CN103740683B - A kind of Wheat ration enzyme containing neutral protease and preparation method thereof - Google Patents

A kind of Wheat ration enzyme containing neutral protease and preparation method thereof Download PDF

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CN103740683B
CN103740683B CN201310738229.0A CN201310738229A CN103740683B CN 103740683 B CN103740683 B CN 103740683B CN 201310738229 A CN201310738229 A CN 201310738229A CN 103740683 B CN103740683 B CN 103740683B
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CN103740683A (en
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李洪兵
李海清
张锦杰
胡永明
易继云
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Hunan Hongying Biological Science & Technology Co Ltd
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Abstract

The invention discloses a kind of Wheat ration enzyme containing neutral protease and preparation method thereof, belong to technical field of enzyme preparation.Described Wheat ration enzyme with subtilis culture, aspartic protease, acidic xylanase, cellulase, beta-glucanase, amylase, 'beta '-mannase, Chinese herbal medicine extract, protective material, activator science is composite forms, subtilis culture can improve animal body immunizing power, Promote immunity organ growth, promote the maturation of animal intestinal structure and function, improve animal day weight gain and improve feed conversion rate.Wheat ration enzyme of the present invention is that livestock and poultry provide safe ingestion enzyme; effectively alleviate digestion burden; improve raw material availability and growth rate; protect environment; appropriate activator under equal conditions can give full play to the effect of zymin simultaneously; save zymin addition, the Chinese herbal medicine extract science composite quality guaranteed period both having extended compound enzymic preparation, the immunizing power of raising livestock and poultry can be improved again.

Description

A kind of Wheat ration enzyme containing neutral protease and preparation method thereof
Technical field
The invention belongs to technical field of enzyme preparation, specifically a kind of Wheat ration enzyme containing neutral protease and preparation method thereof.
Background technology
In feed, add zymin mainly contain following 4 reasons: 1. the antinutritional factor existed in animal-feed of degrading.These materials can not be degraded by animal endogenous enzyme, thus the eubolism of interference animal, cause animal digestion bad, production performance declines.2. improve the utilization ratio of starch, protein and mineral substance.These materials or surrounded by the cell walls of fiber-enriched, or can not be had by the structure formation of animal digestion with some that (such as in plant feed raw material, a large amount of phosphorus exists with the form of phytate phosphorus.）。3. to degrade in the feed some specific chemical bond.These chemical bonds can not degrade by the enzyme of animal self, more nutrition can be discharged after adding exogenous enzyme.4., because young animal own digestive system is also immature, endogenous enzyme deficiency adds exogenous enzyme can improve feed digestibility, prevents indigestion symptom.Except can improving the utilization ratio of daily ration, the enzyme-added difference that can also reduce between feedstuff raw material, improves the accuracy of feed formulation, can also improve the regularity of growth of animal simultaneously, reduces handling cost, increases economic efficiency.Use all right protection of the environment of zymin.Because the utilization ratio of feed improves, corresponding ight soil quantity discharged have dropped.In the obvious situation of effectiveness comparison, the quantity discharged of ight soil can reduce about 20%, and in pig manure, the discharge of nitrogen declines about 15%, and in chicken manure, the discharge of nitrogen declines 20%.For phytate phosphorus, the pollution of phosphorus to environment significantly can be reduced.
The zymin applied in fodder industry at present mainly contains 4 large classes: be used for degraded cellulose, protein, starch and phytic acid respectively.
Fiber degradation enzyme: for monogastric animal, the maximum resistance of digestion is the enzyme that can not produce degradation of fibers, and in the daily ration containing components such as wheat, barley, oats, fiber is araboxylan and beta-glucan greatly.Water miscible fiber can improve the viscosity of small intestine contents, hinders the absorption of nutrient, thus reduces the growth performance of animal.This situation is also relevant with the disease that some cause due to maldigestion simultaneously.As the appetite stimulator of pig, the black toe disease of fowl and piggy have loose bowels.Due to the impact of the factors such as kind, growth place gentle time condition, the altering a great deal of fibre content in barley and wheat, causes the nutritive value of the daily ration containing these components widely different.Fiber degradation enzyme, zytase and beta-glucan can reduce these differences, improve growth performance and the reguarity of animal.Some dyspeptic disease can also be reduced simultaneously.
Proteolytic degradation enzyme: protein is from all feeds raw material in animal diets, they are accumulated in lean meat eventually through the amino acid of degraded.In monogastric animal daily ration, add proteolytic enzyme (DIFFERENT FEED material protein and quality and utilizability) except major part storage protein or Storage protein or for except the available small-molecular peptides of animal fully can be degraded, feed nutritive value can also be improved by degraded anti-nutritional factors.The efficiency variance stockpiled is very large.At plant protein source, as there are some antinutritional factor in soybean cake powder, as a few tannin and trypsin ihhibitor, may cause damage to intestinal absorption surface, affecting nutraceutical absorption.In addition, the incomplete digestion system of young animal makes the protein in vegetable-protein (as soybean cake powder) well not utilized.
Starch degrading enzyme: many nutritionists think cornfeedstuff raw material " golden standard ".Large absolutely number nutritionist thinks that corn does not exist lienteric, digestibility is more than 95%, but Noy and Sklan research recently shows (1994) in the ideal situation, in the daily ration of broiler of 4-12 ages in days, the digestibility of starch rarely exceeds 85%, adds amylase and starch can be made to obtain manyly degrading faster at small intestine.In the weaned piglet phase, due to nutrition, environment and immune change, body weight can decline.In daily ration, add amylase and some other enzyme, the endogenous digestive ferment secretion of animal can be increased, and then improve digesting and assimilating of nutrition, improve food conversion ratio and growth of animal rate.
Phytic acid degrading enzyme: for all animals, phosphorus is all vital for the mineralising of bone, immunity, breeding, growth.Pig and poultry monogastric animal can only in utilize in plant feed 30-40% phosphorus, all the other phytate phosphorus of 60-70% are unserviceable.In many cases, inorganic phosphorus must be supplemented in feed diet to meet the needs of growth of animal.Phosphorus over half in feed is discharged in environment along with ight soil, contaminate environment.Add phytase can to degrade phytic acid, the phosphorus in release phytate molecule.2 benefits can be produced like this: 1. the addition decreasing Dietary phosphorus.2. decrease feces of livestock and poultry to pollute the phosphorus of environment.
Apparent: as four large leading roles of feed enzyme, their mechanism of action and pattern determine or have promoted animal-feed industry to use for the absorption of zymin technology to a great extent.The ratio for input and output example of enzyme is added more than 2:1 at present in broiler chicken material.Comparatively speaking, in pig industry field, the service condition of zymin, with regard to more complicated, seems uncertain.Intensive degree is low, and relate to link many, the result of use of zymin is difficult to carry out business calculating.Although had the imagination using zymin, until just start the eighties in 20th century to understand the strength how playing enzyme in fodder industry in the 1950's.Feed grains, as Wheat and barley all contains the unavailable fiber of higher monogastric animal.As fruit fiber can be degraded, animal just can utilize nutrition better.In Europe, barley is relatively cheap, and bird nutritionist and zymologist have dropped into great effort and have studied add beta-glucan enzyme to reduce the possibility of its negative impact in containing the daily ration of broiler of barley.Its result is proved to be successful, and obtains 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 also obtain successfully.In the mid-90 in 20th century, enzyme obtains at fodder industry generally to be approved.Can not rant out: 1996, containing fiber degradation enzyme in the broiler chicken material (viscosity cereal is energy derive) in Europe 80%.Strengthen thus and accelerate the application of feed industry to new technology.In the world, the poultry feed that can produce viscosity cereal that contains of about 65% with the addition of fiber degradation enzyme.And application percentage in pig feed is much lower, close to 10%.Its major cause is the complicated structure in market, and market is diversification, even cannot calculate.From regional distribution, the area of cellulose degrading enzyme is used mainly to concentrate on the producing region of viscosity cereal as main energetic feed, such as: 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 uses the core of degraded cellulose enzyme to segment market.In order to obtain the accreditation in the whole world, feeding enzyme producer must carry out on a large scale marching based on corn---the North America of soybean meal based diets and the Asian-Pacific area.Corn---soybean meal based diets 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 kind of daily ration problem relevant to robust 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 obtains maximum economy return.It is estimated, this feed a part enzyme market share is 2,000 ten thousand dollars, and the actual broiler fodder at use corn soybean diet only has 5% for enzyme-added feed.1999/2000 year one reduction viscosity and robust fibre are that the feed enzyme marketable value of daily ration is more than 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, about has the animal and fowl fodder of about 8.0% to add phytase in the whole world.Except the reason of economic interests, also have a factor to be the reduction of the content of phytate phosphorus in excrement and be conducive to protection of the environment.
In sum, the market space that the application of Wheat ration enzyme has it wide and huge economic worth, but the thermostability of Wheat ration enzyme, security, composite comprehensive and action effect give full play to the major issue being still zymin manufacturer and numerous raisers and jointly paying close attention to, prepare safer, more comprehensively, the better Wheat ration enzyme of enzyme action effect is corporation responsibility and the pursuit of industry technician.
