CN103695396B - A kind of high vigor complex enzyme for feed - Google Patents
A kind of high vigor complex enzyme for feed Download PDFInfo
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Abstract
The invention discloses a kind of high vigor complex enzyme for feed, belong to fodder enzyme preparation preparing technical field.With the bacterial strain aspergillus niger DM-18 of high yield complex enzyme for feed for starting strain, and carry out medium optimization and zymotechnique improvement, deep liquid mixed fermentation under specifically fermentation condition, the obtained complex enzyme for feed that a kind of enzyme system is complete, enzyme activity is high, tolerable temperature is stronger, resistance to pH value is more wide in range.Containing multiple enzyme, wherein proteinase activity 6500-6700U/ml, mannosans enzyme activity 1500-1700U/ml, α-galactase vigor 1200-1300U/ml, pectinase activity 1000-1200U/ml in height vigor complex enzyme for feed fermented liquid crude enzyme liquid of the present invention.Fermented liquid crude enzyme liquid is carried out to heat stability test and the pH stability test of enzyme, test-results shows: at 30-75 DEG C, and during pH value 2-7, each enzyme all has higher enzyme activity, reaches more than 80%.
Description
Technical field
The present invention relates to fodder enzyme preparation, particularly the high vigor complex enzyme for feed of one.
Background technology
Large quantity research both domestic and external shows, in feed, add zymin can promote animal digesting and assimilating nutritive substance, improve efficiency of feed utilization, strengthen animal disease resistance ability, its major function has the following aspects: (1) supplements the deficiency of animal endogenous enzyme, improves digestibility and the utilization ratio of feed.Zymin facilitates the absorption and digestion of digestive tube to carbohydrate and other nutritive substance, improves the transformation efficiency of feed, is conducive to the growth of animal.(2) eliminate the antinutritional factor in feed, reduce the viscosity of animal digestive tract chyme, improve digestive function, be conducive to absorption of nutrient ingredients.(3) improve intestinal microflora distribution, improve animal health condition.The manna oligosaccharide produced after enzyme preparation degrades non-starch polysaccharide can promote the profitable strain propagation such as bifidus bacillus, Lactobacillus paracasei, Lactobacterium acidophilum, lactobacillus delbruckii, and then limits the malignant bacteria growths such as Salmonellas, Campylobacter and shuttle acid genus bacillus.(4) participate in animal endocrine regulation, improve livestock and poultry hormone in vivo metaboilic level, promote poultry growth by the hormone metabolism affecting body.(5) pollution of husbandry sector to environment is alleviated.Almost in all plant feeds, there is (phytinic acid) with phytic acid form in most phosphoric acid salt.Because in monogastric animal digestive tube, the vigor of phytase is very low, most of phytate phosphorus is excreted, and so not only wastes phosphorus source, and causes environmental pollution.Add phytase, in alternative feed formulation 50% ~ 70% secondary calcium phosphate, to environment excretion the corresponding minimizing more than 30% of phosphorus, play a great role in environment protection.(6) release of the starch promoting cell to surround, protein, fat, by cell wall hydrolysis, makes nutritive substance be fully absorbed.
Digestibility and the utilization ratio that fodder enzyme preparation can improve feed is applied in livestock product cultivation; improve the production performance of livestock and poultry and fish; the excretion of the nitrogen in farm animal excrement, phosphorus can be reduced again; protection water body and soil from pollution, thus fodder enzyme preparation efficient as a class, to have no side effect and " green " fodder additives of environment-friendly type will have very wide application prospect in 21 century.
The commercial applications of fodder enzyme preparation is abroad also only less than vicennial history.Britain is before 1988, and zymin is in the adding rate of chicken feed no better than zero, and by 1993, the adding rate of chicken feed enzyme preparation reached more than 95%.Fodder enzyme preparation starts from 1992 in the application of China, and the annual turnover of China's fodder enzyme preparation in 2005 has reached more than 10,000 tons.Fodder enzyme preparation is efficient, nontoxic as a class, have no side effect and " green " fodder additives of environment-friendly type will have very wide application prospect in 21 century.China's mixed feed 2009 annual production reaches 1.4 hundred million tons, wherein by about 50% enzyme-added 0.1%, then with enzyme about 70,000 tons.According to domestic market forecast analysis, the market capacity of complex enzyme preparation for feeding in 2010 is estimated to reach 7.5 ten thousand tons, and the production capacity of domestic fodder enzyme preparation is less than 20,000 tons, and therefore, fodder enzyme preparation also has the very large market space and potentiality in China.The market of current China fodder enzyme preparation begins to take shape, and progressively develops, and enzyme classes preparation has good growth momentum and prospect at home.
