CN108893457A - The production method and device of a kind of cellulase and its application - Google Patents

The production method and device of a kind of cellulase and its application Download PDF

Info

Publication number
CN108893457A
CN108893457A CN201810931349.5A CN201810931349A CN108893457A CN 108893457 A CN108893457 A CN 108893457A CN 201810931349 A CN201810931349 A CN 201810931349A CN 108893457 A CN108893457 A CN 108893457A
Authority
CN
China
Prior art keywords
cellulase
fermentor
sterilizer
crushing
raw material
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CN201810931349.5A
Other languages
Chinese (zh)
Other versions
CN108893457B (en
Inventor
张强
李忠玲
胡云红
韩珊珊
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Shaanxi Institute Of Biological Agriculture
Original Assignee
Shaanxi Institute Of Biological Agriculture
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Shaanxi Institute Of Biological Agriculture filed Critical Shaanxi Institute Of Biological Agriculture
Priority to CN201810931349.5A priority Critical patent/CN108893457B/en
Publication of CN108893457A publication Critical patent/CN108893457A/en
Application granted granted Critical
Publication of CN108893457B publication Critical patent/CN108893457B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • 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)
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/189—Enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M21/00—Bioreactors or fermenters specially adapted for specific uses
    • C12M21/14—Bioreactors or fermenters specially adapted for specific uses for producing enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
    • C12M29/06—Nozzles; Sprayers; Spargers; Diffusers
    • C12M29/08—Air lift
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/12—Means for regulation, monitoring, measurement or control, e.g. flow regulation of temperature
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M45/00—Means for pre-treatment of biological substances
    • C12M45/02—Means for pre-treatment of biological substances by mechanical forces; Stirring; Trituration; Comminuting
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
    • C12M47/10—Separation or concentration of fermentation products

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • Genetics & Genomics (AREA)
  • General Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Sustainable Development (AREA)
  • Molecular Biology (AREA)
  • Polymers & Plastics (AREA)
  • Mechanical Engineering (AREA)
  • Physics & Mathematics (AREA)
  • Thermal Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Animal Husbandry (AREA)
  • Medicinal Chemistry (AREA)
  • Food Science & Technology (AREA)
  • Processing Of Solid Wastes (AREA)
  • Fodder In General (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

The invention discloses a kind of production methods of cellulase, including Spawn incubation：Collected bacterium source is coated on CMC-Na media surface culture, irradiates culture medium using wave ultraviolet light, continues culture after irradiation and obtains cellulase strain；Pretreatment of raw material：By high-temperature sterilization after corn straw smashing；Fermentation：Cellulase strain and raw materials for production are added in fermentor, adjusts the parameters such as pH, temperature and time, is passed through filtrated air into tank using air cleaning compressor during fermentation；It is concentrated and dried：PH is adjusted after fermentation liquid is diluted, and is filtered using vacuum filter is entered after the concussion of ultrasonic vibration machine, filtrate enters in ultrafiltration concentration machine and is concentrated after filtering, is drying to obtain cellulase by freeze drier after concentration.In short, the present invention has many advantages, such as that simple process, raw material availability are high, not easy to pollute, producing enzyme is high-efficient, producing enzyme quality is stablized.

