CN103695396A - High-activity composite enzyme for feed - Google Patents
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Abstract
The invention discloses a high-activity composite enzyme for feed, belonging to the technical field of preparation of an enzyme preparation for feed. An Aspergillus niger DM-18 strain for a high-yield composite enzyme for feed, which is used as an initial strain, is subjected to liquid submerged mixed fermentation in an optimized culture medium under specific fermentation conditions by an improve fermentation technique to obtain the composite enzyme for feed, which has the advantages of complete enzymatic system, high enzyme activity, high temperature resistance and wide pH value resistance range. The crude enzyme fermentation liquid of the high-activity composite enzyme for feed contains multiple enzymes, wherein the proteinase activity is 6500-6700 U/ml, the mannase activity is 1500-1700 U/ml, the alpha-galactase activity is 1200-1300 U/ml, and the pectinase activity is 1000-1200 U/ml. When the crude enzyme fermentation liquid is subjected to the heat stability test and pH stability test, the test result indicates that all the enzymes have higher enzyme activity (up to higher than 80%) at 30-75 DEG C under the pH value of 2-7.
Description
Technical field
The present invention relates to fodder enzyme preparation, particularly a kind of high vigor complex enzyme for feed.
Background technology
Large quantity research both domestic and external shows, in feed, add zymin and 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 has promoted digestion and the absorption of digestive tube to carbohydrate and other nutritive substance, has improved 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 and distribute, improve animal health condition.The manna oligosaccharide producing after enzyme preparation degrades non-starch polysaccharide can promote the profitable strain propagation such as bifidus bacillus, buttermilk bacillus, 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, by affecting the hormone metabolism of body, promote that poultry grows.(5) alleviate herding and produce the pollution to environment.Almost, in all plant feeds, there is (phytinic acid) with phytic acid form in most phosphoric acid salt.Because the vigor of phytase in monogastric animal digestive tube is very low, most of phytate phosphorus is excreted, and so not only wastes phosphorus source, and causes environmental pollution.Add phytase, 50%～70% secondary calcium phosphate in alternative feed formulation, the corresponding minimizing of phosphorus of draining to environment more than 30%, is played a great role in environment protection.(6) release of the starch that promotion cell surrounds, protein, fat, by hydrolysis cell walls, is fully absorbed nutritive substance.
In livestock product cultivation, apply digestibility and the utilization ratio that fodder enzyme preparation can improve feed; improve the production performance of livestock and poultry and fish; can reduce again the nitrogen in farm animal excrement, the excretion of phosphorus; protection water body and soil are avoided polluting, thus fodder enzyme preparation efficient as a class, 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.2009 annual production of China's mixed feed reach 1.4 hundred million tons, wherein enzyme-added 0.1% by 50% left and right, with approximately 70,000 tons of enzymes.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 20,000 tons of the production capacity deficiencies of domestic fodder enzyme preparation, therefore, fodder enzyme preparation also has the very large market space and potentiality in China.The market of China's fodder enzyme preparation begins to take shape at present, and progressively development, and enzyme classes preparation has good growth momentum and prospect at home.
China's various places staple food crop differs greatly, and zymin has larger effect to the exploitation of the utilization of potential feed resource and new feedstuff resource.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 actively by forbidding ensureing by modes such as microbiotic, hormones the safety of feed and food in feed, maintaining ecological balance.In future 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 particularly raising of genetic engineering technique and production technology of biotechnology, 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, without resistance, the advantage such as free from environmental pollution, will further be applied.
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 preparations, and composite by formula, then add dispersion agent, thinner etc. and process.Main drawback is that cost is high, during use, be difficult for enzyme work compared with high and dosage zymin seldom mixes with a large amount of feeds, thereby had a strong impact on the effect of using, this is increasing again use cost virtually, many managers are hung back, to promotion and application, brought obstacle.2. more than bacterial strain mixed fermentation, naturally complementary enzyme is method: adopt several bacterial classification mixed fermentation, the complementary enzyme process that produces obtains the prozyme product comparatively approaching with method of double crossing, only need adjust a little and be product afterwards, but the technical difficulty of multi-strain fermentation is very large, because the physiological property separately of different bacterial classifications, mixed culture influences each other unavoidably, and make them in same medium, grow and synthesize different enzymes has certain difficulty.3. single strain fermentation, add enzyme process: by bacterial screening, obtain strain excellent, this bacterial strain can high yield plurality of enzymes, and these enzymes form rationally, can meet simple feed formulation requirement, with obtaining prozyme after such strain fermentation, then added and adjusted by the growth needs of the different ages of different animals.