China's various places staple food crop differs greatly, and the exploitation of zymin to the utilization of potential feed resource and new feedstuff resource has larger effect.People are in the urgent need to environment-friendly and energy-efficient green feed additives such as development zymins thus.The Chinese government is also just positive by forbidding using the mode such as microbiotic, hormone to ensure the safety of feed and food, maintaining ecological balance in feed.Estimate that in coming 10 years, along with scientific and technological level improves constantly, the decline of production cost, the volume of production and marketing of feeding enzyme must increase substantially.Although fodder enzyme preparation generally application aborning is also faced with many problems, along with the raising of biotechnology particularly genetic engineering technique and production technology, all will settle one by one.Along with the raising of people's living standard and the enhancing of Environmental awareness, fodder enzyme preparation with its do not produce residual, will be applied further without resistance, the advantage such as free from environmental pollution.
At present, the production of complex enzyme for feed mainly contains following three kinds of methods: 1. complete method of double crossing: both produced and bought various single enzyme preparation, composite by formula, then adds dispersion agent, thinner etc. and process.Main drawback is that cost is high, not easily live higher by enzyme during use and dosage is little zymin mixes with a large amount of feed, thus had a strong impact on the effect used, this is turn increasing use cost virtually, many managers are hung back, brings obstacle to promotion and application.2. more than bacterial strain mixed fermentation, naturally complementary enzyme system method: adopt several bacterial classification mixed fermentation, the complementary enzyme process that produces obtains the prozyme product comparatively close with method of double crossing, only need adjust a little afterwards and be product, but the technical difficulty of multi-strain fermentation is very large, because the respective physiological property of different bacterial classifications, mixed culture influences each other unavoidably, and them be made to grow in same medium and synthesize different enzymes has certain difficulty.3. single strain fermentation, add enzyme process: obtain strain excellent by bacterial screening, this bacterial strain can the multiple enzyme of high yield, and these enzymes composition rationally, simple feed formulation requirement can be met, obtain prozyme with after such strain fermentation, then added by the growth needs of the different ages of different animals and adjust.
In sum, single culture fermentative production complex enzyme for feed is adopted to be the simplest, feeding effect is best, cost is minimum a kind of production method, Chinese patent CN101608175B discloses a kind of compound enzymic preparation and its preparation method and application, take aspergillus niger as starting strain, the prozyme prepared through liquid submerged fermentation contains following enzyme and activity: liquid acidic 'beta '-mannase, 1300u/ml, liquid alpha-galactosidase, 300u/ml, is applicable to feed and petroleum industry.Chinese patent CN101591619B discloses a kind of Aspergillus niger strain and application thereof, this bacterial strain can simultaneously high yield zytase, cellulase, polygalacturonase, amylase, 'beta '-mannase etc., can produce detoxification prozyme, various enzyme optimum pH alive is 4-6.
In view of this prepare that a kind of enzyme activity is high, the complex enzyme for feed of Heat stability is good, optimal pH wide scope is still the industry and vast livestock and poultry cultivation family in the urgent need to.
Summary of the invention
Technical problem solved by the invention is for starting strain with the bacterial strain aspergillus niger of high yield complex enzyme for feed (Aspergillus niger) DM-18, and carry out medium optimization and zymotechnique improvement, deep liquid mixed fermentation under specifically fermentation condition, the obtained complex enzyme for feed that a kind of enzyme system is complete, enzyme activity is high, tolerable temperature is stronger, resistance to pH value is more wide in range.
Prozyme of the present invention comprises mannonase α-galactase, polygalacturonase and proteolytic enzyme, defines a kind of multienzyme complex system for feedstuff raw material, i.e. complex enzyme for feed.