Summary of the invention
Technical problem solved by the invention is to contain high enzymatic activity, action condition is wide in range, based on the subtilis culture of the neutral protease that stability is strong, and the composite Chinese herbal medicine extract of science, protective material, activator and other food grade feed enzyme, the obtained Wheat ration enzyme containing neutral protease, not only provide safety for raising livestock and poultry, comprehensive digestive ferment, alleviate digestion burden, improve raw material availability and growth rate, available protecting environment, appropriate activator under equal conditions can give full play to the effect of zymin simultaneously, make the best use of everything, save zymin addition, the science composite quality guaranteed period that both can extend compound enzymic preparation of Chinese herbal medicine extract, the immunizing power of raising livestock and poultry can be improved again, thus 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 Wheat ration enzyme for neutral protease, be made up of the zymin of following parts by weight:
Subtilis culture 20-30 part; aspartic protease 20-30 part; acidic xylanase 20-30 part; cellulase 20-30 part, beta-glucanase 15-20 part, amylase 15-20 part; 'beta '-mannase 10-15 part; Chinese herbal medicine extract 10-15 part, protective material 10-15 part, activator 10-15 part.
Described aspartic protease, acidic xylanase, cellulase, beta-glucanase, amylase, 'beta '-mannase, be food-grade enzyme preparation;
Described subtilis culture is prepared through liquid submerged fermentation by the Strains B. subtilis 1398-2-12 producing heat-flash stability neutral protease, and its composition mainly comprises neutral protease and subtilis thalline;
The preparation method of described subtilis culture is as follows:
Subtilis 1398-2-12 activates through slant strains and enlarged culturing (comprising one, two, three seed culture and first class seed pot cultivation) obtains liquid seeds step by step; By liquid seeds with 6% inoculum size access fermentor tank, culture temperature 30-36 DEG C, stirring velocity 200-700rpm, ventilation (V/V) 1:1-3, incubation time 10-15h; Then with 1-2 DEG C/h rate of temperature fall slow cooling to 10-15 DEG C, constant temperature culture 15-20h; Continue with 1-2 DEG C/h rate of temperature fall slow cooling to 2-5 DEG C, now, liquid seeds is added access fermentor tank with 4% inoculum size, constant temperature culture 20-30h; Finally slowly be warming up to 10-15 DEG C, constant temperature culture 15-20h with 1-2 DEG C/h temperature rise rate; Continue slowly to be warming up to 30-36 DEG C, constant temperature culture 15-20h with 1-2 DEG C/h temperature rise rate; 10-1000 μm, fermentation liquor aperture filter board coarse filtration, then obtains solid content 20-40% concentrated solution in the concentrated moisture of removing of 10-20 DEG C of loop ultrafiltration; Concentrated solution obtains subtilis culture through vacuum lyophilization, micronizing.
Described slant medium consists of: extractum carnis 3-10g, sodium-chlor 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 DEG C of sterilizing 20min;
The preparation method of described Chinese herbal medicine powder is as follows:
With weight parts, 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 less than 2 millimeters, then Homogeneous phase mixing add the water of 3-6 times of weight in container, control temperature 70 DEG C ~ 90 DEG C keeps 2 ~ 4h, then being cooled to 45-60 DEG C, adding mixing enzyme preparation and carry out enzymolysis, is 5.5-6.8 by lactic acid adjust ph, enzymolysis 2-4h, finally add the mixture of mixture 0.5-3 times of w ethanol and propyl alcohol, control temperature to 60 DEG C ~ 78 DEG C keeps 3 ~ 4h, filters; Filter vacuum concentrates postlyophilization and obtains Chinese herbal medicine powder.
Described mixed enzyme addition is the 5-10% of mixture gross weight.
The parts by weight of described mixed enzyme consist of: endo-beta-glucanase 10-20 part, outer 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 protease 10-15 part, aspartic 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.
One, two, three seed culture medium weight described consists of:
Yeast powder 0.3-0.5%, glucose 1-1.5%, peptone 0.3-0.5%, extractum carnis 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%, Trisodium Citrate 0.1-0.3%, insufficient section pure water is supplied, pH value 7.0-7.2,121-123 DEG C of sterilizing 30-40min.
Described first class seed pot substratum weight consists of:
Maltodextrin 5-15%, yeast powder 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%, Trisodium Citrate 0.1-0.5%, insufficient section pure water is supplied, pH value 7.0-7.2,121-123 DEG C of sterilizing 30-40min.
Described seeding tank fermented liquid cell concentration is 7.0x10 8-8.0x10 8individual/ml;
Described fermention medium consists of: maltodextrin 50-150g, Semen Maydis powder 50-60g, soybean cake powder 15-25g, Chinese herbal medicine powder 30-50g, trehalose 30-40g, yeast powder 4-8g, corn steep liquor 1-5g, ammonium sulfate 1-3g, dipotassium hydrogen phosphate 1-2g, potassium primary phosphate 1-2g, Trisodium Citrate 1-5g, defoamer 0.1-1g, pure water l000mL, pH value 7.0-7.2,121 DEG C of sterilizing 20min;
Described supplemented medium weight consists of: maltodextrin 20-30%, Semen Maydis powder 10-20%, bean powder 15-25%, Chinese herbal medicine powder 5-10%, and insufficient section pure water is supplied, pH value 7.0-7.2,121-123 DEG C of sterilizing 30-40min.
The concocting method of described fermention medium is:
Accurately take raw material in proportion, pure water in raw material, Semen Maydis powder, soybean cake powder are dropped in material-compound tank, regulate pH value 7.0-7.2, add middle temperature amylase (3u/g Semen Maydis powder) and alpha-amylase (30u/g Semen Maydis powder), DEG C insulation 15-30min of warming while stirring to 70 simultaneously, be then slowly warming up to 90 DEG C of insulation 15-30min and liquefy, finally add other raw material, stir, adjust initial pH7.0-7.2,121-123 DEG C of sterilizing 30-40min for subsequent use.
The preparation method of described Chinese herbal medicine extract is: respectively Chinese herbal medicine powder being broken to particle diameter is less than 2 millimeters, then Homogeneous phase mixing add the water of 3-6 times of weight in container, control temperature 70 DEG C ~ 90 DEG C keeps 2 ~ 4h, then being cooled to 45-60 DEG C, adding mixing enzyme preparation and carry out enzymolysis, is 5.5-6.8 by lactic acid adjust ph, enzymolysis 2-4h, finally add the mixture of mixture 0.5-3 times of w ethanol and propyl alcohol, control temperature to 60 DEG C ~ 78 DEG C keeps 3 ~ 4h, filters; Filtrate reduced in volume to solid content is more than 20%, and then lyophilize obtains Chinese herbal medicine extract.
The parts by weight of described herbal medicine consist of: sea-buckthorn 20-30 part, Semen Cassiae 20-30 part, matrimony vine 10-15 part, Chinese yam 10-15 part, Root of coastal Glehnia 5-10 part, Fructus Viticis 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 mixture gross weight;
The parts by weight of described mixed enzyme consist of: endo-beta-glucanase 10-20 part, outer 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 protease 10-15 part, aspartic protease 10-15 part, superoxide-dismutase 5-10 part, glucose oxidase 5-10 part, acid phosphatase 5-10 part;
Described protective material 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, halfcystine 10-15 part.
Described activator is formed by the inorganic salt Homogeneous phase mixing of following quality component: zinc chloride 30-40 part, calcium chloride 10-20 part, sodium sulfate 10-20 part, magnesium chloride 5-10 part.
The present invention is containing the preparation method of the Wheat ration enzyme of neutral protease:
By described protective material, Chinese herbal medicine extract micronizing respectively; guarantee that granularity is less than other zymin; then with subtilis culture, aspartic protease, acidic xylanase, cellulase, beta-glucanase, amylase, 'beta '-mannase, Homogeneous phase mixing; finally add activator, after mixing, pack the Wheat ration enzyme got product containing neutral protease.
Described Wheat ration enzyme is applicable to feed-processing plant and plant's autogamy feed, should mix, can the present invention be mixed with a small amount of feed in advance during use with other raw material in feed, remix in large quantities of feed, Direct-fed.Advise that complete diet pellet addition per ton is: when wheat weighs less than 30% in feed, add 100g; When wheat weight is more than 30%, add 120-150g;
Subtilis 1398-2-12 provided by the invention produces subtilis (Bacillus subtilis) 1398-2 of neutral protease through UV-LiCl-ethyl sulfate Mutation screening acquisition by a strain of Laboratories Accession.
The bacterial strain of product heat-flash stability neutral protease provided by the invention is specially subtilis 1398-2-12.This bacterial strain is preserved in China typical 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: subtilis (Bacillus subtilis) 1398-2-12.
