CN103805583A - Wheat diet enzyme containing beta-glucanase and preparation method thereof - Google Patents
Wheat diet enzyme containing beta-glucanase and preparation method thereof Download PDFInfo
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Abstract
The invention relates to a wheat diet enzyme containing beta-glucanase and a preparation method thereof, belonging to the field of feed enzyme preparations. The wheat diet enzyme is prepared from the following enzyme preparations in parts by weight: 100-140 parts of xylanase, 8-12 parts of cellulase, 15-20 parts of beta-glucanase, 6-10 parts of medium-temperature alpha-amylase and 0.5-2 parts of Chinese herbal medicine powder, wherein the beta-glucanase is produced from bacillus licheniformis beta-10-25, has the collection number of CCTCC No: M2013538, and is prepared by a preparation method which comprises the following steps: carrying out inclined-face strain activation and culture, so as to obtain a liquid seed; at a fermentation stage, adopting a low-temperature culture medium supplementing and batched inoculating combined method; filtrating, concentrating, fine-filtrating and drying fermentation liquor, thereby obtaining the beta-glucanase with good thermal stability. According to the compound enzyme, the conversion ratio of feed can be increased, and the cost of the feed can be reduced.
Description
Technical field
The invention belongs to zymin technical field, be specifically related to a kind of Wheat Based Diet enzyme that contains beta-glucanase and preparation method thereof.
Background technology
Along with the development of modern age zymotechnic and biotechnology, high-effect biologically active substance---zymin is suitability for industrialized production on a large scale, and be applied in fodder industry, many experiments and result of practical application all show, fodder enzyme preparation as a kind of fodder additives can effectively improve feed utilization ratio, promote growth of animal and prevent and treat the generation of Animal diseases, compare with hormonal substance with microbiotic, there is remarkable security, caused the great attention of feedstuff industry in global range.
Feed enzyme preparation is in order to improve digestion, the utilization of animal to feed or to improve the metabolism usefulness in animal body and add the enzyme material in feed.The zymin that can add in feed at present comprises amylase, alpha-galactosidase, cellulase, beta-glucanase, glucose oxidase, lipase, maltin, mannonase polygalacturonase, phytase, proteolytic enzyme, M-Zyme, zytase etc.Due to the complicacy of feedstuff raw material structure, in fodder industry production at present, more uses is compound enzymic preparation.Wheat Based Diet enzyme is a kind of novel fodder additives, and its application starts from 20 century 70s abroad, and the fodder industry of China is just carried out the application of zymin in the nineties in 20th century.Therefore, strengthen the research of feed enzyme preparation is had very important significance for the aquaculture development of China.
Feeding enzyme is of a great variety, has all obtained goodish effect in Production of Livestock and Poultry.USSR (Union of Soviet Socialist Republics) scientist adds amylodextrin enzyme in weanling pig daily ration in early days, makes day weight gain improve 14.6%, and carbohydrate digestion rate improves 77.2%.According to U.S.'s pertinent data, Collier supplements zymin (containing proteolytic enzyme, amylase, dextranase) in weanling pig daily ration in early days, and result experimental group has improved 25% than control group day weight gain, and feed conversion rate has improved 15.5%.West Germany scholar once repeatedly carried out growing and fattening pigs and added the experimental study of compound enzymic preparation, take barley in main daily ration, add 0.1% compound enzymic preparation, result is: experimental group improves 5.1% than the control group speed of growth, efficiency of feed utilization improves 4.1%.Visible, zymin can significantly improve livestock and poultry to the digesting and assimilating of feed, and promotes growth, reduce feed cost, and as a kind of green feed additive, its use has security, is conducive to livestock industry and produces the road for development of going on economy type.