Description

The production method and device of a kind of cellulase and its application
Technical field
The invention belongs to enzyme preparation technical field, it is specifically related to a kind of production method of cellulase and device and its answers With.
Background technique
Cellulase is the general name of the glucogenic one group of enzyme of degraded cellulose, its not instead of monomeric enzyme plays collaboration The multicomponent enzyme system of effect is a kind of complex enzyme, mainly by circumscribed 1,4 beta-glucanase, Endo-β-glucanase and beta-glucosidase The composition such as enzyme, there are also the zytases of very high vigor.The product for acting on cellulose and being derived from cellulose.Microorganism Cellulase destroys cell wall at glucose and in juice in conversion insoluble fibrin to improve fruit juice yield etc. Aspect has very important significance.
Cellulase it is from a wealth of sources, bacterium, fungi, microorganism, in animal body etc. can generate cellulose at actinomyces Enzyme.Fungin production of enzyme is high, activity is big, and the cellulase in the source is difficult to purify, generally also containing hemicellulase and Other relevant enzymes, such as amylase, protease.Therefore the cellulase of originated from fungus is mainly answered in animal husbandry and feed industry With；Microorganism fungus kind it is more be a fungi, wherein the stronger strain of enzyme activity be Trichoderma, Aspergillus, Penicillium notatum, especially Trichoderma is presently preferred cellulase production bacterium；In addition, ruminant relies on rumen microorganism digestible cellulose, therefore It can use the crude enzyme preparation that rumen fluid obtains cellulase, but at high cost in actual production, therefore apply few.
Traditional solid fermentation method be using agricultural crop straws such as corns as primary raw material, advantage be simple process small investment, Product price is cheap.But solid fermentation method disadvantage is it is also obvious that the cellulase produced using stalk as the solid fermentation method of raw material It is difficult to refine, filters pressing after obtaining solid polypeptide formulation or being soaked in water can only be crushed using direct drying method and obtain liquid enzyme formulation, Its fermentation level is unstable, and product appearance coarse impurity is more, miscellaneous bacteria easy to pollute, and production yield is lower.
So design a kind of cellulase production method and device and its application very it is necessary to.
Summary of the invention
In view of the above problems, the present invention provides a kind of production method of cellulase and device and its applications.
The technical scheme is that：A kind of production method of cellulase, mainly includes the following steps that：
Step 1：Spawn incubation
It chooses the intensive detritus environment of carbon source and acquires cellulase bacterium source, collected bacterium source is dissolved in ultrapure water, It is uniformly coated on CMC-Na media surface, is cultivated 4-5 days at 27-29 DEG C, picking single colonie is coated on new CMC-Na training Base is supported, after 27-29 DEG C is cultivated 1 day, at interval of 1h using the ultraviolet light that wavelength is 220-240nm apart from media surface 30min is irradiated at 28-33cm, is irradiated 2-3 times, is continued culture after irradiation and is obtained cellulase strain in 2-3 days, at 2-3 DEG C Under be stored in strain adding set；
Step 2：Pretreatment of raw material
Corn stover is sent to crushing in sterilizer, sealing cover is closed, motor is crushed and drives crushing rotor with 2300- The revolving speed of 2800r/min is kept stirring after crushing 30-40min with 80r/min, and electric heater is started to work, and control crushes sterilizing Temperature handles raw material 2-3h between 135-144 DEG C in machine, and moisture in raw material is discharged with vapor form from exhaust outlet, dry Raw material powder after sterilizing enters fermentor through sieve plate；
Step 3：Fermentation
Being added in the fermentor that raw material powder is added with raw material powder mass ratio is 1:1.25 pure water, after mixing evenly Cellulase strain obtained in step 1, control material and strain weight ratio are added into fermentor using strain adding set Example is 1:0.1-0.3, adjusting pH are 4.0-5.0, and it is 25-35 DEG C that temperature controller, which controls temperature in fermentor, fermentation time 115-123h opens air cleaning compressor every 5-7h during fermentation and is passed through nothing into fermentor from the air flue of fermenter base Bacterium air carries out continuing stirring；
