CN103734480A

CN103734480A - Feed compound enzyme capable of improving animal appetite and preparation method thereof

Info

Publication number: CN103734480A
Application number: CN201310717037.1A
Authority: CN
Inventors: 李海清; 张锦杰; 朱永明; 易继云; 刘文明
Original assignee: Hunan Hongying Biological Science & Technology Co Ltd
Current assignee: YANGLING TERI'S BIOLOGICAL TECHNOLOGY Co.,Ltd.
Priority date: 2013-12-23
Filing date: 2013-12-23
Publication date: 2014-04-23
Anticipated expiration: 2033-12-23
Also published as: CN103734480B

Abstract

The invention discloses a feed compound enzyme capable of improving animal appetite and a preparation method of the feed compound enzyme, and belongs to the technical field of preparation of an enzyme preparation. The feed compound enzyme is scientifically compounded from fragrant malt enzyme, feeding complex enzyme, acidic xylanase, cellulose, beta-glucanase, amylase, phytase, Chinese herb extracts, a protective agent and an activating agent, wherein the feed complex enzyme contains protease, mannose, alpha-galactase and pectinase, and is strong in heat stability and pH stability. By adopting the prepared feed compound enzyme, safe digestive enzyme is provided for beasts and birds, the digestion burden is effectively relieved, the utilization rate and the growth rate of the raw materials are improved, the environment is protected, an enjoyable natural malt fragrance is brought about for the feed, the livestock and poultry appetites are increased, meanwhile, proper activating agent can fully exert the efficacy of an enzyme preparation under the equal conditions, the adding amount of the enzyme preparation is saved, the guarantee period of the complex enzyme preparation is prolonged by the Chinese herbal medicine extract, the immunity of beasts and birds is improved, and the multi-purpose effect of the enzyme is achieved.

Description

A kind of composite enzyme of feed that improves animal appetite and preparation method thereof

Technical field

The invention belongs to enzyme preparation technical field, specifically a kind of composite enzyme of feed that improves animal appetite and preparation method thereof.

Background technology

In feed, add enzyme preparation and mainly contain following 4 reasons: 1. the ANFs that degraded exists in animal feed.These materials can not be degraded by animal endogenous enzymes, thereby disturb the eubolism of animal, cause animal digestion bad, and production performance declines.2. improve the utilization rate of starch, protein and mineral matter.These materials or surrounded by rich fibrous cell membrane, or with some, can not be had by the version of animal digestion that (for example, in plant feed raw material, a large amount of phosphorus exists with the form of phytate phosphorus.）。3. some specific chemical bond of degrading in raw material.These chemical bonds can not be degraded by the enzyme of animal self, add exogenous enzymes can discharge more nutrients later.4. because young animal autodigestion system is also immature, the not enough interpolation of endogenous enzymes exogenous enzymes can improve feed digestibility, prevents indigestion symptom.Except can improving the utilization rate of daily ration, the enzyme-added difference that can also reduce between feedstuff, the accuracy of raising feed formula, the while can also be improved the regularity of growth of animal, reduces management cost, increases economic efficiency.Use all right protection of the environment of enzyme preparation.Because the utilization rate of feed has improved, corresponding ight soil discharge capacity has declined.In the situation that effect is obvious, the discharge capacity of ight soil can reduce by 20% left and right, the discharge of nitrogen 15% left and right that declines in pig manure, and in chicken manure, the discharge of nitrogen declines 20%.For phytate phosphorus, can significantly reduce the pollution of phosphorus to environment.

The enzyme preparation of applying in feed industry at present mainly contains 4 large classes: be used for respectively degraded cellulose, protein, starch and phytic acid.

Fiber degradation enzyme: for nonruminant, the maximum resistance of digestion is the enzyme that can not produce degradation of fibers, in the daily ration that contains the components such as wheat, barley, oat, fiber is araboxylan and beta-glucan greatly.Water miscible fiber can improve the viscosity of small intestine contents, hinders the absorption of nutrient, thereby reduces the growth performance of animal.Simultaneously this situation also with some because the disease that indigestion causes is relevant.As black toe disease and the piggy of the anorexia of pig, fowl have loose bowels.Due to the impact of the factors such as kind, growth place gentle time condition, the altering a great deal of fiber content in barley and wheat, the nutritive value of the daily ration that causes containing these components is widely different.Fiber degradation enzyme, zytase and beta-glucan can reduce these differences, improve growth performance and the regularity of animal.Can also reduce some dyspeptic disease simultaneously.

PD enzyme: protein is from all feeds raw material in animal diets, and they finally stockpile in lean meat by the amino acid of degraded.In nonruminant daily ration, add protease (DIFFERENT FEED material protein and quality and utilizability) except fully degrading most of storage protein or Storage protein or for the available small-molecular peptides of animal, can also improve feed nutritive value by degraded anti-nutritional factors.The efficiency variance stockpiling is very large.At plant protein source, as existed some ANFs in beancake powder, as several tannins and trypsin ihhibitor, may cause damage to intestinal absorption surface, affect nutraceutical absorption.In addition, the incomplete digestion system of young animal can not well be utilized the protein in vegetable protein (as beancake powder).

Starch degrading enzyme: many nutritionists think corn" golden standard " of feedstuff.Large absolutely number nutritionist thinks that corn does not exist lienteric, digestibility surpasses 95%, but Noy and Sklan research show (1994) in the ideal situation recently, in the daily ration of broiler of 4-12 ages in days, the digestibility of starch seldom surpasses 85%, adds amylase and can make starch obtain more degradeds faster at small intestine.In the weaned piglet phase, due to nutrition, environment and immune variation, body weight can decline.In daily ration, add amylase and some other enzyme, can increase the endogenous digestive ferment secretion of animal, and then improve digesting and assimilating of nutrition, improve food conversion ratio and growth of animal rate.

Phytic acid digestive enzyme: for all animals, phosphorus is all vital for mineralising, immunity, breeding, the growth of bone.Pig and poultry nonruminant can only in utilize in plant feed 30-40% phosphorus, all the other phytate phosphorus of 60-70% are unserviceable.In many cases, in feed diet, must supplement the needs that Phos meets growth of animal.Phosphorus over half in feed is along with ight soil is discharged in environment, contaminated environment.Add the phytase phytic acid of can degrading, discharge the phosphorus in phytic acid molecule.Can produce like this 2 benefits: 1. the addition that has reduced Dietary phosphorus.2. having reduced feces of livestock and poultry pollutes the phosphorus of environment.

Apparent: as four large leading roles of feed enzyme, their mechanism of action and pattern determine or promoted animal feed industry to use for the absorption of enzyme preparation technology to a great extent.The ratio for input and output example of adding enzyme at present in broiler chicken material surpasses 2:1.Comparatively speaking, in pig industry field, the service condition of enzyme preparation, with regard to more complicated, seems uncertain.Intensive degree is low, relates to link many, and the result of use of enzyme preparation is difficult to carry out business calculating.Although had the imagination of using enzyme preparation in the 1950's, until just start to understand strength how to bring into play enzyme in feed industry the eighties in 20th century.Feed grain, as Wheat and barley all contains the unavailable fiber of higher nonruminant.As fruit fiber can be degraded, animal just can utilize nutrients better.In Europe, barley is more cheap, and bird nutritionist and zymologist have dropped into great effort and studied and in containing the daily ration of broiler of barley, add beta-glucan enzyme to reduce the possibility of its negative effect.Its result is proved to be successfully, and has obtained a gold law: barley+beta-glucan enzyme=wheat.Be subject to above-mentioned successful inspiration, wheat is the research object of second.Theory hypothesis is: wheat+zytase=corn.The research of this step has also obtained success.In the mid-90 in 20th century, enzyme has obtained generally approval at feed industry.Can not rant out: 1996, in the broiler chicken material (viscosity cereal is energy source) in Europe 80%, contain fiber degradation enzyme.Strengthen thus and accelerate the application of feed industry to new technology.In the world, about 65% the poultry feed that can produce viscosity cereal that contains has added fiber degradation enzyme.And application percentage in pig feed is much lower, approaches 10%.Its main cause is the complicated structure in market, and market is diversification, even cannot calculate.From geographical distribution, use the area of cellulose degrading enzyme for example mainly to concentrate on viscosity cereal, as the producing region of main energy feed: Europe, Canada, Australia and New Zealand.In addition, in the U.S., South America and the Asian-Pacific area, service condition depends on the rate of exchange between corn and wheat.In this sense, Europe is to use the core of degraded cellulose enzyme to segment market.In order to obtain global approval, feeding enzyme producer must carry out on a large scale marching take that corn---bean pulp type daily ration is main North America and the Asian-Pacific area.Corn---bean pulp type daily ration is always counted as " golden standard ", although many nutritionists think that the mobility of these raw materials is more much bigger than the mobility of original imagination.Now, increasing evidence shows that this gold daily ration also can improve its production performance by enzyme, although this class daily ration problem relevant to crude fibre or viscosity is not serious.Past 10 years was expended a lot in research and development Corn-soybean first generation feed enzyme, and started successful Application in 1996, and initial stage application result is multifarious, but industry is just starting to become, how more and more understand could be handy, adds zymotechnic and obtain maximum economy return.It is estimated, this feed a part enzyme market share is 2,000 ten thousand dollars, and the actual broiler fodder using Corn-soybean daily ration only has 5% for enzyme-added feed.Within 1999/2000 year one, reduce feed enzyme market value that viscosity and crude fibre are daily ration over 100,000,000 dollars.At present, phytase has obtained admitting and applying of the whole world.The market share of phytase is approximately annual 5000 ten thousand dollars, approximately has the pig fowl feed of 8.0% left and right to add phytase in the whole world.Except the reason of economic interests, also having a factor is to have reduced the content of phytate phosphorus in excrement to be conducive to protection of the environment.

