CN103766611A

CN103766611A - Neutral protease-containing coarse grain daily ration enzyme and preparation method thereof

Info

Publication number: CN103766611A
Application number: CN201310733042.1A
Authority: CN
Inventors: 李洪兵; 张锦杰; 李海清; 朱永明; 刘文明
Original assignee: Hunan Hongying Biological Science & Technology Co Ltd
Current assignee: Hunan Hongying Biological Science & Technology Co Ltd
Priority date: 2013-12-27
Filing date: 2013-12-27
Publication date: 2014-05-07
Anticipated expiration: 2033-12-27
Also published as: CN103766611B

Abstract

The invention discloses a neutral protease-containing coarse grain daily ration enzyme and a preparation method thereof, belonging to the technical field of preparation of enzyme preparation. The coarse grain daily ration enzyme is prepared by scientifically compounding bacillus subtilis culture, acidic xylanase, beta-glucanase, acidic protease, pectinase, cellulose, amylase, Chinese herb extracts, a protective agent and an activating agent, wherein the bacillus subtilis culture has the functions of improving animal organism immunity, promoting the development of immune organs and the maturity of animal intestinal tract structure and function and improving the animal daily gain and the feed conversion rate. After the neutral protease-containing coarse grain daily ration enzyme is adopted, safe digestive enzyme is provided for livestock and poultry, the digestion burden is effectively lightened, the utilization rate and the growth rate of the raw materials are improved, and the environment is protected; furthermore, the right amount of activating agent gives full play to the effect of the enzyme preparation under the same condition, so that the add amount of the enzyme preparation is reduced; the Chinese herb extracts are scientifically compounded, so that the shelf life of the compound enzyme preparation can be prolonged, and the immunity of the raised livestock and poultry can be improved.

Description

A kind of coarse cereals daily ration enzyme containing neutral proteinase and preparation method thereof

Technical field

The invention belongs to enzyme preparation technical field, specifically a kind of coarse cereals daily ration enzyme containing neutral proteinase 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 can not be had by the version of animal digestion with some 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, causes the nutritive value of the daily ration that contains these components 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 exceedes 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 exceedes 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.The nonruminant such as pig and poultry 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, Phos must be supplemented and meet the needs of 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 exceedes 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 that uses 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 the daily ration of broiler that contains 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 mainly to concentrate on the producing region of viscosity cereal as main energy feed, for example: 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 corn---and 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 enzymes, 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 obtains maximum economy return.It is estimated, this feed a part enzyme market share is 2,000 ten thousand dollars, and actual only have 5% for enzyme-added feed at the broiler fodder that uses Corn-soybean daily ration.Within 1999/2000 year one, reduce the feed enzymes market value that viscosity and crude fibre are daily ration and exceed 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 animal and fowl fodder 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 coarse cereals daily ration enzyme has its wide market space and huge economic worth, but the heat endurance of coarse cereals daily ration 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, the better coarse cereals daily ration of enzyme action effect enzyme is industry technical staff's corporation responsibility and pursuit.

Summary of the invention

Technical problem solved by the invention is to contain high enzymatic activity, action condition is wide in range, the bacillus subtilis culture of the neutral proteinase that stability is strong is basis, and the composite Chinese herbal medicine extract of science, protective agent, activator and other food-grade feed enzymes, the coarse cereals daily ration enzyme containing neutral proteinase making, not only provide safety for raising livestock and poultry, comprehensively digestive ferment, alleviate digestion burden, improve raw material availability and growth rate, effectively protection of the environment, appropriate activator can under equal conditions be given full play to the effect of enzyme preparation simultaneously, make the best use of everything, reduce 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:

Containing a coarse cereals daily ration enzyme for neutral proteinase, formed by the enzyme preparation of following parts by weight:

Bacillus subtilis culture 20-30 part, acidic xylanase 30-50 part, 1,4 beta-glucanase 20-30 part, acid protease 20-30 part; pectase 10-15 part, cellulase 10-15 part, amylase 10-15 part; Chinese herbal medicine extract 10-15 part, protective agent 10-15 part, activator 10-15 part.

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

Described bacillus subtilis culture is prepared through liquid deep layer fermenting by the bacterial strain bacillus subtilis 1398-2-12 that produces heat-flash stability neutral proteinase, and its composition mainly comprises neutral proteinase and bacillus subtilis thalline;

The preparation method of described bacillus subtilis culture is as follows:

Bacillus subtilis 1398-2-12 obtains liquid seeds through slant strains activation and expansion cultivation step by step (comprising one, two, three seed culture and first class seed pot cultivation); Liquid seeds is accessed to fermentation tank, cultivation temperature 30-36 ℃, mixing speed 200-700rpm, ventilation (V/V) 1:1-3, incubation time 10-15h with 6% inoculum concentration; 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 slowly be warming up to 10-15 ℃, constant temperature culture 15-20h with 1-2 ℃/h heating rate; Continue to be slowly warming up to 30-36 ℃, constant temperature culture 15-20h with 1-2 ℃/h heating rate; Zymotic fluid via hole diameter 10-1000 μ m filter board coarse filtration, then in the concentrated concentrate of removing moisture and obtain solid content 20-40% of 10-20 ℃ of loop ultrafiltration; Concentrate obtains bacillus subtilis culture through vacuum freeze drying, ultramicro grinding.

Described slant medium consists of: beef extract 3-10g, and sodium chloride 5-12g, peptone 10-20g, glucose 2-5g, (NH) ₂sO ₄3-5g, K ₂hPO ₄6-8g, CaCl ₂1-3g, agar 15-20g, Chinese herbal medicine powder 5-10g, distilled water l000mL, pH value 7.0-7.2,121 ℃ of sterilizing 20min;

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

In parts by weight, 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, 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; Filtrate Vacuum Concentration postlyophilization obtains Chinese herbal medicine powder.

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.