Described subtilis (Bacillus subtilis) 1398-2-12 bacterial strain feature is as follows:
Described bacterial strain colony colour on solid plate is oyster white, and surface drying is opaque, neat in edge, for having the aerobic bacteria of mobility.Microscopy is elongated rod shape, and gramstaining is positive.This bacterium can utilize Citrate trianion, and nitrate reductase, V-P test into the positive.
Subtilis 1398-2-12 provided by the invention has the advantages that produced neutral protease tolerable temperature is high, be suitable for pH value wide scope, fermented liquid crude enzyme liquid 75 DEG C of enzymes complete stability alive, optimal reactive temperature 70 DEG C, 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 DEG C, the suitableeest product enzyme temperature 32-35 DEG C.
By mutagenesis screening scheme, mutant strain step-sizing is eliminated, finally to strain excellent through leavening property test screen, obtain the Strains B. subtilis bacterium 1398-2-12 that heat-flash stability neutral protease is produced in a strain, the work of fermented liquid neutral protease enzyme can reach 5500-7000U/mL, the thermostability of enzyme is analyzed, at crude enzyme liquid is placed in 40 DEG C, 45 DEG C, 50 DEG C, 55 DEG C, 60 DEG C, 65 DEG C, 70 DEG C, 75 DEG C respectively, lives every 10 minutes sampling and measuring enzymes.At 40 DEG C, 45 DEG C, 50 DEG C, 55 DEG C, 60 minutes enzymes are lived and are not declined.At 60 DEG C and 65 DEG C, what within 30 minutes, drop to constitutive enzyme work drops to 85% in 95%, 60 minutes.At 70 DEG C, what within 30 minutes, drop to constitutive enzyme work drops to 80% in 85%, 60 minutes.At 75 DEG C, what within 30 minutes, drop to constitutive enzyme work drops to 70% in 80%, 60 minutes.
Beneficial effect:
1. the neutral protease enzyme work contained in subtilis culture fermentation broth of the present invention can reach 5500-7000U/mL, and between 40-70 DEG C, have higher enzyme live, optimal reactive temperature is 70 DEG C; The enzyme complete stability alive when pH value is 4.5-8.5, optimal reaction pH value is 7.2; This enzyme still can keep more than 80% enzyme to live preserve 1h under 70 DEG C of conditions after, keep more than 70% enzyme to live after preserving 1h under 75 DEG C of conditions.Stronger than existing neutral protein enzyme heat stability, enzyme activity is higher, enzyme effect optimum pH wide scope, and stability in storage is high, is more applicable to the interpolation of raising animal and fowl fodder.
2. the subtilis thalline in subtilis culture of the present invention can improve animal body immunizing power, Promote immunity organ growth, promote the maturation of animal intestinal structure and function, improve animal day weight gain and improve feed conversion rate, be more applicable to the interpolation of raising animal and fowl fodder.
3. the present invention adopts polysaccharide, inorganic salt and amino acid science composite containing the protective material in the Wheat ration enzyme of neutral protease, effectively slow down the moisture regain of compound enzymic preparation; Can strengthen prozyme simultaneously resistance toly to freeze, resistance toheat, keep identical enzyme activity, its heat resisting temperature can improve 20-30 DEG C, resistance to freezing temp can reduce 10-15 DEG C, effectively prevent the loss of prozyme enzyme activity in transport, preservation and use procedure, extend the quality guaranteed period of prozyme, reach same enzyme activity, can 2-3 be extended than the like product quality guaranteed period.
4. the present invention adds inorganic salt as activator containing the Wheat ration enzyme of neutral protease; create the top condition of enzyme catalysis; give full play to the vigor of each enzyme component of prozyme; the macromolecular substance such as starch, protein, Mierocrystalline cellulose, phytic acid are thoroughly effectively decomposed in feed; significantly reduce the digestion burden of livestock and poultry animal; improve the growth rate of raw material availability and livestock and poultry, effectively prevent the environmental pollution that feces of livestock and poultry causes simultaneously, protect feeding environment.
5. the present invention both can extend the quality guaranteed period of compound enzymic preparation containing the Chinese herbal medicine extract that the Wheat ration enzyme of neutral protease is added, and can improve again the immunizing power of raising livestock and poultry, effectively prevent the generation of livestock and poultry epidemic disease.
6. the present invention is containing the synergy of subtilis culture, protective material, activator, Chinese herbal medicine extract and zymin in the Wheat ration enzyme of neutral protease; the enzyme activity of prozyme and effect are played to greatest extent; and improve the utilization ratio of feed and the growth rate of animal accordingly; enhance appetite and the resistance against diseases of animal, extend the quality guaranteed period of prozyme and protect environment.
Embodiment
The present invention is described below by specific embodiment.Unless stated otherwise, technique means used in the present invention 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 only limited by claims.To those skilled in the art, under the prerequisite not deviating from essence of the present invention and scope, the various change carry out the material component in these embodiments and consumption or change also belong to protection scope of the present invention.
Embodiment 1
Containing a Wheat ration enzyme for neutral protease, be made up of the zymin of following parts by weight:
Subtilis culture 25 parts, aspartic protease 25 parts, acidic xylanase 25 parts, cellulase 25 parts, beta-glucanase 17 parts, amylase 17 parts, 'beta '-mannase 12 parts, Chinese herbal medicine extract 12 parts, protective material 13 parts, activator 12 parts.
Described aspartic protease, acidic xylanase, cellulase, beta-glucanase, amylase, 'beta '-mannase, be food-grade enzyme preparation;
The preparation method of described subtilis culture comprises the steps:
(1) slant strains of intact subtilis 1398-2-12 is inoculated in slant medium, cultivates 24h for 30 DEG C and carry out actication of culture, so activation 2 times;
Described slant medium consists of: extractum carnis 3g, sodium-chlor 5g, peptone 10g, glucose 2g, (NH) 2sO 43g, K 2hPO 46g, CaCl 21g, agar 15g, Chinese herbal medicine powder 5g, distilled water l000mL, pH value 7.0,121 DEG C of sterilizing 20min;
The preparation method of described Chinese herbal medicine powder is as follows:
With weight parts, take the Radix Astragali 20 parts; Radix Codonopsis 10 parts; Radix bupleuri 10 parts; The root of large-flowered skullcap 10 parts; Respectively said herbal medicine being crushed to particle diameter is less than 2 millimeters, then Homogeneous phase mixing add the water of 3 times of weight in container, control temperature 70 DEG C keeps 2h, and be then cooled to 45 DEG C, the mixing enzyme preparation adding mixture gross weight 5% carries out enzymolysis, be 5.5 by lactic acid adjust ph, enzymolysis 2h, finally add the mixture of mixture 0.5 times of w ethanol and propyl alcohol, the mass ratio of described ethanol and propyl alcohol is 1:1, control temperature to 60 DEG C keeps 3h, filters; Filter vacuum concentrates postlyophilization and obtains Chinese herbal medicine powder.
The parts by weight of described mixed enzyme consist of: endo-beta-glucanase 10 parts, outer beta-glucanase 10 parts, beta-glucosidase 10 parts, zytase 15 parts, pentosanase 15 parts, Pullulanase 20 parts, beta-amylase 10 parts, neutral protease 10 parts, aspartic protease 10 parts, superoxide-dismutase 5 parts, glucose oxidase 5 parts, acid phosphatase 5 parts.
(2) liquid seeds enlarged culturing
1. first order seed is cultivated: slant strains 1 articulating after step (1) being activated enters in 500 ml shake flasks, substratum loading amount 100 milliliters, rotary shaker 180 revs/min, culture temperature 30 DEG C, incubation time 10h;
2. secondary seed cultivate: by first order seed according to 10% inoculum size access in 500 milliliters of secondary seed shaking flasks, culture condition is identical with first order seed;
3. three grades of seed culture: secondary seed is accessed in 5000 milliliters of three grades of seed flask with 10% inoculum size, substratum loading amount 1000 milliliters, rotary shaker 100 revs/min, culture temperature 30 DEG C, incubation time 10h;
4. first class seed pot is cultivated: the first class seed pot being 150L with 10% inoculum size access cubic capacity by three grades of seeds, fermention medium loading amount 100L, culture temperature 30 DEG C, stirring velocity 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:
Yeast powder 0.3%, glucose 1%, peptone 0.3%, extractum carnis 0.5%, dipotassium hydrogen phosphate 0.8%, Chinese herbal medicine powder 1.5%, trehalose 1%, calcium sulfate 0.1%, magnesium chloride 0.2%, Trisodium Citrate 0.1%, insufficient section pure water is supplied, pH value 7.0,121 DEG C of sterilizing 30min.
Described first class seed pot substratum weight consists of:
Maltodextrin 5%, yeast powder 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%, Trisodium Citrate 0.1%, insufficient section pure water is supplied, pH value 7.0,121 DEG C of sterilizing 30min.
