CN103740683A - Wheat ration enzyme containing neutral protease and preparation method thereof - Google Patents

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

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CN103740683A
CN103740683A CN201310738229.0A CN201310738229A CN103740683A CN 103740683 A CN103740683 A CN 103740683A CN 201310738229 A CN201310738229 A CN 201310738229A CN 103740683 A CN103740683 A CN 103740683A
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parts
culture
beta
enzyme
herbal medicine
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CN103740683B (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 wheat ration enzyme containing neutral protease and a preparation method thereof and belongs to the technical field of enzyme preparation. The wheat ration enzyme is formed by scientific compounding of bacillus subtilis culture, acid protease, acid xylanase, cellulose, beta-glucanase, amylase, beta-mannase, Chinese herb extract, protective agent and activating agent. Due to the bacillus subtilis culture, the immunity of organism of animals can be improved, the growth of the immune organ is enhanced, the maturity of the structure and the function of the intestine of the animal is enhanced, the daily gain of the animal is increased and the feed conversion ratio is improved. The wheat ration enzyme disclosed by the invention has the advantages that safe digestive enzyme is provided for livestock and poultry, the digestion burden is effectively reduced, the material utilization rate and the growth rate are increased, and the environment is protected; simultaneously, due to use of the proper amount of activating agent, the effect of the enzyme preparation can be fully played under the equal conditions, so that the adding amount of the enzyme preparation is saved; due to the scientific compounding of the Chinese herb extract, not only the quality guarantee period of the compound enzyme preparation is prolonged, but also the immunity of the raised livestock and poultry can be improved.

Description

A kind of Wheat Based Diet enzyme containing neutral protease and preparation method thereof
Technical field
The invention belongs to enzyme preparation technical field, specifically a kind of Wheat Based Diet enzyme containing neutral protease and preparation method thereof.
Background technology
In feed, add zymin and mainly contain following 4 reasons: 1. the antinutritional factor that degraded exists in animal-feed.These materials can not be degraded by animal endogenous enzyme, thereby disturb the eubolism of animal, cause animal digestion bad, and 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 with some, can not be had by the structure formation of animal digestion that (for example, in plant feed raw material, a large amount of phosphorus exists with the form of phytate phosphorus.）。3. some specific chemical bond of degrading in raw material.These chemical bonds can not be degraded by the enzyme of animal self, add exogenous enzyme can discharge more nutrition later.4. because young animal autodigestion system is also immature, the not enough interpolation of endogenous enzyme 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, the accuracy of raising feed formulation, the while can also be improved the regularity of growth of animal, reduces handling cost, increases economic efficiency.Use all right protection of the environment of zymin.Because the utilization ratio of feed has improved, corresponding ight soil quantity discharged has declined.In the situation that effect is obvious, the quantity discharged 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 zymin of applying in fodder industry at present mainly contains 4 large classes: be used for respectively degraded cellulose, protein, starch and phytic acid.
Fiber degradation enzyme: for monogastric animal, 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 maldigestion causes is relevant.As black toe disease and the piggy of the appetite stimulator 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 fibre 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 reguarity of animal.Can also reduce some dyspeptic disease simultaneously.
Proteolytic degradation 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 monogastric animal daily ration, add proteolytic enzyme (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 antinutritional factor in soybean cake 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 soybean cake powder).