The zymologic property of height vigor complex enzyme for feed of the present invention is as follows:
(1) temperature: each enzyme thermal adaptation a wider range in this prozyme, Applicable temperature scope is 30-75 DEG C, optimal reactive temperature 65 DEG C, at 75 DEG C of enzymes complete stability alive;
(2) pH: it is 2.0-7.0 that this enzyme is suitable for pH value in reaction scope, the enzyme complete stability alive when pH value is 2.0, optimal reaction pH value is 5-7;
(3) enzymic activity: containing multiple enzyme activity, wherein proteinase activity 6500-6700U/ml, mannosans enzyme activity 1500-1700U/ml, α-galactase vigor 1200-1300U/ml, pectinase activity 1000-1200U/ml in this mixed enzyme fermentation liquid crude enzyme liquid.
(4) thermostability: more than 85% enzyme still can be kept after each enzyme preserves 1h under 70 DEG C of conditions in this mixed enzyme fermentation liquid crude enzyme liquid to live, keeps more than 80% enzyme to live after preserving 1h under 75 DEG C of conditions;
The preparation method of height vigor complex enzyme for feed of the present invention is as follows: by the slant strains of intact aspergillus niger DM-18 through actication of culture and one-level, secondary, three grades of liquid seeds and seeding tank step by step enlarged culturing obtain liquid seeds, with 6% inoculum size access fermentor tank, culture temperature 28-30 DEG C, stirring velocity 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 fermentor tank with 4% inoculum size, constant temperature culture 20-30h; Finally slowly be warming up to 10-15 DEG C, constant temperature culture 15-20h with 1-2 DEG C/h temperature rise rate; Continue slowly to be warming up to 30-33 DEG C, constant temperature culture 15-20h with 1-2 DEG C/h temperature rise rate; Fermentation liquor is filtered, concentrated, allotment, essence filter, dry solid complex enzyme for feed, described allocation process adds concentrated enzyme liquid gross weight 0.5-5% Chinese herbal medicine powder.
Described slant medium consists of: casein food grade 4g, dipotassium hydrogen phosphate 2g, magnesium chloride 0.6g, Repone K 0.8g, ferrous sulfate 0.02g, glucose 20g, agar 20g, Chinese herbal medicine powder 5-20g, distilled water l000mL, pH value 5.8,121 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, add the mixture of mixture 2-3 times of w ethanol and propyl alcohol, control temperature to 60 DEG C ~ 78 DEG C keeps 3 ~ 4h, filters; Filter vacuum concentrates postlyophilization and obtains Chinese herbal medicine powder.
The mass ratio of described ethanol and propyl alcohol is 1:1-1.5.
Described one-level, secondary, three grades of seed culture mediums consist of: dipotassium hydrogen phosphate 2g, magnesium chloride 0.6g, Repone K 0.8g, ferrous sulfate 0.02g, glucose 20g, Chinese herbal medicine powder 15-25g, trehalose 10-30g, distilled water l000mL, pH value 5.5,121 DEG C of sterilizing 20min.
Described seed tank culture base consists of: Semen Maydis powder 50-60g, bean powder 15-25g, wheat bran 10-15g, fish meal 10-15g, calcium chloride 6-10g, ammonium chloride 1-3g, Sodium phosphate dibasic 1-2g, Chinese herbal medicine powder 15-20g, trehalose 10-30g, pure water l000mL, pH value 5-7,121 DEG C of sterilizing 20min.
Described seeding tank fermented liquid cell concentration is 7.0x10
8-8.0x10
8individual/ml;
Describedly to become: Semen Maydis powder 50-60g, bean powder 15-25g, wheat bran 10-15g, fish meal 10-15g, calcium chloride 6-10g, ammonium chloride 1-3g, Sodium phosphate dibasic 1-2g, Chinese herbal medicine powder 30-50g, trehalose 10-30g, pure water l000mL, pH value 5-7,121 DEG C of sterilizing 20min.