For a long time, people are accustomed to using corn as energy feed, but along with the variation of aquaculture development to corn market on the increasing of corn demand and world market, cause corn buying difficulty and price to raise up, especially in non-main maize area, the shortage of corn becomes the important factor of the local animal husbandry development of restriction.And often the source of wheat is wider in non-main maize area, its price is also comparatively stable, and in harvesting wheat season, its price is compared with corn often has certain advantage, particularly along with the increase gradually of China's wheat yield, and the adding of WTO, import wheat pours in the impact bringing, and makes Wheat price on a declining curve.Simultaneously, animal husbandry development is rapid, and feed consumption constantly increases, and manufactures starch and Methionin and also consumes a large amount of corns, the advantages such as the main corn price as energy feed rose steadily in the past, and wheat is wide with its cultivated area, output is high, price is low become the first-selection of replacement of corn.
At present, the shortage of grain is the principal element of puzzlement China feed industrial development.Therefore, the animal husbandry development road of the resource of selecting to be conducive to save food, improves efficiency of feed utilization, makes full use of limited grain resource and obtains livestock product as much as possible.The use of the zymins such as beta-glucanase, can effectively address this problem, but the zymin enzyme activity of the practical application in producing, testing is mostly lower at present, therefore also becomes one of bottleneck improving feeding enzyme application present situation.
Summary of the invention
The object of the present invention is to provide composite Wheat Based Diet enzyme that contains beta-glucanase of a kind of multienzyme kind and preparation method thereof.
Object of the present invention can be achieved through the following technical solutions:
A Wheat Based Diet enzyme that contains beta-glucanase, is made up of the component of following parts by weight: Chinese herbal medicine powder 0.5-2 part, zytase 100-140 part, cellulase 8-12 part, beta-glucanase 15-20 part, mesophilicα-diastase 6-10 part.
Described zytase, cellulase and mesophilicα-diastase are Hunan letter Wings Of The Eagle biotechnology limited-liability company product;
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; The tuber of dwarf lilyturf 10-15 part; Respectively said herbal medicine being crushed to particle diameter is below 2 millimeters, then in container, evenly mix and add the water of 3-6 times of weight, control temperature 70 C～90 ℃ and keep 2～4h, then be cooled to 45-60 ℃, add mixing enzyme preparation to carry out enzymolysis, with newborn acid for adjusting pH value be 5.5-6.8, enzymolysis 2-4h, finally add the mixture of mixture 0.5-3 times of weight ethanol and propyl alcohol, ultrasonic extraction 0.5～1.5h under 110W power, filters; Filtrate vacuum concentration postlyophilization obtains Chinese herbal medicine powder;
Described mixed enzyme addition is the 5-10% of mixture gross weight;
The parts by weight of described mixed enzyme consist of: beta-glucosidase 10-15 part, zytase 15-20 part, pentosanase 15-20 part, Pullulanase 20-30 part, beta-amylase 10-15 part, polygalacturonase 10-15 part, aspartic protease 10-15 part, papoid 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 beta-glucanase preparation method is as follows:
(1) seed culture
Bacillus licheniformis slant strains is fermented through three grades of shake flask fermentations and first class seed pot;
The substratum of described seed culture is: yeast powder 0.5%, peptone 1%, Zulkovsky starch 1%, NaCl1%, pH4.5-7.0,121-123 ℃ of sterilizing 30-40min.