Starch degrading enzyme: many nutritionists think corn" golden standard " of feedstuff raw material.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 degrading enzyme: for all animals, phosphorus is all vital for mineralising, immunity, breeding, the growth of bone.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, in feed diet, inorganic phosphorus 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, contaminate 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 zymin 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 zymin, with regard to more complicated, seems uncertain.Intensive degree is low, relates to link many, and the result of use of zymin is difficult to carry out business calculating.Although had the imagination of using zymin in the 1950's, until just start to understand strength how to bring into play enzyme in fodder industry the eighties in 20th century.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 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 impact.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 fodder industry.Can not rant out: 1996, in the broiler chicken material (viscosity cereal is energy derive) 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 major cause is the complicated structure in market, and market is diversification, even cannot calculate.From regional 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 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 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 enzyme marketable value that viscosity and robust 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 Wheat Based Diet enzyme has its wide market space and huge economic worth, but the thermostability of Wheat Based Diet enzyme, security, composite comprehensive and giving full play to of action effect are still zymin manufacturer and the common major issue of paying close attention to of numerous raisers, prepare safer, more comprehensively, the better Wheat Based Diet enzyme of enzyme action effect is industry technician 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 subtilis culture of the neutral protease that stability is strong is basis, and the composite Chinese herbal medicine extract of science, protective material, activator and other food grade feed enzyme, the Wheat Based Diet enzyme containing neutral protease making, not only for raising livestock and poultry, provide safety, 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 zymin simultaneously, make the best use of everything, save zymin addition, the composite quality guaranteed period that both can extend compound enzymic preparation of science of Chinese herbal medicine extract, can improve again the immunizing power 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 Wheat Based Diet enzyme for neutral protease, by the zymin of following parts by weight, formed:
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 bacterial strain subtilis 1398-2-12 that produces 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 through slant strains activation and step by step enlarged culturing (comprise one, two, three seed culture and first class seed pot cultivate) obtain liquid seeds; Liquid seeds is accessed to fermentor tank, culture temperature 30-36 ℃, stirring velocity 200-700rpm, ventilation (V/V) 1:1-3, incubation time 10-15h with 6% inoculum size; 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 fermentor tank, constant temperature culture 20-30h by liquid seeds with 4% inoculum size with 1-2 ℃/h rate of temperature fall slow cooling; Finally with 1-2 ℃/h temperature rise rate, be slowly warming up to 10-15 ℃, constant temperature culture 15-20h; Continuation is slowly warming up to 30-36 ℃, constant temperature culture 15-20h with 1-2 ℃/h temperature rise rate; Fermented liquid via hole diameter 10-1000 μ m filter board coarse filtration, then obtains solid content 20-40% concentrated solution in the concentrated moisture of removing of 10-20 ℃ of loop ultrafiltration; Concentrated solution obtains subtilis culture through vacuum lyophilization, micronizing.
Described slant medium consists of: extractum carnis 3-10g, and 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 ℃ 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 mixture 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 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.
Described one, two, three seed culture medium weight 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 ℃ 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 ℃ 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 ℃ of sterilizing 20min;
Described supplemented medium weight consists of: maltodextrin 20-30%, and Semen Maydis powder 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 fermention medium is:
Accurately take in proportion raw material, 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), ℃ 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 standby.
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 mixture 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 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 comprised 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 evenly to be mixed by the inorganic salt 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 contains the preparation method of the Wheat Based Diet 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, evenly mix; finally add activator, after mixing, pack the Wheat Based Diet enzyme getting product containing neutral protease.
Described Wheat Based Diet enzyme is applicable to feed-processing plant and plant's autogamy feed, during use, should mix with other raw material in feed, can be in advance the present invention be mixed with a small amount of feed, remix in large quantities of feeds, Direct-fed.Advise that complete diet pellet addition per ton is: when wheat weighs less than 30% in feed, add 100g; When wheat weight exceedes 30%, add 120-150g;
Subtilis 1398-2-12 provided by the invention is obtained through UV-LiCl-ethyl sulfate Mutation screening by subtilis (Bacillus subtilis) 1398-2 of a strain product neutral protease of laboratory preservation.
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 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: 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, and 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 positive.
Subtilis 1398-2-12 provided by the invention has that produced neutral protease tolerable temperature is high, the feature of applicable pH value wide scope, 75 ℃ of enzymes of fermented liquid 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 leavening property test screen, obtain a strain and produce the bacterial strain subtilis bacterium 1398-2-12 of heat-flash stability neutral protease, the work of fermented liquid neutral protease enzyme can reach 5500-7000U/mL, thermostability to enzyme is analyzed, and crude enzyme liquid is placed in respectively at 40 ℃, 45 ℃, 50 ℃, 55 ℃, 60 ℃, 65 ℃, 70 ℃, 75 ℃, every 10 minutes sampling and measuring enzymes, lives.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 protease enzyme work containing in 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 when 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 stability in storage is high, is more applicable to raising the interpolation of animal and fowl fodder.
2. the subtilis thalline in subtilis culture of the present invention can improve the growth of animal body immunizing power, Promote immunity organ, the maturation that promotes animal intestinal structure and function, raising animal day weight 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 salt and amino acid science composite containing the protective material in the Wheat Based Diet enzyme of neutral protease, has effectively slowed down the moisture regain of compound enzymic preparation; Can strengthen prozyme simultaneously resistance toly freeze, resistance toheat, keep identical enzyme activity, its heat resisting temperature can improve 20-30 ℃, resistance to freezing temp can reduce 10-15 ℃, effectively prevented the loss of prozyme enzyme activity in transportation, preservation and use procedure, extend the quality guaranteed period of prozyme, reached same enzyme activity, can extend 2-3 than the like product quality guaranteed period.
