CN105124171A - Complex enzyme preparation with high thermal stability and preparing method thereof - Google Patents
Complex enzyme preparation with high thermal stability and preparing method thereof Download PDFInfo
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Abstract
The invention relates to a complex enzyme preparation with high thermal stability and a preparing method thereof. The complex enzyme preparation is prepared from the following components in parts by weight: 80-120 parts of xylanase, 30-40 parts of cellulase, 40-100 parts of beta-glucanase, 100-150 parts of protease, 20-30 parts of mesothermal alpha-amylase, 3-8 parts of composite vitamin, 10-20 parts of composite trace elements, 10-20 parts of antimicrobial peptide and 20-30 parts of red yeast culture, wherein red yeast is specifically ocean red yeast WYN1 (Rhodotorula sp.WYN1) with a preservation number of CCTCC No. M 2014592. The complex enzyme preparation is rich in carotene and chelating copper, can obviously improve the use rate of fodder as well as thermal stability and preservation activation of the enzyme preparation.
Description
Technical field:
The invention belongs to enzyme preparation technical field, be specifically related to complex enzyme of a kind of Heat stability is good and preparation method thereof.
Background technology:
In animal husbandry, it is the most complicated in all stages pig that sucking pig is raised.The irrational consequence of nutrition supply is pig poor growth, and diarrhea rate is high, and the death rate is high, and then in making, large pig growth period is slow, extends marketing time.
But in order to cut off the vertical transmission route of source of parents disease, shorten the sow breeding cycle, extend the kind time limit, early weaned technology is widely used in the large-scale farming industry of pig by people simultaneously.But due to newborn piglet, to lack congenital immunity, digestive system development not perfect, digestive ferment secretion capacity is weak, and directly supplying to give corn etc. is that main feed is easy to cause the problems such as the digestibility of porkling to feed is low, feed intake is low, the speed of growth is slow, diarrhea rate is high.
In order to overcome the variety of problems that sucking pig occurs after wean, most enterprise from supplementary feeding, food calling, daily ration acidifying before wean, use good palatability and the feedstuff of absorption easy to digest, add exogenous enzymes, reduce the antigenicity of daily ration, reduce the crude protein content of daily ration and use the many aspects such as promoter to improve and optimize, but actual effect is uneven.
Consider that piglet is when weaning, himself the enzyme such as protease, amylase work stress and decline to a great extent wean time 1/3rd, and these enzymes obtain nutrition for piglet and energy plays vital effect.Meanwhile, fodder enzyme has original effect in Production of Livestock and Poultry, and such as former Soviet Union scientist adds amylodextrin enzyme in weanling pig daily ration in early days, and make daily gain improve 14.6%, carbohydrate digestion rate improves 77.2%.According to U.S.'s interrelated data, Collier supplements enzyme preparation (containing protease, amylase, dextranase) in weanling pig daily ration in early days, and result experimental group improves 25% than control group daily gain, and feed conversion rate improves 15.5%.
Add enzyme preparation and can supplement protease and amylase, the albumen in auxiliary decomposition daily ration and starch, ANFs in degraded daily ration, and effectively can decompose the plant cell wall in corn/soybean meal based diets, intracellular nutriment such as starch, protein etc. are discharged.Decomposing soluble fiber simultaneously, reduces the water absorbing capacity of vegetable fibrous material and the viscosity of intestinal contents.
In addition, in field of animal feed, mainly with the source of the Inorganic Coppers such as copper sulphate as copper, but it is low to there is absorptivity in Inorganic Copper, there is the problems such as antagonism with other nutriments, chelated copper then can well solve the problem, it can alleviate the antagonism with other nutriments, promote the utilization of copper and other mineral matter elements, promote growing of animal, improve efficiency of feed utilization, improve carcass quality, improve reproductive performance, also there is antibiooxidation and a series of function such as immunity and good stability simultaneously, preferably economic benefit can be brought.