Described seeding tank fermented liquid cell concentration is 7.0x10 8individual/ml;
(3) ferment tank
By first class seed pot fermented liquid in step (2) with 6% inoculum size access fermentor tank, culture temperature 30 DEG C, stirring velocity 200r/m, ventilation (V/V) 1:1, incubation time 10h; Then with 1 DEG C/h rate of temperature fall slow cooling to 10 DEG C, constant temperature culture 15h; Continue with 1 DEG C/h rate of temperature fall slow cooling to 2 DEG C, now, first class seed pot fermented liquid in step (2) is added access fermentor tank with 4% inoculum size, constant temperature culture 20h; Finally slowly be warming up to 10 DEG C, constant temperature culture 15h with 1 DEG C/h temperature rise rate; Continue slowly to be warming up to 30 DEG C, constant temperature culture 15h with 1 DEG C/h temperature rise rate;
Dissolved oxygen controls: by adjustment mixing speed and ventilation, controls dissolved oxygen 15%;
PH controls: by mending ammoniacal liquor or dilute phosphoric acid, controls pH value in fermenting process and remains on 7.0;
Control of additive raw material: add supplemented medium, to maintain fermented liquid reducing sugar content for 2-5mg/ml;
Put tank standard: the old and feeble self-dissolving of 60-80% thalline, enzyme activity increasess slowly.
Described fermention medium consists of: maltodextrin 50g, Semen Maydis powder 50g, soybean cake powder 15g, Chinese herbal medicine powder 30g, trehalose 30g, yeast powder 4g, corn steep liquor 1g, ammonium sulfate 1g, dipotassium hydrogen phosphate 1g, potassium primary phosphate 1g, Trisodium Citrate 1g, defoamer 0.1g, pure water l000mL, pH value 7.0,121 DEG C of sterilizing 20min;
Described supplemented medium weight consists of: maltodextrin 20%, Semen Maydis powder 10%, bean powder 15%, Chinese herbal medicine powder 5%, and insufficient section pure water is supplied, pH value 7.0,121 DEG C of sterilizing 30min.
The concocting method of described fermention medium is:
Accurately take raw material in proportion, pure water in raw material, Semen Maydis powder, soybean cake powder are dropped in material-compound tank, regulate pH value 7.0, add middle temperature amylase (3u/g Semen Maydis powder) and alpha-amylase (30u/g Semen Maydis powder), DEG C insulation 15min of warming while stirring to 70 simultaneously, be then slowly warming up to 90 DEG C of insulation 15min and liquefy, finally add other raw material, stir, adjust initial pH7.0,121 DEG C of sterilizing 30min are for subsequent use.
(4) 10 μm, fermentation liquor aperture filter board coarse filtration, then obtaining solid content in 10 DEG C of loop ultrafiltrations concentrated removal moisture is 20% concentrated solution; Concentrated solution obtains subtilis culture through vacuum lyophilization, micronizing.
The preparation method of described Chinese herbal medicine extract is: respectively Chinese herbal medicine powder being broken to particle diameter is less than 2 millimeters, then Homogeneous phase mixing add the water of 5 times of weight in container, control temperature 80 DEG C keeps 3h, then being cooled to 53 DEG C, adding mixing enzyme preparation and carry out enzymolysis, is 6.2 by lactic acid adjust ph, enzymolysis 3h, finally add the mixture of mixture 2 times of w ethanol and propyl alcohol, control temperature to 69 DEG C keeps 4h, filters; Filtrate reduced in volume to solid content is more than 20%, and then lyophilize obtains Chinese herbal medicine extract.
The parts by weight of described herbal medicine consist of: sea-buckthorn 25 parts, Semen Cassiae 25 parts, matrimony vine 17 parts, Chinese yam 13 parts, Root of coastal Glehnia 8 parts, Fructus Viticis Negundo 7 parts, radix polygonati officinalis 4 parts, the seed of Job's tears 4 parts, Fructus Hordei Germinatus 4 parts, sweet osmanthus 4 parts, the Radix Astragali 4 parts;
Mixed enzyme addition is the 5-10% of mixture gross weight;
The parts by weight of described mixed enzyme consist of: endo-beta-glucanase 15 parts, outer beta-glucanase 15 parts, beta-glucosidase 13 parts, zytase 13 parts, pentosanase 13 parts, Pullulanase 25 parts, beta-amylase 12 parts, neutral protease 13 parts, aspartic protease 13 parts, superoxide-dismutase 7 parts, glucose oxidase 7 parts, acid phosphatase 7 parts;
Described protective material is made up of the raw material of following parts by weight: trehalose 25 parts, NaCl25 part, (NH 4) 2sO 413 parts, halfcystine 12 parts.
Described activator is formed by the inorganic salt Homogeneous phase mixing of following quality component: zinc chloride 35 parts, 15 parts, calcium chloride, 15 parts, sodium sulfate, 7 parts, magnesium chloride.
Preparation method containing the Wheat ration enzyme of neutral protease:
By described protective material, Chinese herbal medicine extract micronizing respectively; guarantee that granularity is less than other zymin; then with subtilis culture, aspartic protease, acidic xylanase, cellulase, beta-glucanase, amylase, 'beta '-mannase, Homogeneous phase mixing; finally add activator, after mixing, pack the Wheat ration enzyme got product containing neutral protease.
Embodiment 2:
Containing a Wheat ration enzyme for neutral protease, be made up of the zymin of following parts by weight:
Subtilis culture 20 parts, aspartic protease 20 parts, acidic xylanase 20 parts, cellulase 20 parts, beta-glucanase 15 parts, amylase 15 parts, 'beta '-mannase 10 parts, Chinese herbal medicine extract 10 parts, protective material 10 parts, activator 10 parts.
Described aspartic protease, acidic xylanase, cellulase, beta-glucanase, amylase, 'beta '-mannase are food-grade enzyme preparation;
The preparation method of described subtilis culture comprises the steps:
(1) actication of culture
The slant strains of intact subtilis 1398-2-12 is inoculated in slant medium, cultivates 30h for 33 DEG C and carry out actication of culture, so activation 2 times;
Described slant medium consists of: extractum carnis 6g, sodium-chlor 8g, peptone 15g, glucose 4g, (NH) 2sO 44g, K 2hPO 47g, CaCl 22g, agar 18g, Chinese herbal medicine powder 8g, distilled water l000mL, pH value 7.2,121 DEG C of sterilizing 20min;
The preparation method of described Chinese herbal medicine powder is as follows:
With weight parts, take the Radix Astragali 25 parts; Radix Codonopsis 16 parts; Radix bupleuri 12 parts; The root of large-flowered skullcap 12 parts; Respectively said herbal medicine being crushed to particle diameter is less than 2 millimeters, then Homogeneous phase mixing add the water of 5 times of weight in container, control temperature 80 DEG C keeps 3h, and be then cooled to 50 DEG C, the mixing enzyme preparation adding mixture gross weight 8% carries out enzymolysis, be 6.0 by lactic acid adjust ph, enzymolysis 3h, finally add the mixture of mixture 2 times of w ethanol and propyl alcohol, the mass ratio of described ethanol and propyl alcohol is 1:1.2, control temperature to 70 DEG C keeps 4h, filters; Filter vacuum concentrates postlyophilization and obtains Chinese herbal medicine powder.
The parts by weight of described mixed enzyme consist of: endo-beta-glucanase 15 parts, outer beta-glucanase 15 parts, beta-glucosidase 12 parts, zytase 18 parts, pentosanase 18 parts, Pullulanase 25 parts, beta-amylase 12 parts, neutral protease 12 parts, aspartic protease 12 parts, superoxide-dismutase 8 parts, glucose oxidase 8 parts, acid phosphatase 8 parts.
(2) liquid seeds enlarged culturing
1. first order seed is cultivated: slant strains 2 articulating after step (1) being activated enters in 500 ml shake flasks, substratum loading amount 100 milliliters, rotary shaker 180 revs/min, culture temperature 33 DEG C, incubation time 12h;
2. secondary seed cultivate: by first order seed according to 10% inoculum size access in 500 milliliters of secondary seed shaking flasks, culture condition is identical with first order seed;
3. three grades of seed culture: secondary seed is accessed in 5000 milliliters of three grades of seed flask with 10% inoculum size, substratum loading amount 1000 milliliters, rotary shaker 100 revs/min, culture temperature 33 DEG C, incubation time 12h;
4. first class seed pot is cultivated: the first class seed pot being 150L with 10% inoculum size access cubic capacity by three grades of seeds, fermention medium loading amount 100L, culture temperature 33 DEG C, stirring velocity 300rpm, ventilation (V/V) 1:1.5, tank pressure 0.05Mpa, incubation time 15h;
Described one-level, secondary, three grades of seed culture medium weight consist of:
Yeast powder 0.4%, glucose 1.2%, peptone 0.4%, extractum carnis 0.6%, dipotassium hydrogen phosphate 1.0%, Chinese herbal medicine powder 1.8%, trehalose 2%, calcium sulfate 0.1%, magnesium chloride 0.2%, Trisodium Citrate 0.2%, insufficient section pure water is supplied, pH value 7.2,121 DEG C of sterilizing 30min.
Described first class seed pot substratum weight consists of:
Maltodextrin 10%, yeast powder 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%, Trisodium Citrate 0.3%, insufficient section pure water is supplied, pH value 7.2,121 DEG C of sterilizing 30min.
