CN1685924A - Method of producing feed composite enzyme using orange peel as raw material by solid fermentation - Google Patents
Method of producing feed composite enzyme using orange peel as raw material by solid fermentation Download PDFInfo
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- CN1685924A CN1685924A CNA2005100502865A CN200510050286A CN1685924A CN 1685924 A CN1685924 A CN 1685924A CN A2005100502865 A CNA2005100502865 A CN A2005100502865A CN 200510050286 A CN200510050286 A CN 200510050286A CN 1685924 A CN1685924 A CN 1685924A
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- solid fermentation
- enzyme
- oranges
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- 102000004190 Enzymes Human genes 0.000 title claims abstract description 69
- 108090000790 Enzymes Proteins 0.000 title claims abstract description 69
- 239000007787 solid Substances 0.000 title claims abstract description 38
- 238000000855 fermentation Methods 0.000 title claims description 50
- 230000004151 fermentation Effects 0.000 title claims description 50
- 238000000034 method Methods 0.000 title claims description 21
- 239000002994 raw material Substances 0.000 title claims description 12
- 239000002131 composite material Substances 0.000 title abstract 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 35
- 241000675108 Citrus tangerina Species 0.000 claims abstract description 24
- 238000001816 cooling Methods 0.000 claims abstract description 10
- 238000004519 manufacturing process Methods 0.000 claims abstract description 10
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- 235000015099 wheat brans Nutrition 0.000 claims abstract description 10
- 238000001035 drying Methods 0.000 claims abstract description 8
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- 238000002156 mixing Methods 0.000 claims abstract description 3
- 229940088598 enzyme Drugs 0.000 claims description 67
- 239000000725 suspension Substances 0.000 claims description 25
- 239000001963 growth medium Substances 0.000 claims description 19
- 244000046052 Phaseolus vulgaris Species 0.000 claims description 16
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- 238000002360 preparation method Methods 0.000 claims description 15
- 241000207199 Citrus Species 0.000 claims description 13
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 claims description 13
- 239000004202 carbamide Substances 0.000 claims description 13
- 235000020971 citrus fruits Nutrition 0.000 claims description 13
- 235000012054 meals Nutrition 0.000 claims description 13
- 241001112078 Aspergillus usamii Species 0.000 claims description 12
- 239000000843 powder Substances 0.000 claims description 10
- 238000004659 sterilization and disinfection Methods 0.000 claims description 9
- 230000001580 bacterial effect Effects 0.000 claims description 8
- 108091005508 Acid proteases Proteins 0.000 claims description 7
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims description 7
- 235000019800 disodium phosphate Nutrition 0.000 claims description 7
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 6
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- 235000011130 ammonium sulphate Nutrition 0.000 claims description 6
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- 235000007079 manganese sulphate Nutrition 0.000 description 5
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 description 5
- 238000012545 processing Methods 0.000 description 5
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- 238000009835 boiling Methods 0.000 description 4
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- 239000011702 manganese sulphate Substances 0.000 description 4
- 238000004321 preservation Methods 0.000 description 4
- BHZOKUMUHVTPBX-UHFFFAOYSA-M sodium acetic acid acetate Chemical compound [Na+].CC(O)=O.CC([O-])=O BHZOKUMUHVTPBX-UHFFFAOYSA-M 0.000 description 4
- 230000007306 turnover Effects 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 3
- NGFMICBWJRZIBI-JZRPKSSGSA-N Salicin Natural products O([C@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@H](CO)O1)c1c(CO)cccc1 NGFMICBWJRZIBI-JZRPKSSGSA-N 0.000 description 3
- NGFMICBWJRZIBI-UHFFFAOYSA-N alpha-salicin Natural products OC1C(O)C(O)C(CO)OC1OC1=CC=CC=C1CO NGFMICBWJRZIBI-UHFFFAOYSA-N 0.000 description 3
- 239000003674 animal food additive Substances 0.000 description 3
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- NGFMICBWJRZIBI-UJPOAAIJSA-N salicin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=CC=CC=C1CO NGFMICBWJRZIBI-UJPOAAIJSA-N 0.000 description 3