In sum, adopting single culture fermentative production complex enzyme for feed is 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 of preparing 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 is high yield zytase, cellulase, polygalacturonase, amylase, 'beta '-mannase etc. simultaneously, can produce detoxification prozyme, various enzymes 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 that to take bacterial strain aspergillus niger (Aspergillus niger) DM-18 of high yield complex enzyme for feed be starting strain, and carry out medium optimization and zymotechnique and improve, deep liquid mixed fermentation under specifically fermentation condition, makes that a kind of enzyme system is complete, enzyme activity is high, tolerable temperature is stronger, the more wide in range complex enzyme for feed of resistance to pH value.
Prozyme of the present invention comprises mannonase α-galactase, polygalacturonase and proteolytic enzyme, has formed a kind of multienzyme complex for feedstuff raw material to be, i.e. complex enzyme for feed.
The zymologic property of the high 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 ℃, 65 ℃ of optimal reactive temperatures, at 75 ℃ of enzymes complete stability alive;
(2) pH: the applicable pH value in reaction scope of this enzyme is 2.0-7.0, is 2.0 o'clock enzyme complete stabilities alive in pH value, and optimal reaction pH value is 5-7;
(3) enzymic activity: contain plurality of enzymes vigor in this mixed enzyme fermentation liquid crude enzyme liquid, wherein proteinase activity 6500-6700U/ml, mannosans enzyme activity 1500-1700U/ml, α-galactase vigor 1200-1300U/ml, pectinase activity 1000-1200U/ml.
(4) thermostability: in this mixed enzyme fermentation liquid crude enzyme liquid, each enzyme still can keep 85% above enzyme to live preserve 1h under 70 ℃ of conditions after, keeps 80% above enzyme work after preserving 1h under 75 ℃ of conditions;
The preparation method of the high 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 ℃, stirring velocity 200-700r/m, ventilation (V/V) 1:1-3, incubation time 10-15h; Then with 1-2 ℃/h rate of temperature fall slow cooling to 10-15 ℃, constant temperature culture 15-20h; Continuation to 2-5 ℃, now, is appended access fermentor tank, constant temperature culture 20-30h by liquid seeds with 4% inoculum size with 1-2 ℃/h rate of temperature fall slow cooling; Finally with 1-2 ℃/h temperature rise rate, be slowly warming up to 10-15 ℃, constant temperature culture 15-20h; Continuation is slowly warming up to 30-33 ℃, constant temperature culture 15-20h with 1-2 ℃/h temperature rise rate; Fermented liquid after filtration, concentrated, allotment, essence filter, the dry solid complex enzyme for feed that to obtain, 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, 5.8,121 ℃ of sterilizing 20min of pH value.
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 below 2 millimeters, then in container, evenly mix and add the water of 3-6 times of weight, control temperature 70 C～90 ℃ and keep 2～4h, add the mixture of 2-3 times of weight ethanol of mixture and propyl alcohol, control temperature to 60 ℃～78 ℃ and keep 3～4h, filter; Filtrate vacuum concentration postlyophilization 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, 5.5,121 ℃ of sterilizing 20min of pH value.
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 ℃ of sterilizing 20min.
Described seeding tank fermented liquid cell concentration is 7.0x10
8-8.0x10
8individual/ml;
Described becoming: 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 ℃ 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 ℃ of sterilizing 30-40min.
The application of the high vigor complex enzyme for feed of the present invention in preparing 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, aspergillus niger (Aspergillus niger) HYX0022 that the separation of Jinshi City Yang You township, Shi Cong Hunan Province vegetable mould, straw compound sample soil obtains is through through ultraviolet mutagenesis, the mutagenesis of ultraviolet nitrous acid, ultraviolet nitrosoguanidine complex mutation, then mutant strain step-sizing is eliminated, finally strain excellent is obtained producing the aspergillus niger DM-18 of complex enzyme for feed through leavening property test screen.This bacterial strain is preserved in Chinese Typical Representative culture collection center on November 3rd, 2013 and (is called for short CCTCC, address is: Wuchang District, Wuhan City, Hubei Province Luo Jia Shan Wuhan University Life Science College postcode: 430072), preserving number is CCTCC NO:M2013541, and Classification And Nomenclature is aspergillus niger (Aspergillus niger) DM-18.
