CN102392006A - Production method for raising output of acidic cellulose produced by use of Trichoderma spp - Google Patents
Production method for raising output of acidic cellulose produced by use of Trichoderma spp Download PDFInfo
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Abstract
The invention discloses a production method for raising the output of acidic cellulose produced by the use of Trichoderma spp, and relates to the field of microorganism. According to the metabolic rule of microorganisms, the output of acidic cellulose is raised by reasonable design of the carbon source concentration of a fermentation medium and segmental adjustment of pH value during the strain fermentation process.
Description
Technical field
The present invention relates to microorganism field, relate in particular to a kind of raising and utilize Trichoderma to produce the working method of the output of acidic cellulase.
Background technology
Cellulose substances is a polysaccharide material the abundantest on the earth; It also is a kind of renewable resources of the tool potentiality of occurring in nature; Utilize cellulase that it is hydrolyzed into glucose; Further fermentative production of ethanol, organic acid, single cell protein etc., for solving problems such as energy shortage that current mankind faces, environmental pollution, and it is significant to promote sustainable development etc.
Cellulase is a kind of prozyme, and staple is made up of NCE5, exoglucanase and beta-glucosidase, and three kinds of component synergies can be degraded to glucose with cellulose macromolecule.Mikrobe particularly fungi has the ability that produces this prozyme, wherein produces stronger the having of enzyme activity wood is mould, aspergillus, head mold and mould etc., and is in the majority with the Trichoderma bacterial classification especially.Easy, the technology of cellulase production process control efficiently is exactly the precondition that will realize that at present the cellulase suitability for industrialized production is indispensable.
Summary of the invention
The present invention has designed and developed a kind of raising and has utilized Trichoderma to produce the working method of the output of acidic cellulase.In the present invention; According to the metabolic rule of mikrobe, appropriate design the carbon source concentration of fermention medium, and according to the Changing Pattern of pH value in the fermenting process; PH value in the strain fermentation process is carried out sectional-regulated, reach and improve the purpose that Trichoderma produces the output of acidic cellulase.
Technical scheme provided by the invention is:
A kind of raising utilizes Trichoderma to produce the working method of the output of acidic cellulase, includes following steps:
Step 1, preparation Trichoderma kind seed liquor;
Step 2, preparatory production phase: said Trichoderma kind seed liquor is inoculated in the preparatory production fermention medium; Regulate original ph 3.0～7.0, begin to ferment, measure during the fermentation; Produce the flex point of the pH value beginning sustainable growth of fermention medium in advance, confirm the flex point time T;
Step 3, production phase: with said Trichoderma kind seed liquor be inoculated in said preparatory production fermention medium component and the identical production fermention medium of component concentration in; It is also identical to regulate original ph; Begin to ferment, in the fermenting process, carry out sectional-regulated the pH value of said production fermention medium; Control method is following: (1) fermentation beginning to said flex point time T was first period; PH value to said production fermention medium does not add control, and (2) were second period from said flex point time T to fermentation ends, and the pH value of controlling said production fermention medium is 4.0～5.3.
Preferably, described raising utilizes Trichoderma to produce in the working method of output of acidic cellulase, wherein; By mass percentage, said preparatory production fermention medium also comprises: Microcrystalline Cellulose 3%～7%, organic nitrogen source 2～4%; Inorganic nitrogen-sourced 0.3～0.6%; Inorganic salt 0.6～1.3%, glycerine 0.15～0.4%, and aqueous solvent.
Preferably, described raising utilizes Trichoderma to produce in the working method of output of acidic cellulase, and in the said step 2, the original ph of regulating said preparatory production fermention medium is 4.5～5.5.
Preferably; Described raising utilizes Trichoderma to produce in the working method of output of acidic cellulase, in the said step 3, in the fermenting process; Second period from time flex point T to fermentation ends, the pH value of controlling said production fermention medium is 4.7～5.1.
Preferably; Described raising utilizes Trichoderma to produce in the working method of output of acidic cellulase, in the said step 3, in said fermenting process; Fermentation 20h to 80h; Control said production fermention medium in the volume percent of dissolved oxygen 30%～40%, 80h is to fermentation ends in fermentation, the volume percent of dissolved oxygen of controlling said production fermention medium is 20%～30%.
Preferably, described raising utilizes Trichoderma to produce in the working method of output of acidic cellulase, in the step 2, Microcrystalline Cellulose account for said preparatory production fermention medium total mass 5%.
Preferably, described raising utilizes Trichoderma to produce in the working method of output of acidic cellulase, and in said step 2 and the step 3, the temperature in the fermenting process is controlled at 25～30 ℃.