- 229940120668 salicin Drugs 0.000 description 3
- 239000011734 sodium Substances 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 2
- 108091005804 Peptidases Proteins 0.000 description 2
- 239000004365 Protease Substances 0.000 description 2
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 2
- SRBFZHDQGSBBOR-LECHCGJUSA-N alpha-D-xylose Chemical compound O[C@@H]1CO[C@H](O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-LECHCGJUSA-N 0.000 description 2
- 239000005018 casein Substances 0.000 description 2
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 description 2
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- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 description 2
- 229920001221 xylan Polymers 0.000 description 2
- 150000004823 xylans Chemical class 0.000 description 2
- 229960003487 xylose Drugs 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 1
- 241000609240 Ambelania acida Species 0.000 description 1
- 241001019179 Cingula Species 0.000 description 1
- AEMOLEFTQBMNLQ-YMDCURPLSA-N D-galactopyranuronic acid Chemical compound OC1O[C@H](C(O)=O)[C@H](O)[C@H](O)[C@H]1O AEMOLEFTQBMNLQ-YMDCURPLSA-N 0.000 description 1
- 206010012735 Diarrhoea Diseases 0.000 description 1
- 206010013786 Dry skin Diseases 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 101710121765 Endo-1,4-beta-xylanase Proteins 0.000 description 1
- IAJILQKETJEXLJ-UHFFFAOYSA-N Galacturonsaeure Natural products O=CC(O)C(O)C(O)C(O)C(O)=O IAJILQKETJEXLJ-UHFFFAOYSA-N 0.000 description 1
- 229920002488 Hemicellulose Polymers 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- 108010059820 Polygalacturonase Proteins 0.000 description 1
- 244000061456 Solanum tuberosum Species 0.000 description 1
- 235000002595 Solanum tuberosum Nutrition 0.000 description 1
- 240000008042 Zea mays Species 0.000 description 1
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 1
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 239000010905 bagasse Substances 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
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- 108010085318 carboxymethylcellulase Proteins 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 235000005822 corn Nutrition 0.000 description 1
- 235000005911 diet Nutrition 0.000 description 1
- 230000037213 diet Effects 0.000 description 1
- 235000019621 digestibility Nutrition 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 201000006549 dyspepsia Diseases 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 108010093305 exopolygalacturonase Proteins 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000010200 folin Substances 0.000 description 1
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- 230000031700 light absorption Effects 0.000 description 1
- 244000144972 livestock Species 0.000 description 1
- 239000013028 medium composition Substances 0.000 description 1
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- 230000003020 moisturizing effect Effects 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
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- 235000015097 nutrients Nutrition 0.000 description 1
- 238000000247 postprecipitation Methods 0.000 description 1
- 230000013777 protein digestion Effects 0.000 description 1
- 238000010298 pulverizing process Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 239000002893 slag Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- KMPHTYSTEHXSTL-UHFFFAOYSA-M sodium;2-hydroxypropanoate;2-hydroxypropanoic acid Chemical compound [Na+].CC(O)C(O)=O.CC(O)C([O-])=O KMPHTYSTEHXSTL-UHFFFAOYSA-M 0.000 description 1
- 238000010563 solid-state fermentation Methods 0.000 description 1
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- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 1
- 238000009423 ventilation Methods 0.000 description 1
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Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
- Y02A40/81—Aquaculture, e.g. of fish
- Y02A40/818—Alternative feeds for fish, e.g. in aquacultures
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
- Y02P60/80—Food processing, e.g. use of renewable energies or variable speed drives in handling, conveying or stacking
- Y02P60/87—Re-use of by-products of food processing for fodder production
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
A process for preparing the composite enzyme for the feed includes such steps as mixing tangerine peel with soybean dregs and wheat bran, adding less inorganic salt and water, stirring, sterilizing, cooling, inoculating a kind of Aspergillus, solid fermenting and drying.