Step 4：It is concentrated and dried
The pure water agitation and dilution with phase homogenous quantities in step 3 is added into the fermentor after fermentation, then adjusts hair Zymotic fluid pH to 5.0, using ultrasonic vibration machine with the frequency oscillation 10-13min of 1.3-1.5MHz, the then discharge port of fermented tank Vacuum filter is carried out into vacuum filter, filtrate enters in ultrafiltration concentration machine and is concentrated after filtering, passes through freezing after concentration Drying machine is drying to obtain cellulase.
Further, the proportion of CMC-Na culture medium is in the step 1：CMC-Na13.8-14.0g,Na2HPO1.2- 1.4g、KH2PO1.3-1.5g, yeast extract 0.8-0.9g, peptone 2.1-2.5g, Congo red 0.3-0.32g, agar 10-14g, Microcrystalline cellulose 1.25-1.27g, 800-900ml distilled water, be added in culture medium it is Congo red, can culture CELLULOLYTIC BACTERIUM Degradation circle is shown when falling, and is convenient for bacterium, a small amount of microcrystalline cellulose is added and avoids culture medium hardened.
Further, the screen-aperture of the sieve plate is 40-80 mesh.
Further, the pure water temperature added in the step 3 and step 4 is 16-41 DEG C, too low and excessive temperature Pure water can destroy yeasting, reduce cellulase activity.
A kind of process units of cellulase, it is mainly net including strain adding set, crushing sterilizer, fermentor, air Change compressor, ultrasonic vibration machine, vacuum filter, machine and freeze drier be concentrated by ultrafiltration, is equipped with temperature inside strain adding set Device is controlled, sterilizer is crushed and includes sealing cover, crush motor, crush rotor, electric heater, exhaust outlet and sieve plate, the crushing The upper surface of sealing cover is arranged in motor, and the crushing rotor setting is on crushing sterilizer central axes, and upper end and crushing electricity The output end of machine connects, and on crushing sterilizer inner wall, the exhaust outlet setting is crushing sterilizer for the electric heater setting Outer wall on, sieve plate setting is crushing sterilizer bottom end, strain adding set and crushing sterilizer respectively with fermentor Feeding inlet connection, the fermentor further include internal temperature controller being arranged in the multiple of fermenter base are arranged in spiral Air flue, the air outlet of the air cleaning compressor and the air flue of fermentor are connected by pipeline, the ultrasonic vibration machine and hair The discharge port of fermentation tank connects, and the vacuum filter is connect with ultrasonic vibration machine, and the ultrafiltration concentration machine and vacuum filter connect It connects, the freeze drier is connect with the machine of ultrafiltration concentration, and the process units high degree of automation, working condition is controllable, is suitable for The use of large-scale production of cellulase.
Further, the application of cellulase obtained is in the preparation of animal feed, and preparation cost is low, raw material availability High, preparation efficiency height, animal feed obtained can be enhanced animal immunizing power, promote growth of animal, improve its disease resistance.
The beneficial effects of the invention are as follows：A kind of production method of cellulase provided by the invention is suitable for efficient animal Feed preparation aspect, by the way that collected bacterium source to be obtained to the highest bacterium of enzymatic productivity after culture, screening, mutagenesis and secondary screening Culture is fallen for producing cellulase, is crushed after raw materials for production are carried out dehydration sterilizing, miscellaneous bacteria is avoided to influence the life of cellulase It produces, and during the fermentation, by the plenty of time, cellulase fermentations optimum temperature, pH and time is determined, in fermentation process In, aseptic gas is carried out at regular intervals and is passed through stirring, and the product after fermentation is dense by ultrasonic vibration, vacuum filter, ultrafiltration Up to cellulase after contracting and drying.In short, the present invention has, simple process, raw material availability be high, not easy to pollute, producing enzyme efficiency The advantages that high, producing enzyme quality is stablized.