Preferably, described raising utilizes Trichoderma to produce in the working method of output of acidic cellulase, and in the said step 2, the volume of said preparatory production fermention medium is 0.1L; In the said step 3, the volume of said production fermention medium is 3L.
Preferably, described raising utilizes Trichoderma to produce in the working method of output of acidic cellulase, and in said step 2 and the step 3, fermenting process is concussion cultivation on 250～350r/min constant temperature shaking table.
Preferably, described raising utilizes Trichoderma to produce in the working method of output of acidic cellulase, in the said step 1; The method for preparing Trichoderma kind seed liquor is that Trichoderma kind spore suspension is inserted in the activation medium, at 28～30 ℃; Concussion is cultivated 24～36h and is obtained the bacterial classification seed liquor on 170～200r/min constant temperature shaking table, and wherein, said activation medium comprises Microcrystalline Cellulose 1～4%; Steeping water 1～2%, and aqueous solvent are regulated original ph 4.0～5.0.
Raising of the present invention utilizes Trichoderma to produce the working method of the output of acidic cellulase; Metabolic rule according to mikrobe; Appropriate design the carbon source concentration of fermention medium; And according to the Changing Pattern of pH value in the fermenting process, the pH value in the strain fermentation process is carried out sectional-regulated, reach and improve the purpose that Trichoderma produces the output of acidic cellulase.
Description of drawings
Fig. 1 is fermention medium pH value change with time figure (Trichodermareesei and viride) in the Trichoderma kind fermenting process
Embodiment
Below in conjunction with accompanying drawing the present invention is done further detailed description, can implement according to this with reference to the specification sheets literal to make those skilled in the art.
As shown in Figure 1, the present invention provides a kind of raising to utilize Trichoderma to produce the working method of the output of acidic cellulase, includes following steps: step 1, preparation Trichoderma kind seed liquor; Step 2, preparatory production phase: said Trichoderma kind seed liquor is inoculated in the preparatory production fermention medium; Regulate original ph 3.0～7.0, begin to ferment, measure during the fermentation; Produce the flex point of the pH value beginning sustainable growth of fermention medium in advance, confirm the flex point time T; Step 3, production phase: with said Trichoderma kind seed liquor be inoculated in said preparatory production fermention medium component and the identical production fermention medium of component concentration in; It is also identical to regulate original ph; Begin to ferment, in the fermenting process, carry out sectional-regulated the pH value of said production fermention medium; Control method is following: (1) fermentation beginning to said flex point time T was first period; PH value to said production fermention medium does not add control, and (2) were second period from said flex point time T to fermentation ends, and the pH value of controlling said production fermention medium is 4.0～5.3.
Described raising utilizes Trichoderma to produce in the working method of output of acidic cellulase, wherein, and by mass percentage; Said preparatory production fermention medium also comprises: Microcrystalline Cellulose 3%～7%; Organic nitrogen source 2～4%, inorganic nitrogen-sourced 0.3～0.6%, inorganic salt 0.6～1.3%; Glycerine 0.15～0.4%, and aqueous solvent.
Described raising utilizes Trichoderma to produce in the working method of output of acidic cellulase, and in the said step 2, the original ph of regulating said preparatory production fermention medium is 4.5～5.5.
Described raising utilizes Trichoderma to produce in the working method of output of acidic cellulase, and in the said step 3, in the fermenting process, second period from time flex point T to fermentation ends, the pH value of controlling said production fermention medium is 4.7～5.1.
Described raising utilizes Trichoderma to produce in the working method of output of acidic cellulase; In the said step 3; In said fermenting process, fermentation 20h to 80h, control said production fermention medium in the volume percent of dissolved oxygen 30%～40%; 80h is to fermentation ends in fermentation, and the volume percent of dissolved oxygen of controlling said production fermention medium is 20%～30%.
Described raising utilizes Trichoderma to produce in the working method of output of acidic cellulase, in the step 2, Microcrystalline Cellulose account for said preparatory production fermention medium total mass 5%.
Described raising utilizes Trichoderma to produce in the working method of output of acidic cellulase, and in said step 2 and the step 3, the temperature in the fermenting process is controlled at 25～30 ℃.
Described raising utilizes Trichoderma to produce in the working method of output of acidic cellulase, and in the said step 2, the volume of said preparatory production fermention medium is 0.1L; In the said step 3, the volume of said production fermention medium is 3L.
Described raising utilizes Trichoderma to produce in the working method of output of acidic cellulase, and in said step 2 and the step 3, fermenting process is concussion cultivation on 250～350r/min constant temperature shaking table.
