CN104017790A - Preparation method of liquid complex enzyme from solid fermentation source and application thereof - Google Patents
Preparation method of liquid complex enzyme from solid fermentation source and application thereof Download PDFInfo
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Abstract
The invention relates to a preparation method of a liquid complex enzyme from a solid fermentation source and an application thereof. The method comprises the following steps: solid fermentation: performing high-pressure moist heat sterilization of a culture medium, adding the prepared strain and fermenting to obtain solid fermented mash; liquid mash preparation: adding a buffer solution into the solid fermented mash to obtain the liquid mash; filtering: filtering the liquid mash to obtain enzyme liquid; ultrafiltration concentration: concentrating the enzyme liquid through ultrafiltration to obtain concentrated enzyme liquid; enzyme activity detection and compounding: measuring the enzyme activity of the concentrated enzyme liquid, calculating the addition amount of each single enzyme according to the formula of the complex enzyme, compounding, and diluting to the regulated level to obtain a semi-finished product of a liquid enzyme; addition of a protective agent: adding a stable protective agent into the semi-finished product of the liquid enzyme to obtain the liquid complex enzyme. The method provided by the invention can guarantee the advantage of the solid-fermentation complex enzyme in high degradation rate and ensure that the enzyme activity is not damaged by high-temperature granulation, thereby greatly improving the enzymolysis efficiency.
Description
Technical field
The preparation method and its usage that the present invention relates to the liquid complex enzyme in a kind of solid fermentation source, belongs to feed complex enzyme.
Background technology
The use of complex enzyme for feed in actual production is more and more extensive.Complex enzyme for feed is divided into two kinds in form: solid complex enzyme and liquid complex enzyme.The solid complex enzyme production of granulating after being directly added on and mixing in feedstuff raw material, liquid complex enzyme is sprayed on feed surface after feed granulating.In the mode of production in enzyme source, divide two kinds: solid fermentation and liquid fermenting.Solid complex enzyme has two kinds of modes of production simultaneously: solid fermentation and liquid fermenting; Liquid complex enzyme only derives from liquid fermenting.Solid fermentation prozyme because of its substratum be the reason of all feeds raw material, its result of use is much better than liquid fermenting prozyme.Solid complex enzyme in granulated feed is compared with liquid complex enzyme, and the loss of living of the enzyme of solid complex enzyme in high temperature granulating process is larger, have up to 90%, specifically in Table 1; Liquid complex enzyme sprays after high temperature granulating again, and its enzyme work is without any loss.Therefore the liquid enzymes result of use of solid fermentation is obviously better than solid fermentation solid enzyme and liquid fermenting liquid enzyme.Therefore good liquid complex enzyme is very necessary to develop a kind of result of use.The enzymatic hydrolyzation of liquid fermenting prozyme is 1.88% in addition, and enzymatic hydrolyzation is very low.
The enzyme of table 1 solid enzyme preparation in granulated feed pelletization lived and retained
Summary of the invention
The preparation method and its usage that the invention provides the liquid complex enzyme in a kind of solid fermentation source, has solved existing liquid complex enzyme and has only derived from liquid fermenting, the problem that result of use is bad.
For solving the problems of the technologies described above, the technical solution used in the present invention is:
A preparation method for the liquid complex enzyme in solid fermentation source, comprises the following steps:
The first step, solid fermentation: substratum is mixed in stirrer, then divide and install in cloth bag, 121 ℃, 30min, high pressure moist heat sterilization, treat that substratum is cooled to 25-35 ℃, substratum is poured in stirrer and then added the bacterial classification preparing to stir, after stirring, be put in koji tray, tiling evenly, thickness is moderate, for 3-5cm, finally cover wet cloth, open temperature control switch, controlling temperature is 29 ℃, after fermentation 40-60h, turn over song and moisturizing, rate of water make-up is 8-15%, and then fermentation completes whole fermenting process for 72 hours, obtain solid fermentation wine with dregs,
Second step, liquid mash: in solid fermentation wine with dregs, add damping fluid and obtain liquid mash, damping fluid add-on is 2-3 times of solid fermentation wine with dregs weight;
The 3rd step, filtration: liquid mash is filtered, obtain enzyme liquid;
The 4th step, ultrafiltration and concentration: by ultrafiltration mode, enzyme liquid is concentrated, concentrated multiple is 2-3 times, obtain concentrated enzyme liquid;
The 5th step, enzyme biopsy survey, composite: the enzyme work to concentrated enzyme liquid is measured, and then according to the addition of the various single enzymes of formula calculation of prozyme, carries out compositely, is diluted to prescribed level, obtains liquid enzymes work in-process;
The 6th step, interpolation protective material: in liquid enzymes work in-process, add stability protective agent, obtain liquid complex enzyme.