4. the present invention adds inorganic salt as activator containing the Wheat Based Diet enzyme of neutral protease; created the top condition of enzyme catalysis; given full play to the vigor of the each enzyme component of prozyme; the macromolecular substance such as starch in feed, protein, Mierocrystalline cellulose, phytic acid have thoroughly effectively been decomposed; greatly alleviated the digestion burden of livestock and poultry animal; improved the growth rate of raw material availability and livestock and poultry, effectively prevented the environmental pollution that feces of livestock and poultry causes, protected feeding environment simultaneously.
5. the Chinese herbal medicine extract that the present invention adds containing the Wheat Based Diet enzyme of neutral protease both can extend the quality guaranteed period of compound enzymic preparation, can improve again the immunizing power of raising livestock and poultry, effectively prevented 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 Based Diet enzyme of neutral protease; enzyme activity and the effect of prozyme are brought into play to greatest extent; and the utilization ratio of feed and the growth rate of animal have been improved accordingly; strengthened appetite and the resistance against diseases of animal, extended the quality guaranteed period of prozyme and protected environment.
Embodiment
Below by specific embodiment narration the present invention.Unless stated otherwise, in the present invention, technique 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
Containing a Wheat Based Diet enzyme for neutral protease, by the zymin of following parts by weight, formed:
25 parts of subtilis cultures, 25 parts of aspartic proteases, 25 parts of acidic xylanases, 25 parts of cellulases, 17 parts of beta-glucanases, 17 parts of amylase, 12 parts of 'beta '-mannases, 12 parts of Chinese herbal medicine extracts, 13 parts of protective materials, 12 parts of activator.
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 to slant medium, cultivates 24h for 30 ℃ and carry out actication of culture, so activate 2 times;
Described slant medium consists of: extractum carnis 3g, and 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, 7.0,121 ℃ of sterilizing 20min of pH value;
The preparation method of described Chinese herbal medicine powder is as follows:
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 mixture 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 mixture 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 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 proteases, 10 parts of aspartic proteases, 5 parts of superoxide-dismutases, 5 parts of glucose oxidases, 5 parts of acid phosphatases.
(2) liquid seeds enlarged culturing
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 substratum loading amounts, 180 revs/min of rotary shaking tables, 30 ℃ of culture temperature, incubation time 10h;
2. secondary seed is cultivated: by first order seed, according in 500 milliliters of secondary seed shaking flasks of inoculum size access of 10%, 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 shaking flasks to 1000 milliliters of substratum loading amounts, 100 revs/min of rotary shaking tables, 30 ℃ of culture temperature, incubation time 10h with 10% inoculum size;
4. first class seed pot is cultivated: the first class seed pot by three grades of seeds take 10% inoculum size access cubic capacity as 150L, fermention medium loading amount 100L, 30 ℃ of culture temperature, 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, 7.0,121 ℃ of sterilizing 30min of pH value.
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, 7.0,121 ℃ of sterilizing 30min of pH value.
Described seeding tank fermented liquid cell concentration is 7.0x10 8individual/ml;
(3) ferment tank
First class seed pot fermented liquid in step (2) is accessed to fermentor tank, 30 ℃ of culture temperature, stirring velocity 200r/m, ventilation (V/V) 1:1, incubation time 10h with 6% inoculum size; Then with 1 ℃/h rate of temperature fall slow cooling to 10 ℃, constant temperature culture 15h; Continuation, with 1 ℃/h rate of temperature fall slow cooling to 2 ℃, now, is appended access fermentor tank, constant temperature culture 20h by first class seed pot fermented liquid in step (2) with 4% inoculum size; Finally with 1 ℃/h temperature rise rate, be slowly warming up to 10 ℃, constant temperature culture 15h; Continuation is slowly warming up to 30 ℃ with 1 ℃/h temperature rise rate, constant temperature culture 15h;
Dissolved oxygen control: by adjusting mixing speed and ventilation, control dissolved oxygen 15%;
PH controls: by mending ammoniacal liquor or dilute phosphoric acid, in controlled fermentation process, pH value remains on 7.0;
Control of additive raw material: add supplemented medium, to maintain fermented liquid reducing sugar content as 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, 7.0,121 ℃ of sterilizing 20min of pH value;
Described supplemented medium weight consists of: maltodextrin 20%, and Semen Maydis powder 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 fermention medium is:
Accurately take in proportion raw material, 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), ℃ 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 standby.