Detailed description of the invention
Fig. 1 is process flow chart of the invention；
Fig. 2 is the device of the invention structural schematic diagram；
Fig. 3 is the growth curve chart that different wave length ultraviolet light irradiates lower strain；
Fig. 4 is the activity curve figure of different pH fermentation juice cellulases；
Fig. 5 is the activity curve figure of different fermentations temperature cellulase；
Fig. 6 is the activity curve figure of different fermentations time cellulase.
Wherein, 1- strain adding set, 11- temperature control device, 2- crush sterilizer, 21- sealing cover, 22- crush motor, It is empty that 23- crushes rotor, 24- electric heater, 25- exhaust outlet, 26- sieve plate, 3- fermentor, 31- temperature controller, 32- air flue, 4- Gas purifies compressor, 41- air outlet, 5- ultrasonic vibration machine, 6- vacuum filter, 7- and machine, 8- freeze drier is concentrated by ultrafiltration.
Specific embodiment
For convenient for the understanding to technical solution of the present invention, with reference to the accompanying drawing 1-6 and specific embodiment to the present invention do into The explanation of one step, embodiment do not constitute the restriction to invention protection scope.
Embodiment 1
As shown in Figure 1, a kind of production method of cellulase, mainly includes the following steps that：
Step 1：Spawn incubation
It chooses the intensive detritus environment of carbon source and acquires cellulase bacterium source, collected bacterium source is dissolved in ultrapure water, It is uniformly coated on CMC-Na media surface, is cultivated 4 days at 27 DEG C, picking single colonie is coated on new CMC-Na culture medium, After 27 DEG C are cultivated 1 day, 30min is being irradiated at media surface 28cm at interval of the 1h ultraviolet light for the use of wavelength being 220, Irradiation 2 times continues culture and obtains cellulase strain in 2 days, strain adding set 1 is stored at 2 DEG C after irradiation；
The proportion of CMC-Na culture medium is：CMC-Na13.8g,Na2HPO1.2g、KH2PO1.3g, yeast extract 0.8g, albumen Peptone 2.1g, Congo red 0.3g, agar 10g, microcrystalline cellulose 1.25g, 800ml distilled water；
Step 2：Pretreatment of raw material
By corn stover send to crush sterilizer 2 in, close sealing cover 21, crush motor 22 drive crush rotor 23 with The revolving speed of 2300r/min is kept stirring after crushing 30min with 80r/min, and electric heater 24 is started to work, and control crushes sterilizer Temperature moisture in raw material is discharged with vapor form from exhaust outlet 25, after dry sterilization in 135 DEG C of processing raw material 2h in 2 Raw material powder enters fermentor 3 through sieve plate 26；
Step 3：Fermentation
Being added in the fermentor 3 that raw material powder is added with raw material powder mass ratio is 1:1.25 pure water, pure water temperature It is 16 DEG C, cellulase strain obtained in step 1 is added into fermentor 3 using strain adding set 1 after mixing evenly, It controls material and strain weight ratio is 1:0.1, adjusting pH is 4.0, and it is 25 that temperature controller 31, which controls temperature in fermentor 3, DEG C, fermentation time 115h opens air cleaning compressor 4 from the air flue 32 of 3 bottom of fermentor to fermentation every 5h during fermentation Filtrated air is passed through in tank 3 to carry out continuing stirring；
Step 4：It is concentrated and dried
The pure water agitation and dilution with phase homogenous quantities in step 3, pure water temperature are added into the fermentor 3 after fermentation It is 16 DEG C, fermentation liquid pH to 5.0 is then adjusted, using ultrasonic vibration machine 5 with the frequency oscillation 10min of 1.3MHz, then through sending out The discharge port of fermentation tank 3 enters vacuum filter 6 and carries out vacuum filter, and filtrate enters in ultrafiltration concentration machine 7 and is concentrated after filtering, Cellulase is drying to obtain by freeze drier 8 after concentration, application of cellulase obtained is in the preparation of animal feed.
Embodiment 2
As shown in Figure 1, a kind of production method of cellulase, mainly includes the following steps that：
Step 1：Spawn incubation