Described raising utilizes Trichoderma to produce in the working method of output of acidic cellulase, and in the said step 1, the method for preparing Trichoderma kind seed liquor is; Trichoderma kind spore suspension is inserted in the activation medium, and at 28～30 ℃, concussion is cultivated 24～36h and is obtained the bacterial classification seed liquor on 170～200r/min constant temperature shaking table; Wherein, said activation medium comprises Microcrystalline Cellulose 1～4%, steeping water 1～2%; And aqueous solvent, regulate original ph 4.0～5.0.
Experiment material
In this experiment; Respectively with Trichodermareesei Rut C-30 (Trichoderma reesei Rut C-30; Preserving number is CICC13052; Buy in Chinese industrial microbial strains preservation administrative center), two kinds of virides (Trichoderma viride, preserving number is CGMCC3.2941, buys the common DSMZ in Institute of Microorganism, Academia Sinica) are strains tested.
Activation medium comprises Microcrystalline Cellulose 1～4%, and steeping water 1～2%, and aqueous solvent are regulated original ph 4.0～5.0.
Fermention medium of the present invention comprises preparatory production fermention medium and produces fermention medium.As carbon source, steeping water is as organic nitrogen source with Microcrystalline Cellulose for fermention medium, (NH
4)
2SO
4As inorganic nitrogen-sourced, glycerine is as the direct carbon source at strain fermentation initial stage, KH
2PO
4, CaCO
3, MgSO
4As the inorganic salt composition, wherein, KH
2PO
4, CaCO
3Available buffer solution, MgSO
4Can promote enzymic synthesis.Therefore, the fermention medium of being determined comprises: Microcrystalline Cellulose 3～7%, steeping water 2～4%, (NH
4)
2SO
40.3～0.6%, KH
2PO
40.4～0.8%, glycerine 0.15～0.4%, CaCO
30.15～0.4%, MgSO
40.06～0.14%, and aqueous solvent, original ph 4.5～5.5 regulated.Wherein, organic nitrogen source can also be that albumen freezes, and the organic nitrogen source of using always in other this areas; Inorganic nitrogen-sourced can also be NH
4That uses always in Cl and this area is inorganic nitrogen-sourced.
In following examples, the International Standards Method that the detection method of cellulase activity adopts IUPAC (IUPAC) to recommend is through measuring the activity level that filter paper enzyme activity is used to characterize cellulase.
Embodiment one
Experiment finds that cellulosic substrate is as carbon source, and its concentration can produce very big influence to the fermenting process of mikrobe, especially can influence the pH value in the fermenting process of mikrobe.Among the present invention with Microcrystalline Cellulose as carbon source, investigate and to produce fermention medium in advance or to produce in the preparatory fermention medium carbon source concentration the influence of the output of the cellulase of microbial fermentation gained.
Microcrystalline Cellulose concentration changes (other compositions are all constant) between 2%～7%, obtain the Changing Pattern along with the variation filter paper enzyme activity of Microcrystalline Cellulose concentration.In conjunction with factors such as production cost and economic benefits, the Microcrystalline Cellulose mass percent was the working condition of the best at 5% o'clock, and filter paper enzyme activity is higher relatively.Above-mentioned conclusion is applicable to the situation that the fermention medium scale is less and bigger simultaneously.
Investigate the Changing Pattern (see figure 1) of pH value in the Trichoderma kind fermenting process.In the fermenting process, there is flex point in the variation of pH value.And the appearance position of above-mentioned flex point is along with the difference of carbon source concentration also can difference.Carbon source concentration is big more, and the time flex point occurs more late.In the fermenting process of bacterial classification; The fermentation culture initial stage (fermentation began to first period of time flex point T); Mycelium absorbs and consumes nutritive ingredient in the fermention medium; Fermented liquid pH value rises in the fermention medium, and along with the minimizing and the product acid of nutritive substance, the rising of fermented liquid pH value slows down and subsides a little.Above-mentioned fermenting process fermented liquid pH value changes, but amplitude is limited, is difficult to also needn't do too tight control to the pH value during this period.Mikrobe gets into the logarithmic growth later stage or produce enzyme (time, flex point T was to fermentation ends) stationary phase, and the pH value occurs continuing to rise since time flex point T, and ascensional range is bigger, and the fermented liquid pH value of controlling well this moment is most important.The pH value low or too high meeting cause that the bacterial metabolism process is different, the quality of meta-bolites and ratio are changed, mycelia stability is had disadvantageous effect, and influences the activity of cellulase protein.