Carotenoid is a class in Polyenes that is yellow, orange or red, and it is the most ubiquity of occurring in nature is also the most stable natural colouring matter, is extensively present in many animals and plants.Carotenoid as a kind of excellent additive and the nutritional supplement improving nutrition, its fine quality and effect, for a long time just generally acknowledge by countries in the world, be described as most promising antioxidant.At present, carotenoid is widely used in feed additive industry, the application prospect good due to it and the function of brilliance, for a long time extremely people's favor.It is significant to feed additive industry development how effective exploitation obtains green feed additive product with natural plants and fungi.
Therefore, we, through repeatedly administering transgenic, have developed a kind of complex enzyme, its can effectively improve feed stability, utilization rate, reduction diarrhea rate, promote growth of animal, with strong points, effective, remarkable in economical benefits.
Summary of the invention:
The object of the present invention is to provide complex enzyme that a kind of good stability, utilization rate are high and preparation method thereof.
Object of the present invention is achieved through the following technical solutions:
A kind of complex enzyme formulation, be made up of the component of following parts by weight: zytase 80-120 part, cellulase 30-40 part, 1,4 beta-glucanase 40-100 part, protease 100-150 part, mesophilicα-diastase 20-30 part, B B-complex 3-8 part, composite trace element 10-20 part, antibacterial peptide 10-20 part, rhodotorula culture 20-30 part;
Rhodotorula used in the present invention, be the rich copper ocean rhodotorula WYN1 of a strain High Yield of Carotenoid, described bacterial strain on November 25th, 2014 be preserved in China typical culture collection center (address: China. Wuhan. Wuhan University, postcode 430072), deposit number is: CCTCCNo:M2014592, Classification And Nomenclature: ocean rhodotorula WYN1 (Rhodotorulasp.WYN1).
Described zytase, cellulase, 1,4 beta-glucanase, protease, mesophilicα-diastase and antibacterial peptide are commercially available prod;
Described composite trace element per kilogram comprises iodine 1000mg, potassium 180mg, iron 1800mg, selenium 80mg, cobalt 200mg, magnesium 145mg, manganese 1400mg, chromium 20mg, zinc 1100mg, copper 800mg; Its surplus is carrier, and described carrier is wood chip;
The preparation method of described wood chip comprises the steps: to remove foreign material by without mouldy, anosis worm sawmilling end or waste wood, be placed in supersonic wave cleaning machine and clean 10min in 200W, 30KHz, rinse, drain, put into steam still in 0.3-0.4MPa boiling 20-30min, then put microwave dryer in 2000W, 120 DEG C carry out intermittent drying 3min, make it moisture and reach 8-10%, last 2.0 sieves are pulverized, are packed and obtain wood chip;
In described supersonic wave cleaning machine, cleaning fluid is the sodium bicarbonate solution of mass percent concentration 0.5%;
Described intermittent drying technique is: microwave 10s, stops 20s, so repeats, cyclic drying.
Described B B-complex per kilogram comprises dehydroretinol 800mg, orotic acid 000mg, Catergen 000mg, riboflavin 1600mg, pantothenic acid 1900mg, vitamin B6 1800mg, vitamin B12 1200mg, inositol 5000mg, vitamin E 600mg, vitamine D3 5500mg, Vitamin K3 4000mg; Its surplus is carrier, and described carrier is the wood chip of above-mentioned preparation.
Described rhodotorula culture preparation method is as follows:
Seed culture is cultivated by yeast conventional culture methods;
Fermented and cultured: initial pH is 8, and seed culture fluid is inoculated in fermentation medium by inoculum concentration 3-8%, and shaking speed is 50-80r/min, cultivate for 24-28 DEG C and to adjust temperature after 10-15 hour and be 16-20 DEG C and carry out low temperature static gas wave refrigerator, stop stirring, pH is adjusted to 3-5, keeps 2-4 hour; Stairstepping is warmed up to 24-30 DEG C afterwards, and pH is adjusted to 4.6-5.5, and the addition according to 2.5% adds peptone, and the amount according to 2% adds dusty yeast; Shaking speed is 50-100r/min, continues fermentation 10-15h.