In sum, the application of feed enzyme has its wide market space and huge economic worth, but the heat endurance of feed enzyme, security, composite comprehensive and giving full play to of action effect are still enzyme preparation manufacturer and the common major issue of paying close attention to of numerous raisers, prepare safer, more comprehensively, enzyme action effect is better, and the composite enzyme of feed that can improve raising appetite of livestock and poultry is industry technical staff's corporation responsibility and pursuit.

Summary of the invention

Technical problem solved by the invention is with enzyme system comprehensively, natural safety, fragrance maltase and the heat endurance with Fructus Hordei Germinatus fragrance, the complex enzyme for feed that pH stability is strong is basis, and the composite Chinese herbal medicine extract of science, protective agent, activator and other food stage feed enzyme, the composite enzyme of feed making not only provides safety for raising livestock and poultry, comprehensive digestive ferment, alleviate digestion burden, improve raw material availability and growth rate, effective protection of the environment, and can bring joyful native malt fragrance to feed, strengthen the appetite of livestock and poultry, appropriate activator can under equal conditions be given full play to the effect of enzyme preparation simultaneously, make the best use of everything, save enzyme preparation addition, the composite shelf-life that both can extend complex enzyme formulation of science of Chinese herbal medicine extract, can improve again the immunity of raising livestock and poultry, thereby reach the multiplex effect of an enzyme.

In order to achieve the above object, the present invention is by the following technical solutions:

Improve the composite enzyme of feed of animal appetite, by the enzyme preparation of following parts by weight, formed:

Fragrance maltase 30-40 part, complex enzyme for feed 30-50 part, acidic xylanase 20-30 part, cellulase 20-30 part; 1,4 beta-glucanase 15-20 part, amylase 15-20 part, phytase 10-15 part; Chinese herbal medicine extract 10-15 part, protective agent 10-15 part, activator 10-15 part.

Described acidic xylanase, cellulase, 1,4 beta-glucanase, amylase, phytase are food-grade enzyme preparation;

In described complex enzyme for feed zymotic fluid crude enzyme liquid, contain plurality of enzymes vigor, wherein, prolease activity 6500-6700U/ml, mannosan enzyme activity 1500-1700U/ml, α-galactase vigor 1200-1300U/ml, pectinase activity 1000-1200U/ml.Zymotic fluid crude enzyme liquid is carried out to heat stabilization test and the pH stability test of enzyme, result of the test shows: at 30-75 ℃, during pH value 2-7, each enzyme all has higher enzyme activity, reaches more than 80%.

The preparation method of described complex enzyme for feed is as follows: the slant strains of intact aspergillus niger DM-18 is expanded to cultivate through actication of culture and one-level, secondary, three grades of liquid seeds and seeding tank step by step obtain liquid seeds, with 6% inoculum concentration access fermentation tank culture medium, cultivation temperature 28-30 ℃, mixing speed 200-700r/m, ventilation (V/V) 1:1-3, incubation time 10-15h; Then with 1-2 ℃/h rate of temperature fall slow cooling to 10-15 ℃, constant temperature culture 15-20h; Continuation to 2-5 ℃, now, is appended access fermentation tank, constant temperature culture 20-30h by liquid seeds with 4% inoculum concentration with 1-2 ℃/h rate of temperature fall slow cooling; Finally with 1-2 ℃/h heating rate, be slowly warming up to 10-15 ℃, constant temperature culture 15-20h; Continuation is slowly warming up to 30-33 ℃, constant temperature culture 15-20h with 1-2 ℃/h heating rate; Zymotic fluid after filtration, concentrated, allotment, essence filter, the dry solid complex enzyme for feed that to obtain, described allocation process adds concentrated enzyme liquid gross weight 0.5-5% Chinese herbal medicine powder.

Described slant medium consists of: casein 4g, dipotassium hydrogen phosphate 2g, magnesium chloride 0.6g, potassium chloride 0.8g, ferrous sulfate 0.02g, glucose 20g, agar 20g, Chinese herbal medicine powder 5-20g, distilled water l000mL, 5.8,121 ℃ of sterilizing 20min of pH value.

The preparation method of described Chinese herbal medicine powder is as follows:

Take Radix Astragali 20-30 part; Radix Codonopsis 10-18 part; Radix bupleuri 10-15 part; Root of large-flowered skullcap 10-15 part; Respectively said herbal medicine being crushed to particle diameter is below 2 millimeters, then in container, evenly mix and add the water of 3-6 times of weight, control temperature 70 C-90 ℃ and keep 2-4h, add the mixture of 2-3 times of weight ethanol of mixed material and propyl alcohol, control temperature to 60 ℃-78 ℃ of maintenance 3-4h, filter; Filtrate Vacuum Concentration postlyophilization obtains Chinese herbal medicine powder.

The mass ratio of described ethanol and propyl alcohol is 1:1-1.5.

Described one-level, secondary, three grades of seed culture mediums consist of: dipotassium hydrogen phosphate 2g, magnesium chloride 0.6g, potassium chloride 0.8g, ferrous sulfate 0.02g, glucose 20g, Chinese herbal medicine powder 15-25g, trehalose 10-30g, distilled water l000mL, 5.5,121 ℃ of sterilizing 20min of pH value.

Described seed tank culture base consists of: corn flour 50-60g, bean powder 15-25g, wheat bran 10-15g, fish meal 10-15g, calcium chloride 6-10g, ammonium chloride 1-3g, sodium hydrogen phosphate 1-2g, Chinese herbal medicine powder 15-20g, trehalose 10-30g, pure water l000mL, pH value 5-7,121 ℃ of sterilizing 20min.

Described seeding tank zymotic fluid cell concentration is 7.0x10 ⁸-8.0x10 ⁸individual/ml;

Described fermentation medium consists of: corn flour 50-60g, bean powder 15-25g, wheat bran 10-15g, fish meal 10-15g, calcium chloride 6-10g, ammonium chloride 1-3g, sodium hydrogen phosphate 1-2g, Chinese herbal medicine powder 30-50g, trehalose 10-30g, pure water 1000mL, pH value 5-7,121 ℃ of sterilizing 20min.

Described ferment tank process supplemented medium percentage by weight consists of: maltodextrin 20-30%, corn flour 10-20%, bean powder 15-25%, fish meal 1.0-1.5%, calcium chloride 0.6-1.0%, ammonium chloride 0.1-0.3%, sodium hydrogen phosphate 0.1-0.2%, Chinese herbal medicine powder 5-10%, insufficient section pure water is supplied, pH value 5-7,121-123 ℃ of sterilizing 30-40min.

Described fragrance maltase preparation method is as follows:

(1) Fructus Hordei Germinatus moisture regain: use malt weight 3-4%, temperature is 40-50 ℃ of warm water even spraying Fructus Hordei Germinatus grain surface, puts the even 3-5min of mixing in mixer.

(2) cure: pack moisture regain Fructus Hordei Germinatus into drum-type and fry stove, in 10-15min, temperature rises to 170-200 ℃, insulation 10-15min, is then cooled to 100-110 ℃, and insulation 20-30min, finally takes out spreading for cooling, obtains fragrance Fructus Hordei Germinatus;

(3) pulverize: add maltcrusher to pulverize in fragrance Fructus Hordei Germinatus, adjusting roller spacing is 0.5mm, and rotating speed is 250rpm, obtains fragrance malt flour;

(4) ultrasonic, PEF extracts: fragrance malt flour is placed in to pill tank, adds 3-6 water doubly, limit is slowly stirred limit and with probe type ultrasonic extraction apparatus, under current strength 0.6A/W-1.5A/W condition, carried out ultrasonic extraction 10-15min; Then carry out high-pressure pulse electric (PEF) and extract 15-20min, electric-field intensity 20-40kV/cm, burst length 400-600 μ s, pulse frequency 200-300Hz;

(5) filter: in room temperature, with 500-800 order filter cloth, adopt flame filter press to filter extract, operating pressure is 0.14-0.24MPa;

(6) ultrafiltration concentration: adopt the daltonian milipore filter circulation of molecular cut off <100000 concentrated, until concentrate enzyme content is 10-15 times before ultrafiltration;

(7) dry: to the protective agent of the present invention that adds concentrate weight 3-5% in concentrate, then dry with the special-purpose spray dryer of enzyme preparation, EAT 150-160 ℃; temperature of outgoing air 75-85 ℃; drying time 5-15s, product moisture < 5%, obtains fragrance maltase.