For Trichodermareesei, the position that time flex point T occurs is at 16～20h, and the position that viride time flex point T occurs is then at 23～26h.
Embodiment two
Can know that by embodiment one the pH value of regulating fermenting process can satisfy the demand that improves fermentation yield and subsequent extracted purifying process simultaneously.
Activation medium comprises Microcrystalline Cellulose 4%, and steeping water 2%, and aqueous solvent are regulated original ph 5.0.
Producing fermention medium in advance comprises: Microcrystalline Cellulose 7%, steeping water 4%, (NH
4)
2SO
40.6%, KH
2PO
40.8%, glycerine 0.4%, CaCO
30.4%, MgSO
40.14%, and aqueous solvent, original ph 5.5 regulated.
With bacterial classification spore suspension (10
7～10
8Individual/as ml) to insert activation medium, on 30 ℃, 200r/min shaking table, cultivate 36h, obtain seed liquor.Seed liquor is seeded to fresh preparatory production fermention medium by 5% inoculum size.At 30 ℃, concussion is cultivated on the 350r/min constant temperature shaking table, intermittently cultivates the fermentative prodn cellulase.Fermentation total time is 135h.
The preparatory production phase: fermenting process is not carried out the control of pH value, and the flex point (time flex point T) that obtains the variation of pH value relative time is respectively Trichodermareesei 18h, viride 25h.The volume of producing fermention medium in advance is 0.1L (placing 250ml to shake in the bottle).
Production phase: in the fermenting process; PH value to producing fermention medium is carried out sectional-regulated; Control method is following: (1) first period, the pH value of producing fermention medium is not added control, (2) second periods; Add sulfuric acid or ammoniacal liquor adjusting pH value through stream in producing fermention medium, control is produced the pH value of fermention medium 4.7～5.1.Wherein, the volume of producing fermention medium is 3L (placing the 5L fermentor tank), and other conditions are identical with the preparatory production phase.
In addition; Also compare experiment; In the fermenting process of production phase, remove and carry out the adjusting of pH value, carry out the sectional-regulated of dissolved oxygen content, (1) is during fermentation 20 to 80h; Through regulating shaking speed and ventilation, the volume percent that dissolved oxygen in the fermention medium is produced in control maintains 30～40%; (2) fermentation 80h maintains 20～30% to the volume percent of fermentation ends dissolved oxygen.
Blank assay is a situation about in the production phase fermenting process not being controlled.
The filter paper enzyme activity contrast (Microcrystalline Cellulose concentration be 7%, three kind fermentation condition for fermenting process pH value segmentation control, fermenting process pH value segmentation control+dissolved oxygen segmentation control and fermenting process pH value do not add control) of two kinds of Trichoderma kinds of table 1 under production phase three kinds of fermentation conditions
Can know that by table 1 Microcrystalline Cellulose concentration is at 7% o'clock, than to the complete out-of-control situation of fermenting process, only the pH value of the fermenting process of production phase regulated that for Trichodermareesei, filter paper enzyme activity has improved 48%; For viride, filter paper enzyme activity has improved 23%.Than to the complete out-of-control situation of fermenting process, the fermenting process of production phase is carried out the adjusting of pH value and dissolved oxygen, for Trichodermareesei, filter paper enzyme activity has improved 60%; For viride, filter paper enzyme activity has improved 32%.
Embodiment three
Activation medium comprises Microcrystalline Cellulose 1%, and steeping water 1%, and aqueous solvent are regulated original ph 4.0.
Producing fermention medium in advance comprises: Microcrystalline Cellulose 3%, steeping water 2%, (NH
4)
2SO
40.3%, KH
2PO
40.4%, glycerine 0.15%, CaCO
30.15%, MgSO
40.06%, and aqueous solvent, original ph 4.5 regulated.
With bacterial classification spore suspension (10
7～10
8Individual/as ml) to insert activation medium, on 28 ℃, 170r/min shaking table, cultivate 24h, obtain seed liquor.Seed liquor is seeded to fresh preparatory production fermention medium by 5% inoculum size.At 25 ℃, concussion is cultivated on the 250r/min constant temperature shaking table, intermittently cultivates the fermentative prodn cellulase.Fermentation total time is 120h.
The preparatory production phase: fermenting process is not carried out the control of pH value, and the flex point that obtains the variation of pH value relative time is respectively Trichodermareesei 17h, viride 23h.The volume of producing fermention medium in advance is 0.1L (placing 250ml to shake in the bottle).