Described ferment tank process supplemented medium weight percent consists of: maltodextrin 20-30%, Semen Maydis powder 10-20%, bean powder 15-25%, fish meal 1.0-1.5%, calcium chloride 0.6-1.0%, ammonium chloride 0.1-0.3%, Sodium phosphate dibasic 0.1-0.2%, Chinese herbal medicine powder 5-10%, insufficient section pure water is supplied, pH value 5-7,121-123 DEG C of sterilizing 30-40min.
Height vigor complex enzyme for feed of the present invention is preparing the application in feed complex enzyme.
The Aspergillus niger strain of high yield complex enzyme for feed of the present invention is specially aspergillus niger (Aspergillus niger) DM-18, be separated aspergillus niger (Aspergillus niger) HYX0022 that obtains through through ultraviolet mutagenesis, ultraviolet HNO_2 mutagenesis, ultraviolet nitrosoguanidine complex mutation from Jinshi City Yang You township of Hunan Province vegetable mould, straw compound sample soil, then mutant strain step-sizing is eliminated, finally through leavening property test screen, the aspergillus niger DM-18 producing complex enzyme for feed is obtained to strain excellent.This bacterial strain is preserved in China typical culture collection center on November 3rd, 2013 and (is called for short CCTCC, address is: Wuchang District, Wuhan City, Hubei Province Luo Jia Shan Wuhan University Life Science College postcode: 430072), preserving number is CCTCC NO:M2013541, and Classification And Nomenclature is aspergillus niger (Aspergillus niger) DM-18.
The aspergillus niger DM-18 (CCTCC NO:M2013541) of high yield complex enzyme for feed provided by the invention has following microbial characteristic:
1, morphological feature:
Aspergillus niger DM-18, biology morphology is for comprising several parts such as conidium, falx, top capsule, conidial fructification.Conidial head is spherical to Radiation, and diameter 150-450 μm, conidiophore betides matrix.Falx stem 1000-3000 (length) × 12-20 (diameter) μm, yellow or tawny, wall is level and smooth; Spherical or the almost spherical of top capsule, diameter 35-50 μm, surface can be educated comprehensively; Conidial fructification is double-deck, metulae 15-20 (length) × 3-4.0 (diameter) μm, falx 6-8 (length) × 2-4 (diameter) μm; conidium is spherical or subsphaeroidal, less, diameter 3.5-5.0 μm; brown, wall is coarse.
2, feature is cultivated:
Aspergillus niger DM-18 grows rapidly on wort agar substratum, 28 DEG C of 4 days diameter 65mm; Quality velvet shape or be slightly with cotton-shaped; Conidium structure is a large amount of, and brown-black, has a small amount of transudate; Bacterium colony reverse side yellowish.
3, physiological and biochemical property:
Aspergillus niger DM-18 can at potato, Semen Maydis powder, Zulkovsky starch, the upper growth such as molasses, optimum pH 4.6, optimum growth temperature 28-34 DEG C, the suitableeest product enzyme temperature 28-30 DEG C.
The triage techniques route of aspergillus niger DM-18 is: original strain is separated, the preparation → mutagenic treatment → plate isolation → primary dcreening operation of selection systems → starting strain → slant culture → spore suspension → multiple sieve → sieve → expand experiment (leavening property mensuration) more again.
Beneficial effect:
1. height vigor complex enzyme for feed of the present invention with the bacterial strain aspergillus niger DM-18 of high yield complex enzyme for feed for starting strain, and carry out medium optimization and zymotechnique improvement, adopt the zymotechnique of gradient cooling and gradient increased temperature, added inoculation and in good time feed supplement simultaneously, especially the zymotechnique of gradient cooling and gradient increased temperature significantly improves the anti-stress ability of starting strain, causes the enzymatic productivity of bacterial classification to manifest to greatest extent.And the present invention implements full optimization to substratum composition, with the addition of the root of large-flowered skullcap, the radix bupleuri with anti-heat stress original work, with the addition of and there is adjustment and repair body function, the immunologic function of enhancing body, the Radix Astragali with Tiny ecosystem regulatory function, Radix Codonopsis etc., further enhancing the body function of microorganism under same yeasting, adaptation of virus and collaborate, and then enhancing the metabolic function of microorganism, the present invention is produced, and complex enzyme for feed vigor is high, tolerable temperature is higher, stability is strong, be suitable for suitability for industrialized production.