The preparation method of described Chinese herbal medicine extract is: respectively Chinese herbal medicine powder being broken to particle diameter is below 2 millimeters, then in container, evenly mix and add the water of 3-6 times of weight, control temperature 70 C-90 ℃ and keep 2-4h, then being cooled to 45-60 ℃, adding mixing enzyme preparation to carry out enzymolysis, is 5.5-6.8 with newborn acid for adjusting pH value, enzymolysis 2-4h, finally add the mixture of mixed material 0.5-3 times of weight ethanol and propyl alcohol, control temperature to 60 ℃-78 ℃ of maintenance 3-4h, filter; It is more than 20% that filtrate decompression is concentrated into solid content, and then freeze drying obtains Chinese herbal medicine extract.

The parts by weight of described Chinese herbal medicine consist of: sea-buckthorn 20-30 part, cassia seed 20-30 part, matrimony vine 10-15 part, Chinese yam 10-15 part, radix glehniae 5-10 part, fruit of negundo 5-10 part, radix polygonati officinalis 3-5 part, seed of Job's tears 3-5 part, fructus hordei germinatus 3-5 part, sweet osmanthus 3-5 part, Radix Astragali 3-5 part;

Mixed enzyme addition is the 5-10% of mixed material gross weight;

The parts by weight of described mixed enzyme consist of: endo-beta-glucanase 10-20 part, outer 1,4 beta-glucanase 10-20 part, beta-glucosidase 10-15 part, zytase 15-20 part, pentosanase 15-20 part, Pullulanase 20-30 part, beta amylase 10-15 part, neutral proteinase 10-15 part, acid protease 10-15 part, superoxide dismutase 5-10 part, glucose oxidase 5-10 part, acid phosphatase 5-10 part;

Described protective agent is comprised of the raw material of following parts by weight: trehalose 20-30 part, NaCl20-30 part, (NH ₄) ₂sO ₄

10-15 part, cysteine 10-15 part.

Described activator is that the inorganic salts by following quality component evenly mix: zinc chloride 30-40 part, calcium chloride 10-20 part, sodium sulphate 10-20 part, magnesium chloride 5-10 part.

The preparation method of the composite enzyme of feed of the present invention:

By described protective agent, Chinese herbal medicine extract, fragrance maltase, complex enzyme for feed difference ultramicro grinding; guarantee that granularity is less than described acidic xylanase and other enzyme preparation; then with acidic xylanase, cellulase, 1,4 beta-glucanase, amylase, phytase; evenly mix; finally add activator, after mixing, pack the composite enzyme of the feed that gets product.

The Aspergillus niger strain of high yield complex enzyme for feed of the present invention is specially aspergillus niger (Aspergillus niger) DM-18, aspergillus niger (Aspergillus niger) HYX0022 that Jinshi City Yang You township, Shi Cong Hunan Province fertile soil, the separation of straw aggregate sample soil obtain is through ultraviolet mutagenesis, the mutagenesis of ultraviolet nitrous acid, ultraviolet nitrosoguanidine complex mutation, then mutant strain step-sizing is eliminated, finally strain excellent is obtained producing the aspergillus niger DM-18 of complex enzyme for feed through fermenting property test screen.This bacterial strain is preserved in Chinese Typical Representative culture collection center on November 3rd, 2013 and (is called for short CCTCC, address is: China. Wuhan. and Wuhan University, postcode: 430072), preserving number is CCTCC NO:M2013541, Classification And Nomenclature is aspergillus niger DM-18Aspergillus nigerDM-18.

The aspergillus niger DM-18 of high yield complex enzyme for feed provided by the invention (CCTCC NO:M2013541) has following microbial characteristic:

1, morphological feature:

Aspergillus niger DM-18, biology morphology is for comprising several parts such as conidium, falx, top capsule, conidial fructification.Conidial head is spherical to Radiation, diameter 150-450 μ m, and conidiophore betides matrix.Falx stem 1000-3000 (length) * 12-20 (diameter) μ m, yellow or yellowish-brown, wall is level and smooth; Spherical or the almost spherical of top capsule, diameter 35-50 μ m, surface can be educated comprehensively; Conidial fructification is double-deck, metulae 15-20 (length) * 3-4.0 (diameter) μ m, and falx 6-8 (length) * 2-4 (diameter) μ m, conidium is spherical or subsphaeroidal, less, diameter 3.5-5.0 μ m, brown, wall is coarse.

2, cultivate and learn feature:

Aspergillus niger DM-18 grows rapidly on wort agar culture medium, 28 ℃ of 4 days diameter 65mm; Quality velvet shape or be slightly with cotton-shaped; Conidium structure is a large amount of, and brown-black has a small amount of diffusate; Bacterium colony reverse side is slightly yellow.

3, physiological and biochemical property:

Aspergillus niger DM-18 can be at potato, corn flour, soluble starch, the upper growth such as molasses, optimum PH 4.6, optimum growth temperature 28-34 ℃, the suitableeest product enzyme temperature 28-30 ℃.

The triage techniques route of aspergillus niger DM-18 is: the preparation → mutagenic treatment → plate isolation → primary dcreening operation of original strain separation, screening and evaluation → starting strain → inclined-plane cultivation → spore suspension → multiple sieve → sieve → expansion experiment (fermenting property mensuration) again again.

Beneficial effect:

1. to take the bacterial strain aspergillus niger DM-18 of high yield complex enzyme for feed be starting strain to the complex enzyme for feed in the present invention, and carry out medium optimization and zymotechnique and improve, adopt the zymotechnique of gradient cooling and gradient increased temperature, appended inoculation and feed supplement in good time simultaneously, especially the zymotechnique of gradient cooling and gradient increased temperature has significantly improved the anti-stress ability of starting strain, causes the enzymatic productivity of bacterial classification to manifest to greatest extent.And the present invention forms and implements full optimization culture medium, the root of large-flowered skullcap, the radix bupleuri with the former effect of anti-heat stress have been added, added to have and adjusted and repaired body function, the immunologic function that strengthens body, the Radix Astragali with micro-Ecological regulation services, Radix Codonopsis etc., further strengthened body function, adaptation of virus and the common collaborative of microorganism under same yeasting, and then strengthened the metabolic function of microorganism, make the present invention produce that complex enzyme for feed vigor is high, tolerable temperature is higher, stability is strong, be suitable for suitability for industrialized production.In described complex enzyme for feed zymotic fluid crude enzyme liquid, contain plurality of enzymes vigor, wherein, prolease activity 6500-6700U/ml, mannosan enzyme activity 1500-1700U/ml, α-galactase vigor 1200-1300U/ml, pectinase activity 1000-1200U/ml.Zymotic fluid crude enzyme liquid is carried out to heat stabilization test and the pH stability test of enzyme, result of the test shows: at 30-75 ℃, during pH value 2-7, each enzyme all has higher enzyme activity, reaches more than 80%.Complex enzyme for feed heat endurance of the present invention and pH stability are strong, are more applicable to feed industry processing technology demand, and the feed enzyme loss alive after processing is low.

2. to take high-quality import Fructus Hordei Germinatus be raw material to the fragrance maltase in feed of the present invention, adopting the methods such as ultrasonic wave, high-voltage pulse electric field technology (pulsed electric field is called for short " PEF ") to extract to greatest extent maltase is enzyme source, it is a kind of natural complex enzyme, its enzyme source kind and single enzyme characteristic 100% are from brewers malt, and hydrolysis result is far superior to the single enzyme and the combination thereof that adopt microorganism fermentation to make; Fragrance maltase wherein not only can provide enzyme source, and most importantly its strong, unique Fructus Hordei Germinatus fragrance can obviously improve the appetite of raising livestock and poultry.

3. it is composite that the protective agent in the composite enzyme of feed of the present invention adopts polysaccharide, inorganic salts and amino acid science, effectively slowed down the moisture regain of fragrance maltase and compounded enzyme preparate; Can strengthen composite enzyme simultaneously resistance toly freeze, heat resistance, keep identical enzyme activity, its heat resisting temperature can improve 20-30 ℃, resistance to cryogenic temperature can reduce 10-15 ℃, effectively prevented the loss of composite enzyme enzyme activity in transportation, preservation and use procedure, extended the shelf-life of composite enzyme, reached same enzyme activity, the like product shelf-life can extend 2-3.