Preferably: described bacterial classification is a kind of in long handle Trichoderma powder, aspergillus oryzae powder, black-koji mould powder and bacillus amyloliquefaciens bacterium powder or more than one mixture.
Preferably: described damping fluid is citric acid-sodium citrate damping fluid, pH is 5.0～6.0.
Preferably: three-stage filtration is taked in described filtration, liquid mash is successively by Plate Filtration, centrifuging and ceramic membrane filter.The aperture of ceramic filter membrane used is 0.45 μ m.
Preferably: described stability protective agent is that massfraction is 60% sodium lactate aqueous solution, and addition is the 20-25% of liquid enzymes work in-process weight.
The present invention also provides a kind of purposes of liquid complex enzyme in addition, and the prepared liquid complex enzyme of the present invention, for the preparation of the additive of feed, adopts the method for spraying to add in the middle of feed.
The preparation method of the long handle Trichoderma powder that the present invention is used: by mould being inoculated on PDA solid medium of long handle wood, cultivate 24-72h in 28 ℃; After growing spore, by spore inoculating in 50mlPDA liquid nutrient medium, 28 ℃ of shaking tables are cultivated 24-72h, grow up to the flocculence mycelia floating on a liquid, then this liquid and the bran mass of sterilizing are in advance mixed, be placed on 28 ℃, cultivate 24-72h, after growing spore, according to spore and moisture, how much add calcium carbonate and starch (calcium carbonate: the weight ratio of starch is 3:2) to mix, make spore count reach 10
12individual/g, vacuum drying at 30-45 ℃, is long handle Trichoderma powder, and viable count reaches 10
12individual/g, normal temperature is preserved stand-by.
Aspergillus oryzae powder is similar to the preparation method of long handle Trichoderma powder with black-koji mould powder, is not discussing.
The preparation method of bacillus amyloliquefaciens bacterium powder: the bacillus amyloliquefaciens that slant tube is preserved is protected bacterium inclined-plane with the washing of 5-8mL stroke-physiological saline solution, to shake up in aseptic triangular flask bacteria suspension 150mL, by 10% volume, slant culture is inoculated into seed bottle, at 28 ℃, 222rpm cultivates 24-48h, the seed liquor of 6-10% is inoculated into enlarged culturing 24-72h in fermentation flask and obtains fermented liquid.The ratio of volume ratio 1:1 of take adds calcium carbonate and starch (calcium carbonate: the weight ratio of starch is 3:2) to mix, and vacuum drying at 30-45 ℃, is bacillus amyloliquefaciens bacterium powder, and viable count reaches 10
12individual/g, normal temperature is preserved stand-by.
The measuring method of enzymatic hydrolyzation, with reference to application publication number CN103308413A, name is called the patent of the method for the feeding non-starch polysaccharide enzyme hydrolysis result of a kind of external test and carries out.
