CN104017790B - A kind of preparation method and its usage of the liquid complex enzyme in solid fermentation source - Google Patents

A kind of preparation method and its usage of the liquid complex enzyme in solid fermentation source Download PDF

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CN104017790B
CN104017790B CN201410275822.0A CN201410275822A CN104017790B CN 104017790 B CN104017790 B CN 104017790B CN 201410275822 A CN201410275822 A CN 201410275822A CN 104017790 B CN104017790 B CN 104017790B
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颜鹏飞
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Abstract

The present invention relates to the preparation method and its usage of the liquid complex enzyme in a kind of solid fermentation source, described method includes: solid fermentation: by culture medium high pressure moist heat sterilization, adds the strain fermentation prepared and obtains solid fermentation wine with dregs；Liquid mash: add buffer in solid fermentation wine with dregs and obtain liquid mash, filter: by liquid mash filtrations, obtain enzyme liquid；It is concentrated by ultrafiltration: by ultrafiltration mode, enzyme liquid is concentrated, obtains concentrating enzyme liquid；Enzyme activity assay, compounding: the enzyme concentrating enzyme liquid to be lived and is measured, then according to the addition of the various single enzyme of recipe calculation of compound enzyme, compound, be diluted to prescribed level, obtain liquid enzymes semi-finished product；Add protective agent: in liquid enzymes semi-finished product, add stability protective agent, obtain liquid complex enzyme.The method of the present invention, both can guarantee that the advantage of solid fermentation compound enzyme high degradation rate, can guarantee that again enzyme is lived and is not lost by high temperature granulating, can greatly submit the efficiency of enzymolysis to.

Description

A kind of preparation method and its usage of the liquid complex enzyme in solid fermentation source
Technical field
The present invention relates to the preparation method and its usage of the liquid complex enzyme in a kind of solid fermentation source, belong to feedstuff and be combined Enzyme.
Background technology
Complex enzyme for feed use in actual production is more and more extensive.Complex enzyme for feed is morphologically divided into two kinds: Gu Bluk recombination enzyme and liquid complex enzyme.Solid complex enzyme carries out after being directly added in feedstuff mixing pelletizing producing, and liquid is multiple Synthase is sprayed on feed surface after feed granulating.The mode of production in enzyme source divides two kinds: solid fermentation and liquid fermentation.Gu Bluk recombination enzyme has two kinds of modes of production simultaneously: solid fermentation and liquid fermentation；Liquid complex enzyme is only derived from liquid fermentation.Gu The compoiste fermented enzyme of body is much better than liquid fermentation compound enzyme because of the reason that its culture medium is all feeds raw material, its using effect.? Solid complex enzyme in grain feedstuff is compared with liquid complex enzyme, and solid complex enzyme enzyme during high temperature granulating lives loss relatively Greatly, up to 90% had, specifically it is shown in Table 1；Liquid complex enzyme sprays after high temperature granulating again, and its enzyme is lived does not has any damage Consumption.Therefore the liquid enzymes using effect of solid fermentation is substantially better than solid fermentation solid enzyme and liquid fermentation liquid enzyme.Therefore open Good liquid complex enzyme is the most necessary to send out using effect a kind of.Additionally the enzymatic hydrolyzation of liquid fermentation compound enzyme is 1.88%, Enzymatic hydrolyzation is the lowest.
Table 1 solid polypeptide formulation enzyme in pellet pelletization is lived and is retained
Summary of the invention
The invention provides the preparation method and its usage of the liquid complex enzyme in a kind of solid fermentation source, solve existing Liquid complex enzyme is only derived from liquid fermentation, the problem that using effect is bad.