2. contain multiple enzyme activity in height vigor complex enzyme for feed fermented liquid crude enzyme liquid of the present invention, wherein, proteinase activity 6500-6700U/ml, mannosans enzyme activity 1500-1700U/ml, α-galactase vigor 1200-1300U/ml, pectinase activity 1000-1200U/ml.Fermented liquid crude enzyme liquid is carried out to heat stability test and the pH stability test of enzyme, test-results shows: at 30-75 DEG C, and during pH value 2-7, each enzyme all has higher enzyme activity, reaches more than 80%.
3. height vigor complex enzyme for feed of the present invention comprises mannonase α-galactase, polygalacturonase and proteolytic enzyme; thus form a kind of multienzyme complex system for feedstuff raw material; complex enzyme for feed can be produced; and the various enzymes in this prozyme live optimum pH between 2-7; be applicable to the absorption and digestion environment of animal gastrointestinal tract; this complex enzyme for feed can be widely used in animal and fowl fodder; antinutritional factor can be reduced; improve digestibility; there is good economic benefit; also be conducive to preserving the ecological environment simultaneously, promote the sound development of livestock industry.
4. height vigor complex enzyme for feed thermostability of the present invention and pH stability by force, are applicable to fodder industry complete processing demand more, and the loss alive of the feed enzyme after processing is low.
Embodiment
The present invention is described below by specific embodiment.Unless stated otherwise, technique means used in the present invention is method known in those skilled in the art.In addition, embodiment is interpreted as illustrative, but not limits the scope of the invention, and the spirit and scope of the invention only limited by claims.To those skilled in the art, under the prerequisite not deviating from essence of the present invention and scope, the various change carry out the material component in these embodiments and consumption or change also belong to protection scope of the present invention.
Embodiment 1 complex enzyme for feed enzyme activity determination
1) enzyme activity unit definition:
Proteinase activity unit of force defines: 40 DEG C, under pH5.5. condition, the enzyme amount that 1min caseinhydrolysate produces 1 μm of ol tyrosine is 1 enzyme activity unit, represents with U/ml.
Mannosans enzyme activity unit defines: 40 DEG C, under pH5.5 condition, 1min be hydrolyzed from the Viscogum BE solution that concentration is 5mg/ml that to discharge the enzyme amount required for reducing sugar being equivalent to 1 μm of ol seminose be 1 enzyme activity unit, represents with U/ml.
α-galactase unit of activity definition: 40 DEG C, under pH5.5 condition, it is that 1 alpha-galactosidase enzyme is lived unit that 1min decomposes the pnitrophenylα Dgalactospyranoside enzyme amount generated needed for 1 μm of ol p-nitrophenol, represents with U/ml.
Pectinase activity unit definition: 40 DEG C, under pH5.5 condition, it is 1 enzyme activity unit that 1min is hydrolyzed the enzyme amount required for reducing sugar that release is equivalent to 1 μm of ol from the pectin solution that concentration is 10mg/ml, represents with U/ml.
2) measure: embodiment 2 crude enzyme liquid distilled water is suitably diluted, get four brace plug test tubes, the diluted complex enzyme for feed crude enzyme liquid of 0.1ml is added respectively in three (three repetitions), adding 0.1ml in 4th test tube through boiling the inactivator liquid of 5min in contrast, arising from preheating 5min in 40 DEG C of water-baths with the substrate prepared by the 0.2mol/l acetic acid-sodium acetate buffer solution of pH5.5 (concrete concentration of substrate is shown in that the enzyme activity unit described in step 1 defines); In each test tube, add 0.1ml substrate, 40 DEG C are accurately reacted 30min, add 0.6mlDNS reagent after having reacted in four test tubes, shake up, boil 10min deactivation colour developing, rapid cool to room temperature, with water constant volume to 5.0ml, under 550nm wavelength, measure absorbancy.The extension rate (with distilled water diluting) of adjustment complex enzyme for feed crude enzyme liquid, makes the absorption values of mensuration within standard curve determination scope.