It chooses the intensive detritus environment of carbon source and acquires cellulase bacterium source, collected bacterium source is dissolved in ultrapure water, It is uniformly coated on CMC-Na media surface, is cultivated 5 days at 28 DEG C, picking single colonie is coated on new CMC-Na culture medium, After 28 DEG C are cultivated 1 day, irradiated at media surface 30cm at interval of 1h using the ultraviolet light that wavelength is 234nm 30min irradiates 3 times, continues culture after irradiation and obtains cellulase strain in 3 days, strain adding set is stored at 3 DEG C 1；
The proportion of CMC-Na culture medium is：CMC-Na13.9g,Na2HPO1.3g、KH2PO1.4g, yeast extract 0.85g, albumen Peptone 2.35g, Congo red 0.31g, agar 12g, microcrystalline cellulose 1.26g, 850ml distilled water；
Step 2：Pretreatment of raw material
By corn stover send to crush sterilizer 2 in, close sealing cover 21, crush motor 22 drive crush rotor 23 with The revolving speed of 2600r/min is kept stirring after crushing 35min with 80r/min, and electric heater 24 is started to work, and control crushes sterilizer Temperature moisture in raw material is discharged with vapor form from exhaust outlet 25, after dry sterilization in 140 DEG C of processing raw material 3h in 2 Raw material powder enters fermentor 3 through sieve plate 26；
Step 3：Fermentation
Being added in the fermentor 3 that raw material powder is added with raw material powder mass ratio is 1:1.25 pure water, pure water temperature It is 30 DEG C, cellulase strain obtained in step 1 is added into fermentor 3 using strain adding set 1 after mixing evenly, It controls material and strain weight ratio is 1:0.2, adjusting pH is 4.3, and it is 30 that temperature controller 31, which controls temperature in fermentor 3, DEG C, fermentation time 120h opens air cleaning compressor 4 from the air flue 32 of 3 bottom of fermentor to fermentation every 6h during fermentation Filtrated air is passed through in tank 3 to carry out continuing stirring；
Step 4：It is concentrated and dried
The pure water agitation and dilution with phase homogenous quantities in step 3, pure water temperature are added into the fermentor 3 after fermentation It is 30 DEG C, fermentation liquid pH to 5.0 is then adjusted, using ultrasonic vibration machine 5 with the frequency oscillation 12min of 1.4MHz, then through sending out The discharge port of fermentation tank 3 enters vacuum filter 6 and carries out vacuum filter, and filtrate enters in ultrafiltration concentration machine 7 and is concentrated after filtering, Cellulase is drying to obtain by freeze drier 8 after concentration, application of cellulase obtained is in the preparation of animal feed.
Embodiment 3
As shown in Figure 1, a kind of production method of cellulase, mainly includes the following steps that：
Step 1：Spawn incubation
It chooses the intensive detritus environment of carbon source and acquires cellulase bacterium source, collected bacterium source is dissolved in ultrapure water, It is uniformly coated on CMC-Na media surface, is cultivated 5 days at 29 DEG C, picking single colonie is coated on new CMC-Na culture medium, After 29 DEG C are cultivated 1 day, irradiated at media surface 33cm at interval of 1h using the ultraviolet light that wavelength is 240nm 30min irradiates 3 times, continues culture after irradiation and obtains cellulase strain in 3 days, strain adding set is stored at 3 DEG C 1；
The proportion of CMC-Na culture medium is：CMC-Na14.0g,Na2HPO1.4g、KH2PO1.5g, yeast extract 0.9g, albumen Peptone 2.5g, Congo red 0.32g, agar 14g, microcrystalline cellulose 1.27g, 900ml distilled water；
Step 2：Pretreatment of raw material
By corn stover send to crush sterilizer 2 in, close sealing cover 21, crush motor 22 drive crush rotor 23 with The revolving speed of 2800r/min is kept stirring after crushing 40min with 80r/min, and electric heater 24 is started to work, and control crushes sterilizer Temperature moisture in raw material is discharged with vapor form from exhaust outlet 25, after dry sterilization in 144 DEG C of processing raw material 3h in 2 Raw material powder enters fermentor 3 through sieve plate 26；
Step 3：Fermentation