Beneficial effect of the present invention:
The long handle mould main product cellulase of wood and beta-glucanase that the present invention adopts, aspergillus oryzae main product zytase, aspergillus niger main product mannase and alpha-galactosidase, bacillus amyloliquefaciens main product α-amylase, be the bacterial classification that in fodder additives kind catalogue (2013), regulation is used, utilize them to produce liquid complex enzyme, can solve well the low problem of efficiency of feed utilization, enzymatic hydrolyzation reaches 14.05%
The present invention adopts the method for solid fermentation to produce liquid complex enzyme, can guarantee the advantage of solid fermentation prozyme high degradation rate, can guarantee again the enzyme loss that is not subject to high temperature granulating alive, can greatly submit the efficiency of enzymolysis to.
Embodiment
Below by specific embodiment, technical scheme of the present invention is elaborated.
Embodiment 1
The mould solid fermentation of long handle wood, production of cellulose enzyme and beta-glucanase
The first step, by substratum (6000g wheat bran, 2000g dregs of beans, 8000ml nutritive medium, wherein nutritive medium is containing 3% ammonium sulfate (massfraction), 1.5% potassium primary phosphate (massfraction)) in stirrer, mix, then divide and install in cloth bag, 121 ℃, 30min, high pressure moist heat sterilization, treat that substratum is cooled to 25-35 ℃, substratum is poured in stirrer and then added the long handle Trichoderma powder preparing to stir, after stirring, be put in koji tray, tiling evenly, thickness is moderate, for 3-5cm, finally cover wet cloth, open temperature control switch, controlling temperature is 29 ℃, after fermentation 40-60h, turn over song and moisturizing, rate of water make-up is 8-15%, and then fermentation completes whole fermenting process for 72 hours, obtain solid fermentation wine with dregs,
Second step, liquid mash: in solid fermentation wine with dregs, add damping fluid and obtain liquid mash, damping fluid add-on is 2 times of solid fermentation wine with dregs weight, buffered soln used is citric acid-sodium citrate damping fluid, pH is 5.0, specific configuration is: the citric acid solution 8.2ml of 0.1mol/L: the sodium citrate solution 11.8ml of 0.1mol/L;
The 3rd step, filtration: liquid mash is filtered, obtain enzyme liquid, three-stage filtration is taked in described filtration, liquid mash is successively by Plate Filtration, centrifuging and ceramic membrane filter;
The 4th step, ultrafiltration and concentration: by ultrafiltration mode, enzyme liquid is concentrated, concentrated multiple is 3 times, obtain concentrated enzyme liquid.
Embodiment 2
Aspergillus oryzae solid fermentation, produces zytase
The first step, by substratum (6000g wheat bran, 2000g dregs of beans, 8000ml nutritive medium, wherein nutritive medium is containing 3% ammonium sulfate (massfraction), 1.5% potassium primary phosphate (massfraction)) in stirrer, mix, then divide and install in cloth bag, 121 ℃, 30min, high pressure moist heat sterilization, treat that substratum is cooled to 25-35 ℃, substratum is poured in stirrer and then added the aspergillus oryzae powder preparing to stir, after stirring, be put in koji tray, tiling evenly, thickness is moderate, for 3-5cm, finally cover wet cloth, open temperature control switch, controlling temperature is 29 ℃, after fermentation 40-60h, turn over song and moisturizing, rate of water make-up is 8-15%, and then fermentation completes whole fermenting process for 72 hours, obtain solid fermentation wine with dregs,
Second step, liquid mash: in solid fermentation wine with dregs, add damping fluid and obtain liquid mash, damping fluid add-on is 3 times of solid fermentation wine with dregs weight, buffered soln used is citric acid-sodium citrate damping fluid, pH is 5.4, specific configuration is: the citric acid solution 6.4ml of 0.1mol/L: the sodium citrate solution 13.6ml of 0.1mol/L;
The 3rd step, filtration: liquid mash is filtered, obtain enzyme liquid, three-stage filtration is taked in described filtration, liquid mash is successively by Plate Filtration, centrifuging and ceramic membrane filter;
The 4th step, ultrafiltration and concentration: by ultrafiltration mode, enzyme liquid is concentrated, concentrated multiple is 2 times, obtain concentrated enzyme liquid.