Described seeding tank fermented liquid cell concentration is 7.5x10 8individual/ml;
(3) ferment tank
By first class seed pot fermented liquid in step (2) with 6% inoculum size access fermentor tank, culture temperature 35 DEG C, stirring velocity 400r/m, ventilation (V/V) 1:2, incubation time 12h; Then with 2 DEG C/h rate of temperature fall slow cooling to 12 DEG C, constant temperature culture 18h; Continue with 2 DEG C/h rate of temperature fall slow cooling to 4 DEG C, now, first class seed pot fermented liquid in step (2) is added access fermentor tank with 4% inoculum size, constant temperature culture 25h; Finally slowly be warming up to 12 DEG C, constant temperature culture 18h with 2 DEG C/h temperature rise rate; Continue slowly to be warming up to 33 DEG C, constant temperature culture 18h with 2 DEG C/h temperature rise rate;
Dissolved oxygen controls: by adjustment mixing speed and ventilation, controls dissolved oxygen 20%;
PH controls: by mending ammoniacal liquor or dilute phosphoric acid, controls pH value in fermenting process and remains on 7.2;
Control of additive raw material: add supplemented medium, to maintain fermented liquid reducing sugar content for 2-5mg/ml;
Put tank standard: the old and feeble self-dissolving of 60-80% thalline, enzyme activity increasess slowly.
Described fermention medium consists of: maltodextrin 100g, Semen Maydis powder 55g, soybean cake powder 20g, Chinese herbal medicine powder 40g, trehalose 35g, yeast powder 6g, corn steep liquor 3g, ammonium sulfate 2g, dipotassium hydrogen phosphate 2g, potassium primary phosphate 2g, Trisodium Citrate 3g, defoamer 0.5g, pure water l000mL, pH value 7.2,121 DEG C of sterilizing 20min;
Described supplemented medium weight consists of: maltodextrin 25%, Semen Maydis powder 15%, bean powder 20%, Chinese herbal medicine powder 8%, and insufficient section pure water is supplied, pH value 7.2,121 DEG C of sterilizing 30min.
The concocting method of described fermention medium is:
Accurately take raw material in proportion, pure water in raw material, Semen Maydis powder, soybean cake powder are dropped in material-compound tank, regulate pH value 7.2, add middle temperature amylase (3u/g Semen Maydis powder) and alpha-amylase (30u/g Semen Maydis powder), DEG C insulation 20min of warming while stirring to 70 simultaneously, be then slowly warming up to 90 DEG C of insulation 20min and liquefy, finally add other raw material, stir, adjust initial pH7.2,121 DEG C of sterilizing 30min are for subsequent use.
(4) 500 μm, fermentation liquor aperture filter board coarse filtration, then obtaining solid content in 15 DEG C of loop ultrafiltrations concentrated removal moisture is 30% concentrated solution; Concentrated solution obtains subtilis culture through vacuum lyophilization, micronizing.
The preparation method of described Chinese herbal medicine extract is: respectively Chinese herbal medicine powder being broken to particle diameter is less than 2 millimeters, then Homogeneous phase mixing add the water of 3 times of weight in container, control temperature 70 DEG C keeps 2h, then being cooled to 45 DEG C, adding mixing enzyme preparation and carry out enzymolysis, is 5.5 by lactic acid adjust ph, enzymolysis 2h, finally add the mixture of mixture 0.5 times of w ethanol and propyl alcohol, control temperature to 60 DEG C keeps 3h, filters; Filtrate reduced in volume to solid content is more than 20%, and then lyophilize obtains Chinese herbal medicine extract.
The parts by weight of described herbal medicine consist of: sea-buckthorn 20 parts, Semen Cassiae 20 parts, matrimony vine 10 parts, Chinese yam 10 parts, Root of coastal Glehnia 5 parts, Fructus Viticis Negundo 5 parts, radix polygonati officinalis 3 parts, the seed of Job's tears 3 parts, Fructus Hordei Germinatus 3 parts, sweet osmanthus 3 parts, the Radix Astragali 3 parts;
Mixed enzyme addition is 5% of mixture gross weight;
The parts by weight of described mixed enzyme consist of: endo-beta-glucanase 10 parts, outer beta-glucanase 10 parts, beta-glucosidase 10 parts, zytase 15 parts, pentosanase 15 parts, Pullulanase 20 parts, beta-amylase 10 parts, neutral protease 10 parts, aspartic protease 10 parts, superoxide-dismutase 5 parts, glucose oxidase 5 parts, acid phosphatase 5 parts;
Described protective material is made up of the raw material of following parts by weight: trehalose 20 parts, NaCl20 part, (NH 4) 2sO 410 parts, halfcystine 10 parts.
Described activator is formed by the inorganic salt Homogeneous phase mixing of following quality component: zinc chloride 30 parts, 10 parts, calcium chloride, 10 parts, sodium sulfate, 5 parts, magnesium chloride.
Containing the preparation method of the Wheat ration enzyme of neutral protease as embodiment 1.
Embodiment 3:
Containing a Wheat ration enzyme for neutral protease, be made up of the zymin of following parts by weight:
Subtilis culture 30 parts, aspartic protease 30 parts, acidic xylanase 30 parts, cellulase 30 parts, beta-glucanase 20 parts, amylase 20 part, 'beta '-mannase 15 parts, Chinese herbal medicine extract 15 parts, protective material 15 parts, activator 15 parts.
Described aspartic protease, acidic xylanase, cellulase, beta-glucanase, amylase, 'beta '-mannase, be food-grade enzyme preparation;
The preparation method of described subtilis culture comprises the steps:
(1) actication of culture
The slant strains of intact subtilis 1398-2-12 is inoculated in slant medium, cultivates 36h for 36 DEG C and carry out actication of culture, so activation 3 times;
Described slant medium consists of: extractum carnis 10g, sodium-chlor 12g, peptone 20g, glucose 5g, (NH) 2sO 45g, K 2hPO 48g, CaCl 23g, agar 20g, Chinese herbal medicine powder 10g, distilled water l000mL, pH value 7.2,121 DEG C of sterilizing 20min;
The preparation method of described Chinese herbal medicine powder is as follows:
With weight parts, take the Radix Astragali 30 parts; Radix Codonopsis 18 parts; Radix bupleuri 15 parts; The root of large-flowered skullcap 15 parts; Respectively said herbal medicine being crushed to particle diameter is less than 2 millimeters, then Homogeneous phase mixing add the water of 6 times of weight in container, control temperature 90 DEG C keeps 4h, and be then cooled to 60 DEG C, the mixing enzyme preparation adding mixture gross weight 10% carries out enzymolysis, be 6.8 by lactic acid adjust ph, enzymolysis 4h, finally add the mixture of mixture 3 times of w ethanol and propyl alcohol, the mass ratio of described ethanol and propyl alcohol is 1:1.5, control temperature to 78 DEG C keeps 4h, filters; Filter vacuum concentrates postlyophilization and obtains Chinese herbal medicine powder.
The parts by weight of described mixed enzyme consist of: endo-beta-glucanase 20 parts, outer beta-glucanase 20 parts, beta-glucosidase 15 parts, zytase 20 parts, pentosanase 20 parts, Pullulanase 30 parts, beta-amylase 15 parts, neutral protease 15 parts, aspartic protease 15 parts, superoxide-dismutase 10 parts, glucose oxidase 10 parts, acid phosphatase 10 parts.
(2) liquid seeds enlarged culturing
1. first order seed is cultivated: slant strains 2 articulating after step (1) being activated enters in 500 ml shake flasks, substratum loading amount 100 milliliters, rotary shaker 180 revs/min, culture temperature 36 DEG C, incubation time 15h;
2. secondary seed cultivate: by first order seed according to 10% inoculum size access in 500 milliliters of secondary seed shaking flasks, culture condition is identical with first order seed;
3. three grades of seed culture: secondary seed is accessed in 5000 milliliters of three grades of seed flask with 10% inoculum size, substratum loading amount 1000 milliliters, rotary shaker 100 revs/min, culture temperature 36 DEG C, incubation time 15h;
4. first class seed pot is cultivated: the first class seed pot being 150L with 10% inoculum size access cubic capacity by three grades of seeds, fermention medium loading amount 100L, culture temperature 36 DEG C, stirring velocity 400rpm, ventilation (V/V) 1:2, tank pressure 0.05Mpa, incubation time 20h;
Described one-level, secondary, three grades of seed culture medium weight consist of:
Yeast powder 0.5%, glucose 1.5%, peptone 0.5%, extractum carnis 0.8%, dipotassium hydrogen phosphate 1.5%, Chinese herbal medicine powder 2%, trehalose 3%, calcium sulfate 0.1%, magnesium chloride 0.2%, Trisodium Citrate 0.3%, insufficient section pure water is supplied, pH value 7.2,123 DEG C of sterilizing 40min.