Being added in the fermentor 3 that raw material powder is added with raw material powder mass ratio is 1:1.25 pure water, pure water temperature It is 41 DEG C, cellulase strain obtained in step 1 is added into fermentor 3 using strain adding set 1 after mixing evenly, It controls material and strain weight ratio is 1:0.3, adjusting pH is 5.0, and it is 35 that temperature controller 31, which controls temperature in fermentor 3, DEG C, fermentation time 123h opens air cleaning compressor 4 from the air flue 32 of 3 bottom of fermentor to fermentation every 7h during fermentation Filtrated air is passed through in tank 3 to carry out continuing stirring；
Step 4：It is concentrated and dried
The pure water agitation and dilution with phase homogenous quantities in step 3, pure water temperature are added into the fermentor 3 after fermentation It is 41 DEG C, fermentation liquid pH to 5.0 is then adjusted, using ultrasonic vibration machine 5 with the frequency oscillation 13min of 1.5MHz, then through sending out The discharge port of fermentation tank 3 enters vacuum filter 6 and carries out vacuum filter, and filtrate enters in ultrafiltration concentration machine 7 and is concentrated after filtering, Cellulase is drying to obtain by freeze drier 8 after concentration, application of cellulase obtained is in the preparation of animal feed.
Embodiment 4
As shown in figure 3, the method provided using embodiment 1, shadow of the measurement different wave length ultraviolet light to growth situation It rings.
Conclusion：When ultraviolet wavelength is 234nm, irradiation CELLULOLYTIC BACTERIUM obtains after dropping into row cellulase-producing induction mutation of bacterium Thallus quality it is maximum, microbial activity highest.
Embodiment 5
As shown in figure 4, the method provided using embodiment 1, measures influence of the fermentation liquid difference pH to cellulase activity.
Conclusion：When fermentation liquid pH is 4.3, the active highest of obtained cellulase.
Embodiment 6
As shown in figure 5, the method provided using embodiment 1, measures influence of the different fermentations temperature to cellulase activity.
Conclusion：When fermentation temperature is 30 DEG C, the active highest of obtained cellulase.
Embodiment 7
As shown in fig. 6, the method provided using embodiment 1, measures influence of the different fermentations time to cellulase activity.
Conclusion：When fermentation time is 120h, the active highest of obtained cellulase.
Embodiment 8
As shown in Fig. 2, a kind of process units of cellulase, mainly includes strain adding set 1, crushes sterilizer 2, hair Air cleaning compressor 4, ultrasonic vibration machine 5, vacuum filter 6, machine 7 and freeze drier 8 is concentrated by ultrafiltration in fermentation tank 3, and strain adds Temperature control device 11 is equipped with inside feeder apparatus 1, crushing sterilizer 2 adds including sealing cover 21, crushing motor 22, crushing rotor 23, electricity Hot device 24, exhaust outlet 25 and sieve plate 26 crush the upper surface that sealing cover 21 is arranged in motor 22, crush the setting of rotor 23 and are crushing On 2 central axes of sterilizer, and upper end is connect with the output end for crushing motor 22, and the setting of electric heater 24 is crushing in sterilizer 2 On wall, exhaust outlet 25 is arranged on the outer wall for crushing sterilizer 2, and the setting of sieve plate 26 is crushing 2 bottom end of sterilizer, the sieve of sieve plate 26 Hole aperture is 50 mesh, and strain adding set 1 and crushing sterilizer 2 are connect with the feeding inlet of fermentor 3 respectively, and fermentor 3 also wraps It includes and multiple air flues 32 that 3 bottom of fermentor is arranged in internal temperature controller 31 and spiral is set, air cleaning compressor 4 Air outlet 41 connect with the air flue 32 of fermentor 3 by pipeline, ultrasonic vibration machine 5 is connect with the discharge port of fermentor 3, vacuum Filter 6 is connect with ultrasonic vibration machine 5, and machine 7 is concentrated by ultrafiltration and connect with vacuum filter 6, freeze drier 8 and ultrafiltration concentration machine 7 connections.
Finally it should be noted that：The above embodiments are merely illustrative of the technical solutions of the present invention, rather than its limitations；Although Present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that：It still may be used To modify to technical solution documented by previous embodiment or equivalent replacement of some of the technical features；And These are modified or replaceed, the spirit and model of technical solution of the embodiment of the present invention that it does not separate the essence of the corresponding technical solution It encloses.