After fermentation ends, 10-1000 μm, fermentation liquor aperture coarse filtration, then concentratedly with 10-20 DEG C of loop ultrafiltration removes moisture and obtains solid content 20-40% concentrate, and concentrate obtains rhodotorula culture through vacuum freeze drying, ultramicro grinding.
Fermentation medium mass volume ratio consists of: glucose 1%, enzymolysis corn flour 1-3%, peptone 2.5%, magnesium sulfate 0.02%, potassium dihydrogen phosphate 0.15%, copper content 150-200mg/L, sodium chloride 20%, and all the other are water;
The preparation method of described enzymolysis corn flour is: corn flour is placed in material-compound tank, be that 2:1 adds pure water with V:m, adjust ph 3-7, add middle temperature amylase, enzyme is lived as 30u/g corn flour, lives as the protease of 30u/g corn flour with enzyme, and warming while stirring is to 45-55 DEG C of insulation 15-30min, then be slowly warming up to 60-65 DEG C of insulation 15-30min, freeze drying is for subsequent use.
Described complex enzyme is applicable to feed-processing plant and plant's autogamy feed, should mix, can the present invention be mixed with a small amount of feed in advance, then be mixed in large quantities of feed, Direct-fed during use with other raw material in feed.Advise that complete diet pellet addition per ton is 80-120g.
Beneficial effect:
1, the present invention utilizes the rich copper ocean rhodotorula WYN1 being separated from marine environment and obtaining a plant height product carrotene, effectively raises carrotene and copper output in yeast by static gas wave refrigerator method.Adopt said method fermented and cultured ocean rhodotorula WYN1, in its biomass, carotenoid output and born of the same parents, copper content is respectively more than 27g/L, the dry bacterium of 35-40mg/L and 7240-8000 μ g/g.In research and development, inventor surprisingly finds that low temperature static gas wave refrigerator effectively can improve carrot and copper content, is important achievement of the present invention.
2, because rhodotorula used in the present invention can high yield carrotene, and the Inorganic Copper in culture medium is converted into chelated copper and carries out enrichment, better facilitate growing of animal, improve efficiency of feed utilization, weanling pig daily gain improves 48.91%, feedstuff-meat ratio improves 14.1%, and diarrhea rate reduces 10%, and death rate reduces by 3.7%.
3, because chelated copper has good heat endurance, the interpolation of rhodotorula makes complex enzyme provided by the present invention have and preserves active and heat endurance preferably, and preserve activity and improve 25-30%, heat endurance improves more than 35%.
4, the present invention is according to pig digestive physiological characteristics, and through zoopery screening compound prescription, complex enzyme zymogram is set, effectively supplements the deficiency of the endogenous enzymes such as alimentary canal protease, amylase, effective raising, to the digestive utilization ratio of feed, prevents the generation of trophism diarrhea.
Detailed description of the invention:
Embodiment 1: the acquisition of ocean rhodotorula bacterium WYN1 and the domestication of resistance to copper ability
(1) by gathering the fresh biological specimen such as extra large shrimp, starfish, marine alga from Huanghai Sea seashore, be placed in aseptic YEPD culture medium, picking redness or pink circular colonies, be further purified cultivation.According to the accumulation of each bacterial classification carotenoid, the bacterial classification that screening carotenoid output is high.The ocean rhodotorula WYN1 that wherein carotenoid output is higher, cell is oval, does not have pseudohypha; The YEPD culture medium of seawater configuration forms red colonies, smooth surface, neat in edge; Product spore culture medium does not produce ascospore, and polygon budding, without ballistopore; Azymic sugar, does not form kind of starch compound, does not assimilate inositol.According to These characteristics, tentatively determine to belong to Rhodotorula (Rhodotorula).Inclined-plane is stored in refrigerator.