Production phase: in the fermenting process; PH value to producing fermention medium is carried out sectional-regulated; Control method is following: (1) first period, the pH value of producing fermention medium is not added control, (2) second periods; Add sulfuric acid or ammoniacal liquor adjusting pH value through stream in producing fermention medium, control is produced the pH value of fermention medium 4.0～5.3.Wherein, the volume of producing fermention medium is 3L (placing the 5L fermentor tank), and other conditions are identical with the preparatory production phase.
In addition; Also compare experiment; In the fermenting process of production phase, remove and carry out the adjusting of pH value, carry out the sectional-regulated of dissolved oxygen content, (1) is during fermentation 20 to 80h; Through regulating shaking speed and ventilation, the volume percent that dissolved oxygen in the fermention medium is produced in control maintains 30～40%; (2) fermentation 80h maintains 20～30% to the volume percent of fermentation ends dissolved oxygen.
The filter paper enzyme activity contrast (Microcrystalline Cellulose concentration be 3%, three kind fermentation condition for fermenting process pH value segmentation control, fermenting process pH value segmentation control+dissolved oxygen segmentation control and fermenting process pH value do not add control) of two kinds of Trichoderma kinds of table 2 under production phase three kinds of fermentation conditions
Can know that by table 2 Microcrystalline Cellulose concentration is at 3% o'clock, than to the complete out-of-control situation of fermenting process, only the pH value carried out sectional-regulatedly in the fermenting process of production phase that for Trichodermareesei, filter paper enzyme activity has improved 17%; For viride, filter paper enzyme activity has improved 23%.Than to the complete out-of-control situation of fermenting process, in the fermenting process of production phase the pH value is carried out sectional-regulatedly, and also dissolved oxygen is carried out sectional-regulatedly, for Trichodermareesei, filter paper enzyme activity has improved 22%; For viride, filter paper enzyme activity has improved 27%.
Embodiment four
Activation medium comprises Microcrystalline Cellulose 4%, and steeping water 2%, and aqueous solvent are regulated original ph 5.0.
Producing fermention medium in advance comprises: Microcrystalline Cellulose 7%, steeping water 4%, (NH
4)
2SO
40.6%, KH
2PO
40.8%, glycerine 0.4%, CaCO
30.4%, MgSO
40.14%, and aqueous solvent, original ph 7.0 regulated.
With bacterial classification spore suspension (10
7～10
8Individual/as ml) to insert activation medium, on 30 ℃, 200r/min shaking table, cultivate 36h, obtain seed liquor.Seed liquor is seeded to fresh preparatory production fermention medium by 5% inoculum size.At 30 ℃, concussion is cultivated on the 350r/min constant temperature shaking table, intermittently cultivates the fermentative prodn cellulase.Fermentation total time is 135h.
The preparatory production phase: fermenting process is not carried out the control of pH value, and the flex point that obtains the variation of pH value relative time is respectively Trichodermareesei 19h, viride 26h.The volume of producing fermention medium in advance is 0.1L (placing 250ml to shake in the bottle).
Production phase: in the fermenting process, the pH value of producing fermention medium is carried out sectional-regulated, the time flex point that the segmentation of pH value is controlled is Trichodermareesei 19h, viride 26h.Control method is following: (1) first period, the pH value of producing fermention medium is not added control, and (2) second periods, add sulfuric acid or ammoniacal liquor adjusting pH value through stream in producing fermention medium, control is produced the pH value of fermention medium 4.0～5.3.Wherein, the volume of producing fermention medium is 3L (placing the 5L fermentor tank), and other conditions are identical with the preparatory production phase.
In addition; Also compare experiment; In the fermenting process of production phase, remove and carry out the adjusting of pH value, also carry out the sectional-regulated of dissolved oxygen content, (1) is during fermentation 20 to 80h; Through regulating shaking speed and ventilation, the volume percent that dissolved oxygen in the fermention medium is produced in control maintains 30～40%; (2) fermentation 80h maintains 20～30% to the volume percent of fermentation ends dissolved oxygen.
The filter paper enzyme activity contrast (Microcrystalline Cellulose concentration be 7%, three kind fermentation condition for fermenting process pH value segmentation control, fermenting process pH value segmentation control+dissolved oxygen segmentation control and fermenting process pH value do not add control) of two kinds of Trichoderma kinds of table 3 under production phase three kinds of fermentation conditions
Can know that by table 3 than to the complete out-of-control situation of fermenting process, only sectional-regulated to the pH value of the fermenting process of production phase, for Trichodermareesei, filter paper enzyme activity has improved 49%; For viride, filter paper enzyme activity has improved 20%.Than to the complete out-of-control situation of fermenting process, the pH value of the fermenting process of production phase and dissolved oxygen content are carried out sectional-regulated, for Trichodermareesei, filter paper enzyme activity has improved 58%; For viride, filter paper enzyme activity has improved 42%.