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

Described one, two, three seed culture medium weight consists of:

Dusty yeast 0.3-0.5%, glucose 1-1.5%, peptone 0.3-0.5%, beef extract 0.5-0.8%, dipotassium hydrogen phosphate 0.8-1.5%, Chinese herbal medicine powder 1.5-2%, trehalose 1-3%, calcium sulfate 0.1%, magnesium chloride 0.2%, natrium citricum 0.1-0.3%, insufficient section pure water is supplied, pH value 7.0-7.2,121-123 ℃ of sterilizing 30-40min.

Described first class seed pot culture medium weight consists of:

Maltodextrin 5-15%, dusty yeast 0.4-0.8%, Chinese herbal medicine powder 1.5-2%, trehalose 1-3%, peptone 0.1-0.5%, corn steep liquor 0.1-0.5%, dipotassium hydrogen phosphate 0.8-1.5%, magnesium sulfate 0.05-0.1%, natrium citricum 0.1-0.5%, insufficient section pure water is supplied, pH value 7.0-7.2,121-123 ℃ of sterilizing 30-40min.

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

Described fermentation medium consists of: maltodextrin 50-150g, corn flour 50-60g, beancake powder 15-25g, Chinese herbal medicine powder 30-50g, trehalose 30-40g, dusty yeast 4-8g, corn steep liquor 1-5g, ammonium sulfate 1-3g, dipotassium hydrogen phosphate 1-2g, potassium dihydrogen phosphate 1-2g, natrium citricum 1-5g, defoamer 0.1-1g, pure water l000mL, pH value 7.0-7.2,121 ℃ of sterilizing 20min;

Described supplemented medium weight consists of: maltodextrin 20-30%, and corn flour 10-20%, bean powder 15-25%, Chinese herbal medicine powder 5-10%, insufficient section pure water is supplied, pH value 7.0-7.2,121-123 ℃ of sterilizing 30-40min.

The concocting method of described fermentation medium is:

Accurately take in proportion raw material, pure water in raw material, corn flour, beancake powder are dropped in material-compound tank, regulate pH value 7.0-7.2, add middle temperature amylase (3u/g corn flour) and alpha-amylase (30u/g corn flour), ℃ insulation 15-30min of warming while stirring to 70 simultaneously, is then slowly warming up to 90 ℃ of insulation 15-30min and liquefies, finally add other raw material, stir, adjust initial pH7.0-7.2,121-123 ℃ of sterilizing 30-40min is for subsequent use.

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.

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;

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;

Described protective agent is made up 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.

Described activator is evenly mixed by the inorganic salts of following mass fraction: zinc chloride 30-40 part, calcium chloride 10-20 part, sodium sulphate 10-20 part, magnesium chloride 5-10 part.

The present invention contains the preparation method of the coarse cereals daily ration enzyme of neutral proteinase:

By described protective agent, Chinese herbal medicine extract ultramicro grinding respectively; guarantee that granularity is less than other enzyme preparation; then evenly mix with bacillus subtilis culture, acidic xylanase, 1,4 beta-glucanase, acid protease, pectase, cellulase, amylase; finally add activator, after mixing, pack the coarse cereals daily ration enzyme getting product containing neutral proteinase.

Described coarse cereals daily ration enzyme is applicable to feed-processing plant and plant's autogamy feed, when use, should mix with other raw material in feed, can in advance the present invention be mixed with a small amount of feed, then be mixed in large quantities of feeds Direct-fed.Advise that complete diet pellet addition per ton is 100-120g.

Bacillus subtilis 1398-2-12 provided by the invention is obtained through UV-LiCl-dithyl sulfate Mutation screening by bacillus subtilis (Bacillus subtilis) 1398-2 of a strain product neutral proteinase of laboratory preservation.

The bacterial strain of product heat-flash stability neutral proteinase provided by the invention is specially bacillus subtilis 1398-2-12.This bacterial strain is preserved in Chinese Typical Representative culture collection center on November 3rd, 2013 and (is called for short CCTCC, address is: Wuchang District, Wuhan City, Hubei Province Luo Jia Shan Wuhan University Life Science College postcode: 430072), preserving number is CCTCC NO:M2013539, and Classification And Nomenclature is: bacillus subtilis (Bacillus subtilis) 1398-2-12.

Described bacillus subtilis (Bacillus subtilis) 1398-2-12 bacterial strain feature is as follows:

Described bacterial strain colony colour on solid plate is milky, and dry tack free is opaque, and neat in edge, for having the aerobic bacteria of motility.Microscopy is elongated rod shape, and Gram's staining is positive.This bacterium can utilize citrate, and nitrate reductase, V-P test into positive.

Bacillus subtilis 1398-2-12 provided by the invention has that produced neutral proteinase tolerable temperature is high, the feature of applicable pH value wide scope, 75 ℃ of enzymes of zymotic fluid crude enzyme liquid complete stability alive, 70 ℃ of optimal reactive temperatures, pH value 4.5-8.5 enzyme is lived stable, optimal reaction pH value 7.2.This bacterial strain the most suitable growth pH value 7.0-7.2, optimum growth temperature 30-36 ℃, the suitableeest product enzyme temperature 32-35 ℃.

Press mutagenesis screening scheme, mutant strain step-sizing is eliminated, finally to strain excellent through fermenting property test screen, obtain a strain and produce the bacterial strain bacillus subtilis bacterium 1398-2-12 of heat-flash stability neutral proteinase, the work of zymotic fluid neutral proteinase enzyme can reach 5500-7000U/mL, heat endurance to enzyme is analyzed, and crude enzyme liquid is placed in respectively at 40 ℃, 45 ℃, 50 ℃, 55 ℃, 60 ℃, 65 ℃, 70 ℃, 75 ℃, lives every 10 minutes sampling and measuring enzymes.At 40 ℃, 45 ℃, 50 ℃, 55 ℃, 60 minutes enzymes are lived and are not declined.At 60 ℃ and 65 ℃, what within 30 minutes, drop to constitutive enzyme work drops to 85% in 95%, 60 minute.At 70 ℃, what within 30 minutes, drop to constitutive enzyme work drops to 80% in 85%, 60 minute.At 75 ℃, what within 30 minutes, drop to constitutive enzyme work drops to 70% in 80%, 60 minute.