The aspergillus niger DM-18 of high yield complex enzyme for feed provided by the invention (CCTCC NO:M2013541) 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, diameter 150-450 μ m, and conidiophore betides matrix.Falx stem 1000-3000 (length) * 12-20 (diameter) μ m, yellow or tawny, wall is level and smooth; Spherical or the almost spherical of top capsule, diameter 35-50 μ m, surface can be educated comprehensively; Conidial fructification is double-deck, metulae 15-20 (length) * 3-4.0 (diameter) μ m, and falx 6-8 (length) * 2-4 (diameter) μ m, conidium is spherical or subsphaeroidal, less, diameter 3.5-5.0 μ m, brown, wall is coarse.
2, cultivate and learn feature:
Aspergillus niger DM-18 grows rapidly on wort agar substratum, 28 ℃ 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 is slightly yellow.
3, physiological and biochemical property:
Aspergillus niger DM-18 can be at potato, Semen Maydis powder, Zulkovsky starch, the upper growth such as molasses, optimum pH 4.6, optimum growth temperature 28-34 ℃, the suitableeest product enzyme temperature 28-30 ℃.
The triage techniques route of aspergillus niger DM-18 is: the preparation → mutagenic treatment → plate isolation → primary dcreening operation of original strain separation, screening and evaluation → starting strain → slant culture → spore suspension → multiple sieve → sieve → expansion experiment (leavening property mensuration) again again.
Beneficial effect:
1. to take the bacterial strain aspergillus niger DM-18 of high yield complex enzyme for feed be starting strain to the high vigor complex enzyme for feed of the present invention, and carry out medium optimization and zymotechnique and improve, adopt the zymotechnique of gradient cooling and gradient increased temperature, appended inoculation and feed supplement in good time simultaneously, especially the zymotechnique of gradient cooling and gradient increased temperature has significantly improved the anti-stress ability of starting strain, causes the enzymatic productivity of bacterial classification to manifest to greatest extent.And the present invention forms and implements full optimization substratum, the root of large-flowered skullcap, the radix bupleuri with the former effect of anti-heat stress have been added, added to have and adjusted and repaired body function, the immunologic function of enhancing body, the Radix Astragali with micro-Ecological regulation services, Radix Codonopsis etc., further strengthened body function, adaptation of virus and the common interoperability of microorganism under same yeasting, and then strengthened the metabolic function of microorganism, make the present invention produce that complex enzyme for feed vigor is high, tolerable temperature is higher, stability is strong, be suitable for suitability for industrialized production.
2. in the high vigor complex enzyme for feed of the present invention fermented liquid crude enzyme liquid, contain plurality of enzymes vigor, 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 ℃, during pH value 2-7, each enzyme all has higher enzyme activity, reaches more than 80%.
3. the high vigor complex enzyme for feed of the present invention comprises mannonase α-galactase, polygalacturonase and proteolytic enzyme; thereby form a kind of multienzyme complex for feedstuff raw material, be; can produce complex enzyme for feed; and the various enzymes in this prozyme are lived optimum pH between 2-7; the digestion and the absorbing environmental that are applicable to animal gastrointestinal tract; this complex enzyme for feed can be widely used in animal and fowl fodder; can reduce antinutritional factor; improve digestibility; there is good economic benefit; also be conducive to preserve the ecological environment, promote the sound development of livestock industry simultaneously.
4. the present invention's high vigor complex enzyme for feed thermostability and pH stability are strong, are more applicable to fodder industry complete processing demand, and the feed enzyme loss alive after processing is low.
Embodiment
Below by specific embodiment narration the present invention.Unless stated otherwise, in the present invention, technique means used is method known in those skilled in the art.In addition, embodiment is interpreted as illustrative, but not limits the scope of the invention, and the spirit and scope of the invention are only limited by claims.To those skilled in the art, do not deviating under the prerequisite of essence of the present invention and scope various changes that the material component in these embodiments and consumption are carried out or change and also belong to protection scope of the present invention.