3) result: after measured, containing multiple enzyme activity in the fermented liquid fermented liquid crude enzyme liquid that embodiment 2 obtains, wherein, proteinase activity 6700U/ml, mannosans enzyme activity 1700U/ml, α-galactase vigor 1300U/ml, pectinase activity 1200U/ml.
Embodiment 2
A kind of high vigor complex enzyme for feed preparation method comprises the steps:
(1) actication of culture
The slant strains of intact aspergillus niger DM-18 is inoculated in slant medium, cultivates 42h for 30 DEG C and carry out actication of culture, so activation 3 times;
Described slant medium consists of: casein food grade 4g, dipotassium hydrogen phosphate 2g, magnesium chloride 0.6g, Repone K 0.8g, ferrous sulfate 0.02g, glucose 20g, agar 20g, Chinese herbal medicine powder 12g, distilled water l000mL, pH value 5.8,121 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 15 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, adds the mixture of mixture 3 times of w ethanol and propyl alcohol, control temperature to 70 DEG C keeps 4h, filters; Filter vacuum concentrates postlyophilization and obtains Chinese herbal medicine powder.
The mass ratio of described ethanol and propyl alcohol is 1:1.2.
(2) liquid seeds enlarged culturing
1. first order seed is cultivated: the slant strains after step (1) being activated, with spore under aseptic washing, accesses in 500 ml shake flasks, liquid seed culture medium loading amount 100 milliliters, 30 DEG C, 100rpm shaking table cultivation 42h;
2. secondary seed cultivate: by first order seed according to 10% inoculum size access in 500 milliliters of secondary seed shaking flasks, culture condition is identical with first order seed;
3. three grades of seed culture: secondary seed is accessed in 5000 milliliters of three grades of seed flask with 8% inoculum size, liquid nutrient medium loading amount 1000 milliliters, 30 DEG C, 100rpm shaking table cultivation 42h;
4. seed tank culture: the first class seed pot being 150L with 8% inoculum size access cubic capacity by three grades of seeds, seed tank culture base loading amount 100L, control ph is 6, culture temperature 30 DEG C, stirring velocity 300rpm, ventilation (V/V) 1:1, incubation time 42h, dissolved oxygen 20%;
Described one-level, secondary, three grades of seed culture mediums consist of: dipotassium hydrogen phosphate 2g, magnesium chloride 0.6g, Repone K 0.8g, ferrous sulfate 0.02g, glucose 20g, Chinese herbal medicine powder 20g, trehalose 20g, distilled water l000mL, pH value 5.5,121 DEG C of sterilizing 20min.
Described seed tank culture base consists of: Semen Maydis powder 55g, bean powder 20g, wheat bran 12g, fish meal 12g, calcium chloride 8g, ammonium chloride 2g, Sodium phosphate dibasic 2g, Chinese herbal medicine powder 18g, trehalose 20g, pure water l000mL, pH value 6,121 DEG C of sterilizing 20min.
Described seeding tank fermented liquid cell concentration is 7.5x10
8individual/ml;
(3) ferment tank
By seeding tank liquid seeds in step (2) with 6% inoculum size access fermentor tank, culture temperature 30 DEG C, stirring velocity 350r/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, seeding tank liquid seeds in step (2) is added access fermentor tank with 4% inoculum size, constant temperature culture 25h; Finally slowly be warming up to 12 DEG C, constant temperature culture 18h with 2 DEG C/h temperature rise rate; Continue slowly to be warming up to 30 DEG C, constant temperature culture 18h with 2 DEG C/h temperature rise rate;
Dissolved oxygen controls: by adjustment mixing speed and ventilation, controls dissolved oxygen 20%;
PH controls: by mending ammoniacal liquor or dilute phosphoric acid, controls pH value in fermenting process and remains on 5.2;
Control of additive raw material: when the reducing sugar content in fermented liquid is down to 3mg/ml-8mg/ml, start to add supplemented medium, feed supplement amount is to maintain fermented liquid reducing sugar content for 2mg/ml-5mg/ml;
Put tank standard: the old and feeble self-dissolving of 70% thalline, enzyme activity increasess slowly.