Beneficial effect:

1. the neutral proteinase enzyme work containing in bacillus subtilis culture fermentation broth of the present invention can reach 5500-7000U/mL, has higher enzyme and live between 40-70 ℃, and optimal reactive temperature is 70 ℃; Enzyme complete stability alive in the time that pH value is 4.5-8.5, optimal reaction pH value is 7.2; This enzyme still can keep 80% above enzyme to live preserve 1h under 70 ℃ of conditions after, keep 70% above enzyme work after preserving 1h under 75 ℃ of conditions.Stronger than existing neutral protein enzyme heat stability, enzyme activity is higher, enzyme effect optimum pH wide scope, and storage stability is high, is more applicable to raising the interpolation of animal and fowl fodder.

2. the bacillus subtilis thalline in bacillus subtilis culture of the present invention can improve the growth of animal body immunity, Promote immunity organ, the maturation that promotes animal intestinal structure and function, raising animal daily gain and improve feed conversion rate, is more applicable to raising the interpolation of animal and fowl fodder.

3. the present invention adopts polysaccharide, inorganic salts and amino acid science composite containing the protective agent in the coarse cereals daily ration enzyme of neutral proteinase, has effectively slowed down the moisture regain of complex enzyme formulation; Can strengthen complex 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 prevent the loss of complex enzyme enzyme activity in transportation, preservation and use procedure, extend the shelf-life of complex enzyme, reached same enzyme activity, can extend 2-3 than the like product shelf-life.

4. the present invention adds inorganic salts as activator containing the coarse cereals daily ration enzyme of neutral proteinase; create the optimum condition of enzyme catalysis; give full play to the vigor of the each enzyme component of complex enzyme; the macromolecular substances such as starch in feed, protein, cellulose, phytic acid are thoroughly effectively decomposed; greatly alleviate the digestion burden of livestock and poultry animal; improve 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 present invention adds containing the coarse cereals daily ration enzyme of neutral proteinase both can extend the shelf-life of complex enzyme formulation, can improve again the immunity of raising livestock and poultry, effectively prevented the generation of livestock and poultry epidemic disease.

6. the present invention is containing the synergy of bacillus subtilis culture, protective agent, activator, Chinese herbal medicine extract and enzyme preparation in the coarse cereals daily ration enzyme of neutral proteinase; enzyme activity and the effect of complex enzyme are brought into play to greatest extent; and the utilization rate of feed and the growth rate of animal are improved accordingly; strengthen appetite and the resistance against diseases of animal, extended the shelf-life of complex 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

25 parts of bacillus subtilis cultures, 40 parts of acidic xylanases, 25 parts of 1,4 beta-glucanases, 25 parts of acid proteases, 12 parts of pectases, 12 parts of cellulases, 12 parts of amylase, 12 parts of Chinese herbal medicine extracts, 12 parts of protective agents, 12 parts of activator.

The preparation method of described bacillus subtilis culture comprises the steps:

(1) slant strains of intact bacillus subtilis 1398-2-12 is inoculated in to slant medium, cultivates 24h for 30 ℃ and carry out actication of culture, so activate 2 times;

Described slant medium consists of: beef extract 3g, and sodium chloride 5g, peptone 10g, glucose 2g, (NH) ₂sO ₄3g, K ₂hPO ₄6g, CaCl ₂1g, agar 15g, Chinese herbal medicine powder 5g, distilled water l000mL, 7.0,121 ℃ of sterilizing 20min of pH value;

In parts by weight, 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, be then cooled to 45 ℃, add the mixing enzyme preparation of mixed material gross weight 5% to carry out enzymolysis, with newborn acid for adjusting pH value be 5.5, enzymolysis 2h, finally adds the mixture of 0.5 times of weight ethanol of mixed material and propyl alcohol, and the mass ratio of described ethanol and propyl alcohol is 1:1, control temperature to 60 ℃ and keep 3h, filter; Filtrate Vacuum Concentration postlyophilization obtains Chinese herbal medicine powder.

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.

(2) liquid seeds expands cultivation

1. first order seed is cultivated: slant strains 1 articulating after step (1) activation is entered in 500 ml shake flasks to 100 milliliters of culture medium loading amounts, 180 revs/min of rotary shaking tables, 30 ℃ of cultivation temperature, incubation time 10h;

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: secondary seed is accessed in 5000 milliliters of three grades of seed shaking flasks to 1000 milliliters of culture medium loading amounts, 100 revs/min of rotary shaking tables, 30 ℃ of cultivation temperature, incubation time 10h with 10% inoculum concentration;

4. first class seed pot is cultivated: the first class seed pot by three grades of seeds take 10% inoculum concentration access total measurement (volume) as 150L, fermentation medium loading amount 100L, 30 ℃ of cultivation temperature, mixing speed 200rpm, ventilation (V/V) 1:1, tank pressure 0.05Mpa, incubation time 10h;

Described one-level, secondary, three grades of seed culture medium weight consist of:

Dusty yeast 0.3%, glucose 1%, peptone 0.3%, beef extract 0.5%, dipotassium hydrogen phosphate 0.8%, Chinese herbal medicine powder 1.5%, trehalose 1%, calcium sulfate 0.1%, magnesium chloride 0.2%, natrium citricum 0.1%, insufficient section pure water is supplied, 7.0,121 ℃ of sterilizing 30min of pH value.