(2) ferment tank
First class seed pot fermented liquid is accessed in the fermentor tank that contains 3L fermention medium to culture temperature 35-45 ℃, stirring velocity 200-300r/min, ventilation (V/V) 1:1-3, incubation time 10-15h with 3% inoculum size; Then by 1L through 121 ℃ of sterilizing 20min, temperature is that the fermention medium of 10 ℃ fills into fermentor tank, constant temperature culture 15-20h in the time that temperature rises to 35-45 ℃; Now, first class seed pot fermented liquid is appended to access fermentor tank, constant temperature culture 10-15h with 2% inoculum size; Continuation is by 1L through 121 ℃ of sterilizing 20min, and temperature is that the fermention medium of 10 ℃ fills into fermentor tank, constant temperature culture 10-15h in the time that temperature rises to 35-45 ℃;
Described fermention medium consists of: wheat bran 75g, Semen Maydis powder 55g, rice bran 25g, soybean cake powder 20g, beta-glucan 2g, herbal mediciment powder 40g, trehalose 35g, yeast powder 6g, corn steep liquor 3g, ammonium sulfate 2g, dipotassium hydrogen phosphate 2g, potassium primary phosphate 2g, Trisodium Citrate 3g, defoamer 0.5g, pure water l000mL, pH value 4.5-7.0,121 ℃ of sterilizing 20min;
The concocting method of described fermention medium is: accurately take in proportion raw material, pure water in raw material, Semen Maydis powder, soybean cake powder are dropped in material-compound tank, regulate pH value 3-7, add middle temperature amylase (3u/g Semen Maydis powder) and alpha-amylase (30u/g Semen Maydis powder), ℃ insulation 15-30min of warming while stirring to 70 simultaneously, is then slowly warming up to 90 ℃ of insulation 15-30min and liquefies, finally add other raw material, stir, pH3-7,121-123 ℃ of sterilizing 30-40min is for subsequent use.
Dissolved oxygen control: by adjusting mixing speed and ventilation, control dissolved oxygen 15-30%;
PH controls: by mending ammoniacal liquor or dilute phosphoric acid, in controlled fermentation process, pH value remains on 3-7;
Control of additive raw material: in the time that 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 as 2mg/ml-5mg/ml;
Put tank standard: the old and feeble self-dissolving of 60-80% thalline, enzyme activity increasess slowly.
(3) fermented liquid after filtration, concentrated, essence is filtered, the dry beta-glucanase that to obtain.
Described Bacillus licheniformis is specially Bacillus licheniformis (Β acillus licheniformis) β-10-25.This bacterial strain is preserved in Chinese Typical Representative culture collection center (be called for short CCTCC, address is: Wuchang District, Wuhan City, Hubei Province Luo Jia Shan Wuhan University Life Science College) on November 3rd, 2013, and preserving number is CCTCC NO:M2013538.
This bacterial strain feature is as follows:
Described bacterial strain bacterium colony on solid plate is white, and edge is irregular, and surface drying is opaque, and microscopy is gram positive bacterium, and cellular form is rod-short, and raw in gemma, ellipse, does not expand.
Described bacterial strain physiological and biochemical property: V-P tests (+), Starch Hydrolysis (+), casein hydrolysis (+), gelatin hydrolysis (+), nitrate reduction (+), indole test (-), utilizes Citrate trianion (+), nitrate reductase (+), N.F,USP MANNITOL (+), wood sugar (+).
Preferably, a kind of Wheat Based Diet enzyme is made up of following parts by weight raw material: 120 parts of zytases, 10 parts of cellulases, 18 parts of beta-glucanases, middle temperature amylase 8 part, 1 part of Chinese herbal medicine powder.
Described Wheat Based Diet enzyme is applicable to feed-processing plant and plant's autogamy feed, when use, should mix with other raw material in feed.Advise that complete diet pellet addition per ton is that in 80-150g(feed, wheat consumption is lower than 30%, addition is 80-100g/t, and wheat consumption exceedes 30%, and addition is 100-150g/t).
Beneficial effect:
1, beta-glucanase enzyme activity used in the present invention, up to 8000-10000u/ml, is 2.5 times of original strain; Under 50-70 ℃ of condition, have high enzyme and live, optimal reactive temperature is 65 ℃; Being suitable for pH value in reaction scope is 5.0; At room temperature preserving after 6 months the enzyme loss of living is less than 25%, 4 ℃ and preserves after 12 months the enzyme loss of living and be less than 20%.Higher than existing beta-glucanase enzyme activity, enzyme effect optimum pH wide scope, thermostability and stability in storage are all higher, are particularly suitable for that temperature of reaction is high, liquefaction process and Mashing process the industrialization demand of depositing.