4. the composite enzyme of feed of the present invention adds inorganic salts as activator; created the optimum condition of enzyme catalysis; given full play to the vigor of each enzyme component of composite enzyme; the macromolecular substances such as starch in feed, protein, cellulose, phytic acid have thoroughly effectively been decomposed; greatly alleviated the digestion burden of livestock and poultry animal; improved the growth rate of raw material availability and livestock and poultry, effectively prevented the environmental pollution that feces of livestock and poultry causes, protected feeding environment simultaneously.

5. the Chinese herbal medicine extract that the composite enzyme of feed of the present invention adds both can extend the shelf-life of compounded enzyme preparate, can improve again the immunity of raising livestock and poultry, effectively prevented the generation of livestock and poultry epidemic disease.

6. the common synergy of protective agent and enzyme preparation, activator and enzyme preparation, Chinese herbal medicine extract and enzyme preparation in the composite enzyme of feed of the present invention; enzyme activity and the effect of composite enzyme are brought into play to greatest extent; and the utilization rate of feed and the growth rate of animal have been improved accordingly; strengthened appetite and the resistance against diseases of animal, extended the shelf-life of composite enzyme and protected environment.

The specific embodiment

Below by specific embodiment narration the present invention.Unless stated otherwise, in the present invention, technological means used is method known in those skilled in the art.In addition, embodiment is interpreted as illustrative, but not limits the scope of the invention, and the spirit and scope of the invention are only limited by claims.To those skilled in the art, do not deviating under the prerequisite of essence of the present invention and scope various changes that the material component in these embodiments and consumption are carried out or change and also belong to protection scope of the present invention.

Embodiment 1

35 parts of fragrance maltases, 40 parts of complex enzyme for feed, 25 parts of acidic xylanases, 25 parts of cellulases, 17 parts of 1,4 beta-glucanases, 17 parts of amylase, 13 parts of phytases, 12 parts of Chinese herbal medicine extracts, 13 parts of protective agents, 12 parts of activator.

Described complex enzyme for feed preparation method comprises the steps:

(1) actication of culture

The slant strains of intact aspergillus niger DM-18 is inoculated in to slant medium, cultivates 36h for 28 ℃ and carry out actication of culture, so activate 2 times;

Described slant medium consists of: casein 4g, dipotassium hydrogen phosphate 2g, magnesium chloride 0.6g, potassium chloride 0.8g, ferrous sulfate 0.02g, glucose 20g, agar 20g, Chinese herbal medicine powder 5g, distilled water l000mL, 5.8,121 ℃ of sterilizing 20min of pH value;

Take 20 parts of the Radixs Astragali; 10 parts of Radix Codonopsis; 10 parts of radix bupleuri; 10 parts of the roots of large-flowered skullcap; Respectively said herbal medicine being crushed to particle diameter is below 2 millimeters, then in container, evenly mix and add the water of 3 times of weight, control temperature 70 C and keep 2h, add the mixture of 2 times of weight ethanol of mixed material and propyl alcohol, control temperature to 60 ℃ and keep 3h, filter; Filtrate Vacuum Concentration postlyophilization obtains Chinese herbal medicine powder.

The mass ratio of described ethanol and propyl alcohol is 1:1.

(2) liquid seeds expands cultivation

1. first order seed is cultivated: the Aspergillus usamii slant strains after step (1) activation, with spore under aseptic washing, is accessed in 500 ml shake flasks to 100 milliliters of liquid seed culture medium loading amounts, 28 ℃, 80rpm shaking table cultivation 36h;

2. secondary seed is cultivated: by first order seed, according in 500 milliliters of secondary seed shaking flasks of inoculum concentration access of 10%, condition of culture is identical with first order seed;

3. three grades of seed culture: by secondary seed with in 5000 milliliters of three grades of seed shaking flasks of 8% inoculum concentration access, 1000 milliliters of fluid nutrient medium loading amounts, 28 ℃, 80rpm shaking table are cultivated 36h;

4. seed tank culture: three grades of seeds be take to the first class seed pot that 8% inoculum concentration access total measurement (volume) is 150L, seed tank culture base loading amount 100L, controlling pH value is 5,28 ℃ of cultivation temperature, mixing speed 200rpm, ventilation (V/V) 1:0.8, incubation time 36h, dissolved oxygen 10%;

Described one-level, secondary, three grades of seed culture mediums consist of: dipotassium hydrogen phosphate 2g, magnesium chloride 0.6g, potassium chloride 0.8g, ferrous sulfate 0.02g, glucose 20g, Chinese herbal medicine powder 15g, trehalose 10g, distilled water l000mL, 5.5,121 ℃ of sterilizing 20min of pH value.

Described seed tank culture base consists of: corn flour 50-60g, bean powder 15-25g, wheat bran 10-15g, fish meal 10g, calcium chloride 6g, ammonium chloride 1g, sodium hydrogen phosphate 1g, Chinese herbal medicine powder 15g, trehalose 10g, pure water l000mL, 5,121 ℃ of sterilizing 20min of pH value.

Described seeding tank zymotic fluid cell concentration is 7.0x10 ⁸individual/ml;

(3) ferment tank

Seeding tank liquid seeds in step (2) is accessed to fermentation tank, 28 ℃ of cultivation temperature, mixing speed 200r/m, ventilation (V/V) 1:1, incubation time 10h with 6% inoculum concentration; Then with 1 ℃/h rate of temperature fall slow cooling to 10 ℃, constant temperature culture 15h; Continuation, with 1 ℃/h rate of temperature fall slow cooling to 2 ℃, now, is appended access fermentation tank, constant temperature culture 20h by seeding tank liquid seeds in step (2) with 4% inoculum concentration; Finally with 1 ℃/h heating rate, be slowly warming up to 10 ℃, constant temperature culture 15h; Continuation is slowly warming up to 30 ℃ with 1 ℃/h heating rate, constant temperature culture 15h;

Dissolved oxygen is controlled: by adjusting speed of agitator and ventilation, control dissolved oxygen 15%;

PH controls: by mending ammoniacal liquor or phosphoric acid,diluted, in controlled fermentation process, pH value remains on 5;

Control of additive raw material: when the content of reducing sugar in zymotic fluid is down to 3mg/ml-8mg/ml, start to add supplemented medium, feed supplement amount be take and maintained zymotic fluid content of reducing sugar as 2mg/ml-5mg/ml;

Put tank standard: the old and feeble self-dissolving of 60% thalline, enzyme activity increasess slowly.

Described fermentation medium consists of: corn flour 50g, bean powder 15g, wheat bran 10g, fish meal 10g, calcium chloride 6g, ammonium chloride 1g, sodium hydrogen phosphate 1g, Chinese herbal medicine powder 30g, trehalose 10g, pure water l000mL, 5,121 ℃ of sterilizing 20min of pH value.

Described supplemented medium percentage by weight consists of: maltodextrin 20%, corn flour 10%, bean powder 15%, fish meal 1.0%, calcium chloride 0.6%, ammonium chloride 0.1%, sodium hydrogen phosphate 0.1%, Chinese herbal medicine powder 5%, insufficient section pure water is supplied, 5,121 ℃ of sterilizing 30min of pH value.

(4) zymotic fluid after filtration, concentrated, allotment, essence filter, the dry solid complex enzyme for feed that to obtain.

Described allocation process adds concentrated enzyme liquid gross weight 0.5% Chinese herbal medicine powder.

In the zymotic fluid crude enzyme liquid obtaining through above-mentioned preparation method, contain plurality of enzymes vigor, wherein, prolease activity 6500U/ml, mannosan enzyme activity 1500U/ml, α-galactase vigor 1200U/ml, pectinase activity 1000U/ml.

Described fragrance maltase preparation method is as follows:

(1) Fructus Hordei Germinatus moisture regain: with malt weight 3.5%, temperature is 45 ℃ of warm water even spraying Fructus Hordei Germinatus grain surfaces, puts the even 4min of mixing in mixer.

(2) cure: pack moisture regain Fructus Hordei Germinatus into drum-type and fry stove, in 12min, temperature rises to 185 ℃, insulation 12min, is then cooled to 105 ℃, and insulation 25min, finally takes out spreading for cooling, obtains fragrance Fructus Hordei Germinatus;

(4) ultrasonic, PEF extracts: fragrance malt flour is placed in to pill tank, adds the water of 4 times, limit is slowly stirred limit and with probe type ultrasonic extraction apparatus, under current strength 1.0A/W condition, carried out ultrasonic extraction 12min; Then carry out high-pressure pulse electric (PEF) and extract 17min, electric-field intensity 30kV/cm, burst lengths 500 μ s, pulse frequency 250Hz;

(5) filter: in room temperature, with 650 order filter clothes, adopt flame filter press to filter extract, operating pressure is 0.19MPa;

(6) ultrafiltration concentration: adopt the daltonian milipore filter circulation of molecular cut off <100000 concentrated, until concentrate enzyme content is 12 times before ultrafiltration;

(7) dry: to the protective agent of the present invention that adds concentrate weight 4% in concentrate, then dry with the special-purpose spray dryer of enzyme preparation, 155 ℃ of EATs; 80 ℃ of temperature of outgoing airs; drying time 10s, product moisture < 5%, obtains fragrance maltase.