For solving above-mentioned technical problem, the technical solution used in the present invention is:
The preparation method of the liquid complex enzyme in a kind of solid fermentation source, comprises the following steps:
The first step, solid fermentation: by culture medium mix homogeneously in blender, be then dispensed in cloth bag, 121 DEG C, 30min, high pressure moist heat sterilization, treat that culture medium is cooled to 25-35 DEG C, pour into culture medium in blender to be subsequently adding and prepare Strain stirs, and is put in koji tray after stirring, and uniformly, thickness is moderate, for 3-5cm, finally covers wet cloth in tiling, opens Temperature control switch, controlling temperature is 29 DEG C, after fermentation 40-60h, carries out turning over song and moisturizing, and rate of water make-up is 8-15%, ferments the most again Within 72 hours, complete whole sweat, obtain solid fermentation wine with dregs；
Second step, liquid mash: adding buffer in solid fermentation wine with dregs and obtain liquid mash, buffer addition is solid 2-3 times of body karusen weight；
3rd step, filtration: by liquid mash filtrations, obtain enzyme liquid；
4th step, ultrafiltration concentration: concentrated enzyme liquid by ultrafiltration mode, the multiple of concentration is 2-3 times, is concentrated Enzyme liquid；
5th step, Enzyme activity assay, compounding: the enzyme concentrating enzyme liquid to be lived and is measured, then according to the formula meter of compound enzyme Calculate the addition of various single enzyme, compound, be diluted to prescribed level, obtain liquid enzymes semi-finished product；
6th step, interpolation protective agent: in liquid enzymes semi-finished product, add stability protective agent, obtain liquid complex enzyme.
Preferably: described strain is long handle Trichoderma spp. mycopowder, aspergillus oryzae mycopowder, aspergillus niger mycopowder and bacillus amyloliquefaciens The mixture of the one or more than one in mycopowder.
Preferably: described buffer is citric acid-sodium citrate buffer, pH is 5.0～6.0.
Preferably: described filtration takes three-stage filtration, liquid mash to pass sequentially through plate-and-frame filtration, centrifugal filtration and pottery Membrane filtration.The aperture of ceramic filter membrane used is 0.45 μm.
Preferably: described stability protective agent be mass fraction be the sodium lactate aqueous solution of 60%, addition is liquid enzymes The 20-25% of semi-finished product weight.
Additionally present invention provides the purposes of a kind of liquid complex enzyme, the liquid complex enzyme prepared by the present invention is used for making The additive of standby feedstuff, uses the method for spraying to add in the middle of feedstuff.
The preparation method of the long handle Trichoderma spp. mycopowder used by the present invention: long handle Trichoderma spp. is inoculated on PDA solid medium, in Cultivate 24-72h for 28 DEG C；After growing spore, by spore inoculating in 50mlPDA fluid medium, 24-cultivated by 28 DEG C of shaking tables 72h, grows up to the flocculence mycelia floated on a liquid, then by the bran mass mixing of this liquid with prior sterilizing, places At 28 DEG C, cultivate 24-72h, after growing spore, how much add calcium carbonate and starch (calcium carbonate: starch according to spore and moisture Weight ratio be 3:2) mixing, make spore count reach 1012Individual/g, vacuum drying at 30-45 DEG C, it is long handle Trichoderma spp. mycopowder, Viable count reaches 1012Individual/g, room temperature preserves stand-by.
Aspergillus oryzae mycopowder is similar to the preparation method of long handle Trichoderma spp. mycopowder with aspergillus niger mycopowder, is not discussing.
The preparation method of bacillus amyloliquefaciens mycopowder: by slant tube preserve bacillus amyloliquefaciens 5-8mL without Bacterium brine protects bacterium inclined-plane, to aseptic triangular flask shakes up bacteria suspension 150mL, by 10% volume by slant culture Thing is inoculated into seed bottle, and at 28 DEG C, 222rpm cultivates 24-48h, and the seed liquor of 6-10% is inoculated into amplification culture in fermentation flask 24-72h obtains fermentation liquid.Calcium carbonate and starch (calcium carbonate: the weight ratio of starch is as 3:2) is added with the ratio of volume ratio 1:1 Mixing, vacuum drying at 30-45 DEG C, it is bacillus amyloliquefaciens mycopowder, viable count reaches 1012Individual/g, room temperature preserves and treats With.