Described first class seed pot culture medium weight consists of:

Maltodextrin 5%, dusty yeast 0.4%, Chinese herbal medicine powder 1.5%, trehalose 1%, peptone 0.1%, corn steep liquor 0.1%, dipotassium hydrogen phosphate 0.8%, magnesium sulfate 0.05%, natrium citricum 0.1%, insufficient section pure water is supplied, 7.0,121 ℃ of sterilizing 30min of pH value.

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

(3) ferment tank

First class seed pot zymotic fluid in step (2) is accessed to fermentation tank, 30 ℃ 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; Continue with 1 ℃/h rate of temperature fall slow cooling to 2 ℃, now, first class seed pot zymotic fluid in step (2) is appended to access fermentation tank, constant temperature culture 20h with 4% inoculum concentration; Finally slowly be warming up to 10 ℃ with 1 ℃/h heating rate, constant temperature culture 15h; Continue to be slowly warming up to 30 ℃ with 1 ℃/h heating rate, constant temperature culture 15h;

Dissolved oxygen control: 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 7.0;

Control of additive raw material: add supplemented medium, to maintain zymotic fluid content of reducing sugar as 2-5mg/ml;

Put tank standard: the old and feeble self-dissolving of 60-80% thalline, enzyme activity increasess slowly.

Described fermentation medium consists of: maltodextrin 50g, corn flour 50g, beancake powder 15g, Chinese herbal medicine powder 30g, trehalose 30g, dusty yeast 4g, corn steep liquor 1g, ammonium sulfate 1g, dipotassium hydrogen phosphate 1g, potassium dihydrogen phosphate 1g, natrium citricum 1g, defoamer 0.1g, pure water l000mL, 7.0,121 ℃ of sterilizing 20min of pH value;

Described supplemented medium weight consists of: maltodextrin 20%, and corn flour 10%, bean powder 15%, Chinese herbal medicine powder 5%, insufficient section pure water is supplied, 7.0,121 ℃ of sterilizing 30min of pH value.

The concocting method of described fermentation medium is:

Accurately take in proportion raw material, pure water in raw material, corn flour, beancake powder are dropped in material-compound tank, regulate pH value 7.0, add middle temperature amylase (3u/g corn flour) and alpha-amylase (30u/g corn flour), ℃ insulation 15min of warming while stirring to 70 simultaneously, is then slowly warming up to 90 ℃ of insulation 15min and liquefies, finally add other raw material, stir, adjust initial pH7.0,121 ℃ of sterilizing 30min are for subsequent use.

(4) zymotic fluid via hole diameter 10 μ m filter board coarse filtration, then obtaining solid content in the concentrated removal of 10 ℃ of loop ultrafiltrations moisture is 20% concentrate; Concentrate obtains bacillus subtilis culture through vacuum freeze drying, ultramicro grinding.

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 be cooled to 53 ℃, add mixing enzyme preparation to carry out enzymolysis, with newborn acid for adjusting pH value be 6.2, 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 made up 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 evenly to be mixed by the inorganic salts of following quality component: 35 parts of zinc chloride, 15 parts, calcium chloride, 15 parts, sodium sulphate, 7 parts, magnesium chloride.

Contain the preparation method of the coarse cereals daily ration enzyme of neutral proteinase:

Embodiment 2:

20 parts of bacillus subtilis cultures, 30 parts of acidic xylanases, 20 parts of 1,4 beta-glucanases, 20 parts of acid proteases, 10 parts of pectases, 10 parts of cellulases, 10 parts of amylase, 10 parts of Chinese herbal medicine extracts, 10 parts of protective agents, 10 parts of activator.

(1) actication of culture

The slant strains of intact bacillus subtilis 1398-2-12 is inoculated in to slant medium, cultivates 30h for 33 ℃ and carry out actication of culture, so activate 2 times;

Described slant medium consists of: beef extract 6g, and sodium chloride 8g, peptone 15g, glucose 4g, (NH) ₂sO ₄4g, K ₂hPO ₄7g, CaCl ₂2g, agar 18g, Chinese herbal medicine powder 8g, distilled water l000mL, 7.2,121 ℃ of sterilizing 20min of pH value;

In parts by weight, take 25 parts of the Radixs Astragali; 16 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, be then cooled to 50 ℃, add the mixing enzyme preparation of mixed material gross weight 8% to carry out enzymolysis, with newborn acid for adjusting pH value be 6.0, enzymolysis 3h, finally adds the mixture of 2 times of weight ethanol of mixed material and propyl alcohol, and the mass ratio of described ethanol and propyl alcohol is 1:1.2, control temperature to 70 ℃ and keep 4h, filter; Filtrate Vacuum Concentration postlyophilization obtains Chinese herbal medicine powder.

The parts by weight of described mixed enzyme consist of: 15 parts of endo-beta-glucanases, 15 parts of outer 1,4 beta-glucanases, 12 parts of beta-glucosidases, 18 parts of zytases, 18 parts of pentosanases, 25 parts of Pullulanases, 12 parts of beta amylases, 12 parts of neutral proteinases, 12 parts of acid proteases, 8 parts of superoxide dismutases, 8 parts of glucose oxidases, 8 parts of acid phosphatases.

(2) liquid seeds expands cultivation

1. first order seed is cultivated: slant strains 2 articulatings after step (1) activation are entered in 500 ml shake flasks to 100 milliliters of culture medium loading amounts, 180 revs/min of rotary shaking tables, 33 ℃ of cultivation temperature, incubation time 12h;

3. three grades of seed culture: secondary seed is accessed in 5000 milliliters of three grades of seed shaking flasks to 1000 milliliters of culture medium loading amounts, 100 revs/min of rotary shaking tables, 33 ℃ of cultivation temperature, incubation time 12h with 10% inoculum concentration;

4. first class seed pot is cultivated: the first class seed pot by three grades of seeds take 10% inoculum concentration access total measurement (volume) as 150L, fermentation medium loading amount 100L, 33 ℃ of cultivation temperature, mixing speed 300rpm, ventilation (V/V) 1:1.5, tank pressure 0.05Mpa, incubation time 15h;

Dusty yeast 0.4%, glucose 1.2%, peptone 0.4%, beef extract 0.6%, dipotassium hydrogen phosphate 1.0%, Chinese herbal medicine powder 1.8%, trehalose 2%, calcium sulfate 0.1%, magnesium chloride 0.2%, natrium citricum 0.2%, insufficient section pure water is supplied, 7.2,121 ℃ of sterilizing 30min of pH value.