(4) fermented liquid via hole diameter 10 μ m filter board coarse filtration, then in the concentrated removal of 10 ℃ of loop ultrafiltrations moisture, obtaining solid content 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 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 mixture 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 lyophilize obtains Chinese herbal medicine extract.
The parts by weight of described herbal medicine consist of: 25 parts of sea-buckthorns, 25 parts of Semen Cassiaes, 17 parts of matrimony vines, 13 parts of Chinese yams, 8 parts of Root of coastal Glehnia, 7 parts of Fructus Viticis 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 mixture gross weight;
The parts by weight of described mixed enzyme consist of: 15 parts of endo-beta-glucanases, 15 parts of outer 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 proteases, 13 parts of aspartic proteases, 7 parts of superoxide-dismutases, 7 parts of glucose oxidases, 7 parts of acid phosphatases;
Described protective material is comprised of the raw material of following parts by weight: 25 parts of trehaloses, NaCl25 part, (NH 4) 2sO 413 parts, 12 parts of halfcystines.
Described activator is evenly to be mixed by the inorganic salt of following quality component: 35 parts of zinc chloride, 15 parts, calcium chloride, 15 parts, sodium sulfate, 7 parts, magnesium chloride.
Contain the preparation method of the Wheat Based Diet 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, evenly mix; finally add activator, after mixing, pack the Wheat Based Diet enzyme getting product containing neutral protease.
Embodiment 2:
Containing a Wheat Based Diet enzyme for neutral protease, by the zymin of following parts by weight, formed:
20 parts of subtilis cultures, 20 parts of aspartic proteases, 20 parts of acidic xylanases, 20 parts of cellulases, 15 parts of beta-glucanases, 15 parts of amylase, 10 parts of 'beta '-mannases, 10 parts of Chinese herbal medicine extracts, 10 parts of protective materials, 10 parts of activator.
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 to slant medium, cultivates 30h for 33 ℃ and carry out actication of culture, so activate 2 times;
Described slant medium consists of: extractum carnis 6g, and 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, 7.2,121 ℃ of sterilizing 20min of pH value;
The preparation method of described Chinese herbal medicine powder is as follows:
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 mixture 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 mixture 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 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 proteases, 12 parts of aspartic proteases, 8 parts of superoxide-dismutases, 8 parts of glucose oxidases, 8 parts of acid phosphatases.
(2) liquid seeds enlarged culturing
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 substratum loading amounts, 180 revs/min of rotary shaking tables, 33 ℃ of culture temperature, incubation time 12h;
2. secondary seed is cultivated: by first order seed, according in 500 milliliters of secondary seed shaking flasks of inoculum size access of 10%, 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 shaking flasks to 1000 milliliters of substratum loading amounts, 100 revs/min of rotary shaking tables, 33 ℃ of culture temperature, incubation time 12h with 10% inoculum size;
4. first class seed pot is cultivated: the first class seed pot by three grades of seeds take 10% inoculum size access cubic capacity as 150L, fermention medium loading amount 100L, 33 ℃ of culture temperature, 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, 7.2,121 ℃ of sterilizing 30min of pH value.
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, 7.2,121 ℃ of sterilizing 30min of pH value.
Described seeding tank fermented liquid cell concentration is 7.5x10 8individual/ml;
(3) ferment tank
First class seed pot fermented liquid in step (2) is accessed to fermentor tank, 35 ℃ of culture temperature, stirring velocity 400r/m, ventilation (V/V) 1:2, incubation time 12h with 6% inoculum size; Then with 2 ℃/h rate of temperature fall slow cooling to 12 ℃, constant temperature culture 18h; Continuation, with 2 ℃/h rate of temperature fall slow cooling to 4 ℃, now, is appended access fermentor tank, constant temperature culture 25h by first class seed pot fermented liquid in step (2) with 4% inoculum size; Finally with 2 ℃/h temperature rise rate, be slowly warming up to 12 ℃, constant temperature culture 18h; Continuation is slowly warming up to 33 ℃ with 2 ℃/h temperature rise rate, constant temperature culture 18h;
Dissolved oxygen control: by adjusting mixing speed and ventilation, control dissolved oxygen 20%;
PH controls: by mending ammoniacal liquor or dilute phosphoric acid, in controlled fermentation process, pH value remains on 7.2;
Control of additive raw material: add supplemented medium, to maintain fermented liquid reducing sugar content as 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, 7.2,121 ℃ of sterilizing 20min of pH value;
Described supplemented medium weight consists of: maltodextrin 25%, and Semen Maydis powder 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 fermention medium is:
Accurately take in proportion raw material, 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), ℃ 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 standby.
(4) fermented liquid via hole diameter 500 μ m filter board coarse filtration, then in the concentrated removal of 15 ℃ of loop ultrafiltrations moisture, obtaining solid content 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 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 mixture 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 lyophilize obtains Chinese herbal medicine extract.
The parts by weight of described herbal medicine consist of: 20 parts of sea-buckthorns, 20 parts of Semen Cassiaes, 10 parts of matrimony vines, 10 parts of Chinese yams, 5 parts of Root of coastal Glehnia, 5 parts of Fructus Viticis 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 mixture gross weight;
The parts by weight of described mixed enzyme consist of: 10 parts of endo-beta-glucanases, 10 parts of outer 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 proteases, 10 parts of aspartic proteases, 5 parts of superoxide-dismutases, 5 parts of glucose oxidases, 5 parts of acid phosphatases;
Described protective material is comprised of the raw material of following parts by weight: 20 parts of trehaloses, NaCl20 part, (NH 4) 2sO 410 parts, 10 parts of halfcystines.
Described activator is evenly to be mixed by the inorganic salt of following quality component: 30 parts of zinc chloride, 10 parts, calcium chloride, 10 parts, sodium sulfate, 5 parts, magnesium chloride.
Containing the preparation method of the Wheat Based Diet enzyme of neutral protease as embodiment 1.
Embodiment 3:
Containing a Wheat Based Diet enzyme for neutral protease, by the zymin of following parts by weight, formed:
30 parts of subtilis cultures, 30 parts of aspartic proteases, 30 parts of acidic xylanases, 30 parts of cellulases, 20 parts of beta-glucanases, 0 part of amylase 2,15 parts of 'beta '-mannases, 15 parts of Chinese herbal medicine extracts, 15 parts of protective materials, 15 parts of activator.
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 to slant medium, cultivates 36h for 36 ℃ and carry out actication of culture, so activate 3 times;
Described slant medium consists of: extractum carnis 10g, and 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, 7.2,121 ℃ of sterilizing 20min of pH value;
The preparation method of described Chinese herbal medicine powder is as follows:
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 mixture 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 mixture 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 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 proteases, 15 parts of aspartic proteases, 10 parts of superoxide-dismutases, 10 parts of glucose oxidases, 10 parts of acid phosphatases.
(2) liquid seeds enlarged culturing
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 substratum loading amounts, 180 revs/min of rotary shaking tables, 36 ℃ of culture temperature, incubation time 15h;
2. secondary seed is cultivated: by first order seed, according in 500 milliliters of secondary seed shaking flasks of inoculum size access of 10%, 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 shaking flasks to 1000 milliliters of substratum loading amounts, 100 revs/min of rotary shaking tables, 36 ℃ of culture temperature, incubation time 15h with 10% inoculum size;
4. first class seed pot is cultivated: the first class seed pot by three grades of seeds take 10% inoculum size access cubic capacity as 150L, fermention medium loading amount 100L, 36 ℃ of culture temperature, 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, 7.2,123 ℃ of sterilizing 40min of pH value.
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, 7.2,123 ℃ of sterilizing 40min of pH value.
Described seeding tank fermented liquid cell concentration is 8.0x10 8individual/ml;
(3) ferment tank
First class seed pot fermented liquid in step (2) is accessed to fermentor tank, 36 ℃ of culture temperature, stirring velocity 700r/m, ventilation (V/V) 1:3, incubation time 15h with 6% inoculum size; Then with 2 ℃/h rate of temperature fall slow cooling to 15 ℃, constant temperature culture 20h; Continuation, with 2 ℃/h rate of temperature fall slow cooling to 5 ℃, now, is appended access fermentor tank, constant temperature culture 30h by first class seed pot fermented liquid in step (2) with 4% inoculum size; Finally with 2 ℃/h temperature rise rate, be slowly warming up to 15 ℃, constant temperature culture 20h; Continuation is slowly warming up to 36 ℃ with 2 ℃/h temperature rise rate, constant temperature culture 20h;
Dissolved oxygen control: by adjusting mixing speed and ventilation, control dissolved oxygen 30%;
PH controls: by mending ammoniacal liquor or dilute phosphoric acid, in controlled fermentation process, pH value remains on 7.2;
Control of additive raw material: add supplemented medium, to maintain fermented liquid reducing sugar content as 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, 7.2,121 ℃ of sterilizing 20min of pH value;
Described supplemented medium weight consists of: maltodextrin 30%, and Semen Maydis powder 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 fermention medium is:
Accurately take in proportion raw material, 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), ℃ 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 standby.
(4) fermented liquid via hole diameter 1000 μ m filter board coarse filtration, then in the concentrated removal of 20 ℃ of loop ultrafiltrations moisture, obtaining solid content 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 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 mixture 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 lyophilize obtains Chinese herbal medicine extract.
The parts by weight of described herbal medicine consist of: 30 parts of sea-buckthorns, 30 parts of Semen Cassiaes, 15 parts of matrimony vines, 15 parts of Chinese yams, 10 parts of Root of coastal Glehnia, 10 parts of Fructus Viticis 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 mixture gross weight;
The parts by weight of described mixed enzyme consist of: 20 parts of endo-beta-glucanases, 20 parts of outer 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 proteases, 15 parts of aspartic proteases, 10 parts of superoxide-dismutases, 10 parts of glucose oxidases, 10 parts of acid phosphatases;
Described protective material is comprised of the raw material of following parts by weight: 30 parts of trehaloses, NaCl30 part, (NH 4) 2sO 415 parts, 15 parts of halfcystines.
Described activator is evenly to be mixed by the inorganic salt of following quality component: 40 parts of zinc chloride, 20 parts, calcium chloride, 20 parts, sodium sulfate, 10 parts, magnesium chloride.
Containing the preparation method of the Wheat Based Diet enzyme of neutral protease as embodiment 1.
The result of use test of embodiment 4 embodiment of the present invention 1 Wheat Based Diet enzymes
1. feed kind: full price Wheat Based Diet, wherein in feed, wheat weight is 40%;
2. Wheat Based Diet enzyme addition: feed per ton adds 120g;
3. Wheat Based Diet enzyme test group design: Wheat Based Diet enzyme parts by weight composition is divided into test group, control group 1, control group 2; Wherein test group is Wheat Based Diet 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 that commercially available Wheat Based Diet 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
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 at random 3 groups, and every group of 2 hurdles repeat, 6 of every repetitions, galt and gilt half and half.The official 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 envrionment conditions 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 Wheat Based Diet feed as basis, feed per ton adds the Wheat Based Diet enzyme 120g of test group, control group 1 and control group 2; The raw material of Wheat Based Diet 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, take hurdle, as its every daily material consumption of unit record, sickness rate, calculate daily ingestion amount and food utilization efficiency simultaneously, and above-mentioned data are carried out to statistical study, growing swine growth performance is in Table 2.
Table 2
Figure BDA0000447805440000171
4.1.5 the full phase test result of above-mentioned test is analyzed as table 3
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)
Material anharmonic 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 scoring 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, and in the situation that the test conditionss such as test materials, testing circumstance, test method and Experimental Establishment are identical, day weight gain has improved respectively 17.02% and 32.88%; Food consumption has reduced respectively 1.92% and 9.7%; Material anharmonic ratio has reduced respectively 14.5% and 30.84%; Diarrhea rate has reduced respectively 75% and 94%; Outward appearance hair color quality has improved respectively 30.76% and 112.5%; Use Wheat Based Diet enzyme growing swine of the present invention to there is good growth performance and significant immunological competence, improved cultured output and quality, reduced aquaculture cost, improved fanning economics.

Claims (10)

1. the Wheat Based Diet enzyme containing neutral protease, zymin by following parts by weight forms: 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:M2013539 cultivates enlarged culturing step by step through slant strains activation and one, two, three seed culture and first class seed pot and obtains seed liquor; Liquid seeds is accessed to fermentor tank, culture temperature 30-36 ℃, stirring velocity 200-700rpm, ventilation (V/V) 1:1-3, incubation time 10-15h with 6% inoculum size; 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 fermentor tank, constant temperature culture 20-30h by liquid seeds with 4% inoculum size with 1-2 ℃/h rate of temperature fall slow cooling; Finally with 1-2 ℃/h temperature rise rate, be slowly warming up to 10-15 ℃, constant temperature culture 15-20h; Continuation is slowly warming up to 30-36 ℃, constant temperature culture 15-20h with 1-2 ℃/h temperature rise rate; Fermented liquid via hole diameter 10-1000 μ m filter board coarse filtration, then in the concentrated removal of 10-20 ℃ of loop ultrafiltration moisture, obtaining solid content is 20-40% concentrated solution; Concentrated solution obtains subtilis culture through vacuum lyophilization, micronizing;
Described protective material is comprised 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 evenly to be mixed by the inorganic salt 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 Based Diet enzyme containing neutral protease as claimed in claim 1, 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 ℃ of sterilizing 20min.
3. a kind of Wheat Based Diet enzyme containing neutral protease as claimed in claim 1, 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 ℃ of sterilizing 30-40min.
4. a kind of Wheat Based Diet enzyme containing neutral protease as claimed in claim 1, 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 ℃ of sterilizing 30-40min.
5. a kind of Wheat Based Diet enzyme containing neutral protease as claimed in claim 1, 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 Based Diet 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 ℃ of sterilizing 20min.
7. the preparation method containing the Wheat Based Diet enzyme of neutral protease; 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, evenly mix; finally add activator, after mixing, pack the Wheat Based Diet enzyme getting product containing neutral protease.
8. the preparation method of a kind of Wheat Based Diet enzyme containing neutral protease as claimed in claim 7, it is characterized in that, the described Wheat Based Diet enzyme containing neutral protease is comprised 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 1398-2-12 cultivates enlarged culturing step by step through slant strains activation and one, two, three seed culture and first class seed pot and obtains seed liquor; Liquid seeds is accessed to fermentor tank, culture temperature 30-36 ℃, stirring velocity 200-700rpm, ventilation (V/V) 1:1-3, incubation time 10-15h with 6% inoculum size; 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 fermentor tank, constant temperature culture 20-30h by liquid seeds with 4% inoculum size with 1-2 ℃/h rate of temperature fall slow cooling; Finally with 1-2 ℃/h temperature rise rate, be slowly warming up to 10-15 ℃, constant temperature culture 15-20h; Continuation is slowly warming up to 30-36 ℃, constant temperature culture 15-20h with 1-2 ℃/h temperature rise rate; Fermented liquid via hole diameter 10-1000 μ m filter board coarse filtration, then in the concentrated removal of 10-20 ℃ of loop ultrafiltration moisture, obtaining solid content is 20-40% concentrated solution; Concentrated solution obtains subtilis culture through vacuum lyophilization, micronizing;
Described protective material is comprised 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 evenly to be mixed by the inorganic salt of following quality component: zinc chloride 30-40 part, calcium chloride 10-20 part, sodium sulfate 10-20 part, magnesium chloride 5-10 part.
9. the preparation method of a kind of Wheat Based Diet enzyme containing neutral protease 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 mixture 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 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.
10. the preparation method of a kind of Wheat Based Diet enzyme containing neutral protease as claimed in claim 8, is characterized in that, the described Wheat Based Diet enzyme containing neutral protease, zymin by following parts by weight forms: 25 parts of subtilis cultures, 25 parts of aspartic proteases, 25 parts of acidic xylanases, 25 parts of cellulases, 17 parts of beta-glucanases, 17 parts of amylase, 12 parts of 'beta '-mannases, 12 parts of Chinese herbal medicine extracts, 13 parts of protective materials, 12 parts of activator;
The preparation method of described subtilis culture comprises the steps:
(1) slant strains of intact 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: extractum carnis 3g, and 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, 7.0,121 ℃ of sterilizing 20min of pH value;
The preparation method of described Chinese herbal medicine powder is as follows:
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 mixture 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 mixture 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 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 proteases, 10 parts of aspartic proteases, 5 parts of superoxide-dismutases, 5 parts of glucose oxidases, 5 parts of acid phosphatases;
(2) liquid seeds enlarged culturing
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 substratum loading amounts, 180 revs/min of rotary shaking tables, 30 ℃ of culture temperature, incubation time 10h;
2. secondary seed is cultivated: by first order seed, according in 500 milliliters of secondary seed shaking flasks of inoculum size access of 10%, 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 shaking flasks to 1000 milliliters of substratum loading amounts, 100 revs/min of rotary shaking tables, 30 ℃ of culture temperature, incubation time 10h with 10% inoculum size;
4. first class seed pot is cultivated: the first class seed pot by three grades of seeds take 10% inoculum size access cubic capacity as 150L, fermention medium loading amount 100L, 30 ℃ of culture temperature, 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, 7.0,121 ℃ of sterilizing 30min of pH value;
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, 7.0,121 ℃ of sterilizing 30min of pH value;
Described seeding tank fermented liquid cell concentration is 7.0x10 8individual/ml;
(3) ferment tank
First class seed pot fermented liquid in step (2) is accessed to fermentor tank, 30 ℃ of culture temperature, stirring velocity 200r/m, ventilation (V/V) 1:1, incubation time 10h with 6% inoculum size; Then with 1 ℃/h rate of temperature fall slow cooling to 10 ℃, constant temperature culture 15h; Continuation, with 1 ℃/h rate of temperature fall slow cooling to 2 ℃, now, is appended access fermentor tank, constant temperature culture 20h by first class seed pot fermented liquid in step (2) with 4% inoculum size; Finally with 1 ℃/h temperature rise rate, be slowly warming up to 10 ℃, constant temperature culture 15h; Continuation is slowly warming up to 30 ℃ with 1 ℃/h temperature rise rate, constant temperature culture 15h;
Dissolved oxygen control: by adjusting mixing speed and ventilation, control dissolved oxygen 15%;
PH controls: by mending ammoniacal liquor or dilute phosphoric acid, in controlled fermentation process, pH value remains on 7.0;
Control of additive raw material: add supplemented medium, to maintain fermented liquid reducing sugar content as 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, 7.0,121 ℃ of sterilizing 20min of pH value;
Described supplemented medium weight consists of: maltodextrin 20%, and Semen Maydis powder 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 fermention medium is:
Accurately take in proportion raw material, 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), ℃ 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 standby;
(4) fermented liquid via hole diameter 10 μ m filter board coarse filtration, then in the concentrated removal of 10 ℃ of loop ultrafiltrations moisture, obtaining solid content 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 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 mixture 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 lyophilize obtains Chinese herbal medicine extract;
The parts by weight of described herbal medicine consist of: 25 parts of sea-buckthorns, 25 parts of Semen Cassiaes, 17 parts of matrimony vines, 13 parts of Chinese yams, 8 parts of Root of coastal Glehnia, 7 parts of Fructus Viticis 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 mixture gross weight;
The parts by weight of described mixed enzyme consist of: 15 parts of endo-beta-glucanases, 15 parts of outer 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 proteases, 13 parts of aspartic proteases, 7 parts of superoxide-dismutases, 7 parts of glucose oxidases, 7 parts of acid phosphatases;
Described protective material is comprised of the raw material of following parts by weight: 25 parts of trehaloses, NaCl25 part, (NH 4) 2sO 413 parts, 12 parts of halfcystines;
Described activator is evenly to be mixed by the inorganic salt of following quality component: 35 parts of zinc chloride, 15 parts, calcium chloride, 15 parts, sodium sulfate, 7 parts, magnesium chloride;
Contain the preparation method of the Wheat Based Diet 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, evenly mix; finally add activator, after mixing, pack the Wheat Based Diet enzyme getting product containing neutral protease.
CN201310738229.0A 2013-12-27 2013-12-27 A kind of Wheat ration enzyme containing neutral protease and preparation method thereof Active CN103740683B (en)

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CN111187763A (en) * 2019-11-22 2020-05-22 武汉新华扬生物股份有限公司 Complex enzyme preparation for oat hydrolysis and application thereof

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CN104264262A (en) * 2014-09-01 2015-01-07 郸城财鑫糖业有限责任公司 Method for extracting araboxylan and protein fiber from wheat bran
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CN111187763A (en) * 2019-11-22 2020-05-22 武汉新华扬生物股份有限公司 Complex enzyme preparation for oat hydrolysis and application thereof

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