The assay method of enzymatic hydrolyzation, with reference to application publication number CN103308413A, entitled a kind of external test is feeding non- The patent of the method for starch-polysaccharides enzyme hydrolysis result is carried out.
Beneficial effects of the present invention:
The long handle Trichoderma spp. main product cellulase of present invention employing and 1,4 beta-glucanase, aspergillus oryzae main product xylanase, black fermented preparation Mould main product mannase and alpha-galactosidase, bacillus amyloliquefaciens main product α-amylase, it is feed additive kind The strain that in catalogue (2013), regulation uses, utilizes them to produce liquid complex enzyme, can solve efficiency of feed utilization well low Under problem, enzymatic hydrolyzation reaches 14.05%,
The present invention uses the method for solid fermentation to produce liquid complex enzyme, both can guarantee that solid fermentation compound enzyme height was degraded The advantage of rate, can guarantee that again enzyme is lived and is not lost by high temperature granulating, can greatly submit the efficiency of enzymolysis to.
Detailed description of the invention
Below by specific embodiment, technical scheme is described in detail.
Embodiment 1
Long handle Trichoderma spp. solid fermentation, produces cellulase and 1,4 beta-glucanase
The first step, by culture medium (6000g wheat bran, 2000g bean cake, 8000ml nutritional solution, wherein nutritional solution contain 3% sulphuric acid Ammonium (mass fraction), 1.5% potassium dihydrogen phosphate (mass fraction)) mix homogeneously in blender, then it is dispensed in cloth bag, 121 DEG C, 30min, high pressure moist heat sterilization, treat that culture medium is cooled to 25-35 DEG C, culture medium is poured in blender the system that is subsequently adding The long handle Trichoderma spp. mycopowder got ready stirs, and is put in koji tray after stirring, and uniformly, thickness is moderate in tiling, for 3-5cm, Wet cloth on bonnet, opens temperature control switch, and controlling temperature is 29 DEG C, after fermentation 40-60h, carries out turning over song and moisturizing, and rate of water make-up is 8- 15%, ferment the most again 72 hours and complete whole sweat, obtain solid fermentation wine with dregs；
Second step, liquid mash: adding buffer in solid fermentation wine with dregs and obtain liquid mash, buffer addition is solid 2 times of body karusen weight, buffer solution used is citric acid-sodium citrate buffer, and pH is 5.0, and concrete configuration is: The citric acid solution 8.2ml of 0.1mol/L: the sodium citrate solution 11.8ml of 0.1mol/L；
3rd step, filtration: by liquid mash filtrations, obtain enzyme liquid, and described filtration takes three-stage filtration, liquid mash to depend on Secondary by plate-and-frame filtration, centrifugal filtration and ceramic membrane filter；
4th step, ultrafiltration concentration: concentrated enzyme liquid by ultrafiltration mode, the multiple of concentration is 3 times, obtains concentrating enzyme Liquid.
Embodiment 2
Aspergillus oryzae solid fermentation, produces xylanase
The first step, by culture medium (6000g wheat bran, 2000g bean cake, 8000ml nutritional solution, wherein nutritional solution contain 3% sulphuric acid Ammonium (mass fraction), 1.5% potassium dihydrogen phosphate (mass fraction)) mix homogeneously in blender, then it is dispensed in cloth bag, 121 DEG C, 30min, high pressure moist heat sterilization, treat that culture medium is cooled to 25-35 DEG C, culture medium is poured in blender the system that is subsequently adding The aspergillus oryzae mycopowder got ready stirs, and is put in koji tray after stirring, and uniformly, thickness is moderate in tiling, for 3-5cm, finally Covering wet cloth, open temperature control switch, controlling temperature is 29 DEG C, after fermentation 40-60h, carries out turning over song and moisturizing, and rate of water make-up is 8- 15%, ferment the most again 72 hours and complete whole sweat, obtain solid fermentation wine with dregs；
Second step, liquid mash: adding buffer in solid fermentation wine with dregs and obtain liquid mash, buffer addition is solid 3 times of body karusen weight, buffer solution used is citric acid-sodium citrate buffer, and pH is 5.4, and concrete configuration is: The citric acid solution 6.4ml of 0.1mol/L: the sodium citrate solution 13.6ml of 0.1mol/L；
3rd step, filtration: by liquid mash filtrations, obtain enzyme liquid, and described filtration takes three-stage filtration, liquid mash to depend on Secondary by plate-and-frame filtration, centrifugal filtration and ceramic membrane filter；
4th step, ultrafiltration concentration: concentrated enzyme liquid by ultrafiltration mode, the multiple of concentration is 2 times, obtains concentrating enzyme Liquid.