Described first class seed pot substratum 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%, Trisodium Citrate 0.5%, insufficient section pure water is supplied, pH value 7.2,123 DEG C of sterilizing 40min.
Described seeding tank fermented liquid cell concentration is 8.0x10 8individual/ml;
(3) ferment tank
By first class seed pot fermented liquid in step (2) with 6% inoculum size access fermentor tank, culture temperature 36 DEG C, stirring velocity 700r/m, ventilation (V/V) 1:3, incubation time 15h; Then with 2 DEG C/h rate of temperature fall slow cooling to 15 DEG C, constant temperature culture 20h; Continue with 2 DEG C/h rate of temperature fall slow cooling to 5 DEG C, now, first class seed pot fermented liquid in step (2) is added access fermentor tank with 4% inoculum size, constant temperature culture 30h; Finally slowly be warming up to 15 DEG C, constant temperature culture 20h with 2 DEG C/h temperature rise rate; Continue slowly to be warming up to 36 DEG C, constant temperature culture 20h with 2 DEG C/h temperature rise rate;
Dissolved oxygen controls: by adjustment mixing speed and ventilation, controls dissolved oxygen 30%;
PH controls: by mending ammoniacal liquor or dilute phosphoric acid, controls pH value in fermenting process and remains on 7.2;
Control of additive raw material: add supplemented medium, to maintain fermented liquid reducing sugar content for 2-5mg/ml;
Put tank standard: the old and feeble self-dissolving of 60-80% thalline, enzyme activity increasess slowly.
Described fermention medium consists of: maltodextrin 150g, Semen Maydis powder 60g, soybean cake powder 25g, Chinese herbal medicine powder 50g, trehalose 40g, yeast powder 8g, corn steep liquor 5g, ammonium sulfate 3g, dipotassium hydrogen phosphate 2g, potassium primary phosphate 2g, Trisodium Citrate 5g, defoamer 1g, pure water l000mL, pH value 7.2,121 DEG C of sterilizing 20min;
Described supplemented medium weight consists of: maltodextrin 30%, Semen Maydis powder 20%, bean powder 25%, Chinese herbal medicine powder 10%, and insufficient section pure water is supplied, pH value 7.2,123 DEG C of sterilizing 40min.
The concocting method of described fermention medium is:
Accurately take raw material in proportion, pure water in raw material, Semen Maydis powder, soybean cake powder are dropped in material-compound tank, regulate pH value 7.2, add middle temperature amylase (3u/g Semen Maydis powder) and alpha-amylase (30u/g Semen Maydis powder), DEG C insulation 30min of warming while stirring to 70 simultaneously, be then slowly warming up to 90 DEG C of insulation 30min and liquefy, finally add other raw material, stir, adjust initial pH7.2,123 DEG C of sterilizing 40min are for subsequent use.
(4) 1000 μm, fermentation liquor aperture filter board coarse filtration, then obtaining solid content in 20 DEG C of loop ultrafiltrations concentrated removal moisture is 40% concentrated solution; Concentrated solution obtains subtilis culture through vacuum lyophilization, micronizing.
The preparation method of described Chinese herbal medicine extract is: respectively Chinese herbal medicine powder being broken to particle diameter is less than 2 millimeters, then Homogeneous phase mixing add the water of 6 times of weight in container, control temperature 90 DEG C keeps 4h, then being cooled to 60 DEG C, adding mixing enzyme preparation and carry out enzymolysis, is 6.8 by lactic acid adjust ph, enzymolysis 4h, finally add the mixture of mixture 3 times of w ethanol and propyl alcohol, control temperature to 78 DEG C keeps 4h, filters; Filtrate reduced in volume to solid content is more than 20%, and then lyophilize obtains Chinese herbal medicine extract.
The parts by weight of described herbal medicine consist of: sea-buckthorn 30 parts, Semen Cassiae 30 parts, matrimony vine 15 parts, Chinese yam 15 parts, Root of coastal Glehnia 10 parts, Fructus Viticis Negundo 10 parts, radix polygonati officinalis 5 parts, the seed of Job's tears 5 parts, Fructus Hordei Germinatus 5 parts, sweet osmanthus 5 parts, the Radix Astragali 5 parts;
Mixed enzyme addition is 10% of mixture gross weight;
The parts by weight of described mixed enzyme consist of: endo-beta-glucanase 20 parts, outer beta-glucanase 20 parts, beta-glucosidase 15 parts, zytase 20 parts, pentosanase 20 parts, Pullulanase 30 parts, beta-amylase 15 parts, neutral protease 15 parts, aspartic protease 15 parts, superoxide-dismutase 10 parts, glucose oxidase 10 parts, acid phosphatase 10 parts;
Described protective material is made up of the raw material of following parts by weight: trehalose 30 parts, NaCl30 part, (NH 4) 2sO 415 parts, halfcystine 15 parts.
Described activator is formed by the inorganic salt Homogeneous phase mixing of following quality component: zinc chloride 40 parts, 20 parts, calcium chloride, 20 parts, sodium sulfate, 10 parts, magnesium chloride.
Containing the preparation method of the Wheat ration enzyme of neutral protease as embodiment 1.
The result of use test of embodiment 4 embodiment of the present invention 1 Wheat ration enzyme
1. feed kind: full price Wheat Based Diet, wherein in feed, wheat weight is 40%;
2. Wheat ration enzyme addition: feed per ton adds 120g;
3. Wheat ration enzyme test group design: Wheat ration enzyme parts by weight composition is divided into test group, control group 1, control group 2; Wherein test group is Wheat ration enzyme prepared by the embodiment of the present invention 1; Control group 1 is all the other components except subtilis culture in test group component; Control group 2 is commercially available Wheat ration enzyme and its each enzyme class comprised, enzyme activity are identical with control group 1 with test group with enzyme parts by weight; The concrete parts by weight composition of each test group is as table 1
Table 1
Project Test group Control group 1 Control group 2 Remarks
Subtilis culture (part) 25 0 0
Aspartic protease (part) 25 25 25
Acidic xylanase (part) 25 25 25
Cellulase (part) 25 25 25
Beta-glucanase (part) 17 17 17
Amylase (part) 17 17 17
'beta '-mannase (part) 12 12 12
Chinese herbal medicine extract (part) 12 12 0
Protective material (part) 13 13 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, chooses the healthy DLY three way cross growing swine that 36 body weight are (30 ± 2) kg, is divided into 3 groups at random, often organizes 2 hurdles and repeats, often repeat 6, galt and gilt half and half.The prerun of 1 week official test advance behavior phase, and complete expelling parasite and routine immunization work.Test point two phases of front and back carry out, in earlier stage (30kg-60kg), and the later stage (61kg ~ 90kg).
4.1.2 feeding and management
Feed dry mash, free choice feeding, is limited cannot not have enough surplusly, and automatic drinking bowl is drunk water.Day in early stage feeds three times, and day in later stage feeds secondary, in units of hurdle, record feed consumption rate.Pig house is brick structure, cement flooring, single-column type, and examination pig is raised respectively in 9 hurdles, and the envrionment conditions of each column home is consistent, cleans colony house morning and afternoon every day each once, observes the behavior of pig, appetite, ight soil simultaneously.Duration of test record disease and treatment situation.
4.1.3 feed and formula
Based on full price Wheat Based Diet feed, feed per ton adds the Wheat ration enzyme 120g of test group, control group 1 and control group 2; The raw material composition of Wheat ration enzyme is specifically in table 1.
4.1.4 test index: before the test at the end of (30kg-60kg), mid-term and later stage (61kg ~ 90kg), point another name individual weight, weighs and carries out on an empty stomach all in the morning.Stage by stage, record its every daily material consumption, sickness rate in units of hurdle, calculate daily ingestion amount and food utilization efficiency simultaneously, and carry out statistical study to above-mentioned data, growing swine growth performance is in table 2.
Table 2
4.1.5 analysis is carried out as table 3 to the full phase test result of above-mentioned test
Table 3
Project Test group Control group 1 Deviation (%) Control group 2 Deviation (%)
Day weight gain (g) 873 746 127(17.02) 657 216(32.88)
Food consumption (kg) 189.73 193.46 -3.73(-1.92) 210.17 -20.44(-9.7)
Feed-weight ratio 2.87 3.36 -0.49(-14.5) 4.15 -1.28(-30.84)
Diarrhea rate (%) 1 4 -3(-75) 17 -16(-94)
Hair color is marked 8.5 6.5 2(30.76) 4 4.5(112.5)
From above-mentioned analysis of statistical results: test group is compared with control group 2 with control group 1, when the test conditionss such as test materials, testing circumstance, test method and Experimental Establishment are identical, day weight gain improves 17.02% and 32.88% respectively; Food consumption reduces 1.92% and 9.7% respectively; Feed-weight ratio reduces 14.5% and 30.84% respectively; Diarrhea rate reduces 75% and 94% respectively; Outward appearance hair color quality improves 30.76% and 112.5% respectively; Use Wheat ration enzyme growing swine of the present invention to have excellent growth performance and significant immunological competence, improve cultured output and quality, reduce aquaculture cost, improve fanning economics.