2, beta-glucanase used in the present invention has good thermostability, still can keep after preserving 1h 80% above enzyme to live under 60 ℃ of conditions, keeps 20% above enzyme work after preserving 10min under 70 ℃ of conditions.
3, compared to the Wheat Based Diet enzyme that is added with common beta-glucanase, under same consumption the present invention higher to the enzymatic hydrolyzation of beta-glucan, thereby improve food conversion ratio, reduce feed cost.
4, in the present invention, added Chinese herbal medicine powder, it has the effect of natural antibacterial and strengthening immunity, can effectively improve anti-stress ability, immunologic function and the Defense response function of livestock and poultry animal.In addition, the root of large-flowered skullcap, the radix bupleuri with the former effect of anti-heat stress in Chinese herbal medicine powder, are added, make product of the present invention have better stability and preserve actively, the preservation activity of the product that does not more add Chinese herbal medicine powder under long-time hot conditions improved 15%.
Embodiment
The preparation method of embodiment 1 Chinese herbal medicine powder
A kind of preparation method of 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 tubers of dwarf lilyturf; 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, then be cooled to 50 ℃, add mixing enzyme preparation to carry out enzymolysis, with newborn acid for adjusting pH value be 6, enzymolysis 3h, finally add the mixture of 2 times of weight ethanol of mixture and propyl alcohol, 11W ultrasonic extraction 1h, filters; Filtrate vacuum concentration postlyophilization obtains Chinese herbal medicine powder.
Described mixed enzyme addition is 8% of mixture gross weight.
The parts by weight of described mixed enzyme consist of: 12 parts of beta-glucosidases, 18 parts of zytases, 18 parts of pentosanases, 25 parts of Pullulanases, 18 parts of beta-amylases, 12 parts of polygalacturonases, 12 parts of aspartic proteases, 8 parts of papoids, 8 parts of glucose oxidases, 8 parts of acid phosphatases.
The mass ratio of described ethanol and propyl alcohol is 1:1.2.
The preparation method of 2 one kinds of beta-glucanases of embodiment
A preparation method for beta-glucanase, comprises the steps:
(1) liquid seeds enlarged culturing
1. first order seed is cultivated: slant strains 2 articulatings after activation are entered in 500 ml shake flasks to 100 milliliters of substratum loading amounts, 180 revs/min of rotary shaking tables, 40 ℃ of culture temperature, incubation time 10h;
2. secondary seed is cultivated: by first order seed, according in 500 milliliters of secondary seed shaking flasks of inoculum size access of 10%, culture condition is identical with first order seed;
3. three grades of seed culture: secondary seed is accessed in 5000 milliliters of three grades of seed shaking flasks to 1000 milliliters of substratum loading amounts, 100 revs/min of rotary shaking tables, 40 ℃ of culture temperature, incubation time 12h with 10% inoculum size;
4. first class seed pot is cultivated: the first class seed pot by three grades of seeds take 10% inoculum size access cubic capacity as 150L, fermention medium loading amount 100L, 41 ℃ of culture temperature, stirring velocity 300rpm, ventilation (V/V) 1:1.5, tank pressure 0.05Mpa, incubation time 15h;
Described seed culture medium weight consists of: yeast powder 0.5%, peptone 1%, Zulkovsky starch 1%, NaCl1%pH5.5,121 ℃ of sterilizing 35min.