Implement five
Activation medium comprises Microcrystalline Cellulose 1%, and steeping water 1%, and aqueous solvent are regulated original ph 4.0.
Producing fermention medium in advance comprises: Microcrystalline Cellulose 3%, steeping water 2%, (NH
4)
2SO
40.3%, KH
2PO
40.4%, glycerine 0.15%, CaCO
30.15%, MgSO
40.06%, and aqueous solvent, original ph 3.0 regulated.
With bacterial classification spore suspension (10
7～10
8Individual/as ml) to insert activation medium, on 28 ℃, 170r/min shaking table, cultivate 24h, obtain seed liquor.Seed liquor is seeded to fresh preparatory production fermention medium by 5% inoculum size.At 25 ℃, concussion is cultivated on the 250r/min constant temperature shaking table, intermittently cultivates the fermentative prodn cellulase.Fermentation total time is 120h.
The preparatory production phase: fermenting process is not carried out the control of pH value, and the flex point that obtains the variation of pH value relative time is respectively Trichodermareesei 17h, viride 24h.The volume of producing fermention medium in advance is 0.1L (placing 250ml to shake in the bottle).
Production phase: in the fermenting process, the pH value of producing fermention medium is carried out sectional-regulated, the time flex point that the segmentation of pH value is controlled is Trichodermareesei 17h, viride 24h.Control method is following: (1) fermentation begins to the time flex point; PH value to producing fermention medium does not add control; (2) flex point is to fermentation ends the time, adds sulfuric acid or ammoniacal liquor is regulated pH value through stream in producing fermention medium, and the pH value of controlling fermention medium is 4.0～5.3.Wherein, the volume of producing fermention medium is 3L (placing the 5L fermentor tank), and other conditions are identical with the preparatory production phase.
In addition; Also compare experiment; In the fermenting process of production phase, remove and carry out the adjusting of pH value, carry out the sectional-regulated of dissolved oxygen content, (1) is during fermentation 20 to 80h; Through regulating shaking speed and ventilation, the volume percent that dissolved oxygen in the fermention medium is produced in control maintains 30～40%; (2) fermentation 80h maintains 20～30% to the volume percent of fermentation ends dissolved oxygen.
The filter paper enzyme activity contrast (Microcrystalline Cellulose concentration be 3%, three kind fermentation condition for fermenting process pH value segmentation control, fermenting process pH value segmentation control+dissolved oxygen segmentation control and fermenting process pH value do not add control) of two kinds of Trichoderma kinds of table 4 under production phase three kinds of fermentation conditions
Can know that by table 4 than to the complete out-of-control situation of fermenting process, only the fermenting process of production phase is carried out the pH value when sectional-regulated, for Trichodermareesei, filter paper enzyme activity has improved 17%; For viride, filter paper enzyme activity has improved 28%.Than to the complete out-of-control situation of fermenting process, the fermenting process pH value of production phase is sectional-regulated sectional-regulated with dissolved oxygen, and for Trichodermareesei, filter paper enzyme activity has improved 26%; For viride, filter paper enzyme activity has improved 33%.Can know that by data after the fermentation ends, filter paper enzyme activity all has growth.
Embodiment six
Activation medium comprises Microcrystalline Cellulose 4%, and steeping water 2%, and aqueous solvent are regulated original ph 5.0.
Producing fermention medium in advance comprises: Microcrystalline Cellulose 5%, steeping water 4%, (NH
4)
2SO
40.6%, KH
2PO
40.8%, glycerine 0.4%, CaCO
30.4%, MgSO
40.14%, and aqueous solvent, original ph 7.0 regulated.
With bacterial classification spore suspension (10
7～10
8Individual/as ml) to insert activation medium, on 30 ℃, 200r/min shaking table, cultivate 36h, obtain seed liquor.Seed liquor is seeded to fresh preparatory production fermention medium by 5% inoculum size.At 30 ℃, concussion is cultivated on the 350r/min constant temperature shaking table, intermittently cultivates the fermentative prodn cellulase.Fermentation total time is 135h.
The preparatory production phase: fermenting process is not carried out the control of pH value, and the flex point that obtains the variation of pH value relative time is respectively Trichodermareesei 18h, viride 25h.The volume of producing fermention medium in advance is 0.1L (placing 250ml to shake in the bottle).