The preparation method of described Chinese herbal medicine extract is: respectively Chinese herbal medicine powder being broken to particle diameter is below 2 millimeters, then in container, evenly mix and add the water of 5 times of weight, control 80 ℃ of temperature and keep 3h, then being cooled to 53 ℃, adding mixing enzyme preparation to carry out enzymolysis, is 6.2 with newborn acid for adjusting pH value, enzymolysis 3h, finally add the mixture of 2 times of weight ethanol of mixed material and propyl alcohol, control temperature to 69 ℃ and keep 4h, filter; It is more than 20% that filtrate decompression is concentrated into solid content, and then freeze drying obtains Chinese herbal medicine extract.

The parts by weight of described Chinese herbal medicine consist of: 25 parts of sea-buckthorns, 25 parts of cassia seeds, 17 parts of matrimony vines, 13 parts of Chinese yams, 8 parts of radix glehniaes, 7 parts of fruits of negundo, 4 parts of radix polygonati officinalis, 4 parts of the seeds of Job's tears, 4 parts of fructus hordei germinatus, 4 parts of sweet osmanthus, 4 parts of the Radixs Astragali;

Mixed enzyme addition is the 5-10% of mixed material gross weight;

The parts by weight of described mixed enzyme consist of: 15 parts of endo-beta-glucanases, 15 parts of outer 1,4 beta-glucanases, 13 parts of beta-glucosidases, 13 parts of zytases, 13 parts of pentosanases, 25 parts of Pullulanases, 12 parts of beta amylases, 13 parts of neutral proteinases, 13 parts of acid proteases, 7 parts of superoxide dismutases, 7 parts of glucose oxidases, 7 parts of acid phosphatases;

Described protective agent is comprised of the raw material of following parts by weight: 25 parts of trehaloses, NaCl25 part, (NH ₄) ₂sO ₄13 parts, 12 parts of cysteines.

Described activator is that the inorganic salts by following quality component evenly mix: 35 parts of zinc chloride, 15 parts, calcium chloride, 15 parts, sodium sulphate, 7 parts, magnesium chloride.

Embodiment 2

30 parts of fragrance maltases, 30 parts of complex enzyme for feed, 20 parts of acidic xylanases, 20 parts of cellulases, 15 parts of 1,4 beta-glucanases, 15 parts of amylase, 10 parts of phytases, 10 parts of Chinese herbal medicine extracts, 10 parts of protective agents, 10 parts of activator.

Described complex enzyme for feed preparation method comprises the steps:

(1) actication of culture

The slant strains of intact aspergillus niger DM-18 is inoculated in to slant medium, cultivates 48h for 34 ℃ and carry out actication of culture, so activate 4 times;

Described slant medium consists of: casein 4g, dipotassium hydrogen phosphate 2g, magnesium chloride 0.6g, potassium chloride 0.8g, ferrous sulfate 0.02g, glucose 20g, agar 20g, Chinese herbal medicine powder 20g, distilled water l000mL, 5.8,121 ℃ of sterilizing 20min of pH value.

Take 30 parts of the Radixs Astragali; 18 parts of Radix Codonopsis; 15 parts of radix bupleuri; 15 parts of the roots of large-flowered skullcap; Respectively said herbal medicine being crushed to particle diameter is below 2 millimeters, then in container, evenly mix and add the water of 6 times of weight, control 90 ℃ of temperature and keep 4h, add the mixture of 3 times of weight ethanol of mixed material and propyl alcohol, control temperature to 78 ℃ and keep 4h, filter; Filtrate Vacuum Concentration postlyophilization obtains Chinese herbal medicine powder.

The mass ratio of described ethanol and propyl alcohol is 1:1.5.

(2) liquid seeds expands cultivation

1. first order seed is cultivated: the slant strains after step (1) activation, with spore under aseptic washing, is accessed in 500 ml shake flasks to 100 milliliters of liquid seed culture medium loading amounts, 34 ℃, 120rpm shaking table cultivation 48h;

3. three grades of seed culture: by secondary seed with in 5000 milliliters of three grades of seed shaking flasks of 8% inoculum concentration access, 1000 milliliters of fluid nutrient medium loading amounts, 34 ℃, 120rpm shaking table are cultivated 48h;

4. seed tank culture: three grades of seeds be take to the first class seed pot that 8% inoculum concentration access total measurement (volume) is 150L, seed tank culture base loading amount 100L, controlling pH value is 5-7,30 ℃ of cultivation temperature, mixing speed 400rpm, ventilation (V/V) 1:1.2, incubation time 48h, dissolved oxygen 20%;

Described one-level, secondary, three grades of seed culture mediums consist of: dipotassium hydrogen phosphate 2g, magnesium chloride 0.6g, potassium chloride 0.8g, ferrous sulfate 0.02g, glucose 20g, Chinese herbal medicine powder 25g, trehalose 30g, distilled water l000mL, 5.5,121 ℃ of sterilizing 20min of pH value.

Described seed tank culture base consists of: corn flour 60g, bean powder 25g, wheat bran 15g, fish meal 15g, calcium chloride 10g, ammonium chloride 3g, sodium hydrogen phosphate 2g, Chinese herbal medicine powder 20g, trehalose 30g, pure water l000mL, 7,121 ℃ of sterilizing 20min of pH value.

Described seeding tank zymotic fluid cell concentration is 8.0x10 ⁸individual/ml;

(3) ferment tank

Seeding tank liquid seeds in step (2) is accessed to fermentation tank, 30 ℃ of cultivation temperature, mixing speed 700r/m, ventilation (V/V) 1:3, incubation time 15h with 6% inoculum concentration; Then with 2 ℃/h rate of temperature fall slow cooling to 15 ℃, constant temperature culture 20h; Continuation, with 2 ℃/h rate of temperature fall slow cooling to 5 ℃, now, is appended access fermentation tank, constant temperature culture 30h by seeding tank liquid seeds in step (2) with 4% inoculum concentration; Finally with 2 ℃/h heating rate, be slowly warming up to 15 ℃, constant temperature culture 20h; Continuation is slowly warming up to 34 ℃ with 2 ℃/h heating rate, constant temperature culture 20h;

Dissolved oxygen is controlled: by adjusting speed of agitator and ventilation, control dissolved oxygen 30%;

PH controls: by mending ammoniacal liquor or phosphoric acid,diluted, in controlled fermentation process, pH value remains on 6;

Put tank standard: the old and feeble self-dissolving of 80% thalline, enzyme activity increasess slowly.

Described fermentation medium consists of: corn flour 60g, bean powder 25g, wheat bran 15g, fish meal 15g, calcium chloride 10g, ammonium chloride 3g, sodium hydrogen phosphate 2g, Chinese herbal medicine powder 50g, trehalose 30g, pure water l000mL, 7,121 ℃ of sterilizing 20min of pH value.

Described supplemented medium percentage by weight consists of: maltodextrin 30%, corn flour 20%, bean powder 25%, fish meal 1.5%, calcium chloride 1.0%, ammonium chloride 0.3%, sodium hydrogen phosphate 0.2%, Chinese herbal medicine powder 10%, insufficient section pure water is supplied, 7,123 ℃ of sterilizing 40min of pH value.

Described allocation process adds concentrated enzyme liquid gross weight 5% Chinese herbal medicine powder.

In the zymotic fluid zymotic fluid crude enzyme liquid obtaining through above-mentioned preparation method, contain plurality of enzymes vigor, wherein, prolease activity 6600U/ml, mannosan enzyme activity 1600U/ml, α-galactase vigor 1240U/ml, pectinase activity 1080U/ml.

Described fragrance maltase preparation method is as follows:

(1) Fructus Hordei Germinatus moisture regain: with malt weight 3%, temperature is 40 ℃ of warm water even spraying Fructus Hordei Germinatus grain surfaces, puts the even 3min of mixing in mixer.

(2) cure: pack moisture regain Fructus Hordei Germinatus into drum-type and fry stove, in 105min, temperature rises to 170 ℃, insulation 10min, is then cooled to 100 ℃, and insulation 20min, finally takes out spreading for cooling, obtains fragrance Fructus Hordei Germinatus;

(4) ultrasonic, PEF extracts: fragrance malt flour is placed in to pill tank, adds the water of 3 times, limit is slowly stirred limit and with probe type ultrasonic extraction apparatus, under current strength 0.6A/W condition, carried out ultrasonic extraction 10min; Then carry out high-pressure pulse electric (PEF) and extract 15min, electric-field intensity 20kV/cm, burst lengths 400 μ s, pulse frequency 200Hz;

(5) filter: in room temperature, with 500 order filter clothes, adopt flame filter press to filter extract, operating pressure is 0.14MPa;

(7) dry: to the protective agent of the present invention that adds concentrate weight 3% in concentrate, then dry with the special-purpose spray dryer of enzyme preparation, 150 ℃ of EATs; 75 ℃ of temperature of outgoing airs; drying time 5s, product moisture < 5%, obtains fragrance maltase.