Described first class seed pot culture medium weight consists of:

Maltodextrin 10%, dusty yeast 0.5%, Chinese herbal medicine powder 1.8%, trehalose 2%, peptone 0.2%, corn steep liquor 0.3%, dipotassium hydrogen phosphate 1.0%, magnesium sulfate 0.08%, natrium citricum 0.3%, insufficient section pure water is supplied, 7.2,121 ℃ of sterilizing 30min of pH value.

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

(3) ferment tank

First class seed pot zymotic fluid in step (2) is accessed to fermentation tank, 35 ℃ of cultivation temperature, mixing speed 400r/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; Continue with 2 ℃/h rate of temperature fall slow cooling to 4 ℃, now, first class seed pot zymotic fluid in step (2) is appended to access fermentation tank, constant temperature culture 25h with 4% inoculum concentration; Finally slowly be warming up to 12 ℃ with 2 ℃/h heating rate, constant temperature culture 18h; Continue to be slowly warming up to 33 ℃ with 2 ℃/h heating rate, constant temperature culture 18h;

Dissolved oxygen control: 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 7.2;

Described fermentation medium consists of: maltodextrin 100g, corn flour 55g, beancake powder 20g, Chinese herbal medicine powder 40g, trehalose 35g, dusty yeast 6g, corn steep liquor 3g, ammonium sulfate 2g, dipotassium hydrogen phosphate 2g, potassium dihydrogen phosphate 2g, natrium citricum 3g, defoamer 0.5g, pure water l000mL, 7.2,121 ℃ of sterilizing 20min of pH value;

Described supplemented medium weight consists of: maltodextrin 25%, and corn flour 15%, bean powder 20%, Chinese herbal medicine powder 8%, insufficient section pure water is supplied, 7.2,121 ℃ of sterilizing 30min of pH value.

The concocting method of described fermentation medium is:

Accurately take in proportion raw material, pure water in raw material, corn flour, beancake powder are dropped in material-compound tank, regulate pH value 7.2, add middle temperature amylase (3u/g corn flour) and alpha-amylase (30u/g corn flour), ℃ insulation 20min of warming while stirring to 70 simultaneously, is then slowly warming up to 90 ℃ of insulation 20min and liquefies, finally add other raw material, stir, adjust initial pH7.2,121 ℃ of sterilizing 30min are for subsequent use.

(4) zymotic fluid via hole diameter 500 μ m filter board coarse filtration, then obtaining solid content in the concentrated removal of 15 ℃ of loop ultrafiltrations moisture is 30% concentrate; Concentrate obtains bacillus subtilis culture through vacuum freeze drying, ultramicro grinding.

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 be cooled to 45 ℃, add mixing enzyme preparation to carry out enzymolysis, with newborn acid for adjusting pH value be 5.5, 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 made up 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 evenly to be mixed by the inorganic salts of following quality component: 30 parts of zinc chloride, 10 parts, calcium chloride, 10 parts, sodium sulphate, 5 parts, magnesium chloride.

Containing the preparation method of the coarse cereals daily ration enzyme of neutral proteinase as embodiment 1.

Embodiment 3:

30 parts of bacillus subtilis cultures, 50 parts of acidic xylanases, 30 parts of 1,4 beta-glucanases, 30 parts of acid proteases, 15 parts of pectases, 15 parts of cellulases, 15 parts of amylase, 15 parts of Chinese herbal medicine extracts, 15 parts of protective agents, 15 parts of activator.

(1) actication of culture

The slant strains of intact bacillus subtilis 1398-2-12 is inoculated in to slant medium, cultivates 36h for 36 ℃ and carry out actication of culture, so activate 3 times;

Described slant medium consists of: beef extract 10g, and sodium chloride 12g, peptone 20g, glucose 5g, (NH) ₂sO ₄5g, K ₂hPO ₄8g, CaCl ₂3g, agar 20g, Chinese herbal medicine powder 10g, distilled water l000mL, 7.2,121 ℃ of sterilizing 20min of pH value;

In parts by weight, 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, be then cooled to 60 ℃, add the mixing enzyme preparation of mixed material gross weight 10% to carry out enzymolysis, with newborn acid for adjusting pH value be 6.8, enzymolysis 4h, finally adds the mixture of 3 times of weight ethanol of mixed material and propyl alcohol, and the mass ratio of described ethanol and propyl alcohol is 1:1.5, control temperature to 78 ℃ and keep 4h, filter; Filtrate Vacuum Concentration postlyophilization obtains Chinese herbal medicine powder.

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.

(2) liquid seeds expands cultivation

1. first order seed is cultivated: slant strains 2 articulatings after step (1) activation are entered in 500 ml shake flasks to 100 milliliters of culture medium loading amounts, 180 revs/min of rotary shaking tables, 36 ℃ of cultivation temperature, incubation time 15h;

3. three grades of seed culture: secondary seed is accessed in 5000 milliliters of three grades of seed shaking flasks to 1000 milliliters of culture medium loading amounts, 100 revs/min of rotary shaking tables, 36 ℃ of cultivation temperature, incubation time 15h with 10% inoculum concentration;

4. first class seed pot is cultivated: the first class seed pot by three grades of seeds take 10% inoculum concentration access total measurement (volume) as 150L, fermentation medium loading amount 100L, 36 ℃ of cultivation temperature, mixing speed 400rpm, ventilation (V/V) 1:2, tank pressure 0.05Mpa, incubation time 20h;

Dusty yeast 0.5%, glucose 1.5%, peptone 0.5%, beef extract 0.8%, dipotassium hydrogen phosphate 1.5%, Chinese herbal medicine powder 2%, trehalose 3%, calcium sulfate 0.1%, magnesium chloride 0.2%, natrium citricum 0.3%, insufficient section pure water is supplied, 7.2,123 ℃ of sterilizing 40min of pH value.