Production phase: in the fermenting process, the pH value of producing fermention medium is carried out sectional-regulated, the time flex point that the segmentation of pH value is controlled is Trichodermareesei 18h, viride 25h.Control method is following: (1) first period, the pH value of producing fermention medium is not added control, and (2) second periods, add sulfuric acid or ammoniacal liquor adjusting pH value through stream in producing fermention medium, control is produced the pH value of fermention medium 4.7～5.1.Wherein, the volume of producing fermention medium is 3L (placing the 5L fermentor tank), and other conditions are identical with the preparatory production phase.
In addition; Also compare experiment; In the fermenting process of production phase, remove and carry out the adjusting of pH value, also carry out the sectional-regulated of dissolved oxygen content, (1) is during fermentation 20 to 80h; Through regulating shaking speed and ventilation, the volume percent that dissolved oxygen in the fermention medium is produced in control maintains 30～40%; (2) fermentation 80h maintains 20～30% to the volume percent of fermentation ends dissolved oxygen.
The filter paper enzyme activity contrast (Microcrystalline Cellulose concentration be 5%, three kind fermentation condition for fermenting process pH value segmentation control, fermenting process pH value segmentation control+dissolved oxygen segmentation control and fermenting process pH value do not add control) of two kinds of Trichoderma kinds of table 5 under production phase three kinds of fermentation conditions
Can know that by table 5 than to the complete out-of-control situation of fermenting process, only sectional-regulated to the pH value of the fermenting process of production phase, for Trichodermareesei, filter paper enzyme activity has improved 49%; For viride, filter paper enzyme activity has improved 46%.Than to the complete out-of-control situation of fermenting process, the pH value of the fermenting process of production phase and dissolved oxygen content are carried out sectional-regulated, for Trichodermareesei, filter paper enzyme activity has improved 67%; For viride, filter paper enzyme activity has improved 54%.
Embodiment seven
Activation medium comprises Microcrystalline Cellulose 1%, and steeping water 1%, and aqueous solvent are regulated original ph 4.0.
Producing fermention medium in advance comprises: Microcrystalline Cellulose 5%, steeping water 2%, (NH
4)
2SO
40.3%, KH
2PO
40.4%, glycerine 0.15%, CaCO
30.15%, MgSO
40.06%, and aqueous solvent, original ph 3.0 regulated.
With bacterial classification spore suspension (10
7～10
8Individual/as ml) to insert activation medium, on 28 ℃, 170r/min shaking table, cultivate 24h, obtain seed liquor.Seed liquor is seeded to fresh preparatory production fermention medium by 5% inoculum size.At 25 ℃, concussion is cultivated on the 250r/min constant temperature shaking table, intermittently cultivates the fermentative prodn cellulase.Fermentation total time is 120h.
The preparatory production phase: fermenting process is not carried out the control of pH value, and the flex point that obtains the variation of pH value relative time is respectively Trichodermareesei 18h, viride 25h.The volume of producing fermention medium in advance is 0.1L (placing 250ml to shake in the bottle).
Production phase: in the fermenting process, the pH value of producing fermention medium is carried out sectional-regulated, the time flex point that the segmentation of pH value is controlled is Trichodermareesei 18h, viride 25h.Control method is following: (1) first period, the pH value of producing fermention medium is not added control, and (2) second periods, add sulfuric acid or ammoniacal liquor adjusting pH value through stream in producing fermention medium, control is produced the pH value of fermention medium 4.0～5.3.Wherein, the volume of producing fermention medium is 3L (placing the 5L fermentor tank), and other conditions are identical with the preparatory production phase.
In addition; Also compare experiment; In the fermenting process of production phase, remove and carry out the adjusting of pH value, also carry out the sectional-regulated of dissolved oxygen content, (1) is during fermentation 20 to 80h; Through regulating shaking speed and ventilation, the volume percent that dissolved oxygen in the fermention medium is produced in control maintains 30～40%; (2) fermentation 80h maintains 20～30% to the volume percent of fermentation ends dissolved oxygen.
The filter paper enzyme activity contrast (Microcrystalline Cellulose concentration be 5%, three kind fermentation condition for fermenting process pH value segmentation control, fermenting process pH value segmentation control+dissolved oxygen segmentation control and fermenting process pH value do not add control) of two kinds of Trichoderma kinds of table 6 under production phase three kinds of fermentation conditions
Can know that by table 6 than to the complete out-of-control situation of fermenting process, only sectional-regulated to the pH value of the fermenting process of production phase, for Trichodermareesei, filter paper enzyme activity has improved 40%; For viride, filter paper enzyme activity has improved 32%.Than to the complete out-of-control situation of fermenting process, the pH value of the fermenting process of production phase and dissolved oxygen content are carried out sectional-regulated, for Trichodermareesei, filter paper enzyme activity has improved 49%; For viride, filter paper enzyme activity has improved 34%.