Embodiment 1 complex enzyme for feed enzyme activity determination
1) enzyme activity unit definition:
Proteinase activity unit of force definition: under 40 ℃, pH5.5. condition, the enzyme amount that 1min caseinhydrolysate produces 1 μ mol tyrosine is 1 enzyme activity unit, represents with U/ml.
Mannosans enzyme activity unit definition: under 40 ℃, pH5.5 condition, the Viscogum BE solution that 1min is 5mg/ml from concentration, to discharge the needed enzyme amount of reducing sugar that is equivalent to 1 μ mol seminose be 1 enzyme activity unit in hydrolysis, represents with U/ml.
α-galactase unit of activity definition: under 40 ℃, pH5.5 condition, 1min decomposes pnitrophenylα Dgalactospyranoside, and to generate the required enzyme amount of 1 μ mol p-nitrophenol be 1 alpha-galactosidase enzyme unit that lives, and represents with U/ml.
Pectinase activity unit definition: under 40 ℃, pH5.5 condition, the pectin solution that 1min is 10mg/ml from concentration, to discharge the needed enzyme amount of reducing sugar that is equivalent to 1 μ mol be 1 enzyme activity unit in hydrolysis, represents with U/ml.
2) measure: embodiment 2 crude enzyme liquids are suitably diluted with distilled water, get four brace plug test tubes, in three (three repetitions), add the diluted complex enzyme for feed crude enzyme liquid of 0.1ml respectively, in the 4th test tube, add the inactivator liquid of 0.1ml through boiling 5min in contrast, arise from preheating 5min in 40 ℃ of water-baths with the substrate (concrete concentration of substrate is shown in the enzyme activity unit definition described in step 1) of 0.2mol/l acetic acid-sodium acetate buffer preparation with pH5.5; In each test tube, add 0.1ml substrate, 40 ℃ are accurately reacted 30min, after having reacted, in four test tubes, add 0.6mlDNS reagent, shake up, boil 10min deactivation colour developing, rapidly cool to room temperature, water constant volume, to 5.0ml, is measured absorbancy under 550nm wavelength.The extension rate (with distilled water diluting) of adjusting complex enzyme for feed crude enzyme liquid, makes the absorbancy numerical value of measuring within standard curve determination scope.
3) result: after measured, in the fermented liquid fermented liquid crude enzyme liquid that embodiment 2 obtains, contain plurality of enzymes vigor, 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 to slant medium, cultivates 42h for 30 ℃ and carry out actication of culture, so activate 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, 5.8,121 ℃ of sterilizing 20min of pH value.
The preparation method of described Chinese herbal medicine powder is as follows:
Take 25 parts of the Radixs Astragali; 15 parts of Radix Codonopsis; 12 parts of radix bupleuri; 12 parts of the roots of large-flowered skullcap; Respectively said herbal medicine being crushed to particle diameter is below 2 millimeters, then in container, evenly mix and add the water of 5 times of weight, control 80 ℃ of temperature and keep 3h, add the mixture of 3 times of weight ethanol of mixture and propyl alcohol, control temperature to 70 ℃ and keep 4h, filter; Filtrate vacuum concentration postlyophilization 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) activation, with spore under aseptic washing, is accessed in 500 ml shake flasks to 100 milliliters of liquid seed culture medium loading amounts, 30 ℃, 100rpm shaking table cultivation 42h;
2. secondary seed is cultivated: by first order seed, according in 500 milliliters of secondary seed shaking flasks of inoculum size access of 10%, culture condition is identical with first order seed;
3. three grades of seed culture: by secondary seed with in 5000 milliliters of three grades of seed shaking flasks of 8% inoculum size access, 1000 milliliters of liquid nutrient medium loading amounts, 30 ℃, 100rpm shaking table are cultivated 42h;
4. seed tank culture: three grades of seeds be take to the first class seed pot that 8% inoculum size access cubic capacity is 150L, seed tank culture base loading amount 100L, controlling pH value is 6,30 ℃ of culture temperature, 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, 5.5,121 ℃ of sterilizing 20min of pH value.
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, 6,121 ℃ of sterilizing 20min of pH value.