The preparation method of described Chinese herbal medicine extract is: respectively Chinese herbal medicine powder being broken to particle diameter is below 2 millimeters, then in container, evenly mix and add the water of 3 times of weight, control temperature 70 C and keep 2h, then being cooled to 45 ℃, adding mixing enzyme preparation to carry out enzymolysis, is 5.5 with newborn acid for adjusting pH value, enzymolysis 2h, finally add the mixture of 0.5 times of weight ethanol of mixed material and propyl alcohol, control temperature to 60 ℃ and keep 3h, filter; It is more than 20% that filtrate decompression is concentrated into solid content, and then freeze drying obtains Chinese herbal medicine extract.

The parts by weight of described Chinese herbal medicine consist of: 20 parts of sea-buckthorns, 20 parts of cassia seeds, 10 parts of matrimony vines, 10 parts of Chinese yams, 5 parts of radix glehniaes, 5 parts of fruits of negundo, 3 parts of radix polygonati officinalis, 3 parts of the seeds of Job's tears, 3 parts of fructus hordei germinatus, 3 parts of sweet osmanthus, 3 parts of the Radixs Astragali;

Mixed enzyme addition is 5% of mixed material gross weight;

The parts by weight of described mixed enzyme consist of: 10 parts of endo-beta-glucanases, 10 parts of outer 1,4 beta-glucanases, 10 parts of beta-glucosidases, 15 parts of zytases, 15 parts of pentosanases, 20 parts of Pullulanases, 10 parts of beta amylases, 10 parts of neutral proteinases, 10 parts of acid proteases, 5 parts of superoxide dismutases, 5 parts of glucose oxidases, 5 parts of acid phosphatases;

Described protective agent is comprised of the raw material of following parts by weight: 20 parts of trehaloses, NaCl20 part, (NH ₄) ₂sO ₄10 parts, 10 parts of cysteines.

Described activator is that the inorganic salts by following quality component evenly mix: 30 parts of zinc chloride, 10 parts, calcium chloride, 10 parts, sodium sulphate, 5 parts, magnesium chloride.

The preparation method of the composite enzyme of feed of the present invention as above.

Embodiment 3

40 parts of fragrance maltases, 50 parts of complex enzyme for feed, 30 parts of acidic xylanases, 30 parts of cellulases, 20 parts of 1,4 beta-glucanases, 0 part of amylase 2,15 parts of phytases, 15 parts of Chinese herbal medicine extracts, 15 parts of protective agents, 15 parts of activator.

Described complex enzyme for feed preparation method comprises the steps:

(1) actication of culture

The slant strains of intact aspergillus niger DM-18 is inoculated in to slant medium, cultivates 42h for 30 ℃ and carry out actication of culture, so activate 3 times;

Described slant medium consists of: casein 4g, dipotassium hydrogen phosphate 2g, magnesium chloride 0.6g, potassium chloride 0.8g, ferrous sulfate 0.02g, glucose 20g, agar 20g, Chinese herbal medicine powder 12g, distilled water l000mL, 5.8,121 ℃ of sterilizing 20min of pH value.

Take 25 parts of the Radixs Astragali; 15 parts of Radix Codonopsis; 12 parts of radix bupleuri; 12 parts of the roots of large-flowered skullcap; Respectively said herbal medicine being crushed to particle diameter is below 2 millimeters, then in container, evenly mix and add the water of 5 times of weight, control 80 ℃ of temperature and keep 3h, add the mixture of 3 times of weight ethanol of mixed material and propyl alcohol, control temperature to 70 ℃ and keep 4h, filter; Filtrate Vacuum Concentration postlyophilization obtains Chinese herbal medicine powder.

The mass ratio of described ethanol and propyl alcohol is 1:1.2.

(2) liquid seeds expands cultivation

1. first order seed is cultivated: the slant strains after step (1) activation, with spore under aseptic washing, is accessed in 500 ml shake flasks to 100 milliliters of liquid seed culture medium loading amounts, 30 ℃, 100rpm shaking table cultivation 42h;

3. three grades of seed culture: by secondary seed with in 5000 milliliters of three grades of seed shaking flasks of 8% inoculum concentration access, 1000 milliliters of fluid nutrient medium loading amounts, 30 ℃, 100rpm shaking table are cultivated 42h;

4. seed tank culture: three grades of seeds be take to the first class seed pot that 8% inoculum concentration access total measurement (volume) is 150L, seed tank culture base loading amount 100L, controlling pH value is 6,30 ℃ of cultivation temperature, mixing speed 300rpm, ventilation (V/V) 1:1, incubation time 42h, dissolved oxygen 20%;

Described one-level, secondary, three grades of seed culture mediums consist of: dipotassium hydrogen phosphate 2g, magnesium chloride 0.6g, potassium chloride 0.8g, ferrous sulfate 0.02g, glucose 20g, Chinese herbal medicine powder 20g, trehalose 20g, distilled water l000mL, 5.5,121 ℃ of sterilizing 20min of pH value.

Described seed tank culture base consists of: corn flour 55g, bean powder 20g, wheat bran 12g, fish meal 12g, calcium chloride 8g, ammonium chloride 2g, sodium hydrogen phosphate 2g, Chinese herbal medicine powder 18g, trehalose 20g, pure water l000mL, 6,121 ℃ of sterilizing 20min of pH value.

Described seeding tank zymotic fluid cell concentration is 7.5x10 ⁸individual/ml;

(3) ferment tank

Seeding tank liquid seeds in step (2) is accessed to fermentation tank, 30 ℃ of cultivation temperature, mixing speed 350r/m, ventilation (V/V) 1:2, incubation time 12h with 6% inoculum concentration; Then with 2 ℃/h rate of temperature fall slow cooling to 12 ℃, constant temperature culture 18h; Continuation, with 2 ℃/h rate of temperature fall slow cooling to 4 ℃, now, is appended access fermentation tank, constant temperature culture 25h by seeding tank liquid seeds in step (2) with 4% inoculum concentration; Finally with 2 ℃/h heating rate, be slowly warming up to 12 ℃, constant temperature culture 18h; Continuation is slowly warming up to 30 ℃ with 2 ℃/h heating rate, constant temperature culture 18h;

Dissolved oxygen is controlled: by adjusting speed of agitator and ventilation, control dissolved oxygen 20%;

PH controls: by mending ammoniacal liquor or phosphoric acid,diluted, in controlled fermentation process, pH value remains on 5.2;

Put tank standard: the old and feeble self-dissolving of 70% thalline, enzyme activity increasess slowly.

Described fermentation medium consists of: corn flour 55g, bean powder 20g, wheat bran 12g, fish meal 12g, calcium chloride 8g, ammonium chloride 2g, sodium hydrogen phosphate 2g, Chinese herbal medicine powder 40g, trehalose 20g, pure water l000mL, 6,121 ℃ of sterilizing 20min of pH value.

Described supplemented medium percentage by weight consists of: maltodextrin 25%, corn flour 15%, bean powder 18%, fish meal 1.2%, calcium chloride 0.8%, ammonium chloride 0.2%, sodium hydrogen phosphate 0.2%, Chinese herbal medicine powder 8%, insufficient section pure water is supplied, 6,123 ℃ of sterilizing 40min of pH value.

Described allocation process adds concentrated enzyme liquid gross weight 3% Chinese herbal medicine powder.

In the zymotic fluid zymotic fluid crude enzyme liquid obtaining through above-mentioned preparation method, contain plurality of enzymes vigor, wherein, prolease activity 6700U/ml, mannosan enzyme activity 1700U/ml, α-galactase vigor 1300U/ml, pectinase activity 1200U/ml.

Described fragrance maltase preparation method is as follows:

(1) Fructus Hordei Germinatus moisture regain: with malt weight 4%, temperature is 50 ℃ of warm water even spraying Fructus Hordei Germinatus grain surfaces, puts the even 5min of mixing in mixer.

(2) cure: pack moisture regain Fructus Hordei Germinatus into drum-type and fry stove, in 15min, temperature rises to 200 ℃, insulation 15min, is then cooled to 110 ℃, and insulation 30min, finally takes out spreading for cooling, obtains fragrance Fructus Hordei Germinatus;

(4) ultrasonic, PEF extracts: fragrance malt flour is placed in to pill tank, adds the water of 6 times, limit is slowly stirred limit and with probe type ultrasonic extraction apparatus, under current strength 1.5A/W condition, carried out ultrasonic extraction 15min; Then carry out high-pressure pulse electric (PEF) and extract 20min, electric-field intensity 40kV/cm, burst lengths 600 μ s, pulse frequency 300Hz;

(5) filter: in room temperature, with 800 order filter clothes, adopt flame filter press to filter extract, operating pressure is 0.14-0.24MPa;

(7) dry: to the protective agent of the present invention that adds concentrate weight 5% in concentrate, then dry with the special-purpose spray dryer of enzyme preparation, 160 ℃ of EATs; 85 ℃ of temperature of outgoing airs; drying time 15s, product moisture < 5%, obtains fragrance maltase.

