农业大学学报（自然科学版）》 20080430 胡仕凤等 康氏木霉REMI突变株产纤维素酶固态发酵条件 第207-211页 1-2 第34卷, 第2期 2 * |
| 《纤维素科学与技术》 20051231 王菁莎等 康宁木霉固态发酵秸秆生产纤维素酶的研究 第26-31页 1-2 第13卷, 第4期 2 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103283953A (en) * | 2013-06-22 | 2013-09-11 | 河南歌瑞农牧股份有限公司 | Beef cattle health care type feed production method |
| CN104164463A (en) * | 2014-06-09 | 2014-11-26 | 泰安生力源生物工程有限公司 | Low-moisture raw material solid fermentation method for assimilating inorganic nitrogen of fiber byproducts |
| CN108299029A (en) * | 2018-03-01 | 2018-07-20 | 商丘职业技术学院 | A kind of maize straw quick rotting agent and preparation method thereof |
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Open date: 20100901 |