Described seeding tank fermented liquid cell concentration is 7.5x10
8individual/ml;
(3) ferment tank
Seeding tank liquid seeds in step (2) is accessed to fermentor tank, 30 ℃ of culture temperature, stirring velocity 350r/m, ventilation (V/V) 1:2, incubation time 12h with 6% inoculum size; Then with 2 ℃/h rate of temperature fall slow cooling to 12 ℃, constant temperature culture 18h; Continuation, with 2 ℃/h rate of temperature fall slow cooling to 4 ℃, now, is appended access fermentor tank, constant temperature culture 25h by seeding tank liquid seeds in step (2) with 4% inoculum size; Finally with 2 ℃/h temperature rise rate, be slowly warming up to 12 ℃, constant temperature culture 18h; Continuation is slowly warming up to 30 ℃ with 2 ℃/h temperature rise rate, constant temperature culture 18h;
Dissolved oxygen is controlled: by adjusting mixing speed and ventilation, control dissolved oxygen 20%;
PH controls: by mending ammoniacal liquor or dilute phosphoric acid, in controlled fermentation process, pH value remains on 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 be take and maintained fermented liquid reducing sugar content as 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, 6,121 ℃ of sterilizing 20min of pH value.
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, 6,123 ℃ of sterilizing 40min of pH value.
(4) fermented liquid after filtration, concentrated, allotment, essence filter, the dry solid complex enzyme for feed that to obtain.
Described allocation process adds concentrated enzyme liquid gross weight 3% Chinese herbal medicine powder.
In the fermented liquid fermented liquid crude enzyme liquid obtaining through above-mentioned preparation method, contain plurality of enzymes vigor, 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
Thermostability to complex enzyme for feed is analyzed, and embodiment 4 crude enzyme liquids are placed in respectively at 30 ℃, 40 ℃, 45 ℃, 50 ℃, 55 ℃, 60 ℃, 65 ℃, 70 ℃, 75 ℃, and in 10 minutes sampling and measuring complex enzyme for feed, the enzyme of each constitutive enzyme is lived.At 30 ℃, 40 ℃, 45 ℃, 50 ℃, 60 minutes each constitutive enzyme enzymes are lived and are not declined.At 55 ℃, 60 ℃ and 65 ℃, each constitutive enzyme enzyme work in 30 minutes drops to constitutive enzyme each constitutive enzyme enzyme work in 95%, 60 minute alive and drops to 85%.At 70 ℃, each constitutive enzyme enzyme work in 30 minutes drops to constitutive enzyme each constitutive enzyme enzyme work in 90%, 60 minute alive and drops to 85%.At 75 ℃, each constitutive enzyme enzyme work in 85%, 60 minute that each constitutive enzyme enzyme work in 30 minutes drops to constitutive enzyme work drops to 80% of constitutive enzyme work, and compared with prior art, similarity condition is issued to same enzyme tolerable temperature alive and has on average improved 5-10 ℃.
The pH stability analysis of the high vigor complex enzyme for feed of embodiment 4
PH stability to complex enzyme for feed enzyme is analyzed, and embodiment 4 crude enzyme liquids are placed in respectively to pH value 2.0,2.5,3.5,4.5,5.5,6.0,6.5,7.0 times, and 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, each constitutive enzyme enzyme work in 95%, 60 minute that each constitutive enzyme enzyme work in 30 minutes drops to constitutive enzyme work drops to 85% of constitutive enzyme work.PH value 2.5 times, each constitutive enzyme enzyme work in 30 minutes drops to that constitutive enzyme lives within 90%, 60 minute, drop to that constitutive enzyme lives 85%.PH value 2.0 times, each constitutive enzyme enzyme work in 30 minutes drops to that constitutive enzyme lives within 85%, 60 minute, drop to that constitutive enzyme lives 80%, compared with prior art, it is more wide in range that similarity condition is issued to the same enzyme resistance to pH value of living.
Claims (10)
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 ℃, 65 ℃ of optimal reactive temperatures, at 75 ℃ of enzymes complete stability alive;
(2) pH: the applicable pH value in reaction scope of this enzyme is 2.0-7.0, is 2.0 o'clock enzyme complete stabilities alive in pH value, and optimal reaction pH value is 5-7;
(3) enzymic activity: contain plurality of enzymes vigor in this mixed enzyme fermentation liquid crude enzyme liquid, wherein proteinase activity 6500-6700U/ml, mannosans enzyme activity 1500-1700U/ml, α-galactase vigor 1200-1300U/ml, pectinase activity 1000-1200U/ml;
(4) thermostability: in this mixed enzyme fermentation liquid crude enzyme liquid, each enzyme keeps 85% above enzyme to live preserve 1h under 70 ℃ of conditions after, keeps 80% above enzyme work after preserving 1h under 75 ℃ of conditions.