Embodiment 3
Aspergillus niger solid fermentation, produces mannase and alpha-galactosidase
The first step, by culture medium (6000g wheat bran, 2000g bean cake, 8000ml nutritional solution, wherein nutritional solution contain 3% sulphuric acid Ammonium (mass fraction), 1.5% potassium dihydrogen phosphate (mass fraction)) mix homogeneously in blender, then it is dispensed in cloth bag, 121 DEG C, 30min, high pressure moist heat sterilization, treat that culture medium is cooled to 25-35 DEG C, culture medium is poured in blender the system that is subsequently adding The aspergillus niger mycopowder got ready stirs, and is put in koji tray after stirring, and uniformly, thickness is moderate in tiling, for 3-5cm, finally Covering wet cloth, open temperature control switch, controlling temperature is 29 DEG C, after fermentation 40-60h, carries out turning over song and moisturizing, and rate of water make-up is 8- 15%, ferment the most again 72 hours and complete whole sweat, obtain solid fermentation wine with dregs；
Second step, liquid mash: adding buffer in solid fermentation wine with dregs and obtain liquid mash, buffer addition is solid 2.5 times of body karusen weight, buffer solution used is citric acid-sodium citrate buffer, and pH is 5.6, and concrete configuration is: The citric acid solution 5.5ml of 0.1mol/L: the sodium citrate solution 14.5ml of 0.1mol/L；
3rd step, filtration: by liquid mash filtrations, obtain enzyme liquid, and described filtration takes three-stage filtration, liquid mash to depend on Secondary by plate-and-frame filtration, centrifugal filtration and ceramic membrane filter；
4th step, ultrafiltration concentration: concentrated enzyme liquid by ultrafiltration mode, the multiple of concentration is 2.5 times, is concentrated Enzyme liquid.
Embodiment 4
Bacillus amyloliquefaciens solid fermentation, produces α-amylase
The first step, by culture medium (6000g wheat bran, 2000g bean cake, 8000ml nutritional solution, wherein nutritional solution contain 3% sulphuric acid Ammonium (mass fraction), 1.5% potassium dihydrogen phosphate (mass fraction)) mix homogeneously in blender, then it is dispensed in cloth bag, 121 DEG C, 30min, high pressure moist heat sterilization, treat that culture medium is cooled to 25-35 DEG C, culture medium is poured in blender the system that is subsequently adding The bacillus amyloliquefaciens mycopowder got ready stirs, and is put in koji tray after stirring, and uniformly, thickness is moderate, for 3-in tiling 5cm, finally covers wet cloth, opens temperature control switch, and controlling temperature is 29 DEG C, after fermentation 40-60h, carries out turning over song and moisturizing, moisturizing Amount is 8-15%, and fermenting 72 hours completes whole sweat the most again, obtains solid fermentation wine with dregs；
Second step, liquid mash: adding buffer in solid fermentation wine with dregs and obtain liquid mash, buffer addition is solid 2 times of body karusen weight, buffer solution used is citric acid-sodium citrate buffer, and pH is 6.0, and concrete configuration is: The citric acid solution 3.8ml of 0.1mol/L: the sodium citrate solution 16.2ml of 0.1mol/L；
3rd step, filtration: by liquid mash filtrations, obtain enzyme liquid, and described filtration takes three-stage filtration, liquid mash to depend on Secondary by plate-and-frame filtration, centrifugal filtration and ceramic membrane filter；
4th step, ultrafiltration concentration: concentrated enzyme liquid by ultrafiltration mode, the multiple of concentration is 2 times, obtains concentrating enzyme Liquid.