(2) fermented and cultured of ocean rhodotorula WYN1
Activate on strain transfer to slant medium, after 28 DEG C of cultivation 24h, be inoculated in YPD liquid seed culture medium, 28 DEG C of shaken cultivation 36h obtain seed liquor, be equipped with in the 250mL triangular flask of 60mL fermentation medium by 10% inoculum concentration access, 28 DEG C, shaken cultivation 48h (shaking speed is 180r/min).Fermentation medium consists of glucose 2%, peptone 1%, yeast extract 1%, magnesium sulfate 0.02%, potassium dihydrogen phosphate 0.15%, salinity 20.48h cultivated by 28 DEG C of shaking tables, and rotating speed is 180r/min.
(3) resistance to copper ability domestication
Ocean rhodotorula bacterium WYN1 being inoculated in content of copper ion is in 50mg/L (copper sulphate configuration) culture medium, is placed in 28 DEG C, and on 180r/min constant-temperature table, 24h is cultivated in concussion.Then from this culture, get 3ml fermentate is inoculated in the malt extract medium of not cupric, cultivates 24h.Repeatedly cultivate several generations, yeast is grown in the culture medium of cupric stable, and when quantity no longer increases, more progressively improve the copper concentration of culture medium.So saccharomycete repeatedly just can be made can to adapt to the environment that content of copper ion is 300mg/l, obtain the characteristic of resistance to copper.The present invention, after ocean rhodotorula being carried out to the domestication of resistance to copper ability, can improve cellular biomass, the output of carotenoid and the rich copper ability of yeast effectively.
(4) mensuration of cellular biomass
By centrifugal for zymotic fluid 3000r/min 10min, abandon supernatant, thalline is again centrifugal after aseptic washing 2 ~ 3 times, and gained thalline is placed in 60 DEG C of baking ovens and is dried to constant weight, correct amount.
(5) extraction of carotenoid:
1g dry mycelium is put into 100mL triangular flask, and add the hydrochloric acid soaking at room temperature 1h of 5mL3mol/L, boiling water bath 4min, cools rapidly, and 3000r/min is centrifugal, and 15min is precipitated as cell residue, is settled to 20mL extraction, obtains carotenoid leaching liquor with acetone.
(6) mensuration of carotenoid
After suitably being diluted by extract, under 475nm condition, measure absorbance value with 722 type spectrophotometers.Be calculated as follows carotenoid content:
In formula: A λ max is the absorbance at 475nm wavelength place; D is extension rate when measuring sample; V is acetone consumption (mL); 0.16 is the molar extinction coefficient of carotenoid; W is rhodotorula dry cell weight (g).
(7) yeast copper content measures
Use aas determination.
(8) culture presevation
The above-mentioned rich copper ocean rhodotorula WYN1 through domestication is preserved in China typical culture collection center, and deposit number is: CCTCCNo:M2014592.
In carotenoid production and born of the same parents, chelated copper content is respectively 27.52g/L, 25.12mg/L and the dry bacterium of 6240 μ g/g.
The preparation method of embodiment 2 one kinds of rhodotorula cultures
Be inoculated in YPD liquid seed culture medium by ocean rhodotorula WYN1 slant strains, 28 DEG C of shaken cultivation 36h obtain seed liquor;
Fermented and cultured: initial pH is 8, and seed culture fluid is inoculated in fermentation medium by inoculum concentration 5%, and shaking speed is 65r/min, cultivate for 26 DEG C and to adjust temperature after 12 hours and be 18 DEG C and carry out low temperature static gas wave refrigerator, stop stirring, pH is adjusted to 4, keeps 3 hours; Intensification per hour afterwards 2 DEG C to 26 DEG C, pH is adjusted to 5, and add peptone according to the addition of zymotic fluid w/v 2.5%, the amount according to 2% adds dusty yeast; Shaking speed is 80r/min, continues fermentation 12h;
After fermentation ends, 500 μm, fermentation liquor aperture coarse filtration, then concentratedly with 15 DEG C of loop ultrafiltrations removes moisture and obtains solid content 30% concentrate, and concentrate obtains rhodotorula culture through vacuum freeze drying, ultramicro grinding.