The preparation method of described Chinese herbal medicine extract is: respectively Chinese herbal medicine powder being broken to particle diameter is below 2 millimeters, then in container, evenly mix and add the water of 6 times of weight, control 90 ℃ of temperature and keep 4h, then being cooled to 60 ℃, adding mixing enzyme preparation to carry out enzymolysis, is 6.8 with newborn acid for adjusting pH value, enzymolysis 4h, finally add the mixture of 3 times of weight ethanol of mixed material and propyl alcohol, control temperature to 78 ℃ and keep 4h, filter; It is more than 20% that filtrate decompression is concentrated into solid content, and then freeze drying obtains Chinese herbal medicine extract.

The parts by weight of described Chinese herbal medicine consist of: 30 parts of sea-buckthorns, 30 parts of cassia seeds, 15 parts of matrimony vines, 15 parts of Chinese yams, 10 parts of radix glehniaes, 10 parts of fruits of negundo, 5 parts of radix polygonati officinalis, 5 parts of the seeds of Job's tears, 5 parts of fructus hordei germinatus, 5 parts of sweet osmanthus, 5 parts of the Radixs Astragali;

Mixed enzyme addition is 10% of mixed material gross weight;

The parts by weight of described mixed enzyme consist of: 20 parts of endo-beta-glucanases, 20 parts of outer 1,4 beta-glucanases, 15 parts of beta-glucosidases, 20 parts of zytases, 20 parts of pentosanases, 30 parts of Pullulanases, 15 parts of beta amylases, 15 parts of neutral proteinases, 15 parts of acid proteases, 10 parts of superoxide dismutases, 10 parts of glucose oxidases, 10 parts of acid phosphatases;

Described protective agent is comprised of the raw material of following parts by weight: 30 parts of trehaloses, NaCl30 part, (NH ₄) ₂sO ₄15 parts, 15 parts of cysteines.

Described activator is that the inorganic salts by following quality component evenly mix: 40 parts of zinc chloride, 20 parts, calcium chloride, 20 parts, sodium sulphate, 10 parts, magnesium chloride.

The thermal stability analysis of embodiment 4 complex enzyme for feed

Heat endurance to complex enzyme for feed is analyzed, and embodiment 3 crude enzyme liquids are placed in respectively at 30 ℃, 40 ℃, 45 ℃, 50 ℃, 55 ℃, 60 ℃, 65 ℃, 70 ℃, 75 ℃, and in 10 minutes sampling and measuring complex enzyme for feed, the enzyme of each constitutive enzyme is lived.At 30 ℃, 40 ℃, 45 ℃, 50 ℃, 60 minutes each constitutive enzyme enzymes are lived and are not declined.At 55 ℃, 60 ℃ and 65 ℃, each constitutive enzyme enzyme work in 30 minutes drops to constitutive enzyme each constitutive enzyme enzyme work in 95%, 60 minute alive and drops to 85%.At 70 ℃, each constitutive enzyme enzyme work in 30 minutes drops to constitutive enzyme each constitutive enzyme enzyme work in 90%, 60 minute alive and drops to 85%.At 75 ℃, each constitutive enzyme enzyme work in 85%, 60 minute that each constitutive enzyme enzyme work in 30 minutes drops to constitutive enzyme work drops to 80% of constitutive enzyme work, and compared with prior art, similarity condition is issued to same enzyme tolerable temperature alive and has on average improved 5-10 ℃.

The pH stability analysis of embodiment 5 complex enzyme for feed

PH stability to complex enzyme for feed enzyme is analyzed, and embodiment 3 crude enzyme liquids are placed in respectively to pH value 2.0,2.5,3.5,4.5,5.5,6.0,6.5,7.0 times, and in 10 minutes sampling and measuring complex enzyme for feed, the enzyme of each constitutive enzyme is lived.Crude enzyme liquid pH value 5.5,6.0,6.5,7.0 times, 60 minutes each constitutive enzyme enzymes are lived and are not declined.PH value 4.5,3.5 times, each constitutive enzyme enzyme work in 95%, 60 minute that each constitutive enzyme enzyme work in 30 minutes drops to constitutive enzyme work drops to 85% of constitutive enzyme work.PH value 2.5 times, each constitutive enzyme enzyme work in 30 minutes drops to that constitutive enzyme lives within 90%, 60 minute, drop to that constitutive enzyme lives 85%.PH value 2.0 times, each constitutive enzyme enzyme work in 30 minutes drops to that constitutive enzyme lives within 85%, 60 minute, drop to that constitutive enzyme lives 80%, compared with prior art, it is more wide in range that similarity condition is issued to the same enzyme resistance to pH value of living.

Claims

1. the composite enzyme of feed that can improve animal appetite; enzyme preparation by following parts by weight forms: fragrance maltase 30-40 part; complex enzyme for feed 30-50 part, acidic xylanase 20-30 part, cellulase 20-30 part; 1,4 beta-glucanase 15-20 part; amylase 15-20 part, phytase 10-15 part, Chinese herbal medicine extract 10-15 part; protective agent 10-15 part, activator 10-15 part.

2. can improve according to claim 1 the composite enzyme of feed of animal appetite, it is characterized in that, described fragrance maltase preparation method is as follows: use malt weight 3-4%, temperature is 40-50 ℃ of warm water even spraying Fructus Hordei Germinatus grain surface, puts the even 3-5min of mixing in mixer; Pack moisture regain Fructus Hordei Germinatus into drum-type and fry stove, in 10-15min, temperature rises to 170-200 ℃, insulation 10-15min, then be cooled to 100-110 ℃, insulation 20-30min, finally takes out spreading for cooling, add maltcrusher to pulverize, adjusting roller spacing is 0.5mm, and rotating speed is 250rpm, obtains fragrance malt flour; Fragrance malt flour is placed in to pill tank, adds 3-6 water doubly, limit is slowly stirred limit and with probe type ultrasonic extraction apparatus, under current strength 0.6A/W-1.5A/W condition, is carried out ultrasonic extraction 10-15min; Then carry out high-pressure pulse electric and extract 15-20min, electric-field intensity 20-40kV/cm, burst length 400-600 μ s, pulse frequency 200-300Hz; First in room temperature, with 500-800 order filter cloth, adopt flame filter press to filter extract, operating pressure is 0.14-0.24MPa, then adopt the daltonian milipore filter circulation of molecular cut off <100000 concentrated, until concentrate enzyme content is 10-15 times before ultrafiltration; To the protective agent that adds concentrate weight 3-5% in concentrate, then dry with the special-purpose spray dryer of enzyme preparation, EAT 150-160 ℃, temperature of outgoing air 75-85 ℃, drying time 5-15s, product moisture < 5%, obtains fragrance maltase.

3. can improve according to claim 1 the composite enzyme of feed of animal appetite, it is characterized in that, the preparation method of described complex enzyme for feed is as follows: the slant strains of intact aspergillus niger DM-18 is expanded to cultivate through actication of culture and liquid seeds step by step obtain liquid seeds, with 6% inoculum concentration access fermentation tank, cultivation temperature 28-30 ℃, mixing speed 200-700r/m, ventilation (V/V) 1:1-3, incubation time 10-15h; Then with 1-2 ℃/h rate of temperature fall slow cooling to 10-15 ℃, constant temperature culture 15-20h; Continuation to 2-5 ℃, now, is appended access fermentation tank, constant temperature culture 20-30h by liquid seeds with 4% inoculum concentration with 1-2 ℃/h rate of temperature fall slow cooling; Finally with 1-2 ℃/h heating rate, be slowly warming up to 10-15 ℃, constant temperature culture 15-20h; Continuation is slowly warming up to 30-33 ℃, constant temperature culture 15-20h with 1-2 ℃/h heating rate; Zymotic fluid after filtration, concentrated, allotment, essence filter, the dry solid complex enzyme for feed that to obtain, described allocation process adds concentrated enzyme liquid gross weight 0.5-5% Chinese herbal medicine powder.

4. the composite enzyme of feed that can improve according to claim 1 animal appetite, is characterized in that, described Chinese herbal medicine extract is prepared by the Chinese herbal medicine of following parts by weight: sea-buckthorn 20-30 part, cassia seed 20-30 part, matrimony vine 10-15 part, Chinese yam 10-15 part, radix glehniae 5-10 part, fruit of negundo 5-10 part, radix polygonati officinalis 3-5 part, seed of Job's tears 3-5 part, fructus hordei germinatus 3-5 part, sweet osmanthus 3-5 part, Radix Astragali 3-5 part.