Claims (8)

1. the Wheat ration enzyme containing neutral protease, be made up of the zymin of following parts by weight: subtilis culture 20-30 part, aspartic protease 20-30 part, acidic xylanase 20-30 part, cellulase 20-30 part, beta-glucanase 15-20 part, amylase 15-20 part, 'beta '-mannase 10-15 part, Chinese herbal medicine extract 10-15 part, protective material 10-15 part, activator 10-15 part;
The preparation method of described subtilis culture is as follows: subtilis CCTCC NO:M 2013539 cultivates enlarged culturing step by step through slant strains activation with one, two, three seed culture and first class seed pot and obtains seed liquor; By seed liquor with 6% inoculum size access fermentor tank, culture temperature 30-36 DEG C, stirring velocity 200-700rpm, ventilation (V/V) 1:1-3, incubation time 10-15h; Then with 1-2 DEG C/h rate of temperature fall slow cooling to 10-15 DEG C, constant temperature culture 15-20h; Continue with 1-2 DEG C/h rate of temperature fall slow cooling to 2-5 DEG C, now, seed liquor is added access fermentor tank with 4% inoculum size, constant temperature culture 20-30h; Finally slowly be warming up to 10-15 DEG C, constant temperature culture 15-20h with 1-2 DEG C/h temperature rise rate; Continue slowly to be warming up to 30-36 DEG C, constant temperature culture 15-20h with 1-2 DEG C/h temperature rise rate; 10-1000 μm, fermentation liquor aperture filter board coarse filtration, then obtaining solid content in 10-20 DEG C of loop ultrafiltration concentrated removal moisture is 20-40% concentrated solution; Concentrated solution obtains subtilis culture through vacuum lyophilization, micronizing;
The preparation method of described Chinese herbal medicine extract is: in weight fraction, accurately take sea-buckthorn 20-30 part, Semen Cassiae 20-30 part, matrimony vine 10-15 part, Chinese yam 10-15 part, Root of coastal Glehnia 5-10 part, Fructus Viticis 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; Respectively Chinese herbal medicine powder being broken to particle diameter is less than 2 millimeters, then Homogeneous phase mixing add the water of 3-6 times of weight in container, control temperature 70 DEG C ~ 90 DEG C keeps 2 ~ 4h, then be cooled to 45-60 DEG C, the mixing enzyme preparation adding mixture gross weight 5-10% carries out enzymolysis, is 5.5-6.8 by lactic acid adjust ph, enzymolysis 2-4h, finally add the mixture of mixture 0.5-3 times of w ethanol and propyl alcohol, control temperature to 60 DEG C ~ 78 DEG C keeps 3 ~ 4h, filters; Filtrate reduced in volume to solid content is more than 20%, and then lyophilize obtains Chinese herbal medicine extract;
The parts by weight of described mixing enzyme preparation consist of: endo-beta-glucanase 10-20 part, outer 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 protease 10-15 part, aspartic protease 10-15 part, superoxide-dismutase 5-10 part, glucose oxidase 5-10 part, acid phosphatase 5-10 part;
Described protective material 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, halfcystine 10-15 part;
Described activator is formed by the inorganic salt Homogeneous phase mixing of following quality component: zinc chloride 30-40 part, calcium chloride 10-20 part, sodium sulfate 10-20 part, magnesium chloride 5-10 part.
2. a kind of Wheat ration enzyme containing neutral protease as claimed in claim 1, it is characterized in that, the slant medium of preparation subtilis culture consists of: extractum carnis 3-10g, sodium-chlor 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 DEG C of sterilizing 20min;
The preparation method of described Chinese herbal medicine powder is as follows: with weight parts, takes 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 less than 2 millimeters, then Homogeneous phase mixing add the water of 3-6 times of weight in container, control temperature 70 DEG C ~ 90 DEG C keeps 2 ~ 4h, then be cooled to 45-60 DEG C, the mixing enzyme preparation adding mixture gross weight 5-10% carries out enzymolysis, is 5.5-6.8 by lactic acid adjust ph, enzymolysis 2-4h, finally add the mixture of mixture 0.5-3 times of w ethanol and propyl alcohol, control temperature to 60 DEG C ~ 78 DEG C keeps 3 ~ 4h, filters; Filter vacuum concentrates postlyophilization and obtains Chinese herbal medicine powder.
3. a kind of Wheat ration enzyme containing neutral protease as claimed in claim 1, it is characterized in that, the seed culture medium of preparation subtilis culture consists of: yeast powder 0.3-0.5%, glucose 1-1.5%, peptone 0.3-0.5%, extractum carnis 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%, Trisodium Citrate 0.1-0.3%, insufficient section pure water is supplied, pH value 7.0-7.2,121-123 DEG C of sterilizing 30-40min;
The preparation method of described Chinese herbal medicine powder is as follows: with weight parts, takes 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 less than 2 millimeters, then Homogeneous phase mixing add the water of 3-6 times of weight in container, control temperature 70 DEG C ~ 90 DEG C keeps 2 ~ 4h, then be cooled to 45-60 DEG C, the mixing enzyme preparation adding mixture gross weight 5-10% carries out enzymolysis, is 5.5-6.8 by lactic acid adjust ph, enzymolysis 2-4h, finally add the mixture of mixture 0.5-3 times of w ethanol and propyl alcohol, control temperature to 60 DEG C ~ 78 DEG C keeps 3 ~ 4h, filters; Filter vacuum concentrates postlyophilization and obtains Chinese herbal medicine powder.
4. a kind of Wheat ration enzyme containing neutral protease as claimed in claim 1, it is characterized in that, the seed tank culture base of preparation subtilis culture consists of: maltodextrin 5-15%, yeast powder 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%, Trisodium Citrate 0.1-0.5%, insufficient section pure water is supplied, pH value 7.0-7.2,121-123 DEG C of sterilizing 30-40min;
The preparation method of described Chinese herbal medicine powder is as follows: with weight parts, takes 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 less than 2 millimeters, then Homogeneous phase mixing add the water of 3-6 times of weight in container, control temperature 70 DEG C ~ 90 DEG C keeps 2 ~ 4h, then be cooled to 45-60 DEG C, the mixing enzyme preparation adding mixture gross weight 5-10% carries out enzymolysis, is 5.5-6.8 by lactic acid adjust ph, enzymolysis 2-4h, finally add the mixture of mixture 0.5-3 times of w ethanol and propyl alcohol, control temperature to 60 DEG C ~ 78 DEG C keeps 3 ~ 4h, filters; Filter vacuum concentrates postlyophilization and obtains Chinese herbal medicine powder.
5. a kind of Wheat ration enzyme containing neutral protease as claimed in claim 1, it is characterized in that, during preparation subtilis culture, seeding tank fermented liquid cell concentration is 7.0x10 8-8.0x10 8individual/ml.
6. a kind of Wheat ration enzyme containing neutral protease as claimed in claim 1, it is characterized in that, the fermention medium of preparation subtilis culture consists of: maltodextrin 50-150g, Semen Maydis powder 50-60g, soybean cake powder 15-25g, Chinese herbal medicine powder 30-50g, trehalose 30-40g, yeast powder 4-8g, corn steep liquor 1-5g, ammonium sulfate 1-3g, dipotassium hydrogen phosphate 1-2g, potassium primary phosphate 1-2g, Trisodium Citrate 1-5g, defoamer 0.1-1g, pure water l000mL, pH value 7.0-7.2,121 DEG C of sterilizing 20min;
The preparation method of described Chinese herbal medicine powder is as follows: with weight parts, takes 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 less than 2 millimeters, then Homogeneous phase mixing add the water of 3-6 times of weight in container, control temperature 70 DEG C ~ 90 DEG C keeps 2 ~ 4h, then be cooled to 45-60 DEG C, the mixing enzyme preparation adding mixture gross weight 5-10% carries out enzymolysis, is 5.5-6.8 by lactic acid adjust ph, enzymolysis 2-4h, finally add the mixture of mixture 0.5-3 times of w ethanol and propyl alcohol, control temperature to 60 DEG C ~ 78 DEG C keeps 3 ~ 4h, filters; Filter vacuum concentrates postlyophilization and obtains Chinese herbal medicine powder.
7. prepare as claimed in claim 1 containing the method for the Wheat ration enzyme of neutral protease for one kind; it is characterized in that; by protective material, Chinese herbal medicine extract micronizing respectively; guarantee that granularity is less than other zymin; then with subtilis culture, aspartic protease, acidic xylanase, cellulase, beta-glucanase, amylase, 'beta '-mannase, Homogeneous phase mixing; finally add activator, after mixing, pack the Wheat ration enzyme got product containing neutral protease.