Fermentation medium mass volume ratio consists of: glucose 1%, enzymolysis corn flour 2%, peptone 2.5%, magnesium sulfate 0.02%, potassium dihydrogen phosphate 0.15%, copper content 180mg/L, sodium chloride 20%, and all the other are water;
The preparation method of described enzymolysis corn flour is: by corn flour material-compound tank, be that 2:1 adds pure water with V:m, adjust ph 5, add middle temperature amylase, enzyme is lived as 30u/g corn flour, lives as the protease of 30u/g corn flour with enzyme, warming while stirring to 50 DEG C insulation 25min, then be slowly warming up to 62 DEG C of insulation 25min, freeze drying is for subsequent use.
Adopt said method fermented and cultured ocean rhodotorula WYN1, in zymotic fluid, in biomass, carotenoid output and born of the same parents, chelated copper content is respectively 32.52g/L, 40mg/L and the dry bacterium of 8000 μ g/g.
Embodiment 3 one kinds of complex enzyme formulations and preparation method thereof
A kind of complex enzyme, is made up of the component of following parts by weight:
Zytase 100 parts, cellulase 35 parts, 1,4 beta-glucanase 70 parts, 120 parts, protease, mesophilicα-diastase 25 parts, B B-complex 5 parts, composite trace element 10 parts, antibacterial peptide 15 parts, rhodotorula culture 25 parts;
Described composite trace element per kilogram comprises iodine 1000mg, potassium 180mg, iron 1800mg, selenium 80mg, cobalt 200mg, magnesium 145mg, manganese 1400mg, chromium 20mg, zinc 1100mg, copper 800mg; Its surplus is carrier, and described carrier is wood chip;
The preparation method of described wood chip comprises the steps: to remove foreign material by without mouldy, anosis worm sawmilling end or waste wood, be placed in supersonic wave cleaning machine and clean 10min in 200W, 30KHz, rinse, drain, put into steam still in 0.3MPa boiling 25min, then put microwave dryer in 2000W, 120 DEG C carry out intermittent drying 3min, make it moisture and reach 10%, last 2.0 sieves are pulverized, are packed and obtain wood chip;
In described supersonic wave cleaning machine, cleaning fluid is the sodium bicarbonate solution of mass percent concentration 0.5%;
Described intermittent drying technique is: microwave 10s, stops 20s, so repeats, cyclic drying.
Described B B-complex per kilogram comprises dehydroretinol 800mg, orotic acid 000mg, Catergen 000mg, riboflavin 1600mg, pantothenic acid 1900mg, vitamin B6 1800mg, vitamin B12 1200mg, inositol 5000mg, vitamin E 600mg, vitamine D3 5500mg, Vitamin K3 4000mg; Its surplus is carrier, and described carrier is the wood chip of above-mentioned preparation.
Commercially available zytase is got by above-mentioned parts by weight, cellulase, 1,4 beta-glucanase, mesophilicα-diastase, protease, antibacterial peptide, and above-mentioned composite trace element and B B-complex, rhodotorula culture with embodiment 2 gained, stirs under 25 DEG C of conditions in mixer, and mixing time is 5 minutes.Stir after casing and preserve in normal temperature or cold house.
Embodiment 4 one kinds of complex enzyme formulations and preparation method thereof
A kind of complex enzyme, is made up of the component of following parts by weight:
Zytase 80 parts, cellulase 30 parts, 1,4 beta-glucanase 40 parts, 100 parts, protease, mesophilicα-diastase 20 parts, B B-complex 3 parts, composite trace element 5 parts, antibacterial peptide 10 parts, rhodotorula culture 30 parts;
Described composite trace element is with embodiment 3;
Described B B-complex is with embodiment 3;
Commercially available zytase is got by above-mentioned parts by weight, cellulase, 1,4 beta-glucanase, mesophilicα-diastase, protease, antibacterial peptide, composite trace element and B B-complex, and the rhodotorula culture of embodiment 2 gained, stir in mixer under 25 DEG C of conditions, mixing time is 5 minutes.Stir after casing and preserve in normal temperature or cold house.