5. can improve according to claim 4 the composite enzyme of feed of animal appetite, it is characterized in that, the preparation method of described Chinese herbal medicine extract is: respectively Chinese herbal medicine powder being broken to particle diameter is below 2 millimeters, then in container, evenly mix and add the water of 3-6 times of weight, control temperature 70 C～90 ℃ and keep 2～4h, then be cooled to 45-60 ℃, add mixing enzyme preparation to carry out enzymolysis, with newborn acid for adjusting pH value, be 5.5-6.8, enzymolysis 2-4h, finally add the mixture of mixed material 0.5-3 times of weight ethanol and propyl alcohol, control temperature to 60 ℃～78 ℃ and keep 3～4h, filter, it is more than 20% that filtrate decompression is concentrated into solid content, and then freeze drying obtains Chinese herbal medicine extract,

Described mixed enzyme addition is the 5-10% of mixed material gross weight;

The parts by weight of described mixed enzyme consist of: endo-beta-glucanase 10-20 part, outer 1,4 beta-glucanase 10-20 part, beta-glucosidase 10-15 part, zytase 15-20 part, pentosanase 15-20 part, Pullulanase 20-30 part, beta amylase 10-15 part, neutral proteinase 10-15 part, acid protease 10-15 part, superoxide dismutase 5-10 part, glucose oxidase 5-10 part, acid phosphatase 5-10 part.

6. the composite enzyme of feed that can improve according to claim 1 animal appetite, is characterized in that, described protective agent is comprised of the raw material of following parts by weight: trehalose 20-30 part, NaCl20-30 part, (NH ₄) ₂sO ₄10-15 part, cysteine 10-15 part.

7. can improve according to claim 1 the composite enzyme of feed of animal appetite, it is characterized in that, described activator is that the inorganic salts by following quality component evenly mix: zinc chloride 30-40 part, calcium chloride 10-20 part, sodium sulphate 10-20 part, magnesium chloride 5-10 part.

8. the preparation method that can improve the composite enzyme of feed of animal appetite; it is characterized in that; first by described protective agent, Chinese herbal medicine extract, fragrance maltase, complex enzyme for feed difference ultramicro grinding; guarantee that granularity is less than described acidic xylanase and other enzyme preparation; then with acidic xylanase, cellulase, 1,4 beta-glucanase, amylase, phytase; evenly mix, finally add activator, after mixing, pack the composite enzyme of the feed that gets product.

9. can improve according to claim 8 the preparation method of the composite enzyme of feed of animal appetite, it is characterized in that, described fragrance maltase preparation method is as follows: use malt weight 3-4%, temperature is 40-50 ℃ of warm water even spraying Fructus Hordei Germinatus grain surface, puts the even 3-5min of mixing in mixer; Pack moisture regain Fructus Hordei Germinatus into drum-type and fry stove, in 10-15min, temperature rises to 170-200 ℃, insulation 10-15min, then be cooled to 100-110 ℃, insulation 20-30min, finally takes out spreading for cooling, add maltcrusher to pulverize, adjusting roller spacing is 0.5mm, and rotating speed is 250rpm, obtains fragrance malt flour; Fragrance malt flour is placed in to pill tank, adds 3-6 water doubly, limit is slowly stirred limit and with probe type ultrasonic extraction apparatus, under current strength 0.6A/W-1.5A/W condition, is carried out ultrasonic extraction 10-15min; Then carry out high-pressure pulse electric and extract 15-20min, electric-field intensity 20-40kV/cm, burst length 400-600 μ s, pulse frequency 200-300Hz; First in room temperature, with 500-800 order filter cloth, adopt flame filter press to filter extract, operating pressure is 0.14-0.24MPa, then adopt the daltonian milipore filter circulation of molecular cut off <100000 concentrated, until concentrate enzyme content is 10-15 times before ultrafiltration; To the protective agent that adds concentrate weight 3-5% in concentrate, then dry with the special-purpose spray dryer of enzyme preparation, EAT 150-160 ℃, temperature of outgoing air 75-85 ℃, drying time 5-15s, product moisture < 5%, obtains fragrance maltase.

10. the preparation method that can improve according to claim 8 the composite enzyme of feed of animal appetite, is characterized in that, the composite enzyme of described feed, enzyme preparation by following parts by weight forms: 35 parts of fragrance maltases, 40 parts of complex enzyme for feed, 25 parts of acidic xylanases, 25 parts of cellulases, 17 parts of 1,4 beta-glucanases, 17 parts of amylase, 13 parts of phytases, 12 parts of Chinese herbal medicine extracts, 13 parts of protective agents, 12 parts of activator;

Described complex enzyme for feed preparation method comprises the steps:

(1) actication of culture

Take 20 parts of the Radixs Astragali; 10 parts of Radix Codonopsis; 10 parts of radix bupleuri; 10 parts of the roots of large-flowered skullcap; Respectively said herbal medicine being crushed to particle diameter is below 2 millimeters, then in container, evenly mix and add the water of 3 times of weight, control temperature 70 C and keep 2h, add the mixture of 2 times of weight ethanol of mixed material and propyl alcohol, control temperature to 60 ℃ and keep 3h, filter; Filtrate Vacuum Concentration postlyophilization obtains Chinese herbal medicine powder;

The mass ratio of described ethanol and propyl alcohol is 1:1;

(2) liquid seeds expands cultivation

Described one-level, secondary, three grades of seed culture mediums consist of: dipotassium hydrogen phosphate 2g, magnesium chloride 0.6g, potassium chloride 0.8g, ferrous sulfate 0.02g, glucose 20g, Chinese herbal medicine powder 15g, trehalose 10g, distilled water l000mL, 5.5,121 ℃ of sterilizing 20min of pH value;

Described seed tank culture base consists of: corn flour 50-60g, bean powder 15-25g, wheat bran 10-15g, fish meal 10g, calcium chloride 6g, ammonium chloride 1g, sodium hydrogen phosphate 1g, Chinese herbal medicine powder 15g, trehalose 10g, pure water l000mL, 5,121 ℃ of sterilizing 20min of pH value;

(3) ferment tank

Put tank standard: the old and feeble self-dissolving of 60% thalline, enzyme activity increasess slowly;

Described fermentation medium consists of: corn flour 50g, bean powder 15g, wheat bran 10g, fish meal 10g, calcium chloride 6g, ammonium chloride 1g, sodium hydrogen phosphate 1g, Chinese herbal medicine powder 30g, trehalose 10g, pure water l000mL, 5,121 ℃ of sterilizing 20min of pH value;

Described supplemented medium percentage by weight consists of: maltodextrin 20%, corn flour 10%, bean powder 15%, fish meal 1.0%, calcium chloride 0.6%, ammonium chloride 0.1%, sodium hydrogen phosphate 0.1%, Chinese herbal medicine powder 5%, insufficient section pure water is supplied, 5,121 ℃ of sterilizing 30min of pH value;

(4) zymotic fluid after filtration, concentrated, allotment, essence filter, the dry solid complex enzyme for feed that to obtain;

Described allocation process adds concentrated enzyme liquid gross weight 0.5% Chinese herbal medicine powder;

In the zymotic fluid crude enzyme liquid obtaining through above-mentioned preparation method, contain plurality of enzymes vigor, wherein, prolease activity 6500U/ml, mannosan enzyme activity 1500U/ml, α-galactase vigor 1200U/ml, pectinase activity 1000U/ml;

Described fragrance maltase preparation method is as follows:

(1) Fructus Hordei Germinatus moisture regain: with malt weight 3.5%, temperature is 45 ℃ of warm water even spraying Fructus Hordei Germinatus grain surfaces, puts the even 4min of mixing in mixer;

(7) dry: to the protective agent of the present invention that adds concentrate weight 4% in concentrate, then dry with the special-purpose spray dryer of enzyme preparation, 155 ℃ of EATs, 80 ℃ of temperature of outgoing airs, drying time 10s, product moisture < 5%, obtains fragrance maltase;

The preparation method of described Chinese herbal medicine extract is: respectively Chinese herbal medicine powder being broken to particle diameter is below 2 millimeters, then in container, evenly mix and add the water of 5 times of weight, control 80 ℃ of temperature and keep 3h, then being cooled to 53 ℃, adding mixing enzyme preparation to carry out enzymolysis, is 6.2 with newborn acid for adjusting pH value, enzymolysis 3h, finally add the mixture of 2 times of weight ethanol of mixed material and propyl alcohol, control temperature to 69 ℃ and keep 4h, filter; It is more than 20% that filtrate decompression is concentrated into solid content, and then freeze drying obtains Chinese herbal medicine extract;

Mixed enzyme addition is the 5-10% of mixed material gross weight;

Described protective agent is comprised of the raw material of following parts by weight: 25 parts of trehaloses, NaCl25 part, (NH ₄) ₂sO ₄13 parts, 12 parts of cysteines;