Described first class seed pot culture medium weight consists of:

Maltodextrin 15%, yeast 0.8%, Chinese herbal medicine powder 2%, trehalose 3%, peptone 0.5%, corn steep liquor 0.5%, dipotassium hydrogen phosphate 1.5%, magnesium sulfate 0.1%, natrium citricum 0.5%, insufficient section pure water is supplied, 7.2,123 ℃ of sterilizing 40min of pH value.

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

(3) ferment tank

First class seed pot zymotic fluid in step (2) is accessed to fermentation tank, 36 ℃ 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; Continue with 2 ℃/h rate of temperature fall slow cooling to 5 ℃, now, first class seed pot zymotic fluid in step (2) is appended to access fermentation tank, constant temperature culture 30h with 4% inoculum concentration; Finally slowly be warming up to 15 ℃ with 2 ℃/h heating rate, constant temperature culture 20h; Continue to be slowly warming up to 36 ℃ with 2 ℃/h heating rate, constant temperature culture 20h;

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

Described fermentation medium consists of: maltodextrin 150g, corn flour 60g, beancake powder 25g, Chinese herbal medicine powder 50g, trehalose 40g, dusty yeast 8g, corn steep liquor 5g, ammonium sulfate 3g, dipotassium hydrogen phosphate 2g, potassium dihydrogen phosphate 2g, natrium citricum 5g, defoamer 1g, pure water l000mL, 7.2,121 ℃ of sterilizing 20min of pH value;

Described supplemented medium weight consists of: maltodextrin 30%, and corn flour 20%, bean powder 25%, Chinese herbal medicine powder 10%, insufficient section pure water is supplied, 7.2,123 ℃ of sterilizing 40min of pH value.

The concocting method of described fermentation medium is:

Accurately take in proportion raw material, pure water in raw material, corn flour, beancake powder are dropped in material-compound tank, regulate pH value 7.2, add middle temperature amylase (3u/g corn flour) and alpha-amylase (30u/g corn flour), ℃ insulation 30min of warming while stirring to 70 simultaneously, is then slowly warming up to 90 ℃ of insulation 30min and liquefies, finally add other raw material, stir, adjust initial pH7.2,123 ℃ of sterilizing 40min are for subsequent use.

(4) zymotic fluid via hole diameter 1000 μ m filter board coarse filtration, then obtaining solid content in the concentrated removal of 20 ℃ of loop ultrafiltrations moisture is 40% concentrate; Concentrate obtains bacillus subtilis culture through vacuum freeze drying, ultramicro grinding.

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 be cooled to 60 ℃, add mixing enzyme preparation to carry out enzymolysis, with newborn acid for adjusting pH value be 6.8, 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 made up 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 evenly to be mixed by the inorganic salts of following quality component: 40 parts of zinc chloride, 20 parts, calcium chloride, 20 parts, sodium sulphate, 10 parts, magnesium chloride.

The result of use test of embodiment 4 embodiment of the present invention 1 coarse cereals daily ration enzymes

1. feed kind: full price coarse cereals daily ration;

2. coarse cereals daily ration enzyme addition: feed per ton adds 100g;

3. coarse cereals daily ration enzyme test group design: coarse cereals daily ration enzyme parts by weight composition is divided into test group, control group 1, control group 2; Wherein test group is coarse cereals daily ration enzyme prepared by the embodiment of the present invention 1; Control group 1 is all the other components except bacillus subtilis culture in test group component; Control group 2 is that commercially available coarse cereals daily ration enzyme and its each enzyme class, enzyme activity and enzyme parts by weight that comprise are identical with control group 1 with test group; The concrete parts by weight of each test group form as table 1

Table 1

Project	Test group	Control group 1	Control group 2	Remarks
					Bacillus subtilis culture (part)	25	0	0	?
Acid protease (part)	25	25	25	?
					Acidic xylanase (part)	40	40	40	?
Pectase (part)	12	12	12	?
					Cellulase (part)	12	12	12	?
1,4 beta-glucanase (part)	25	25	25	?
					Amylase (part)	12	12	12	?
Chinese herbal medicine extract (part)	12	12	0	?
					Protective agent (part)	12	12	0	?
Activator (part)	12	12	0	?

4. feeding experiment

4.1 materials and methods

4.1.1 the selection of test pig and grouping

The a certain large-scale regular pig farm in Hunan, choosing 36 body weight is the healthy DLY three way cross growth pig of (30 ± 2) kg, is divided at random 3 groups, every group of 2 hurdles repeat, 6 of every repetitions, galt and gilt half and half.The formal test prerun of 1 week behavior phase of advancing, and complete expelling parasite and routine immunization work.Test point two phases of front and back carry out, (30kg-60kg) in earlier stage, later stage (61kg～90kg).

4.1.2 feeding and management

The dry mash of feeding, free choice feeding, is limited cannot not have enough surplusly, automatic drinking bowl drinking-water.Feed three early stage day, and day in later stage is fed secondary, is unit record feed consumption rate take hurdle.Pig house is brick structure, cement flooring, single-column type, and examination pig is raised respectively in 9 hurdles, and the environmental condition of each column home is consistent, cleans colony house morning and afternoon every day respectively once, observes behavior, appetite, the ight soil of pig simultaneously.Duration of test records disease and treatment situation.

4.1.3 feed and formula

Take full price coarse cereals diet feed as basis, feed per ton adds the coarse cereals daily ration enzyme 100g of test group, control group 1 and control group 2; The raw material of coarse cereals daily ration enzyme forms specifically in table 1.