Claims (6)

1. a kind of production method of cellulase, which is characterized in that mainly include the following steps that：
Step 1：Spawn incubation
It chooses the intensive detritus environment of carbon source and acquires cellulase bacterium source, collected bacterium source is dissolved in ultrapure water, uniformly It is coated on CMC-Na media surface, is cultivated 4-5 days at 27-29 DEG C, picking single colonie is coated on new CMC-Na culture medium, After 27-29 DEG C is cultivated 1 day, at interval of 1h using the ultraviolet light that wavelength is 220-240nm apart from media surface 28-33cm Place's irradiation 30min, irradiates 2-3 times, continues culture after irradiation and obtains cellulase strain in 2-3 days, is stored at 2-3 DEG C Strain adding set (1)；
Step 2：Pretreatment of raw material
Corn stover is sent to crushing in sterilizer (2), is closed sealing cover (21), motor (22) is crushed and drives crushing rotor (23) It is kept stirring after crushing 30-40min with the revolving speed of 2300-2800r/min with 80r/min, electric heater (24) is started to work, control System crushes temperature in sterilizer (2) and handles raw material 2-3h between 135-144 DEG C, by moisture in raw material with vapor form from row Port (25) is discharged, and the raw material powder after dry sterilization enters fermentor (3) through sieve plate (26)；
Step 3：Fermentation
Being added in the fermentor (3) that raw material powder is added with raw material powder mass ratio is 1:1.25 pure water, after mixing evenly Cellulase strain obtained in step 1 is added into fermentor (3) using strain adding set (1), controls material and strain Weight ratio is 1:0.1-0.3, adjusting pH are 4.0-5.0, and it is 25-35 that temperature controller (31), which controls fermentor (3) interior temperature, DEG C, fermentation time 115-123h opens the gas of air cleaning compressor (4) from fermentor (3) bottom every 5-7h during fermentation Road (32) is passed through filtrated air into fermentor (3) and carries out continuing stirring；
Step 4：It is concentrated and dried
The pure water agitation and dilution with phase homogenous quantities in step 3 is added into the fermentor (3) after fermentation, then adjusts hair Zymotic fluid pH to 5.0, using ultrasonic vibration machine (5) with the frequency oscillation 10-13min of 1.3-1.5MHz, then fermented tank (3) Discharge port enters vacuum filter (6) and carries out vacuum filter, and filtrate enters in ultrafiltration concentration machine (7) and is concentrated after filtering, dense Cellulase is drying to obtain by freeze drier (8) after contracting.
2. a kind of production method of cellulase according to claim 1, which is characterized in that the step 3 and step 4 The pure water temperature of middle addition is 16-41 DEG C.
3. a kind of production method of cellulase according to claim 1, which is characterized in that added in the step 4 Pure water temperature is 16-41 DEG C.
4. a kind of process units of cellulase, which is characterized in that mainly include strain adding set (1), crush sterilizer (2), fermentor (3), air cleaning compressor (4), ultrasonic vibration machine (5), vacuum filter (6), ultrafiltration concentration are machine (7) and cold Lyophilizer (8) is equipped with temperature control device (11) inside strain adding set (1), crush sterilizer (2) include sealing cover (21), It crushes motor (22), crush rotor (23), electric heater (24), exhaust outlet (25) and sieve plate (26), the crushing motor (22) Upper surface in sealing cover (21) is set, crushing rotor (23) setting on crushing sterilizer (2) central axes, and upper end with The output end connection of motor (22) is crushed, electric heater (24) setting is on crushing sterilizer (2) inner wall, the exhaust outlet (25) it is arranged on the outer wall for crushing sterilizer (2), sieve plate (26) setting is crushing sterilizer (2) bottom end, strain addition It device (1) and crushes sterilizer (2) and is connect respectively with the feeding inlet of fermentor (3), including the fermentor (3) further includes setting Multiple air flues (32) in fermentor (3) bottom, the air cleaning compressor is arranged in the temperature controller (31) and spiral in portion (4) air outlet (41) is connect with the air flue (32) of fermentor (3) by pipeline, the ultrasonic vibration machine (5) and fermentor (3) Discharge port connection, the vacuum filter (6) connect with ultrasonic vibration machine (5), the ultrafiltration concentration machine (7) and vacuum filter Machine (6) connection, the freeze drier (8) connect with machine (7) are concentrated by ultrafiltration.
5. a kind of production method of cellulase according to claim 1, which is characterized in that the sieve pore of the sieve plate (26) Aperture is 40-80 mesh.
6. a kind of application of cellulase made from method as described in claim 1, which is characterized in that the cellulose obtained Enzyme is applied to the preparation of animal feed.
CN201810931349.5A 2018-08-15 2018-08-15 Production method and device of cellulase and application thereof Active CN108893457B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201810931349.5A CN108893457B (en) 2018-08-15 2018-08-15 Production method and device of cellulase and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201810931349.5A CN108893457B (en) 2018-08-15 2018-08-15 Production method and device of cellulase and application thereof