Embodiment 5 one kinds of complex enzyme formulations and preparation method thereof
A kind of complex enzyme, is made up of the component of following parts by weight:
Zytase 120 parts, cellulase 40 parts, 1,4 beta-glucanase 100 parts, 150 parts, protease, mesophilicα-diastase 30 parts, B B-complex 8 parts, composite trace element 20 parts, antibacterial peptide 20 parts, rhodotorula culture 20 parts;
Described composite trace element is with embodiment 3;
Described B B-complex is with embodiment 3;
Commercially available zytase is got by above-mentioned parts by weight, cellulase, 1,4 beta-glucanase, mesophilicα-diastase, protease, antibacterial peptide, composite trace element and B B-complex, and the rhodotorula culture of embodiment 2 gained, stir in mixer under 25 DEG C of conditions, mixing time is 5 minutes.Stir after casing and preserve in normal temperature or cold house.
The result of use test of embodiment 6 embodiment 3 gained complex enzyme in weanling pig
Test method:
Choose 120 weanling pigs, be divided into experimental group and control group by body weight, blood lineage and no sex difference, often organize 3 repetitions, each repetition 20 pigs.Experiment periods is 28 days, and experimental group feed per ton adds complex enzyme 120g described in embodiment 3, and control group does not add complex enzyme, weighs on an empty stomach, calculate feed conversion rate, daily gain, feedstuff-meat ratio, diarrhea rate and death rate morning day at the whole story of experimental period.Experimental result is as shown in the table:
| Daily gain | Feedstuff-meat ratio | Diarrhea rate | Death rate | |
| Control group | 226.31g | 1.49 | 15％ | 5％ |
| Experimental group | 337.16g | 1.28 | 5％ | 1.7％ |
From experimental result, the feed being added with complex enzyme of the present invention can make weanling pig daily gain improve 48.91%, and feedstuff-meat ratio improves 14.1%, and diarrhea rate reduces 10%, and death rate reduces by 3.7%.
Embodiment 7 rhodotorula preserves active impact to complex enzyme formulation
Rhodotorula in embodiment 3, for sample 1, is replaced with commercially available fodder yeast by the complex enzyme obtained with the preparation method described in embodiment 3, and the preservation of comparing two kinds of samples is active.At room temperature preserve above-mentioned sample 6 months, repeat the experiment of embodiment 6 afterwards, characterized the change of complex enzyme by the change of feedstuff-meat ratio, result shows: the residual activity of sample 1 is more than 85%, and the residual activity of sample 2 remains on about 60%.Repeat above-mentioned experiment equally, preserve 12 months samples 1 for 4 DEG C and compare sample 2 enzyme loss alive reduction 30%.
Embodiment 8 rhodotorula is on the impact of additive heat endurance
Rhodotorula in embodiment 3, for sample 1, is replaced with commercially available fodder yeast, compares the heat endurance of two kinds of samples by the complex enzyme obtained with the preparation method described in embodiment 3.At 45 DEG C, preserve above-mentioned sample 24h, repeat the experiment of embodiment 6 afterwards, characterized the change of complex enzyme by the change of feedstuff-meat ratio, result shows: the residual activity of sample 1 is more than 75%, and the residual activity of sample 2 remains on about 40%.
Claims (7)
1. a complex enzyme formulation, it is characterized in that, be made up of the component of following parts by weight: zytase 80-120 part, cellulase 30-40 part, 1,4 beta-glucanase 40-100 part, protease 100-150 part, mesophilicα-diastase 20-30 part, B B-complex 3-8 part, composite trace element 10-20 part, antibacterial peptide 10-20 part, rhodotorula culture 20-30 part;
Described rhodotorula is specially ocean rhodotorula WYN1 (Rhodotorulasp.WYN1), deposit number CCTCCNo:M2014592.