Embodiment 3
Aspergillus niger solid fermentation, produces mannase and alpha-galactosidase
The first step, by substratum (6000g wheat bran, 2000g dregs of beans, 8000ml nutritive medium, wherein nutritive medium is containing 3% ammonium sulfate (massfraction), 1.5% potassium primary phosphate (massfraction)) in stirrer, mix, then divide and install in cloth bag, 121 ℃, 30min, high pressure moist heat sterilization, treat that substratum is cooled to 25-35 ℃, substratum is poured in stirrer and then added the black-koji mould powder preparing to stir, after stirring, be put in koji tray, tiling evenly, thickness is moderate, for 3-5cm, finally cover wet cloth, open temperature control switch, controlling temperature is 29 ℃, after fermentation 40-60h, turn over song and moisturizing, rate of water make-up is 8-15%, and then fermentation completes whole fermenting process for 72 hours, obtain solid fermentation wine with dregs,
Second step, liquid mash: in solid fermentation wine with dregs, add damping fluid and obtain liquid mash, damping fluid add-on is 2.5 times of solid fermentation wine with dregs weight, buffered soln used is citric acid-sodium citrate damping fluid, pH is 5.6, specific configuration is: the citric acid solution 5.5ml of 0.1mol/L: the sodium citrate solution 14.5ml of 0.1mol/L;
The 3rd step, filtration: liquid mash is filtered, obtain enzyme liquid, three-stage filtration is taked in described filtration, liquid mash is successively by Plate Filtration, centrifuging and ceramic membrane filter;
The 4th step, ultrafiltration and concentration: by ultrafiltration mode, enzyme liquid is concentrated, concentrated multiple is 2.5 times, obtain concentrated enzyme liquid.
Embodiment 4
Bacillus amyloliquefaciens solid fermentation, production α-amylase
The first step, by substratum (6000g wheat bran, 2000g dregs of beans, 8000ml nutritive medium, wherein nutritive medium is containing 3% ammonium sulfate (massfraction), 1.5% potassium primary phosphate (massfraction)) in stirrer, mix, then divide and install in cloth bag, 121 ℃, 30min, high pressure moist heat sterilization, treat that substratum is cooled to 25-35 ℃, substratum is poured in stirrer and then added the bacillus amyloliquefaciens bacterium powder preparing to stir, after stirring, be put in koji tray, tiling evenly, thickness is moderate, for 3-5cm, finally cover wet cloth, open temperature control switch, controlling temperature is 29 ℃, after fermentation 40-60h, turn over song and moisturizing, rate of water make-up is 8-15%, and then fermentation completes whole fermenting process for 72 hours, obtain solid fermentation wine with dregs,
Second step, liquid mash: in solid fermentation wine with dregs, add damping fluid and obtain liquid mash, damping fluid add-on is 2 times of solid fermentation wine with dregs weight, buffered soln used is citric acid-sodium citrate damping fluid, pH is 6.0, specific configuration is: the citric acid solution 3.8ml of 0.1mol/L: the sodium citrate solution 16.2ml of 0.1mol/L;
The 3rd step, filtration: liquid mash is filtered, obtain enzyme liquid, three-stage filtration is taked in described filtration, liquid mash is successively by Plate Filtration, centrifuging and ceramic membrane filter;
The 4th step, ultrafiltration and concentration: by ultrafiltration mode, enzyme liquid is concentrated, concentrated multiple is 2 times, obtain concentrated enzyme liquid.
Embodiment 5
Enzyme work to the concentrated enzyme liquid of embodiment 1-4 gained is measured, and concrete data are in Table 2, then according to the addition of the various single enzymes of formula calculation of different composite enzyme in table 3, concrete addition is in Table 4, carry out compositely, be diluted to prescribed level, obtain liquid enzymes work in-process;
Finally in liquid enzymes work in-process, add stability protective agent, obtain liquid complex enzyme, described stability protective agent is that massfraction is 60% sodium lactate aqueous solution, and addition is the 20-25% of liquid enzymes work in-process weight.Wherein in corn-soybean meal type prozyme, addition is 20% of liquid enzymes work in-process weight, and in corn-assorted dregs of rice type prozyme, addition is 23% of liquid enzymes work in-process weight, and in wheat-bean pulp type prozyme, addition is 25% of liquid enzymes work in-process weight; In wheat-assorted dregs of rice type prozyme, addition is 23% of liquid enzymes work in-process weight.