4.1.4 test index: when (30kg-60kg), mid-term and later stage (61kg～90kg) finish before test, point another name individual weight, weighs and all carry out on an empty stomach in the morning.Stage by stage,, calculate daily ingestion amount and food utilization efficiency simultaneously, and above-mentioned data are carried out to statistical analysis as its every daily material consumption of unit record, the incidence of disease take hurdle, growth pig growth performance is in table 3.

Table 2

Project	Test group	Control group 1	Deviation (%)	Control group 2	Deviation (%)
						Daily gain (g)	878	740	138(18.65)	653	225(34.46)
Feed intake (kg)	189.73	193.46	-3.73(-1.92)	210.17	-20.44(-9.7)
						Material anharmonic ratio	2.87	3.36	-0.49(-14.5)	4.15	-1.28(-30.84)
Diarrhea rate (%)	2	4	-2(-50)	17	-15(-88.2)
						Hair color scoring	7.5	6.5	1(15.38)	4	3.5(87.5)

From above-mentioned analysis of statistical results: test group is compared with control group 2 with control group 1, and in the situation that the experimental conditions such as test material, experimental enviroment, test method and Experimental Establishment are identical, daily gain has improved respectively 18.65% and 34.46%; Feed intake has reduced respectively 1.92% and 9.7%; Material anharmonic ratio has reduced respectively 14.5% and 30.84%; Diarrhea rate has reduced respectively 50% and 88.2%; Outward appearance hair color quality has improved respectively 15.38% and 87.5%; Use coarse cereals daily ration enzyme of the present invention, growth pig has good growth performance and significant immunocompetence, has improved cultured output and quality, has reduced aquaculture cost, has improved fanning economics.

Claims

1. the coarse cereals daily ration enzyme containing neutral proteinase, enzyme preparation by following parts by weight forms: bacillus subtilis culture 20-30 part, acidic xylanase 30-50 part, 1,4 beta-glucanase 20-30 part, acid protease 20-30 part, pectase 10-15 part, cellulase 10-15 part, amylase 10-15 part, Chinese herbal medicine extract 10-15 part, protective agent 10-15 part, activator 10-15 part;

The preparation method of described bacillus subtilis culture is as follows: bacillus subtilis CCTCC NO:M2013539 cultivates to expand step by step to cultivate through slant strains activation and one, two, three seed culture and first class seed pot and obtains seed liquor; Liquid seeds is accessed to fermentation tank, cultivation temperature 30-36 ℃, mixing speed 200-700rpm, ventilation (V/V) 1:1-3, incubation time 10-15h with 6% inoculum concentration; 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 slowly be warming up to 10-15 ℃, constant temperature culture 15-20h with 1-2 ℃/h heating rate; Continue to be slowly warming up to 30-36 ℃, constant temperature culture 15-20h with 1-2 ℃/h heating rate; Zymotic fluid via hole diameter 10-1000 μ m filter board coarse filtration, then obtains in the concentrated moisture of removing of 10-20 ℃ of loop ultrafiltration the concentrate that solid content is 20-40%; Concentrate obtains bacillus subtilis culture through vacuum freeze drying, ultramicro grinding;

Described protective agent is made up 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;

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

2. a kind of coarse cereals daily ration enzyme containing neutral proteinase as claimed in claim 1, is characterized in that, the slant medium of preparation bacillus subtilis culture consists of: beef extract 3-10g, sodium chloride 5-12g, peptone 10-20g, glucose 2-5g, (NH) ₂sO ₄3-5g, K ₂hPO ₄6-8g, CaCl ₂1-3g, agar 15-20g, Chinese herbal medicine powder 5-10g, distilled water l000mL, pH value 7.0-7.2,121 ℃ of sterilizing 20min.

3. a kind of coarse cereals daily ration enzyme containing neutral proteinase as claimed in claim 1, is characterized in that, the seed culture medium of preparation bacillus subtilis culture consists of: dusty yeast 0.3-0.5%, glucose 1-1.5%, peptone 0.3-0.5%, beef extract 0.5-0.8%, dipotassium hydrogen phosphate 0.8-1.5%, Chinese herbal medicine powder 1.5-2%, trehalose 1-3%, calcium sulfate 0.1%, magnesium chloride 0.2%, natrium citricum 0.1-0.3%, insufficient section pure water is supplied, pH value 7.0-7.2,121-123 ℃ of sterilizing 30-40min.

4. a kind of coarse cereals daily ration enzyme containing neutral proteinase as claimed in claim 1, is characterized in that, the seed tank culture base of preparation bacillus subtilis culture consists of: maltodextrin 5-15%, dusty yeast 0.4-0.8%, Chinese herbal medicine powder 1.5-2%, trehalose 1-3%, peptone 0.1-0.5%, corn steep liquor 0.1-0.5%, dipotassium hydrogen phosphate 0.8-1.5%, magnesium sulfate 0.05-0.1%, natrium citricum 0.1-0.5%, insufficient section pure water is supplied, pH value 7.0-7.2,121-123 ℃ of sterilizing 30-40min.

5. a kind of coarse cereals daily ration enzyme containing neutral proteinase as claimed in claim 1, is characterized in that, when preparation bacillus subtilis culture, seeding tank zymotic fluid cell concentration is 7.0x10 ⁸-8.0x10 ⁸individual/ml.

6. a kind of coarse cereals daily ration enzyme containing neutral proteinase as claimed in claim 1, it is characterized in that, the fermentation medium of preparation bacillus subtilis culture consists of: maltodextrin 50-150g, corn flour 50-60g, beancake powder 15-25g, Chinese herbal medicine powder 30-50g, trehalose 30-40g, dusty yeast 4-8g, corn steep liquor 1-5g, ammonium sulfate 1-3g, dipotassium hydrogen phosphate 1-2g, potassium dihydrogen phosphate 1-2g, natrium citricum 1-5g, defoamer 0.1-1g, pure water l000mL, pH value 7.0-7.2,121 ℃ of sterilizing 20min.