2. a kind of complex enzyme formulation as claimed in claim 1, it is characterized in that, the preparation method of described B B-complex is as follows: described composite trace element per kilogram comprises iodine 1000mg, potassium 180mg, iron 1800mg, selenium 80mg, cobalt 200mg, magnesium 145mg, manganese 1400mg, chromium 20mg, zinc 1100mg, copper 800mg; Its surplus is carrier, and described carrier is wood chip;
The preparation method of described wood chip comprises the steps: to remove foreign material by without mouldy, anosis worm sawmilling end or waste wood, be placed in supersonic wave cleaning machine and clean 10min in 200W, 30KHz, rinse, drain, put into steam still in 0.3-0.4MPa boiling 20-30min, then put microwave dryer in 2000W, 120 DEG C carry out intermittent drying 3min, make it moisture and reach 8-10%, last 2.0 sieves are pulverized, are packed and obtain wood chip;
In described supersonic wave cleaning machine, cleaning fluid is the sodium bicarbonate solution of mass percent concentration 0.5%;
Described intermittent drying technique is: microwave 10s, stops 20s, so repeats, cyclic drying;
Described B B-complex per kilogram comprises dehydroretinol 800mg, orotic acid 000mg, Catergen 000mg, riboflavin 1600mg, pantothenic acid 1900mg, vitamin B6 1800mg, vitamin B12 1200mg, inositol 5000mg, vitamin E 600mg, vitamine D3 5500mg, Vitamin K3 4000mg; Its surplus is carrier, and described carrier is the wood chip of above-mentioned preparation.
3. a kind of complex enzyme formulation as claimed in claim 1, is characterized in that, the preparation method of described rhodotorula culture is as follows:
Seed culture is cultivated by yeast conventional culture methods;
Fermented and cultured: initial pH is 8, and seed culture fluid is inoculated in fermentation medium by inoculum concentration 3-8%, and shaking speed is 50-80r/min, cultivate for 24-28 DEG C and to adjust temperature after 10-15 hour and be 16-20 DEG C and carry out low temperature static gas wave refrigerator, stop stirring, pH is adjusted to 3-5, keeps 2-4 hour; Stairstepping is warmed up to 24-30 DEG C afterwards, and pH is adjusted to 4.6-5.5, and the addition according to 2.5% adds peptone, and the amount according to 2% adds dusty yeast; Shaking speed is 50-100r/min, continues fermentation 10-15h;
After fermentation ends, 10-1000 μm, fermentation liquor aperture coarse filtration, then concentratedly with 10-20 DEG C of loop ultrafiltration removes moisture and obtains solid content 20-40% concentrate, and concentrate obtains rhodotorula culture through vacuum freeze drying, ultramicro grinding.
4. a kind of complex enzyme formulation as claimed in claim 3, it is characterized in that, described fermentation medium mass volume ratio consists of: glucose 1%, enzymolysis corn flour 1-3%, peptone 2.5%, magnesium sulfate 0.02%, potassium dihydrogen phosphate 0.15%, copper content 150-200mg/L, sodium chloride 20%, all the other are water;
The preparation method of described enzymolysis corn flour is: corn flour is placed in material-compound tank, be that 2:1 adds pure water with V:m, adjust ph 3-7, add middle temperature amylase, enzyme is lived as 30u/g corn flour, lives as the protease of 30u/g corn flour with enzyme, and warming while stirring is to 45-55 DEG C of insulation 15-30min, then be slowly warming up to 60-65 DEG C of insulation 15-30min, freeze drying is for subsequent use.