Although embodiment of the present invention are open as above; But it is not restricted to listed utilization in specification sheets and the embodiment; It can be applied to various suitable the field of the invention fully, for being familiar with those skilled in the art, can easily realize other modification; Therefore under the universal that does not deviate from claim and equivalency range and limited, the legend that the present invention is not limited to specific details and illustrates here and describe.
Claims (10)
1. a raising utilizes Trichoderma to produce the working method of the output of acidic cellulase, it is characterized in that, includes following steps:
Step 1, preparation Trichoderma kind seed liquor;
Step 2, preparatory production phase: said Trichoderma kind seed liquor is inoculated in the preparatory production fermention medium; Regulate original ph 3.0～7.0, begin to ferment, measure during the fermentation; Produce the flex point of the pH value beginning sustainable growth of fermention medium in advance, confirm the flex point time T;
Step 3, production phase: with said Trichoderma kind seed liquor be inoculated in said preparatory production fermention medium component and the identical production fermention medium of component concentration in; It is also identical to regulate original ph; Begin to ferment, in the fermenting process, carry out sectional-regulated the pH value of said production fermention medium; Control method is following: (1) fermentation beginning to said flex point time T was first period; PH value to said production fermention medium does not add control, and (2) were second period from said flex point time T to fermentation ends, and the pH value of controlling said production fermention medium is 4.0～5.3.
2. raising as claimed in claim 1 utilizes Trichoderma to produce the working method of the output of acidic cellulase, it is characterized in that, wherein; By mass percentage, said preparatory production fermention medium also comprises: Microcrystalline Cellulose 3%～7%, organic nitrogen source 2～4%; Inorganic nitrogen-sourced 0.3～0.6%; Inorganic salt 0.6～1.3%, glycerine 0.15～0.4%, and aqueous solvent.
3. according to claim 1 or claim 2 raising utilizes Trichoderma to produce the working method of the output of acidic cellulase, it is characterized in that in the said step 2, the original ph of regulating said preparatory production fermention medium is 4.5～5.5.
4. raising as claimed in claim 3 utilizes Trichoderma to produce the working method of the output of acidic cellulase; It is characterized in that, in the said step 3, in the fermenting process; Second period from time flex point T to fermentation ends, the pH value of controlling said production fermention medium is 4.7～5.1.
5. raising as claimed in claim 3 utilizes Trichoderma to produce the working method of the output of acidic cellulase; It is characterized in that, in the said step 3, in said fermenting process; Fermentation 20h to 80h; Control said production fermention medium in the volume percent of dissolved oxygen 30%～40%, 80h is to fermentation ends in fermentation, the volume percent of dissolved oxygen of controlling said production fermention medium is 20%～30%.
6. raising as claimed in claim 2 utilizes Trichoderma to produce the working method of the output of acidic cellulase, it is characterized in that, in the step 2, Microcrystalline Cellulose account for said preparatory production fermention medium total mass 5%.
7. raising as claimed in claim 1 utilizes Trichoderma to produce the working method of the output of acidic cellulase, it is characterized in that in said step 2 and the step 3, the temperature in the fermenting process is controlled at 25～30 ℃.
8. raising as claimed in claim 1 utilizes Trichoderma to produce the working method of the output of acidic cellulase, it is characterized in that in the said step 2, the volume of said preparatory production fermention medium is 0.1L; In the said step 3, the volume of said production fermention medium is 3L.
9. raising as claimed in claim 1 utilizes Trichoderma to produce the working method of the output of acidic cellulase, it is characterized in that, in said step 2 and the step 3, fermenting process is that concussion is cultivated on 250～350r/min constant temperature shaking table.
10. raising as claimed in claim 1 utilizes Trichoderma to produce the working method of the output of acidic cellulase, it is characterized in that, in the said step 1; The method for preparing Trichoderma kind seed liquor is that Trichoderma kind spore suspension is inserted in the activation medium, at 28～30 ℃; Concussion is cultivated 24～36h and is obtained the bacterial classification seed liquor on 170～200r/min constant temperature shaking table; Wherein, said activation medium comprises Microcrystalline Cellulose 1～4%, steeping water 1～2%; And aqueous solvent, regulate original ph 4.0～5.0.
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