Described fermention medium consists of: Semen Maydis powder 55g, bean powder 20g, wheat bran 12g, fish meal 12g, calcium chloride 8g, ammonium chloride 2g, Sodium phosphate dibasic 2g, Chinese herbal medicine powder 40g, trehalose 20g, pure water l000mL, pH value 6,121 DEG C of sterilizing 20min.
Described supplemented medium weight percent consists of: maltodextrin 25%, Semen Maydis powder 15%, bean powder 18%, fish meal 1.2%, calcium chloride 0.8%, ammonium chloride 0.2%, Sodium phosphate dibasic 0.2%, Chinese herbal medicine powder 8%, insufficient section pure water is supplied, pH value 6,123 DEG C of sterilizing 40min.
(4) fermentation liquor filter, concentrated, allotment, essence filter, dry solid complex enzyme for feed.
Described allocation process adds concentrated enzyme liquid gross weight 3% Chinese herbal medicine powder.
Containing multiple enzyme activity in the fermented liquid fermented liquid crude enzyme liquid that above-mentioned preparation method obtains, wherein, proteinase activity 6700U/ml, mannosans enzyme activity 1700U/ml, α-galactase vigor 1300U/ml, pectinase activity 1200U/ml.
The thermal stability analysis of the high vigor complex enzyme for feed of embodiment 3
Analyze the thermostability of complex enzyme for feed, at embodiment 4 crude enzyme liquid is placed in 30 DEG C, 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, in 10 minutes sampling and measuring complex enzyme for feed, the enzyme of each constitutive enzyme is lived.At 30 DEG C, 40 DEG C, 45 DEG C, 50 DEG C, 60 minutes each constitutive enzyme enzymes are lived and are not declined.At 55 DEG C, 60 DEG C and 65 DEG C, each constitutive enzyme enzyme work in 30 minutes drops to constitutive enzyme each constitutive enzyme enzymes work in alive 95%, 60 minutes and drops to 85%.At 70 DEG C, each constitutive enzyme enzyme work in 30 minutes drops to constitutive enzyme each constitutive enzyme enzymes work in alive 90%, 60 minutes and drops to 85%.At 75 DEG C, 30 minutes each constitutive enzyme enzymes are lived and are dropped to 85%, 60 minutes each constitutive enzyme enzymes that constitutive enzyme lives and live drop to that constitutive enzyme lives 80%, and compared with prior art, similarity condition is issued to same enzyme tolerable temperature of living and on average improves 5-10 DEG C.
The pH stability analysis of the high vigor complex enzyme for feed of embodiment 4
Analyze the pH stability of complex enzyme for feed enzyme, embodiment 4 crude enzyme liquid is placed in respectively pH value 2.0,2.5,3.5,4.5,5.5,6.0,6.5,7.0 times, in 10 minutes sampling and measuring complex enzyme for feed, the enzyme of each constitutive enzyme is lived.Crude enzyme liquid pH value 5.5,6.0,6.5,7.0 times, 60 minutes each constitutive enzyme enzymes are lived and are not declined.PH value 4.5,3.5 times, 30 minutes each constitutive enzyme enzymes are lived and are dropped to 95%, 60 minutes each constitutive enzyme enzymes that constitutive enzyme lives and live drop to that constitutive enzyme lives 85%.PH value 2.5 times, 30 minutes each constitutive enzyme enzymes live dropping to for 90%, 60 minutes of dropping to that constitutive enzyme lives constitutive enzyme live 85%.PH value 2.0 times, 30 minutes each constitutive enzyme enzymes live dropping to for 85%, 60 minutes of dropping to that constitutive enzyme lives constitutive enzyme live 80%, compared with prior art, it is more wide in range that similarity condition is issued to same enzyme resistance to pH value of living.