5. prepare a method for claim 1-4 any one complex enzyme formulation, comprise the steps:
According to raw material composition by zytase, cellulase, 1,4 beta-glucanase, protease, mesophilicα-diastase, B B-complex, composite trace element, antibacterial peptide, rhodotorula culture mixes;
Described zytase, cellulase, 1,4 beta-glucanase, protease, mesophilicα-diastase and antibacterial peptide are commercially available prod;
Described composite trace element per kilogram comprises iodine 1000mg, potassium 180mg, iron 1800mg, selenium 80mg, cobalt 200mg, magnesium 145mg, manganese 1400mg, chromium 20mg, zinc 1100mg, copper 800mg; Its surplus is carrier, and described carrier is wood chip;
The preparation method of described wood chip comprises the steps: to remove foreign material by without mouldy, anosis worm sawmilling end or waste wood, be placed in supersonic wave cleaning machine and clean 10min in 200W, 30KHz, rinse, drain, put into steam still in 0.3-0.4MPa boiling 20-30min, then put microwave dryer in 2000W, 120 DEG C carry out intermittent drying 3min, make it moisture and reach 8-10%, last 2.0 sieves are pulverized, are packed and obtain wood chip;
In described supersonic wave cleaning machine, cleaning fluid is the sodium bicarbonate solution of mass percent concentration 0.5%;
Described intermittent drying technique is: microwave 10s, stops 20s, so repeats, cyclic drying;
Described B B-complex per kilogram comprises dehydroretinol 800mg, orotic acid 000mg, Catergen 000mg, riboflavin 1600mg, pantothenic acid 1900mg, vitamin B6 1800mg, vitamin B12 1200mg, inositol 5000mg, vitamin E 600mg, vitamine D3 5500mg, Vitamin K3 4000mg; Its surplus is carrier, and described carrier is the wood chip of above-mentioned preparation;
Described rhodotorula culture preparation method is as follows:
Seed culture is cultivated by yeast conventional culture methods;
Fermented and cultured: initial pH is 8, and seed culture fluid is inoculated in fermentation medium by inoculum concentration 3-8%, and shaking speed is 50-80r/min, cultivate for 24-28 DEG C and to adjust temperature after 10-15 hour and be 16-20 DEG C and carry out low temperature static gas wave refrigerator, stop stirring, pH is adjusted to 3-5, keeps 2-4 hour; Stairstepping is warmed up to 24-30 DEG C afterwards, and pH is adjusted to 4.6-5.5, and the addition according to 2.5% adds peptone, and the amount according to 2% adds dusty yeast; Shaking speed is 50-100r/min, continues fermentation 10-15h;
After fermentation ends, 10-1000 μm, fermentation liquor aperture coarse filtration, then concentratedly with 10-20 DEG C of loop ultrafiltration removes moisture and obtains solid content 20-40% concentrate, and concentrate obtains rhodotorula culture through vacuum freeze drying, ultramicro grinding;
Fermentation medium mass volume ratio consists of: glucose 1%, enzymolysis corn flour 1-3%, peptone 2.5%, magnesium sulfate 0.02%, potassium dihydrogen phosphate 0.15%, copper content 150-200mg/L, sodium chloride 20%, and all the other are water;
The preparation method of described enzymolysis corn flour is: corn flour is placed in material-compound tank, be that 2:1 adds pure water with V:m, adjust ph 3-7, add middle temperature amylase, enzyme is lived as 30u/g corn flour, lives as the protease of 30u/g corn flour with enzyme, and warming while stirring is to 45-55 DEG C of insulation 15-30min, then be slowly warming up to 60-65 DEG C of insulation 15-30min, freeze drying is for subsequent use.
6. a kind of complex enzyme formulation as claimed in claim 1, it is characterized in that: be made up of the component of following parts by weight: zytase 100 parts, cellulase 35 parts, 1,4 beta-glucanase 70 parts, 120 parts, protease, mesophilicα-diastase 25 parts, B B-complex 5 parts, composite trace element 10 parts, antibacterial peptide 15 parts, rhodotorula culture 25 parts.
7. the application of a kind of complex enzyme formulation as claimed in claim 1 in its feeding.
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Application publication date: 20151209 |