The activity of the enzyme of producing in the different embodiment of table 2
The formula of table 3 different liqs prozyme kind
The addition of the different embodiment of table 4 and enzymatic hydrolyzation
Embodiment 5
A preparation method for the liquid complex enzyme in solid fermentation source, comprises the following steps:
The first step, solid fermentation: by substratum (6000g wheat bran, 2000g dregs of beans, 8000ml nutritive medium, wherein nutritive medium is containing 3% ammonium sulfate (massfraction), 1.5% potassium primary phosphate (massfraction)) in stirrer, mix, then divide and install in cloth bag, 121 ℃, 30min, high pressure moist heat sterilization, treat that substratum is cooled to 25-35 ℃, substratum is poured in stirrer and then added the bacterial classification preparing to stir, after stirring, be put in koji tray, tiling evenly, thickness is moderate, for 3-5cm, finally cover wet cloth, open temperature control switch, 29 ℃, after fermentation 40-60h, turn over song and moisturizing, rate of water make-up is 8-15%, and then fermentation completes whole fermenting process for 72 hours, obtain solid fermentation wine with dregs, described bacterial classification is long handle Trichoderma powder, rice aspergillus powder is mould, black-koji mould powder and bacillus amyloliquefaciens powder, the additional proportion of various bacterial classifications is 3:2:3:2,
Second step, liquid mash: in solid fermentation wine with dregs, add damping fluid and obtain liquid mash, damping fluid add-on is 3 times of solid fermentation wine with dregs weight, buffered soln used is citric acid-sodium citrate damping fluid, pH is 5.2, specific configuration is: the citric acid solution 7.3ml of 0.1mol/L: the sodium citrate solution 12.7ml of 0.1mol/L;
The 3rd step, filtration: liquid mash is filtered, obtain enzyme liquid, three-stage filtration is taked in described filtration, liquid mash is successively by Plate Filtration, centrifuging and ceramic membrane filter;
The 4th step, ultrafiltration and concentration: by ultrafiltration mode, enzyme liquid is concentrated, concentrated multiple is 2 times, obtain concentrated enzyme liquid;
The 5th step, enzyme biopsy are surveyed, and the enzyme work of concentrated enzyme liquid is measured, and concrete enzyme is lived value in Table 5, obtains liquid enzymes work in-process,
The enzyme activity determination of table 5 embodiment 5
| The kind of enzyme | Enzymic activity |
| Cellulase | 2250U |
| Beta-glucanase | 11050U |
| Zytase | 26800U |
| Mannase | 2980U |
| Alpha-galactosidase | 1280U |
| α-amylase | 32000U |
The 6th step, interpolation protective material: in liquid enzymes work in-process, add stability protective agent, obtain liquid complex enzyme, described stability protective agent is that massfraction is 60% sodium lactate aqueous solution, and addition is 22% of liquid enzymes work in-process weight.
The enzymatic hydrolyzation of the liquid complex enzyme that embodiment 5 is prepared is 14.05%.
In addition can application reference publication No. CN103766612A about the embodiment of many solid type mixed culture fermentations, title: the patent of the complex enzyme for feed that a kind of multiple bacteria compound fermentation is produced, obtain solid fermentation wine with dregs, all the other steps are identical with embodiment five, here no longer narration.
Liquid complex enzyme of the present invention, for the preparation of feed, adopts the method for spraying to add in the middle of feed, and general quantity for spray is 80-150 milliliter/ton feed.