Claims (6)
1. a high vigor complex enzyme for feed, is characterized in that, comprises mannonase α-galactase, polygalacturonase and proteolytic enzyme, and the zymologic property of described high vigor complex enzyme for feed is as follows:
(1) temperature: in this prozyme, each enzyme Applicable temperature scope is 30-75 DEG C, optimal reactive temperature 65 DEG C, at 75 DEG C of enzymes complete stability alive;
(2) pH: it is 2.0-7.0 that this enzyme is suitable for pH value in reaction scope, the enzyme complete stability alive when pH value is 2.0, optimal reaction pH value is 5-7;
(3) enzymic activity: containing multiple enzyme activity, wherein proteinase activity 6500-6700U/ml, mannosans enzyme activity 1500-1700U/ml, α-galactase vigor 1200-1300U/ml, pectinase activity 1000-1200U/ml in this mixed enzyme fermentation liquid crude enzyme liquid;
(4) thermostability: keep more than 85% enzyme to live after each enzyme preserves 1h under 70 DEG C of conditions in this mixed enzyme fermentation liquid crude enzyme liquid, keep more than 80% enzyme to live after preserving 1h under 75 DEG C of conditions;
Described high vigor complex enzyme for feed for starting strain with the bacterial strain aspergillus niger CCTCC NO:M 2013541 of high yield complex enzyme for feed, and carries out medium optimization and zymotechnique and improves, the deep liquid mixed fermentation preparation under specifically fermentation condition;
The preparation method of described high vigor complex enzyme for feed is as follows: the slant strains of intact aspergillus niger CCTCC NO:M 2013541 is obtained liquid seeds through actication of culture and one-level, secondary, three grades of liquid seeds and seeding tank enlarged culturing, with 6% inoculum size access ferment tank substratum, culture temperature 28-30 DEG C, stirring velocity 200-700r/m, ventilation 1:1-3V/V, 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, seeding tank liquid seeds is added access fermentor tank with 4% inoculum size, constant temperature culture 20-30h; Finally slowly be warming up to 10-15 DEG C, constant temperature culture 15-20h with 1-2 DEG C/h temperature rise rate; Continue slowly to be warming up to 30-33 DEG C, constant temperature culture 15-20h with 1-2 DEG C/h temperature rise rate; Fermentation liquor is filtered, concentrated, allotment, essence filter, dry solid complex enzyme for feed, 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: 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, add the mixture of mixture 2-3 times of w ethanol and propyl alcohol, the mass ratio of described ethanol and propyl alcohol is 1:1-1.5, control temperature to 60 DEG C ~ 78 DEG C keeps 3 ~ 4h, filters; Filter vacuum concentrates postlyophilization and obtains Chinese herbal medicine powder.
2. high vigor complex enzyme for feed as claimed in claim 1, it is characterized in that, described slant medium consists of: casein food grade 4g, dipotassium hydrogen phosphate 2g, magnesium chloride 0.6g, Repone K 0.8g, ferrous sulfate 0.02g, glucose 20g, agar 20g, Chinese herbal medicine powder 5-20g, distilled water l000mL, pH value 5.8.
3. high vigor complex enzyme for feed as claimed in claim 1, it is characterized in that, described one-level, secondary, three grades of seed culture mediums consist of: dipotassium hydrogen phosphate 2g, magnesium chloride 0.6g, Repone K 0.8g, ferrous sulfate 0.02g, glucose 20g, Chinese herbal medicine powder 15-25g, trehalose 10-30g, distilled water l000mL, pH value 5.5.
4. high vigor complex enzyme for feed as claimed in claim 1, is characterized in that, described seed tank culture base consists of: Semen Maydis powder 50-60g, bean powder 15-25g, wheat bran 10-15g, fish meal 10-15g, calcium chloride 6-10g, ammonium chloride 1-3g, Sodium phosphate dibasic 1-2g, Chinese herbal medicine powder 15-20g, trehalose 10-30g, pure water l000mL, pH value 5-7.
5. high vigor complex enzyme for feed as claimed in claim 1, is characterized in that, described seeding tank liquid seeds cell concentration is 7.0x 10
8-8.0x 10
8individual/ml.
6. high vigor complex enzyme for feed as claimed in claim 1, it is characterized in that, described fermention medium consists of: Semen Maydis powder 50-60g, bean powder 15-25g, wheat bran 10-15g, fish meal 10-15g, calcium chloride 6-10g, ammonium chloride 1-3g, Sodium phosphate dibasic 1-2g, Chinese herbal medicine powder 30-50g, trehalose 10-30g, pure water l000mL, pH value 5-7.
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