7. the preparation method containing the coarse cereals daily ration enzyme of neutral proteinase; it is characterized in that; by protective agent, Chinese herbal medicine extract ultramicro grinding respectively; guarantee that granularity is less than other enzyme preparation; then evenly mix with bacillus subtilis culture, acidic xylanase, 1,4 beta-glucanase, acid protease, pectase, cellulase, amylase; finally add activator, after mixing, pack the coarse cereals daily ration enzyme getting product containing neutral proteinase.

8. the preparation method of a kind of coarse cereals daily ration enzyme containing neutral proteinase as claimed in claim 7, it is characterized in that, the described coarse cereals daily ration enzyme containing neutral proteinase is made up of the enzyme preparation of following parts by weight: bacillus subtilis culture 20-30 part, acidic xylanase 30-50 part, 1,4 beta-glucanase 20-30 part, acid protease 20-30 part, pectase 10-15 part, cellulase 10-15 part, amylase 10-15 part, Chinese herbal medicine extract 10-15 part, protective agent 10-15 part, activator 10-15 part;

The preparation method of described bacillus subtilis culture is as follows: bacillus subtilis 1398-2-12 cultivates to expand step by step to cultivate through slant strains activation and one, two, three seed culture and first class seed pot and obtains seed liquor; Liquid seeds is accessed to fermentation tank, cultivation temperature 30-36 ℃, mixing speed 200-700rpm, ventilation (V/V) 1:1-3, incubation time 10-15h with 6% inoculum concentration; 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 slowly be warming up to 10-15 ℃, constant temperature culture 15-20h with 1-2 ℃/h heating rate; Continue to be slowly warming up to 30-36 ℃, constant temperature culture 15-20h with 1-2 ℃/h heating rate; Zymotic fluid via hole diameter 10-1000 μ m filter board coarse filtration, then obtaining solid content in the concentrated removal of 10-20 ℃ of loop ultrafiltration moisture is 20-40% concentrate; Concentrate obtains bacillus subtilis culture through vacuum freeze drying, ultramicro grinding;

9. the preparation method of a kind of coarse cereals daily ration enzyme containing neutral proteinase as claimed in claim 8, 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;

10. the preparation method of a kind of coarse cereals daily ration enzyme containing neutral proteinase as claimed in claim 8, is characterized in that, the described coarse cereals daily ration enzyme containing neutral proteinase, enzyme preparation by following parts by weight forms: 25 parts of bacillus subtilis cultures, 40 parts of acidic xylanases, 25 parts of 1,4 beta-glucanases, 25 parts of acid proteases, 12 parts of pectases, 12 parts of cellulases, 12 parts of amylase, 12 parts of Chinese herbal medicine extracts, 12 parts of protective agents, 12 parts of activator;

In parts by weight, 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, be then cooled to 45 ℃, add the mixing enzyme preparation of mixed material gross weight 5% to carry out enzymolysis, with newborn acid for adjusting pH value be 5.5, enzymolysis 2h, finally adds the mixture of 0.5 times of weight ethanol of mixed material and propyl alcohol, and the mass ratio of described ethanol and propyl alcohol is 1:1, control temperature to 60 ℃ and keep 3h, filter; Filtrate Vacuum Concentration postlyophilization obtains Chinese herbal medicine powder;

(2) liquid seeds expands cultivation

Dusty yeast 0.3%, glucose 1%, peptone 0.3%, beef extract 0.5%, dipotassium hydrogen phosphate 0.8%, Chinese herbal medicine powder 1.5%, trehalose 1%, calcium sulfate 0.1%, magnesium chloride 0.2%, natrium citricum 0.1%, insufficient section pure water is supplied, 7.0,121 ℃ of sterilizing 30min of pH value;

Described first class seed pot culture medium weight consists of:

Maltodextrin 5%, dusty yeast 0.4%, Chinese herbal medicine powder 1.5%, trehalose 1%, peptone 0.1%, corn steep liquor 0.1%, dipotassium hydrogen phosphate 0.8%, magnesium sulfate 0.05%, natrium citricum 0.1%, insufficient section pure water is supplied, 7.0,121 ℃ of sterilizing 30min of pH value;

(3) ferment tank

Put tank standard: the old and feeble self-dissolving of 60-80% thalline, enzyme activity increasess slowly;

Described supplemented medium weight consists of: maltodextrin 20%, and corn flour 10%, bean powder 15%, Chinese herbal medicine powder 5%, insufficient section pure water is supplied, 7.0,121 ℃ of sterilizing 30min of pH value;

The concocting method of described fermentation medium is:

Accurately take in proportion raw material, pure water in raw material, corn flour, beancake powder are dropped in material-compound tank, regulate pH value 7.0, add middle temperature amylase (3u/g corn flour) and alpha-amylase (30u/g corn flour), ℃ insulation 15min of warming while stirring to 70 simultaneously, is then slowly warming up to 90 ℃ of insulation 15min and liquefies, finally add other raw material, stir, adjust initial pH7.0,121 ℃ of sterilizing 30min are for subsequent use;

(4) zymotic fluid via hole diameter 10 μ m filter board coarse filtration, then obtaining solid content in the concentrated removal of 10 ℃ of loop ultrafiltrations moisture is 20% concentrate; Concentrate obtains bacillus subtilis culture through vacuum freeze drying, ultramicro grinding;

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 be cooled to 53 ℃, add mixing enzyme preparation to carry out enzymolysis, with newborn acid for adjusting pH value be 6.2, 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 made up 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 evenly to be mixed by the inorganic salts of following quality component: 35 parts of zinc chloride, 15 parts, calcium chloride, 15 parts, sodium sulphate, 7 parts, magnesium chloride;