Embodiment 5
Embodiment 1-4 gained is concentrated enzyme liquid enzyme live be measured, concrete data are shown in Table 2, then according in table 3 not With the addition of the various single enzyme of the recipe calculation of compound enzyme, concrete addition is shown in Table 4, compounds, is diluted to prescribed level, To liquid enzymes semi-finished product；
Last interpolation stability protective agent in liquid enzymes semi-finished product, obtains liquid complex enzyme, and described stability protective agent is Mass fraction is the sodium lactate aqueous solution of 60%, and addition is the 20-25% of liquid enzymes semi-finished product weight.Wherein corn-soybean meal In type compound enzyme, addition is the 20% of liquid enzymes semi-finished product weight, and in Semen Maydis miscellaneous meal type compound enzyme, addition is liquid enzymes half The 23% of finished weight, in Semen Tritici aestivi bean pulp type compound enzyme, addition is the 25% of liquid enzymes semi-finished product weight；The miscellaneous dregs of rice of Semen Tritici aestivi In type compound enzyme, addition is the 23% of liquid enzymes semi-finished product weight.
The activity of the enzyme produced in the different embodiment of table 2
The formula of table 3 different liquids compound enzyme kind
The addition of the different embodiment of table 4 and enzymatic hydrolyzation
Embodiment 5
The preparation method of the liquid complex enzyme in a kind of solid fermentation source, comprises the following steps:
The first step, solid fermentation: by culture medium (6000g wheat bran, 2000g bean cake, 8000ml nutritional solution, wherein nutritional solution Containing 3% ammonium sulfate (mass fraction), 1.5% potassium dihydrogen phosphate (mass fraction)) mix homogeneously in blender, then it is dispensed into In cloth bag, 121 DEG C, 30min, high pressure moist heat sterilization, treat that culture medium is cooled to 25-35 DEG C, culture medium is poured in blender right The strain that rear addition prepares stirs, and is put in koji tray after stirring, and uniformly, thickness is moderate in tiling, for 3-5cm, Wet cloth on bonnet, opens temperature control switch, 29 DEG C, after fermentation 40-60h, carries out turning over song and moisturizing, and rate of water make-up is 8-15%, then Fermenting 72 hours again and complete whole sweat, obtain solid fermentation wine with dregs, described strain is long handle Trichoderma spp. mycopowder, rice-koji mycopowder Mould, aspergillus niger mycopowder and bacillus amyloliquefaciens powder, the additional proportion of various strains is 3:2:3:2；
Second step, liquid mash: adding buffer in solid fermentation wine with dregs and obtain liquid mash, buffer addition is solid 3 times of body karusen weight, buffer solution used is citric acid-sodium citrate buffer, and pH is 5.2, and concrete configuration is: The citric acid solution 7.3ml of 0.1mol/L: the sodium citrate solution 12.7ml of 0.1mol/L；
3rd step, filtration: by liquid mash filtrations, obtain enzyme liquid, and described filtration takes three-stage filtration, liquid mash to depend on Secondary by plate-and-frame filtration, centrifugal filtration and ceramic membrane filter；
4th step, ultrafiltration concentration: concentrated enzyme liquid by ultrafiltration mode, the multiple of concentration is 2 times, obtains concentrating enzyme Liquid；
5th step, Enzyme activity assay, live to the enzyme concentrating enzyme liquid and be measured, and concrete enzyme value alive is shown in Table 5, obtains liquid enzymes half Finished product,
The enzyme activity determination of table 5 embodiment 5
The kind of enzyme Enzymatic activity
Cellulase 2250U
1,4 beta-glucanase 11050U
Xylanase 26800U
Mannase 2980U
Alpha-galactosidase 1280U
α-amylase 32000U
6th step, interpolation protective agent: in liquid enzymes semi-finished product, add stability protective agent, obtain liquid complex enzyme, described Stability protective agent be mass fraction be the sodium lactate aqueous solution of 60%, addition is the 22% of liquid enzymes semi-finished product weight.