Claims (7)
1. a preparation method for the liquid complex enzyme in solid fermentation source, is characterized in that, comprises the following steps:
The first step, solid fermentation: substratum is mixed in stirrer, then divide and install in cloth bag, 121 ℃, 30min, high pressure moist heat sterilization, treat that substratum is cooled to 25-35 ℃, substratum is poured in stirrer and then added the bacterial classification preparing to stir, after stirring, be put in koji tray, tiling evenly, thickness is moderate, for 3-5cm, finally cover wet cloth, open temperature control switch, controlling temperature is 29 ℃, after fermentation 40-60h, turn over song and moisturizing, rate of water make-up is 8-15%, and then fermentation completes whole fermenting process for 72 hours, obtain solid fermentation wine with dregs,
Second step, liquid mash: in solid fermentation wine with dregs, add damping fluid and obtain liquid mash, damping fluid add-on is 2-3 times of solid fermentation wine with dregs weight;
The 3rd step, filtration: liquid mash is filtered, obtain enzyme liquid;
The 4th step, ultrafiltration and concentration: by ultrafiltration mode, enzyme liquid is concentrated, concentrated multiple is 2-3 times, obtain concentrated enzyme liquid;
The 5th step, enzyme biopsy survey, composite: the enzyme work to concentrated enzyme liquid is measured, and then according to the addition of the various single enzymes of formula calculation of prozyme, carries out compositely, is diluted to prescribed level, obtains liquid enzymes work in-process;
The 6th step, interpolation protective material: in liquid enzymes work in-process, add stability protective agent, obtain liquid complex enzyme.
2. the preparation method of the liquid complex enzyme in a kind of solid fermentation according to claim 1 source, is characterized in that: described bacterial classification is a kind of in long handle Trichoderma powder, aspergillus oryzae powder, black-koji mould powder and bacillus amyloliquefaciens bacterium powder or more than one mixture.
3. the preparation method of the liquid complex enzyme in a kind of solid fermentation according to claim 1 source, is characterized in that: described damping fluid is citric acid-sodium citrate damping fluid, and pH is 5.0～6.0.
4. the preparation method of the liquid complex enzyme in a kind of solid fermentation according to claim 1 source, is characterized in that: three-stage filtration is taked in described filtration, and liquid mash is successively by Plate Filtration, centrifuging and ceramic membrane filter.
5. the preparation method of the liquid complex enzyme in a kind of solid fermentation according to claim 1 source, is characterized in that: described stability protective agent is that massfraction is 60% sodium lactate aqueous solution, and addition is the 20-25% of liquid enzymes work in-process weight.
6. the preparation method of the liquid complex enzyme in a kind of solid fermentation according to claim 5 source, is characterized in that: described addition is 23% of liquid enzymes work in-process weight.
7. liquid complex enzyme as prepared in claim 1-6 any one is for the preparation of the additive of feed.
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| CN106061293A (en) * | 2014-03-05 | 2016-10-26 | 帝斯曼知识产权资产管理有限公司 | Liquid lactase compositions |
| CN106036082A (en) * | 2016-05-24 | 2016-10-26 | 浙江伊杰斯生物科技有限公司 | Antibiotic-free feed for resisting porcine diarrhea |
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| CN103766612A (en) * | 2014-01-21 | 2014-05-07 | 颜鹏飞 | Compound enzyme for feed during multi-strain mixed fermentation production |
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Non-Patent Citations (2)
| Title |
|---|
| 胡学智等: "固体培养黑曲霉复合酶在传统发酵上的应用", 《江苏调味副食品》 * |
| 陈清华: "我国饲料酶制剂生产技术研究现状", 《饲料工业-酶制剂的技术理论与应用增刊》 * |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106061293A (en) * | 2014-03-05 | 2016-10-26 | 帝斯曼知识产权资产管理有限公司 | Liquid lactase compositions |
| CN106036082A (en) * | 2016-05-24 | 2016-10-26 | 浙江伊杰斯生物科技有限公司 | Antibiotic-free feed for resisting porcine diarrhea |
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