2. high vigor complex enzyme for feed as claimed in claim 1, it is characterized in that, it is starting strain that this enzyme be take the bacterial strain aspergillus niger DM-18 of high yield complex enzyme for feed, and carries out medium optimization and zymotechnique improves, the deep liquid mixed fermentation preparation under specifically fermentation condition.
3. high vigor complex enzyme for feed as claimed in claim 2, it is characterized in that, preparation method is as follows: the slant strains of intact aspergillus niger DM-18 is obtained to 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 ℃, stirring velocity 200-700r/m, ventilation (V/V) 1:1-3, incubation time 10-15h; Then with 1-2 ℃/h rate of temperature fall slow cooling to 10-15 ℃, constant temperature culture 15-20h; Continuation to 2-5 ℃, now, is appended access fermentor tank, constant temperature culture 20-30h by liquid seeds with 4% inoculum size with 1-2 ℃/h rate of temperature fall slow cooling; Finally with 1-2 ℃/h temperature rise rate, be slowly warming up to 10-15 ℃, constant temperature culture 15-20h; Continuation is slowly warming up to 30-33 ℃, constant temperature culture 15-20h with 1-2 ℃/h temperature rise rate; Fermented liquid after filtration, concentrated, allotment, essence filter, the dry solid complex enzyme for feed that to obtain, described allocation process adds concentrated enzyme liquid gross weight 0.5-5% Chinese herbal medicine powder.
4. high vigor complex enzyme for feed as claimed in claim 3, 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.
5. high vigor complex enzyme for feed as claimed in claim 3, it is characterized in that, described seed culture medium consists 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.
6. high vigor complex enzyme for feed as claimed in claim 3, 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.
7. high vigor complex enzyme for feed as claimed in claim 3, is characterized in that, described seeding tank fermented liquid cell concentration is 7.0x10
8-8.0x10
8individual/ml.
8. high vigor complex enzyme for feed as claimed in claim 3, 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.
9. the high vigor complex enzyme for feed as described in as arbitrary in claim 3-8, is characterized in that, 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 below 2 millimeters, then in container, evenly mix and add the water of 3-6 times of weight, control temperature 70 C～90 ℃ and keep 2～4h, add the mixture of 2-3 times of weight ethanol of mixture and propyl alcohol, the mass ratio of described ethanol and propyl alcohol is 1:1-1.5, control temperature to 60 ℃～78 ℃ and keep 3～4h, filter; Filtrate vacuum concentration postlyophilization obtains Chinese herbal medicine powder.
10. the application of high vigor complex enzyme for feed as claimed in claim 1 or 2 in preparing feed complex enzyme.
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| CN103892134A (en) * | 2014-04-15 | 2014-07-02 | 湖南农业大学 | Compound enzyme preparation for hen feed as well as production method and application thereof |
| CN104686838A (en) * | 2015-02-11 | 2015-06-10 | 青岛根源生物技术集团有限公司 | Composite microecological feed additive capable of increasing freshwater fish growth rate and application of composite microecological feed additive |
| CN105779423A (en) * | 2015-11-17 | 2016-07-20 | 济南诺能生物工程有限公司 | Compound protease as well as production method and applications thereof |
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| CN103892134A (en) * | 2014-04-15 | 2014-07-02 | 湖南农业大学 | Compound enzyme preparation for hen feed as well as production method and application thereof |
| CN103892134B (en) * | 2014-04-15 | 2015-09-16 | 湖南农业大学 | A kind of egg feedstuff complex enzyme formulation and production method thereof and application |
| CN104686838A (en) * | 2015-02-11 | 2015-06-10 | 青岛根源生物技术集团有限公司 | Composite microecological feed additive capable of increasing freshwater fish growth rate and application of composite microecological feed additive |
| CN105779423A (en) * | 2015-11-17 | 2016-07-20 | 济南诺能生物工程有限公司 | Compound protease as well as production method and applications thereof |
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