(2) ferment tank
First class seed pot fermented liquid is accessed in the fermentor tank that contains 3L fermention medium to 37 ℃ of culture temperature, stirring velocity 250r/min, ventilation (V/V) 1:1-3, incubation time 12h with 3% inoculum size; Then by 1L through 121 ℃ of sterilizing 20min, temperature is that the fermention medium of 10 ℃ fills into fermentor tank, constant temperature culture 18h in the time that temperature rises to 37 ℃; Now, first class seed pot fermented liquid is appended to access fermentor tank, constant temperature culture 12h with 2% inoculum size; Continuation is by 1L through 121 ℃ of sterilizing 20min, and temperature is that the fermention medium of 10 ℃ fills into fermentor tank, constant temperature culture 12h in the time that temperature rises to 37 ℃;
Described fermention medium consists of: wheat bran 75g, Semen Maydis powder 55g, rice bran 25g, soybean cake powder 20g, beta-glucan 2g, herbal mediciment powder 40g, trehalose 35g, yeast powder 6g, corn steep liquor 3g, ammonium sulfate 2g, dipotassium hydrogen phosphate 2g, potassium primary phosphate 2g, Trisodium Citrate 3g, defoamer 0.5g, pure water l000mL, 5.5,121 ℃ of sterilizing 20min of pH value;
The concocting method of described fermention medium is: accurately take in proportion raw material, pure water in raw material, Semen Maydis powder, soybean cake powder are dropped in material-compound tank, regulate pH value 5.5, add middle temperature amylase (3u/g Semen Maydis powder) and alpha-amylase (30u/g Semen Maydis powder), ℃ insulation 20min of warming while stirring to 70 simultaneously, is then slowly warming up to 90 ℃ of insulation 20min and liquefies, finally add other raw material, stir, pH5.5,121 ℃ of sterilizing 35min are for subsequent use.
The preparation method of described Chinese herbal medicine powder is with embodiment 1.
(3) fermented liquid after filtration, concentrated, essence is filtered, the dry beta-glucanase that to obtain.
The beta-glucanase liquid enzymes vigor obtaining through above-mentioned preparation method is 10000u/ml.
The preparation method of 3 one kinds of complex enzyme preparation for feeding of embodiment
A kind of Wheat Based Diet enzyme, is made up of the zymin of following parts by weight:
120 parts of zytases, 10 parts of cellulases, 18 parts of beta-glucanases, 8 parts of mesophilicα-diastases, 1 part of Chinese herbal medicine powder.
Get 120 parts of zytases that originate from Hunan letter Wings Of The Eagle biotechnology limited-liability company, 10 parts of cellulases, 8 parts of mesophilicα-diastases, and 1 part of the Chinese herbal medicine powder of embodiment 1 gained, 18 parts of the beta-glucanases of embodiment 2 gained stir under 25 ℃ of conditions in mixing machine, and churning time is 5 minutes.Stir vanning after in normal temperature or cold house, preserve.
The preparation method of 4 one kinds of complex enzyme preparation for feeding of embodiment
A kind of Wheat Based Diet enzyme, is made up of the zymin of following parts by weight:
100 parts of zytases, 8 parts of cellulases, 20 parts of beta-glucanases, 10 parts of mesophilicα-diastases, 0.5 part of Chinese herbal medicine powder.
Get the zytase that originates from Hunan letter Wings Of The Eagle biotechnology limited-liability company by above-mentioned parts by weight, cellulase, mesophilicα-diastase, and the Chinese herbal medicine powder of embodiment 1 gained, the beta-glucanase of embodiment 2 gained stirs under 25 ℃ of conditions in mixing machine, and churning time is 5 minutes.Stir vanning after in normal temperature or cold house, preserve.
The preparation method of 5 one kinds of complex enzyme preparation for feeding of embodiment
A kind of Wheat Based Diet enzyme, is made up of the zymin of following parts by weight:
140 parts of zytases, 12 parts of cellulases, 15 parts of beta-glucanases, 6 parts of mesophilicα-diastases, 2 parts of Chinese herbal medicine powders.
Get the zytase that originates from Hunan letter Wings Of The Eagle biotechnology limited-liability company by above-mentioned parts by weight, cellulase, mesophilicα-diastase, and the Chinese herbal medicine powder of embodiment 1 gained, the beta-glucanase of embodiment 2 gained stirs under 25 ℃ of conditions in mixing machine, and churning time is 5 minutes.Stir vanning after in normal temperature or cold house, preserve.
Embodiment 6 effect comparison experiments
Choose 120 and fatten pig in early stage, be divided into experimental group and control group by body weight, blood lineage and no sex difference, every group of 3 repetitions, 20 pigs of each repetition.Experimental period is 28 days, and experimental group feed per ton adds compound enzymic preparation 120g described in the present embodiment 3, and control group does not add compound enzymic preparation, weighs on an empty stomach morning day at the whole story of trial period, calculates day weight gain and feedstuff-meat ratio, and gives a mark to hair color.Experimental result is as shown in the table:
| ? | Control group | Test group |
| Average daily gain (g/d) | 655.20 | 703.15 |
| Feed/weightening finish | 2.45 | 2.21 |
| Hair color marking (ten point system) | 5.5 | 8.5 |
Embodiment 5 effect comparison
Choose 120 and fatten pig in early stage, be divided into experimental group and control group by body weight, blood lineage and no sex difference, every group of 3 repetitions, 20 pigs of each repetition.Experimental period is 28 days, experimental group and control group feed per ton all adds compound enzymic preparation 120g, experimental group compound enzymic preparation used is compound enzymic preparation described in embodiment 3, control group compound enzymic preparation used is that the beta-glucanase adding in embodiment 3 is replaced with to commercially available beta-glucanase, weigh morning day at the whole story in trial period on an empty stomach, calculate day weight gain and feedstuff-meat ratio, and give a mark to hair color.Experimental result is as shown in the table:
| ? | Control group | Test group |
| Average daily gain (g/d) | 675.26 | 703.15 |
| Feed/weightening finish | 2.37 | 2.21 |
| Hair color marking (ten point system) | 6.5 | 8.5 |
Embodiment 7 Chinese herbal medicine powders are preserved active impact to enzyme
The Wheat Based Diet enzyme obtaining take preparation method described in embodiment 2 is as sample 1, to remove Wheat Based Diet enzyme that the preparation method of Chinese herbal medicine powders all in embodiment 2 obtains as sample 2, the relatively preservation activity of two kinds of samples.At 45 ℃, preserve above-mentioned sample 18h, measure afterwards enzyme and live, result shows: the remnant enzyme activity of sample 1 is more than 75%, and the remnant enzyme activity of sample 2 remains on 60% left and right.
Claims (8)
1. a Wheat Based Diet enzyme that contains beta-glucanase, is made up of the component of following parts by weight: zytase 100-140 part, cellulase 8-12 part, beta-glucanase 15-20 part, mesophilicα-diastase 6-10 part, Chinese herbal medicine powder 0.5-2 part;
Described beta-glucanase is by Bacillus licheniformis (Β acillus licheniformis) β-10-25 through biological fermentation system, and described bacterial strain deposit number is CCTCC NO:M2013538.
2. a kind of Wheat Based Diet enzyme that contains beta-glucanase as claimed in claim 1, 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; The tuber of dwarf lilyturf 10-15 part; Respectively said herbal medicine being crushed to particle diameter is below 2 millimeters, then in container, evenly mix and add the water of 3-6 times of weight, control temperature 70 C～90 ℃ and keep 2～4h, then be cooled to 45-60 ℃, add mixing enzyme preparation to carry out enzymolysis, with newborn acid for adjusting pH value be 5.5-6.8, enzymolysis 2-4h, finally add the mixture of mixture 0.5-3 times of weight ethanol and propyl alcohol, ultrasonic extraction 0.5～1.5h under 110W power, filters; Filtrate vacuum concentration postlyophilization obtains Chinese herbal medicine powder.
3. a kind of Wheat Based Diet enzyme that contains beta-glucanase as claimed in claim 2, it is characterized in that, mixing enzyme preparation is composed as follows: beta-glucosidase 10-15 part, zytase 15-20 part, pentosanase 15-20 part, Pullulanase 20-30 part, beta-amylase 10-15 part, polygalacturonase 10-15 part, aspartic protease 10-15 part, papoid 5-10 part, glucose oxidase 5-10 part, acid phosphatase 5-10 part.
4. a kind of Wheat Based Diet enzyme that contains beta-glucanase as claimed in claim 1, is characterized in that, the preparation method of beta-glucanase, comprises the steps:
Bacillus licheniformis β-10-25 carries out one, two, three liquid seeds and seed tank culture after slant strains activation culture; First class seed pot fermented liquid is accessed in the fermentor tank that contains 3L fermention medium to culture temperature 35-45 ℃, stirring velocity 200-300r/min, ventilation (V/V) 1:1-3, incubation time 10-15h with 3% inoculum size; Then by 1L through 121 ℃ of sterilizing 20min, temperature is that the fermention medium of 10 ℃ fills into fermentor tank, constant temperature culture 15-20h in the time that temperature rises to 35-45 ℃; Now, first class seed pot fermented liquid is appended to access fermentor tank, constant temperature culture 10-15h with 2% inoculum size; Continuation is by 1L through 121 ℃ of sterilizing 20min, and temperature is that the fermention medium of 10 ℃ fills into fermentor tank, constant temperature culture 10-15h in the time that temperature rises to 35-45 ℃;
Fermented liquid after filtration, concentrated, essence is filtered, the dry beta-glucanase that to obtain.
5. a kind of Wheat Based Diet enzyme that contains beta-glucanase as claimed in claim 4, is characterized in that, the consisting of of described seed culture: yeast powder 0.5%, peptone 1%, Zulkovsky starch 1%, NaCl1%, pH4.5-7.0;
Described fermention medium consists of: wheat bran 75g, Semen Maydis powder 55g, rice bran 25g, soybean cake powder 20g, beta-glucan 2g, herbal mediciment powder 40g, trehalose 35g, yeast powder 6g, corn steep liquor 3g, ammonium sulfate 2g, dipotassium hydrogen phosphate 2g, potassium primary phosphate 2g, Trisodium Citrate 3g, defoamer 0.5g, pure water l000mL, pH value 4.5-7.0,121 ℃ of sterilizing 20min;
The concocting method of described fermention medium is: accurately take in proportion raw material, pure water in raw material, Semen Maydis powder, soybean cake powder are dropped in material-compound tank, regulate pH value 3-7, add middle temperature amylase (3u/g Semen Maydis powder) and alpha-amylase (30u/g Semen Maydis powder), ℃ insulation 15-30min of warming while stirring to 70 simultaneously, is then slowly warming up to 90 ℃ of insulation 15-30min and liquefies, finally add other raw material, stir, pH3-7.