Publications (2)

Publication Number Publication Date
CN108893457A true CN108893457A (en) 2018-11-27
CN108893457B CN108893457B (en) 2020-02-14

Family

ID=64354357

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201810931349.5A Active CN108893457B (en) 2018-08-15 2018-08-15 Production method and device of cellulase and application thereof

Country Status (1)

Country Link
CN (1) CN108893457B (en)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109776648A (en) * 2019-03-25 2019-05-21 陕西省生物农业研究所 It is a kind of for preventing and treating the Charantin extraction process and device of TYLCV infection insect
CN111500432A (en) * 2020-04-22 2020-08-07 南京益纤生物科技有限公司 Feed preparation device based on solid state fermentation
CN115058407A (en) * 2022-06-06 2022-09-16 杭州鸿瑞生物工程有限公司 Processing technology for cellulase production

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1157853A (en) * 1996-11-13 1997-08-27 山东大学 Preparing method of high active cellulase
CN101717729A (en) * 2009-11-10 2010-06-02 武汉博普奥多肽生产技术有限公司 Cellulase produced by fermentation of aspergillus niger strains and method for preparing polypeptide protein feed

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1157853A (en) * 1996-11-13 1997-08-27 山东大学 Preparing method of high active cellulase
CN101717729A (en) * 2009-11-10 2010-06-02 武汉博普奥多肽生产技术有限公司 Cellulase produced by fermentation of aspergillus niger strains and method for preparing polypeptide protein feed

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109776648A (en) * 2019-03-25 2019-05-21 陕西省生物农业研究所 It is a kind of for preventing and treating the Charantin extraction process and device of TYLCV infection insect
CN109776648B (en) * 2019-03-25 2021-06-11 陕西省生物农业研究所 A charantin extraction process and device for preventing and treating TYLCV virus-transmitting insects
CN111500432A (en) * 2020-04-22 2020-08-07 南京益纤生物科技有限公司 Feed preparation device based on solid state fermentation
CN111500432B (en) * 2020-04-22 2024-02-06 南京益纤生物科技有限公司 Fodder preparation facilities based on solid state fermentation
CN115058407A (en) * 2022-06-06 2022-09-16 杭州鸿瑞生物工程有限公司 Processing technology for cellulase production

Also Published As

Publication number Publication date
CN108893457B (en) 2020-02-14

Similar Documents

Publication Publication Date Title
CN104372060B (en) A kind of method that solid state fermentation grouts prepare oligopeptide
CN108893457A (en) The production method and device of a kind of cellulase and its application
CN101015379B (en) Process for preparing agaricus blazei healht-care liquid
CN203072844U (en) Fermented soy sauce production equipment
CN102048025A (en) Composite leavening agent combining xylanase with multiple strains and method for fermenting straw feed
CN107022493A (en) A kind of aspergillus oryzae strain of high yield complex enzyme for feed and its application
CN108821867A (en) The method for preparing biological liquid fertilizer using fish scale, fish guts and its cleaning sullage
CN1185336C (en) Li's Trichoderma strains and use thereof
CN112273531A (en) Grape branch fermented feed preparation process and equipment based on biological fermentation technology
RU2163076C1 (en) Method and apparatus for biological conversion of plant wastes
CN103392507A (en) Method for preparing bar-shaped strain
CN1899080A (en) Active protein feed and its preparing method
CN1294861A (en) Mixed solid fermentation and straw puffing process to prepare protein feed
CN101979635B (en) Drum fermentation reactor-based bacterial cellulose production technology
CN103598017A (en) Method for cultivating pleurotus eryngii by directly mixing liquid strains with raw material
CN1033171C (en) Automated method and apparatus for semi-solid fermentation used in production of ancient quality rice vinegar and/or rice wine
CN101638645A (en) Method for producing xylanase by solid mechanical fermentation
US20150191751A1 (en) Zero-waste method for producing ethanol and apparatus for implementing same
CN101974503B (en) Method for preparing chymosin by mixed culture fermentation of bean dregs and waste material from fungal culture (WMFC)
CN106036240A (en) High-protein crab feed additive for raising production speed
CN101161809B (en) Method for making ferment for agricultural byproducts
CN103343151A (en) Preparation method of liquid medium for bacterial cellulose film
CN1174671C (en) Asafetida mushroom culturing process
CN209602512U (en) Muscardine solid culture inoculating facility
CN105884458A (en) Water hyacinth growth medium for edible mushrooms and preparation method of water hyacinth growth medium

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant