CN103859169B - A kind of feed complex enzyme containing neutral proteinase and preparation method thereof - Google Patents

Abstract

The invention discloses a kind of feed complex enzyme containing neutral proteinase and preparation method thereof, belong to technical field of enzyme preparation.The described feed complex enzyme containing neutral proteinase; with concentrated maltase, acid protease, neutral proteinase, acidic xylanase, cellulase, 1,4 beta-glucanase, pectase, amylase, seminase, phytase, Chinese herbal medicine extract, protective agent, activator science is composite forms; enzyme activity is high, action condition is wide in range, stability is strong, is more suitable for Feed Manufacturing.Safe ingestion enzyme can be provided for livestock and poultry; alleviate digestion burden; improve raw material availability and growth rate; available protecting environment, and bring joyful native malt fragrance to feed, strengthen appetite of livestock and poultry; appropriate activator under equal conditions can give full play to the effect of enzyme preparation simultaneously; save enzyme preparation addition, the Chinese herbal medicine extract science composite shelf-life both having extended complex enzyme formulation, the immunity of raising livestock and poultry can be improved again.

Description

A kind of feed complex enzyme containing neutral proteinase and preparation method thereof

Technical field

The invention belongs to technical field of enzyme preparation, specifically a kind of feed complex enzyme containing neutral proteinase and preparation method thereof.

Background technology

In feed, add enzyme preparation mainly contain following 4 reasons: 1. the ANFs existed in animal feed of degrading.These materials can not be degraded by animal endogenous enzymes, thus the eubolism of interference animal, cause animal digestion bad, production performance declines.2. improve the utilization rate of starch, protein and mineral matter.These materials or surrounded by the fibrous cell membrane of richness, or can not be had by the version 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 nutrients can be discharged after adding exogenous enzymes.4., because young animal own digestive system is also immature, endogenous enzymes deficiency adds exogenous enzymes can improve feed digestibility, prevents indigestion symptom.Except can improving the utilization rate of daily ration, the enzyme-added difference that can also reduce between feedstuff, improves the accuracy of feed formula, can also improve the regularity of growth of animal simultaneously, reduces management cost, increases economic efficiency.Use all right protection of the environment of enzyme preparation.Because the utilization rate of feed improves, corresponding ight soil discharge capacity have dropped.In the obvious situation of effectiveness comparison, the discharge capacity 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 enzyme preparation applied in feed industry at present mainly contains 4 large classes: be used for degraded cellulose, protein, starch and phytic acid respectively.

Fiber degradation enzyme: for nonruminant, 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 indigestion simultaneously.As the anorexia 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 fiber 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 regularity of animal.Some dyspeptic disease can also be reduced simultaneously.

PD 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 nonruminant daily ration, add protease (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 ANFs in beancake 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 beancake powder) well not utilized.

Starch degrading enzyme: many nutritionists think that corn is feedstuff " 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 digestive enzyme: for all animals, phosphorus is all vital for the mineralising of bone, immunity, breeding, growth.Pig and poultry nonruminant can only in utilize in plant feed 30-40% phosphorus, all the other phytate phosphorus of 60-70% are unserviceable.In many cases, Phos 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, contaminated 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 enzyme preparation 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 enzyme preparation, with regard to more complicated, seems uncertain.Intensive degree is low, and relate to link many, the result of use of enzyme preparation is difficult to carry out business calculating.Although had the imagination using enzyme preparation, until just start the eighties in 20th century to understand the strength how playing enzyme in feed industry in the 1950's.Feed grain, as Wheat and barley all contains the unavailable fiber of higher nonruminant.As fruit fiber can be degraded, animal just can utilize nutrients better.In Europe, barley is 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 effect 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 feed industry generally to be approved.Can not rant out: 1996, containing fiber degradation enzyme in the broiler chicken material (viscosity cereal is energy source) 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 main cause is the complicated structure in market, and market is diversification, even cannot calculate.From geographical 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 crude fibre or viscosity is not serious.Past 10 years was expended a lot in research and development Corn-soybean first generation feed enzyme, and started successful Application in 1996, and initial stage application result is multifarious, but industry is just starting to become how more and more understand could be handy, adds zymotechnic and 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 crude fibre are that the feed enzyme market 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 pig fowl feed 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 feed enzyme has it wide and huge economic worth, but the heat endurance of feed enzyme, security, composite comprehensive and action effect give full play to the major issue being still enzyme preparation manufacturer and numerous raisers and jointly paying close attention to, prepare safer, more comprehensively, enzyme action effect is better, can improve corporation responsibility and pursuit that the feed complex enzyme of raising appetite of livestock and poultry is industry technical staff.

Summary of the invention

Technical problem solved by the invention is comprehensive with enzyme system, natural safety, there is concentrated maltase and the high enzymatic activity of malt flavor, action condition is wide in range, based on the neutral proteinase that stability is strong, and the composite Chinese herbal medicine extract of science, protective agent, activator and other food-grade feed enzyme, the obtained feed complex enzyme containing neutral proteinase not only provides safety for raising livestock and poultry, comprehensive digestive ferment, alleviate digestion burden, improve raw material availability and growth rate, available protecting environment, and joyful native malt fragrance can be brought to feed, increase the appetite of livestock and poultry, appropriate activator under equal conditions can give full play to the effect of enzyme preparation simultaneously, make the best use of everything, save enzyme preparation addition, the science composite shelf-life that both can extend complex enzyme formulation of Chinese herbal medicine extract, the immunity 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 feed complex enzyme for neutral proteinase, be made up of the enzyme preparation of following parts by weight:

Concentrated maltase 30-40 part, acid protease 20-30 part, neutral proteinase 20-30 part; acidic xylanase 20-30 part; cellulase 20-30 part, 1,4 beta-glucanase 15-20 part, pectase 15-20 part; amylase 15-20 part; seminase 10-15 part, phytase 10-15 part, Chinese herbal medicine extract 10-15 part; protective agent 10-15 part, activator 10-15 part.

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

The preparation method of described neutral proteinase is as follows:

Bacillus subtilis 1398-2-12 is through actication of culture and expand cultivation acquisition liquid seeds step by step; By liquid seeds with 6% inoculum concentration access fermentation tank, cultivation temperature 30-36 DEG C, mixing speed 200-700r/m, 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 fermentation tank with 4% inoculum concentration, 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 heating rate; Continue slowly to be warming up to 30-36 DEG C, constant temperature culture 15-20h with 1-2 DEG C/h heating rate; Fermentation liquor is filtered, concentrated, allotment, essence filter, dry solid neutral protease; Described allocation process adds concentrated enzyme liquid gross weight 0.5-5% Chinese herbal medicine powder.

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

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

Take Radix Astragali 20-30 part; Radix Codonopsis 10-18 part; Radix bupleuri 10-15 part; Root of large-flowered skullcap 10-15 part; Respectively said herbal medicine being crushed to particle diameter is 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 mixed material 0.5-3 times of w ethanol and propyl alcohol, control temperature to 60 DEG C-78 DEG C of maintenance 3-4h, filter; Filter vacuum concentrates postlyophilization and obtains Chinese herbal medicine powder.

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

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

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

Described seed culture medium percentage by weight consists of:

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

Described seed tank culture base percentage by weight consists of:

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

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

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

Described supplemented medium percentage by weight consists of: maltodextrin 20-30%, corn flour 10-20%, bean powder 15-25%, herbal mediciment 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 fermentation medium is:

Accurately take raw material in proportion, pure water in raw material, corn flour, beancake powder are dropped in material-compound tank, regulate pH value 7.0-7.2, add middle temperature amylase (3u/g corn flour) and alpha-amylase (30u/g corn flour), 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 p H7.0-7.2,121-123 DEG C of sterilizing 30-40min for subsequent use.

Described concentrated maltase percentage by weight consists of: former maltase 30-40%, fragrance maltase 60-70%.

Described former maltase preparation method is as follows:

(1) high-pressure pulse electric (PEF) immersion treatment: by Fructus Hordei Germinatus in pre-immersion trough with 3-6 times of 20-50 DEG C of emerge in worm water 10-20min, Fructus Hordei Germinatus moisture is made to reach 25-35%, carry out high-pressure pulse electric (PEF) process simultaneously, electric-field intensity 20-40KV/cm, burst length 150-200 μ S, pulse frequency 200-300Hz;

(2) pulverize: by the Fructus Hordei Germinatus handled well in step (1), to add maltcrusher band pigment broken with soaking water, and adjustment roller spacing is 0.5mm, and rotating speed is 250rpm, obtains Fructus Hordei Germinatus slurries;

(3) ultrasonic, PEF extracts: Fructus Hordei Germinatus slurries are placed in pill tank, and limit is slowly stirred limit probe type ultrasonic extraction apparatus under current strength 0.6A/25w-1.5A/275w condition, carried out ultrasonic extraction 10-15min; Then carry out high-pressure pulse electric (PEF) and extract 15-20min, electric-field intensity 20-40KV/cm, burst length 400-600 μ S, pulse frequency 200-300Hz;

(4) filter: by extract in room temperature 500-800 order filter cloth precoating granularity be 100 order-300 order diatomite, adopt flame filter press filter, operating pressure is 0.14-0.24Mpa;

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

(6) dry: the protective agent of the present invention adding concentrate weight 3-5% in concentrate, then use the special spray dryer of enzyme preparation dry, EAT 150-160 DEG C; temperature of outgoing air 75-85 DEG C; drying time 5-15s, product moisture < 5%, both former maltase.

Described fragrance maltase preparation method is as follows:

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

(2) cure: the Fructus Hordei Germinatus that will get damp again loads drum-type and fries stove, and in 10-15min, temperature rises to 170-200 DEG C, insulation 10-15min, is then cooled to 100-110 DEG C, and insulation 20-30min, finally takes out spreading for cooling, obtain fragrance Fructus Hordei Germinatus;

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

(4) ultrasonic, PEF extracts: fragrance malt flour is placed in pill tank, and add 3-6 water doubly, limit is slowly stirred limit probe type ultrasonic extraction apparatus under current strength 0.6A/25w-1.5A/275w condition, carried out ultrasonic extraction 10-15min; Then carry out high-pressure pulse electric (PEF) and extract 15-20min, electric-field intensity 20-40KV/cm, burst length 400-600 μ S, pulse frequency 200-300Hz;

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

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

(7) dry: the protective agent of the present invention adding concentrate weight 3-5% in concentrate, then use the special spray dryer of enzyme preparation dry, EAT 150-160 DEG C; temperature of outgoing air 75-85 DEG C; drying time 5-15s, product moisture < 5%, both fragrance maltase.

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 mixed material 0.5-3 times of w ethanol and propyl alcohol, control temperature to 60 DEG C-78 DEG C of maintenance 3-4h, filter; Filtrate reduced in volume to solid content is more than 20%, and then freeze drying obtains Chinese herbal medicine extract.

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

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

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

Described protective agent is made up of the raw material of following parts by weight: trehalose 20-30 part, NaCl20-30 part, (NH ₄) SO ₄10-15 part, cysteine 10-15 part.

Described activator is formed by the inorganic salts Homogeneous phase mixing of following quality component: zinc chloride 30-40 part, calcium chloride 10-20 part, sodium sulphate 10-20 part, magnesium chloride 5-10 part.

The present invention is containing the preparation method of the feed complex enzyme of neutral proteinase:

By described protective agent, Chinese herbal medicine extract, concentrated maltase ultramicro grinding respectively; guarantee that granularity is less than described neutral proteinase and other enzyme preparation; then with neutral proteinase, acid protease, acidic xylanase, cellulase, 1,4 beta-glucanase, pectase, amylase, seminase, phytase; Homogeneous phase mixing; finally add activator, after mixing, pack the feed complex enzyme got product containing neutral proteinase.

Bacillus subtilis 1398-2-12 provided by the invention produces bacillus subtilis (Bacillus subtilis) 1398-2 of neutral proteinase through UV-LiCl-dithyl sulfate Mutation screening acquisition by a strain of Laboratories Accession.

The bacterial strain of product heat-flash stability neutral proteinase provided by the invention is specially bacillus subtilis 1398-2-12.This bacterial strain is preserved in China typical culture collection center on November 3rd, 2013 and (is called for short CCTCC, address: China. Wuhan. Wuhan University, postcode: 430072), preserving number is CCTCC NO:M2013539, Classification And Nomenclature: bacillus subtilis 1398-2-12(Bacillus subtilis1398-2-12).

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

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

Bacillus subtilis 1398-2-12 provided by the invention has the advantages that produced neutral proteinase tolerable temperature is high, be suitable for pH value wide scope, zymotic fluid 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 fermenting property test screen, obtain the Strains B. subtilis bacterium 1398-2-12 that heat-flash stability neutral proteinase is produced in a strain, the work of zymotic fluid neutral proteinase enzyme can reach 5500-7000U/mL, the heat endurance 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 present invention can reach 5500-7000U/mL containing the neutral proteinase fermentation broth enzyme work in the feed complex enzyme of neutral proteinase, is 2.5 times of starting strain.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 storage stability is high, is more applicable to the interpolation of raising animal and fowl fodder.

2. the present invention containing neutral proteinase feed complex enzyme in concentrated maltase with high-quality import Fructus Hordei Germinatus for raw material, the method such as ultrasonic wave, high-voltage pulse electric field technology (pulsed electric field is called for short " PEF ") is adopted to extract maltase system enzyme source to greatest extent, it is a kind of natural complex enzyme, its enzyme source kind and single enzyme characteristic 100% are from brewers malt, and hydrolysis result is far superior to the obtained single enzyme of employing fermentable and combination thereof; Fragrance maltase wherein not only can provide enzyme source, and most importantly its strong, unique malt flavor can significantly improve the appetite of raising livestock and poultry.

3. the present invention adopts polysaccharide, inorganic salts and amino acid science composite containing the protective agent in the feed complex enzyme of neutral proteinase, effectively slow down the moisture regain of fragrance maltase and complex enzyme formulation; Can strengthen complex enzyme simultaneously resistance toly to freeze, heat resistance, keep identical enzyme activity, its heat resisting temperature can improve 20-30 DEG C, resistance to cryogenic temperature can reduce 10-15 DEG C, effectively prevent the loss of complex enzyme enzyme activity in transport, preservation and use procedure, extend the shelf-life of complex enzyme, reach same enzyme activity, the like product shelf-life can extend 2-3.

4. the present invention adds inorganic salts as activator containing the feed complex enzyme of neutral proteinase; create the optimum condition of enzyme catalysis; give full play to the vigor of each enzyme component of complex enzyme; the macromolecular substances such as starch, protein, 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 shelf-life of complex enzyme formulation containing the Chinese herbal medicine extract that the feed complex enzyme of neutral proteinase adds, and can improve again the immunity of raising livestock and poultry, effectively prevent the generation of livestock and poultry epidemic disease.

6. the present invention is containing the common synergy of protective agent and enzyme preparation, activator and enzyme preparation in the feed complex enzyme of neutral proteinase, Chinese herbal medicine extract and enzyme preparation; the enzyme activity of complex enzyme and effect are played to greatest extent; and improve the utilization rate of feed and the growth rate of animal accordingly; enhance appetite and the resistance against diseases of animal, extend the shelf-life of complex enzyme and protect environment.

Detailed description of the invention

The present invention is described below by specific embodiment.Unless stated otherwise, technological 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 feed complex enzyme for neutral proteinase, be made up of the enzyme preparation of following parts by weight:

Concentrated maltase 35 parts, acid protease 25 parts, neutral proteinase 25 parts, acidic xylanase 25 parts, cellulase 25 parts; 1,4 beta-glucanase 17 parts, pectase 17 parts, amylase 17 parts, seminase 12 parts; phytase 13 parts, Chinese herbal medicine extract 12 parts, protective agent 13 parts, activator 12 parts.

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

The preparation method of described neutral proteinase comprises the steps:

(1) slant strains of intact bacillus 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: beef extract 3g, sodium chloride 5g, peptone 10g, glucose 2g, (NH) ₂sO ₄3g, K ₂hPO ₄6g, CaCl ₂1g, agar 15g, Chinese herbal medicine powder 5g, distilled water l000mL, pH value 7.0,121 DEG C of sterilizing 20min;

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

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 mixed material gross weight 5% carries out enzymolysis, be 5.5 by lactic acid adjust ph, enzymolysis 2h, finally add the mixture of mixed material 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 1,4 beta-glucanase 10 parts, beta-glucosidase 10 parts, zytase 15 parts, pentosanase 15 parts, Pullulanase 20 parts, beta amylase 10 parts, neutral proteinase 10 parts, acid protease 10 parts, superoxide dismutase 5 parts, glucose oxidase 5 parts, acid phosphatase 5 parts.

(2) liquid seeds expands cultivation

1. first order seed is cultivated: slant strains 1 articulating after step (1) being activated enters in 500 ml shake flasks, culture medium loading amount 100 milliliters, rotary shaker 180 revs/min, cultivation temperature 30 DEG C, incubation time 10h;

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

3. three grades of seed culture: secondary seed is accessed in 5000 milliliters of three grades of seed flask with 10% inoculum concentration, culture medium loading amount 1000 milliliters, rotary shaker 100 revs/min, cultivation temperature 30 DEG C, incubation time 10h;

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

Described one-level, secondary, three grades of seed culture medium percentage by weights consist of:

Dusty yeast 0.3%, glucose 1%, peptone 0.3%, beef extract 0.5%, dipotassium hydrogen phosphate 0.8%, Chinese herbal medicine powder 1.5%, trehalose 1%, calcium sulfate 1g, magnesium chloride 2g, natrium citricum 1g, insufficient section pure water is supplied, pH value 7.0,121 DEG C of sterilizing 30min.

Described seed tank culture base percentage by weight consists of:

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

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

(3) ferment tank

By first class seed pot zymotic fluid in step (2) with 6% inoculum concentration access fermentation tank, cultivation temperature 30 DEG C, mixing speed 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 zymotic fluid in step (2) is added access fermentation tank with 4% inoculum concentration, constant temperature culture 20h; Finally slowly be warming up to 10 DEG C, constant temperature culture 15h with 1 DEG C/h heating rate; Continue slowly to be warming up to 30 DEG C, constant temperature culture 15h with 1 DEG C/h heating rate;

Dissolved oxygen controls: by adjustment speed of agitator and ventilation, controls dissolved oxygen 15%;

PH controls: by mending ammoniacal liquor or phosphoric acid,diluted, controls pH value in sweat and remains on 7.0;

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

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

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

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

The concocting method of described fermentation medium is:

Accurately take raw material in proportion, pure water in raw material, corn flour, beancake powder are dropped in material-compound tank, regulate pH value 7.0, add middle temperature amylase (3u/g corn flour) and alpha-amylase (30u/g corn flour), 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 p H7.0,121 DEG C of sterilizing 30min are for subsequent use.

(4) fermentation liquor filter, concentrated, allotment, essence filter, dry heat-flash stability neutral proteinase, described allocation process adds concentrated enzyme liquid gross weight 0.5% Chinese herbal medicine powder.

Described concentrated maltase percentage by weight consists of: former maltase 35%, fragrance maltase 65%.

Described former maltase preparation method is as follows:

(1) high-pressure pulse electric (PEF) immersion treatment: by Fructus Hordei Germinatus in pre-immersion trough with 5 times of 35 DEG C of emerge in worm water 15min, make Fructus Hordei Germinatus moisture reach 30%, carry out high-pressure pulse electric (PEF) process, electric-field intensity 30KV/cm simultaneously, burst length 175 μ S, pulse frequency 250Hz;

(2) pulverize: by the Fructus Hordei Germinatus handled well in step (1), to add maltcrusher band pigment broken with soaking water, and adjustment roller spacing is 0.5mm, and rotating speed is 250rpm, obtains Fructus Hordei Germinatus slurries;

(3) ultrasonic, PEF extracts: Fructus Hordei Germinatus slurries are placed in pill tank, and limit is slowly stirred limit probe type ultrasonic extraction apparatus under current strength 1.0A/150w condition, carried out ultrasonic extraction 12min; Then carry out high-pressure pulse electric (PEF) and extract 12min, electric-field intensity 30KV/cm, burst length 500 μ S, pulse frequency 250Hz;

(4) filter: be 200 order diatomite in room temperature with 650 order filter cloth precoating granularities by extract, employing flame filter press filters, and operating pressure is 0.19Mpa;

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

(6) dry: the protective agent of the present invention adding concentrate weight 4% in concentrate, then use the special spray dryer of enzyme preparation dry, EAT 155 DEG C; temperature of outgoing air 80 DEG C; drying time 10s, product moisture < 5%, both former maltase.

Described fragrance maltase preparation method is as follows:

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

(2) cure: the Fructus Hordei Germinatus that will get damp again loads drum-type and fries stove, and in 12min, temperature rises to 185 DEG C, insulation 12min, is then cooled to 105 DEG C, and insulation 25min, finally takes out spreading for cooling, obtain fragrance Fructus Hordei Germinatus;

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

(4) ultrasonic, PEF extracts: fragrance malt flour is placed in pill tank, adds the water of 4 times, limit is slowly stirred limit probe type ultrasonic extraction apparatus under current strength 1.0A/150w condition, carried out ultrasonic extraction 12min; Then carry out high-pressure pulse electric (PEF) and extract 17min, electric-field intensity 30KV/cm, burst length 500 μ S, pulse frequency 250Hz;

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

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

(7) dry: the protective agent of the present invention adding concentrate weight 4% in concentrate, then use the special spray dryer of enzyme preparation dry, EAT 155 DEG C; temperature of outgoing air 80 DEG C; drying time 10s, product moisture < 5%, both fragrance maltase.

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 mixed material 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 freeze drying obtains Chinese herbal medicine extract.

The parts by weight of described Chinese herbal medicine consist of: sea-buckthorn 25 parts, cassia seed 25 parts, matrimony vine 17 parts, Chinese yam 13 parts, radix glehniae 8 parts, fruit of 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 mixed material gross weight;

The parts by weight of described mixed enzyme consist of: endo-beta-glucanase 15 parts, outer 1,4 beta-glucanase 15 parts, beta-glucosidase 13 parts, zytase 13 parts, pentosanase 13 parts, Pullulanase 25 parts, beta amylase 12 parts, neutral proteinase 13 parts, acid protease 13 parts, superoxide dismutase 7 parts, glucose oxidase 7 parts, acid phosphatase 7 parts;

Described protective agent is made up of the raw material of following parts by weight: trehalose 25 parts, NaCl25 part, (NH ₄) SO ₄13 parts, cysteine 12 parts.

Described activator is formed by the inorganic salts Homogeneous phase mixing of following quality component: zinc chloride 35 parts, 15 parts, calcium chloride, 15 parts, sodium sulphate, 7 parts, magnesium chloride.

Preparation method containing the feed complex enzyme of neutral proteinase:

By described protective agent, Chinese herbal medicine extract, concentrated maltase ultramicro grinding respectively; guarantee that granularity is less than described neutral proteinase and other enzyme preparation; then with neutral proteinase, acid protease, acidic xylanase, cellulase, 1,4 beta-glucanase, pectase, amylase, seminase, phytase; Homogeneous phase mixing; finally add activator, after mixing, pack the feed complex enzyme got product containing neutral proteinase.

Embodiment 2:

Containing a feed complex enzyme for neutral proteinase, be made up of the enzyme preparation of following parts by weight:

Concentrated maltase 30 parts, acid protease 20 parts, neutral proteinase 20 parts, acidic xylanase 20 parts, cellulase 20 parts; 1,4 beta-glucanase 15 parts, pectase 15 parts, amylase 15 parts, seminase 10 parts; phytase 10 parts, Chinese herbal medicine extract 10 parts, protective agent 10 parts, activator 10 parts.

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

The preparation method of described neutral proteinase comprises the steps:

(1) actication of culture

The slant strains of intact bacillus 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: beef extract 6g, sodium chloride 8g, peptone 15g, glucose 4g, (NH) ₂sO ₄4g, K ₂hPO ₄7g, CaCl ₂2g, agar 18g, Chinese herbal medicine powder 8g, distilled water l000mL, pH value 7.2,121 DEG C of sterilizing 20min;

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

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 mixed material gross weight 8% carries out enzymolysis, be 6.0 by lactic acid adjust ph, enzymolysis 3h, finally add the mixture of mixed material 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 1,4 beta-glucanase 15 parts, beta-glucosidase 12 parts, zytase 18 parts, pentosanase 18 parts, Pullulanase 25 parts, beta amylase 12 parts, neutral proteinase 12 parts, acid protease 12 parts, superoxide dismutase 8 parts, glucose oxidase 8 parts, acid phosphatase 8 parts.

(2) liquid seeds expands cultivation

1. first order seed is cultivated: slant strains 2 articulating after step (1) being activated enters in 500 ml shake flasks, culture medium loading amount 100 milliliters, rotary shaker 180 revs/min, cultivation temperature 33 DEG C, incubation time 12h;

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

3. three grades of seed culture: secondary seed is accessed in 5000 milliliters of three grades of seed flask with 10% inoculum concentration, culture medium loading amount 1000 milliliters, rotary shaker 100 revs/min, cultivation temperature 33 DEG C, incubation time 12h;

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

Described one-level, secondary, three grades of seed culture medium percentage by weights consist of:

Dusty yeast 0.4%, glucose 1.2%, peptone 0.4%, beef extract 0.6%, dipotassium hydrogen phosphate 1.0%, Chinese herbal medicine powder 1.8%, trehalose 2%, calcium sulfate 1g, magnesium chloride 2g, natrium citricum 2g, insufficient section pure water is supplied, pH value 7.2,121 DEG C of sterilizing 30min.

Described seed tank culture base percentage by weight consists of:

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

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

(3) ferment tank

By first class seed pot zymotic fluid in step (2) with 6% inoculum concentration access fermentation tank, cultivation temperature 35 DEG C, mixing speed 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 zymotic fluid in step (2) is added access fermentation tank with 4% inoculum concentration, constant temperature culture 25h; Finally slowly be warming up to 12 DEG C, constant temperature culture 18h with 2 DEG C/h heating rate; Continue slowly to be warming up to 33 DEG C, constant temperature culture 18h with 2 DEG C/h heating rate;

Dissolved oxygen controls: by adjustment speed of agitator and ventilation, controls dissolved oxygen 20%;

PH controls: by mending ammoniacal liquor or phosphoric acid,diluted, controls pH value in sweat and remains on 7.2;

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

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

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

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

The concocting method of described fermentation medium is:

Accurately take raw material in proportion, pure water in raw material, corn flour, beancake powder are dropped in material-compound tank, regulate pH value 7.2, add middle temperature amylase (3u/g corn flour) and alpha-amylase (30u/g corn flour), 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 p H7.2,121 DEG C of sterilizing 30min are for subsequent use.

(4) fermentation liquor filter, concentrated, allotment, essence filter, dry heat-flash stability neutral proteinase, described allocation process adds concentrated enzyme liquid gross weight 3% Chinese herbal medicine powder.

Described concentrated maltase percentage by weight consists of: former maltase 30%, fragrance maltase 70%.

Described former maltase preparation method is as follows:

(1) high-pressure pulse electric (PEF) immersion treatment: by Fructus Hordei Germinatus in pre-immersion trough with 3 times of 20 DEG C of emerge in worm water 10min, make Fructus Hordei Germinatus moisture reach 25%, carry out high-pressure pulse electric (PEF) process, electric-field intensity 20KV/cm simultaneously, burst length 150 μ S, pulse frequency 200Hz;

(2) pulverize: by the Fructus Hordei Germinatus handled well in step (1), to add maltcrusher band pigment broken with soaking water, and adjustment roller spacing is 0.5mm, and rotating speed is 250rpm, obtains Fructus Hordei Germinatus slurries;

(3) ultrasonic, PEF extracts: Fructus Hordei Germinatus slurries are placed in pill tank, and limit is slowly stirred limit probe type ultrasonic extraction apparatus under current strength 0.6A/25w condition, carried out ultrasonic extraction 10min; Then carry out high-pressure pulse electric (PEF) and extract 15min, electric-field intensity 20KV/cm, burst length 400 μ S, pulse frequency 200Hz;

(4) filter: be 100 order diatomite in room temperature with 500 order filter cloth precoating granularities by extract, employing flame filter press filters, and operating pressure is 0.14Mpa;

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

(6) dry: the protective agent of the present invention adding concentrate weight 3% in concentrate, then use the special spray dryer of enzyme preparation dry, EAT 150 DEG C; temperature of outgoing air 75 DEG C; drying time 5s, product moisture < 5%, both former maltase.

Described fragrance maltase preparation method is as follows:

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

(2) cure: the Fructus Hordei Germinatus that will get damp again loads drum-type and fries stove, and in 105min, temperature rises to 170 DEG C, insulation 10min, is then cooled to 100 DEG C, and insulation 20min, finally takes out spreading for cooling, obtain fragrance Fructus Hordei Germinatus;

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

(4) ultrasonic, PEF extracts: fragrance malt flour is placed in pill tank, adds the water of 3 times, limit is slowly stirred limit probe type ultrasonic extraction apparatus under current strength 0.6A/25w condition, carried out ultrasonic extraction 10min; Then carry out high-pressure pulse electric (PEF) and extract 15min, electric-field intensity 20KV/cm, burst length 400 μ S, pulse frequency 200Hz;

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

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

(7) dry: the protective agent of the present invention adding concentrate weight 3% in concentrate, then use the special spray dryer of enzyme preparation dry, EAT 150 DEG C; temperature of outgoing air 75 DEG C; drying time 5s, product moisture < 5%, both fragrance maltase.

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 mixed material 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 freeze drying obtains Chinese herbal medicine extract.

The parts by weight of described Chinese herbal medicine consist of: sea-buckthorn 20 parts, cassia seed 20 parts, matrimony vine 10 parts, Chinese yam 10 parts, radix glehniae 5 parts, fruit of 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 mixed material gross weight;

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

Described protective agent is made up of the raw material of following parts by weight: trehalose 20 parts, NaCl20 part, (NH ₄) SO ₄10 parts, cysteine 10 parts.

Described activator is formed by the inorganic salts Homogeneous phase mixing of following quality component: zinc chloride 30 parts, 10 parts, calcium chloride, 10 parts, sodium sulphate, 5 parts, magnesium chloride.

Containing the preparation method of the feed complex enzyme of neutral proteinase with embodiment 1.

Embodiment 3:

Containing a feed complex enzyme for neutral proteinase, be made up of the enzyme preparation of following parts by weight:

Concentrated maltase 40 parts, acid protease 30 parts, neutral proteinase 30 parts, acidic xylanase 30 parts, cellulase 30 parts; 1,4 beta-glucanase 20 parts, pectase 20 parts, amylase 20 part, seminase 15 parts; phytase 15 parts, Chinese herbal medicine extract 15 parts, protective agent 15 parts, activator 15 parts.

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

The preparation method of described neutral proteinase comprises the steps:

(1) actication of culture

The slant strains of intact bacillus 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: beef extract 10g, sodium chloride 12g, peptone 20g, glucose 5g, (NH) ₂sO ₄5g, K ₂hPO ₄8g, CaCl ₂3g, agar 20g, Chinese herbal medicine powder 10g, distilled water l000mL, pH value 7.2,121 DEG C of sterilizing 20min;

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

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 mixed material gross weight 10% carries out enzymolysis, be 6.8 by lactic acid adjust ph, enzymolysis 4h, finally add the mixture of mixed material 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 1,4 beta-glucanase 20 parts, beta-glucosidase 15 parts, zytase 20 parts, pentosanase 20 parts, Pullulanase 30 parts, beta amylase 15 parts, neutral proteinase 15 parts, acid protease 15 parts, superoxide dismutase 10 parts, glucose oxidase 10 parts, acid phosphatase 10 parts.

(2) liquid seeds expands cultivation

1. first order seed is cultivated: slant strains 2 articulating after step (1) being activated enters in 500 ml shake flasks, culture medium loading amount 100 milliliters, rotary shaker 180 revs/min, cultivation temperature 36 DEG C, incubation time 15h;

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

3. three grades of seed culture: secondary seed is accessed in 5000 milliliters of three grades of seed flask with 10% inoculum concentration, culture medium loading amount 1000 milliliters, rotary shaker 100 revs/min, cultivation temperature 36 DEG C, incubation time 15h;

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

Described one-level, secondary, three grades of seed culture medium percentage by weights consist of:

Dusty yeast 0.5%, glucose 1.5%, peptone 0.5%, beef extract 0.8%, dipotassium hydrogen phosphate 1.5%, Chinese herbal medicine powder 2%, trehalose 3%, calcium sulfate 1g, magnesium chloride 2g, natrium citricum 3g, insufficient section pure water is supplied, pH value 7.2,123 DEG C of sterilizing 40min.

Described seed tank culture base percentage by weight consists of:

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

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

(3) ferment tank

By first class seed pot zymotic fluid in step (2) with 6% inoculum concentration access fermentation tank, cultivation temperature 36 DEG C, mixing speed 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 zymotic fluid in step (2) is added access fermentation tank with 4% inoculum concentration, constant temperature culture 30h; Finally slowly be warming up to 15 DEG C, constant temperature culture 20h with 2 DEG C/h heating rate; Continue slowly to be warming up to 36 DEG C, constant temperature culture 20h with 2 DEG C/h heating rate;

Dissolved oxygen controls: by adjustment speed of agitator and ventilation, controls dissolved oxygen 30%;

PH controls: by mending ammoniacal liquor or phosphoric acid,diluted, controls pH value in sweat and remains on 7.2;

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

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

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

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

The concocting method of described fermentation medium is:

Accurately take raw material in proportion, pure water in raw material, corn flour, beancake powder are dropped in material-compound tank, regulate pH value 7.2, add middle temperature amylase (3u/g corn flour) and alpha-amylase (30u/g corn flour), 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 p H7.2,123 DEG C of sterilizing 40min are for subsequent use.

(4) fermentation liquor filter, concentrated, allotment, essence filter, dry heat-flash stability neutral proteinase, described allocation process adds concentrated enzyme liquid gross weight 5% Chinese herbal medicine powder.

Described concentrated maltase percentage by weight consists of: former maltase 40%, fragrance maltase 60%.

Described former maltase preparation method is as follows:

(1) high-pressure pulse electric (PEF) immersion treatment: by Fructus Hordei Germinatus in pre-immersion trough with 6 times of 50 DEG C of emerge in worm water 20min, make Fructus Hordei Germinatus moisture reach 35%, carry out high-pressure pulse electric (PEF) process, electric-field intensity 40KV/cm simultaneously, burst length 200 μ S, pulse frequency 300Hz;

(2) pulverize: by the Fructus Hordei Germinatus handled well in step (1), to add maltcrusher band pigment broken with soaking water, and adjustment roller spacing is 0.5mm, and rotating speed is 250rpm, obtains Fructus Hordei Germinatus slurries;

(3) ultrasonic, PEF extracts: Fructus Hordei Germinatus slurries are placed in pill tank, and limit is slowly stirred limit probe type ultrasonic extraction apparatus under current strength 1.5A/275w condition, carried out ultrasonic extraction 15min; Then carry out high-pressure pulse electric (PEF) and extract 20min, electric-field intensity 40KV/cm, burst length 600 μ S, pulse frequency 300Hz;

(4) filter: be 300 order diatomite in room temperature with 800 order filter cloth precoating granularities by extract, employing flame filter press filters, and operating pressure is 0.24Mpa;

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

(6) dry: the protective agent of the present invention adding concentrate weight 5% in concentrate, then use the special spray dryer of enzyme preparation dry, EAT 160 DEG C; temperature of outgoing air 85 DEG C; drying time 15s, product moisture < 5%, both former maltase.

Described fragrance maltase preparation method is as follows:

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

(2) cure: the Fructus Hordei Germinatus that will get damp again loads drum-type and fries stove, and in 15min, temperature rises to 200 DEG C, insulation 15min, is then cooled to 110 DEG C, and insulation 30min, finally takes out spreading for cooling, obtain fragrance Fructus Hordei Germinatus;

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

(4) ultrasonic, PEF extracts: fragrance malt flour is placed in pill tank, adds the water of 6 times, limit is slowly stirred limit probe type ultrasonic extraction apparatus under current strength 1.5A/275w condition, carried out ultrasonic extraction 15min; Then carry out high-pressure pulse electric (PEF) and extract 20min, electric-field intensity 40KV/cm, burst length 600 μ S, pulse frequency 300Hz;

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

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

(7) dry: the protective agent of the present invention adding concentrate weight 5% in concentrate, then use the special spray dryer of enzyme preparation dry, EAT 160 DEG C; temperature of outgoing air 85 DEG C; drying time 15s, product moisture < 5%, both fragrance maltase.

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 mixed material 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 freeze drying obtains Chinese herbal medicine extract.

The parts by weight of described Chinese herbal medicine consist of: sea-buckthorn 30 parts, cassia seed 30 parts, matrimony vine 15 parts, Chinese yam 15 parts, radix glehniae 10 parts, fruit of 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 mixed material gross weight;

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

Described protective agent is made up of the raw material of following parts by weight: trehalose 30 parts, NaCl30 part, (NH ₄) SO ₄15 parts, cysteine 15 parts.

Described activator is formed by the inorganic salts Homogeneous phase mixing of following quality component: zinc chloride 40 parts, 20 parts, calcium chloride, 20 parts, sodium sulphate, 10 parts, magnesium chloride.

Containing the preparation method of the feed complex enzyme of neutral proteinase with embodiment 1.

Claims (7)

1. containing a feed complex enzyme for neutral proteinase, be made up of the enzyme preparation of following parts by weight: concentrated maltase 30-40 part, acid protease 20-30 part, neutral proteinase 20-30 part, acidic xylanase 20-30 part, cellulase 20-30 part, 1,4 beta-glucanase 15-20 part, pectase 15-20 part, amylase 15-20 part, seminase 10-15 part, phytase 10-15 part, Chinese herbal medicine extract 10-15 part, protective agent 10-15 part, activator 10-15 part;

The preparation method of described neutral proteinase is as follows: bacillus subtilis CCTCC NO:M 2013539 is through actication of culture and expand cultivation acquisition liquid seeds step by step; By liquid seeds with 6% inoculum concentration access fermentation tank, cultivation temperature 30-36 DEG C, mixing speed 200-700r/m, 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 fermentation tank with 4% inoculum concentration, 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 heating rate; Continue slowly to be warming up to 30-36 DEG C, constant temperature culture 15-20h with 1-2 DEG C/h heating rate; Fermentation liquor is filtered, concentrated, allotment, essence filter, dry solid neutral protease; Described allocation process adds concentrated enzyme liquid gross weight 0.5-5% Chinese herbal medicine powder;

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 mixed material 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 mixed material 0.5-3 times of w ethanol and propyl alcohol, control temperature to 60 DEG C-78 DEG C of maintenance 3-4h, filter; Filter vacuum concentrates postlyophilization and obtains Chinese herbal medicine powder;

The preparation method of described Chinese herbal medicine extract is: with weight parts, takes sea-buckthorn 20-30 part, cassia seed 20-30 part, matrimony vine 10-15 part, Chinese yam 10-15 part, radix glehniae 5-10 part, fruit of negundo 5-10 part, radix polygonati officinalis 3-5 part, seed of Job's tears 3-5 part, fructus hordei germinatus 3-5 part, sweet osmanthus 3-5 part, Radix Astragali 3-5 part; 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 mixed material 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 mixed material 0.5-3 times of w ethanol and propyl alcohol, control temperature to 60 DEG C-78 DEG C of maintenance 3-4h, filter; Filtrate reduced in volume to solid content is more than 20%, and then freeze drying obtains Chinese herbal medicine extract;

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

2. a kind of feed complex enzyme containing neutral proteinase as claimed in claim 1, it is characterized in that, the slant medium preparing neutral proteinase consists of: beef extract 3-10g, sodium chloride 5-12g, peptone 10-20g, glucose 2-5g, (NH) ₂sO ₄3-5g, K ₂hPO ₄6-8g, CaCl ₂1-3g, agar 15-20g, Chinese herbal medicine powder 5-10g, distilled water l000mL, pH value 7.0-7.2.

3. a kind of feed complex enzyme containing neutral proteinase as claimed in claim 1, it is characterized in that, the seed culture medium percentage by weight preparing neutral proteinase consists of: dusty yeast 0.3-0.5%, glucose 1-1.5%, peptone 0.3-0.5%, beef extract 0.5-0.8%, dipotassium hydrogen phosphate 0.8-1.5%, Chinese herbal medicine powder 1.5-2%, trehalose 1-3%, calcium sulfate 1g, magnesium chloride 2g, natrium citricum 1-3g, insufficient section pure water is supplied, pH value 7.0-7.2.

4. a kind of feed complex enzyme containing neutral proteinase as claimed in claim 1, it is characterized in that, the seed tank culture base preparing neutral proteinase consists of: maltodextrin 5-15%, dusty yeast 0.4-0.8%, Chinese herbal medicine powder 1.5-2%, trehalose 1-3%, peptone 0.1-0.5%, corn steep liquor 0.1-0.5%, dipotassium hydrogen phosphate 0.8-1.5%, magnesium sulfate 0.05-0.1%, natrium citricum 0.1-0.5%, insufficient section pure water is supplied, pH value 7.0-7.2.

5. a kind of feed complex enzyme containing neutral proteinase as claimed in claim 1, is characterized in that, when preparing neutral proteinase, seeding tank zymotic fluid cell concentration is 7.0x10 ⁸-8.0x10 ⁸individual/ml.

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

7. prepare as claimed in claim 1 containing the method for the feed complex enzyme of neutral proteinase for one kind; it is characterized in that; by described protective agent, Chinese herbal medicine extract, concentrated maltase ultramicro grinding respectively; guarantee that granularity is less than described neutral proteinase and other enzyme preparation; then with neutral proteinase, acid protease, acidic xylanase, cellulase, 1,4 beta-glucanase, pectase, amylase, seminase, phytase; Homogeneous phase mixing; finally add activator, after mixing, pack the feed complex enzyme got product containing neutral proteinase.