The enzymatic hydrolyzation of the liquid complex enzyme prepared by embodiment 5 is 14.05%.
Embodiment further with regards to the fermentation of multi-cultur es hybrid solid is referred to application publication number CN103766612A, name Claim: the patent of the complex enzyme for feed that a kind of multiple bacteria compound fermentation produces, obtain solid fermentation wine with dregs, remaining step and embodiment five Identical, the most no longer describe.
The liquid complex enzyme of the present invention is used for preparing feedstuff, uses the method for spraying to add in the middle of feedstuff, general spray Painting amount is 80-150 milliliter/ton feedstuff.

Claims (5)

1. the preparation method of the liquid complex enzyme in a solid fermentation source, it is characterised in that comprise the following steps:
The first step, solid fermentation: by culture medium mix homogeneously in blender, be then dispensed in cloth bag, 121 DEG C, 30min, High pressure moist heat sterilization, treats that culture medium is cooled to 25-35 DEG C, culture medium is poured in blender and is subsequently adding the strain prepared and stirs Mixing uniformly, be put in koji tray after stirring, uniformly, thickness is moderate in tiling, for 3-5cm, finally covers wet cloth, opens temperature control and opens Closing, controlling temperature is 29 DEG C, after fermentation 40-60h, carries out turning over song and moisturizing, and rate of water make-up is 8-15%, ferments 72 hours the most again Complete whole sweat, obtain solid fermentation wine with dregs；
Second step, liquid mash: adding buffer in solid fermentation wine with dregs and obtain liquid mash, buffer addition is that solid is sent out 2-3 times of ferment wine with dregs weight；
3rd step, filtration: by liquid mash filtrations, obtain enzyme liquid；
4th step, ultrafiltration concentration: concentrated enzyme liquid by ultrafiltration mode, the multiple of concentration is 2-3 times, obtains concentrating enzyme Liquid；Respectively with long handle Trichoderma spp. mycopowder, aspergillus oryzae mycopowder, aspergillus niger mycopowder and bacillus amyloliquefaciens mycopowder as strain, by with Upper step obtains corresponding concentration enzyme liquid；
5th step, Enzyme activity assay, compounding: the enzyme to described concentration enzyme liquid is lived and is measured respectively, then joining according to compound enzyme Side calculates the addition of various single enzymes, compounds, is diluted to prescribed level, obtains containing fermentation described long handle Trichoderma spp., rice-koji The liquid enzymes semi-finished product of the enzyme liquid that mould, aspergillus niger and bacillus amyloliquefaciens obtain；
6th step, interpolation protective agent: in liquid enzymes semi-finished product, add stability protective agent, obtain liquid complex enzyme；
Described stability protective agent be mass fraction be the sodium lactate aqueous solution of 60%, addition is liquid enzymes semi-finished product weight 20-25%.
The preparation method of the liquid complex enzyme in a kind of solid fermentation the most according to claim 1 source, it is characterised in that: institute The buffer stated is citric acid-sodium citrate buffer, and pH is 5.0～6.0.
The preparation method of the liquid complex enzyme in a kind of solid fermentation the most according to claim 1 source, it is characterised in that: institute The filtration stated takes three-stage filtration, liquid mash to pass sequentially through plate-and-frame filtration, centrifugal filtration and ceramic membrane filter.
The preparation method of the liquid complex enzyme in a kind of solid fermentation the most according to claim 1 source, it is characterised in that: institute State addition is liquid enzymes semi-finished product weight the 23% of stability protective agent.
5. the liquid complex enzyme as prepared by any one of claim 1-4 is for preparing the additive of feedstuff.
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