8. the preparation method of a kind of Wheat ration enzyme containing neutral protease as claimed in claim 7, is characterized in that, the described Wheat ration enzyme containing neutral protease, be made up of the zymin of following parts by weight: subtilis culture 25 parts, aspartic protease 25 parts, acidic xylanase 25 parts, cellulase 25 parts, beta-glucanase 17 parts, amylase 17 parts, 'beta '-mannase 12 parts, Chinese herbal medicine extract 12 parts, protective material 13 parts, activator 12 parts;
The preparation method of described subtilis culture comprises the steps:
(1) slant strains of intact subtilis CCTCC NO:M 2013539 is inoculated in slant medium, cultivates 24h for 30 DEG C and carry out actication of culture, so activation 2 times;
Described slant medium consists of: extractum carnis 3g, sodium-chlor 5g, peptone 10g, glucose 2g, (NH) 2sO 43g, K 2hPO 46g, CaCl 21g, agar 15g, Chinese herbal medicine powder 5g, distilled water l000mL, pH value 7.0,121 DEG C of sterilizing 20min;
The preparation method of described Chinese herbal medicine powder is as follows:
With weight parts, take the Radix Astragali 20 parts; Radix Codonopsis 10 parts; Radix bupleuri 10 parts; The root of large-flowered skullcap 10 parts; Respectively said herbal medicine being crushed to particle diameter is less than 2 millimeters, then Homogeneous phase mixing add the water of 3 times of weight in container, control temperature 70 DEG C keeps 2h, and be then cooled to 45 DEG C, the mixing enzyme preparation adding mixture gross weight 5% carries out enzymolysis, be 5.5 by lactic acid adjust ph, enzymolysis 2h, finally add the mixture of mixture 0.5 times of w ethanol and propyl alcohol, the mass ratio of described ethanol and propyl alcohol is 1:1, control temperature to 60 DEG C keeps 3h, filters; Filter vacuum concentrates postlyophilization and obtains Chinese herbal medicine powder;
The parts by weight of described mixing enzyme preparation consist of: endo-beta-glucanase 10 parts, outer beta-glucanase 10 parts, beta-glucosidase 10 parts, zytase 15 parts, pentosanase 15 parts, Pullulanase 20 parts, beta-amylase 10 parts, neutral protease 10 parts, aspartic protease 10 parts, superoxide-dismutase 5 parts, glucose oxidase 5 parts, acid phosphatase 5 parts;
(2) liquid seeds enlarged culturing
1. first order seed is cultivated: slant strains 1 articulating after step (1) being activated enters in 500 ml shake flasks, substratum loading amount 100 milliliters, rotary shaker 180 revs/min, culture temperature 30 DEG C, incubation time 10h;
2. secondary seed cultivate: by first order seed according to 10% inoculum size access in 500 milliliters of secondary seed shaking flasks, culture condition is identical with first order seed;
3. three grades of seed culture: secondary seed is accessed in 5000 milliliters of three grades of seed flask with 10% inoculum size, substratum loading amount 1000 milliliters, rotary shaker 100 revs/min, culture temperature 30 DEG C, incubation time 10h;
4. first class seed pot is cultivated: the first class seed pot being 150L with 10% inoculum size access cubic capacity by three grades of seeds, fermention medium loading amount 100L, culture temperature 30 DEG C, stirring velocity 200rpm, ventilation (V/V) 1:1, tank pressure 0.05Mpa, incubation time 10h;
One-level, secondary, three grades of seed culture medium weight consist of:
Yeast powder 0.3%, glucose 1%, peptone 0.3%, extractum carnis 0.5%, dipotassium hydrogen phosphate 0.8%, Chinese herbal medicine powder 1.5%, trehalose 1%, calcium sulfate 0.1%, magnesium chloride 0.2%, Trisodium Citrate 0.1%, insufficient section pure water is supplied, pH value 7.0,121 DEG C of sterilizing 30min;
First class seed pot substratum weight consists of:
Maltodextrin 5%, yeast powder 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%, Trisodium Citrate 0.1%, insufficient section pure water is supplied, pH value 7.0,121 DEG C of sterilizing 30min;
Seeding tank fermented liquid cell concentration is 7.0x10 8individual/ml;
(3) ferment tank
By first class seed pot fermented liquid in step (2) with 6% inoculum size access fermentor tank, culture temperature 30 DEG C, stirring velocity 200r/m, ventilation (V/V) 1:1, incubation time 10h; Then with 1 DEG C/h rate of temperature fall slow cooling to 10 DEG C, constant temperature culture 15h; Continue with 1 DEG C/h rate of temperature fall slow cooling to 2 DEG C, now, first class seed pot fermented liquid in step (2) is added access fermentor tank with 4% inoculum size, constant temperature culture 20h; Finally slowly be warming up to 10 DEG C, constant temperature culture 15h with 1 DEG C/h temperature rise rate; Continue slowly to be warming up to 30 DEG C, constant temperature culture 15h with 1 DEG C/h temperature rise rate;
Dissolved oxygen controls: by adjustment mixing speed and ventilation, controls dissolved oxygen 15%;
PH controls: by mending ammoniacal liquor or dilute phosphoric acid, controls pH value in fermenting process and remains on 7.0;
Control of additive raw material: add supplemented medium, to maintain fermented liquid reducing sugar content for 2-5mg/ml;
Put tank standard: the old and feeble self-dissolving of 60-80% thalline, enzyme activity increasess slowly;
Fermention medium consists of: maltodextrin 50g, Semen Maydis powder 50g, soybean cake powder 15g, Chinese herbal medicine powder 30g, trehalose 30g, yeast powder 4g, corn steep liquor 1g, ammonium sulfate 1g, dipotassium hydrogen phosphate 1g, potassium primary phosphate 1g, Trisodium Citrate 1g, defoamer 0.1g, pure water l000mL, pH value 7.0,121 DEG C of sterilizing 20min;
Supplemented medium weight consists of: maltodextrin 20%, Semen Maydis powder 10%, bean powder 15%, Chinese herbal medicine powder 5%, and insufficient section pure water is supplied, pH value 7.0,121 DEG C of sterilizing 30min;
The concocting method of fermention medium is:
Accurately take raw material in proportion, pure water in raw material, Semen Maydis powder, soybean cake powder are dropped in material-compound tank, regulate pH value 7.0, add middle temperature amylase (3u/g Semen Maydis powder) and alpha-amylase (30u/g Semen Maydis powder), DEG C insulation 15min of warming while stirring to 70 simultaneously, be then slowly warming up to 90 DEG C of insulation 15min and liquefy, finally add other raw material, stir, adjust initial pH7.0,121 DEG C of sterilizing 30min are for subsequent use;
(4) 10 μm, fermentation liquor aperture filter board coarse filtration, then obtaining solid content in 10 DEG C of loop ultrafiltrations concentrated removal moisture is 20% concentrated solution; Concentrated solution obtains subtilis culture through vacuum lyophilization, micronizing;
The preparation method of described Chinese herbal medicine extract is: respectively Chinese herbal medicine powder being broken to particle diameter is less than 2 millimeters, then Homogeneous phase mixing add the water of 5 times of weight in container, control temperature 80 DEG C keeps 3h, then being cooled to 53 DEG C, adding mixing enzyme preparation and carry out enzymolysis, is 6.2 by lactic acid adjust ph, enzymolysis 3h, finally add the mixture of mixture 2 times of w ethanol and propyl alcohol, control temperature to 69 DEG C keeps 4h, filters; Filtrate reduced in volume to solid content is more than 20%, and then lyophilize obtains Chinese herbal medicine extract;
The parts by weight of described herbal medicine consist of: sea-buckthorn 25 parts, Semen Cassiae 25 parts, matrimony vine 17 parts, Chinese yam 13 parts, Root of coastal Glehnia 8 parts, Fructus Viticis Negundo 7 parts, radix polygonati officinalis 4 parts, the seed of Job's tears 4 parts, Fructus Hordei Germinatus 4 parts, sweet osmanthus 4 parts, the Radix Astragali 4 parts;
Mixing enzyme preparation addition is the 5-10% of mixture gross weight;
The parts by weight of described mixing enzyme preparation consist of: endo-beta-glucanase 15 parts, outer beta-glucanase 15 parts, beta-glucosidase 13 parts, zytase 13 parts, pentosanase 13 parts, Pullulanase 25 parts, beta-amylase 12 parts, neutral protease 13 parts, aspartic protease 13 parts, superoxide-dismutase 7 parts, glucose oxidase 7 parts, acid phosphatase 7 parts;
Described protective material is made up of the raw material of following parts by weight: trehalose 25 parts, NaCl25 part, (NH 4) 2sO 413 parts, halfcystine 12 parts;
Described activator is formed by the inorganic salt Homogeneous phase mixing of following quality component: zinc chloride 35 parts, 15 parts, calcium chloride, 15 parts, sodium sulfate, 7 parts, magnesium chloride;
Preparation method containing the Wheat ration enzyme of neutral protease:
By described protective material, Chinese herbal medicine extract micronizing respectively; guarantee that granularity is less than other zymin; then with subtilis culture, aspartic protease, acidic xylanase, cellulase, beta-glucanase, amylase, 'beta '-mannase, Homogeneous phase mixing; finally add activator, after mixing, pack the Wheat ration enzyme got product containing neutral protease.
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