6. a method of preparing the Wheat Based Diet enzyme that contains beta-glucanase as claimed in claim 1, comprises the steps:
Form zytase according to raw material, cellulase, beta-glucanase, mesophilicα-diastase, Chinese herbal medicine powder mixes;
Described zytase, cellulase, mesophilicα-diastase is Hongyingxiang Biological Engineering Co., Ltd., Hunan's product;
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; The tuber of dwarf lilyturf 10-15 part; Respectively said herbal medicine being crushed to particle diameter is below 2 millimeters, then in container, evenly mix and add the water of 3-6 times of weight, control temperature 70 C～90 ℃ and keep 2～4h, then be cooled to 45-60 ℃, add mixing enzyme preparation to carry out enzymolysis, with newborn acid for adjusting pH value be 5.5-6.8, enzymolysis 2-4h, finally add the mixture of mixture 0.5-3 times of weight ethanol and propyl alcohol, ultrasonic extraction 0.5～1.5h under 110W power, filters; Filtrate vacuum concentration postlyophilization obtains Chinese herbal medicine powder;
Described mixed enzyme addition is the 5-10% of mixture gross weight;
The parts by weight of described mixed enzyme consist of: beta-glucosidase 10-15 part, zytase 15-20 part, pentosanase 15-20 part, Pullulanase 20-30 part, beta-amylase 10-15 part, polygalacturonase 10-15 part, aspartic protease 10-15 part, papoid 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 beta-glucanase preparation method is as follows:
(1) seed culture
Bacillus licheniformis β-10-25 slant strains is fermented through three grades of shake flask fermentation agent first class seed pots;
The substratum of described seed culture is: yeast powder 0.5%, peptone 1%, Zulkovsky starch 1%, NaCl1%, pH4.5-7.0,121-123 ℃ of sterilizing 30-40min;
(2) ferment tank
First class seed pot fermented liquid is accessed in the fermentor tank that contains 3L fermention medium to culture temperature 35-45 ℃, stirring velocity 200-300r/min, ventilation (V/V) 1:1-3, incubation time 10-15h with 3% inoculum size; Then by 1L through 121 ℃ of sterilizing 20min, temperature is that the fermention medium of 10 ℃ fills into fermentor tank, constant temperature culture 15-20h in the time that temperature rises to 35-45 ℃; Now, first class seed pot fermented liquid is appended to access fermentor tank, constant temperature culture 10-15h with 2% inoculum size; Continuation is by 1L through 121 ℃ of sterilizing 20min, and temperature is that the fermention medium of 10 ℃ fills into fermentor tank, constant temperature culture 10-15h in the time that temperature rises to 35-45 ℃;
Described fermention medium consists of: wheat bran 75g, Semen Maydis powder 55g, rice bran 25g, soybean cake powder 20g, beta-glucan 2g, herbal mediciment powder 40g, trehalose 35g, yeast powder 6g, corn steep liquor 3g, ammonium sulfate 2g, dipotassium hydrogen phosphate 2g, potassium primary phosphate 2g, Trisodium Citrate 3g, defoamer 0.5g, pure water l000mL, pH value 4.5-7.0,121 ℃ of sterilizing 20min;
The concocting method of described fermention medium is: accurately take in proportion raw material, pure water in raw material, Semen Maydis powder, soybean cake powder are dropped in material-compound tank, regulate pH value 3-7, add middle temperature amylase (3u/g Semen Maydis powder) and alpha-amylase (30u/g Semen Maydis powder), ℃ insulation 15-30min of warming while stirring to 70 simultaneously, is then slowly warming up to 90 ℃ of insulation 15-30min and liquefies, finally add other raw material, stir, adjust initial p H3-7,121-123 ℃ of sterilizing 30-40min is for subsequent use;
Dissolved oxygen control: by adjusting mixing speed and ventilation, control dissolved oxygen 15-30%;
PH controls: by mending ammoniacal liquor or dilute phosphoric acid, in controlled fermentation process, pH value remains on 3-7;
Control of additive raw material: in the time that 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 as 2mg/ml-5mg/ml;
Put tank standard: the old and feeble self-dissolving of 60-80% thalline, enzyme activity increasess slowly;
(3) fermented liquid after filtration, concentrated, essence is filtered, the dry beta-glucanase that to obtain.
7. a kind of Wheat Based Diet enzyme that contains beta-glucanase as claimed in claim 1, it is characterized in that: the component by following parts by weight forms: 120 parts of zytases, 10 parts of cellulases, 18 parts of beta-glucanases, 8 parts of mesophilicα-diastases, 1 part of Chinese herbal medicine powder.
8. the application of a kind of Wheat Based Diet enzyme that contains beta-glucanase as claimed in claim 1 in animal rearing.
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| CN105192323A (en) * | 2015-10-29 | 2015-12-30 | 青岛蔚蓝生物集团有限公司 | Special compound enzyme for wheat type feed and application of special compound enzyme |
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