Description
Technical field
The present invention relates to biological technical field, particularly a kind of is the method that raw material by solid fermentation is produced complex enzyme for feed with the oranges and tangerines skin.
Background technology
Development along with the oranges and tangerines processing industry; be accompanied by and produce a large amount of processing byproducts; as oranges and tangerines skin, press residue, account for whole fruit heavy 25%～40%, so the comprehensive utilization of oranges and tangerines skin to the economic benefit that improves oranges and tangerines processing factory and reduce pollute, the protection environment is all significant.
In recent decades, energy problem is one of the main social problems in the world, but people ceaselessly seek new forms of energy, and the reasonable utilization of renewable resource is an important channel that solves energy problem.The main method that solves energy scarcity at feed industry improves efficiency of feed utilization exactly.Feed is generally based on the processing of farm products accessory substance, and the animal absorptivity is lower.One of solution is to add exogenous enzymes and eliminate ANFs, is exactly ANFs in the feed as cellulose, hemicellulose etc.Livestock and poultry its childhood because of its digestive ferment system and enzyme quantity not sufficient, relatively poor to the feed protein digestion power, easily cause diseases such as indigestion, diarrhoea, in feed, add the digestibility that protease (exogenous digestive ferment) helps improving protein in the feed.In addition, add the cell membrane that cellulase, zytase etc. can decompose plant in feed, the stripping quantity of the material that has additional nutrients is eliminated effects such as ANFs.
Complex enzyme for feed by microbial fermentation production is nontoxic, harmless, the pollution-free residual green feed additive of a class, adds an amount of enzyme preparation that is complementary with feed diet and can obviously improve efficiency of feed utilization in feed.
The key technology of microbial fermentation production enzyme preparation is mainly by two aspects: be how to screen the bacterial classification that obtains the high yield enzyme on the one hand; Be how to select suitable condition of culture to make the best production performance of bacterial classification performance on the other hand.There is more report to utilize processing of farm products accessory substances such as bagasse, corn stalk, potato slag to come the production enzyme preparation abroad.
Summary of the invention
The purpose of this invention is to provide a kind of is the method that raw material by solid fermentation is produced complex enzyme for feed with the oranges and tangerines skin, be to be the main medium composition based on oranges and tangerines skin (perhaps oranges and tangerines process residues), with seed selection complex enzyme for feed superior strain is the breach, solid state fermentation is produced, can directly make an addition in the feed, eliminate the ANFs in the feed, improve efficiency of feed utilization.
The technical solution used in the present invention is:
The oranges and tangerines skin is mixed with dregs of beans, wheat bran, add small amounts of inorganic salt and form the solid fermentation culture medium, add the water mixing,, after the cooling, insert the Aspergillus usamii bacterial classification,, promptly obtain the product that enzyme of several kinds of enzyme and with high is lived after the drying through a solid fermentation through sterilization.
Described solid fermentation medium preparation method is: dry citrus peel meal 55～75%, wheat bran 0～15%, bean cake powder 20～35%, urea 1～2%, manganese sulfate 2～3%, sodium hydrogen phosphate 2～3%, material-water ratio is 1: 1.0～1.3,121 ℃ sterilization 30min, cools off standby.
The preparation method of the spore suspension of described Aspergillus usamii bacterial classification is: Aspergillus usamii is inoculated in the potato dextrose agar, cultivates 84-120h down at 27～30 ℃, sterilized water washes, and makes 10
6～10
7The spore suspension of individual/mL, 3～5% the spore suspension inoculation with the seed culture medium quality stirs, and cultivates about 60-84h down in 27～30 ℃, then, washes spore with sterilized water, makes spore suspension, and the adjusting spore concentration is 10
6～10
7Individual/mL.
The preparation method of described seed culture medium is: according to dry citrus peel meal 65～75%, bean cake powder 20～30%, urea 1～2%, ammonium sulfate 2～3%, material-water ratio are 1: 1.0～1.3,121 ℃ sterilization 30min, cool off standby.
Described solid fermentation process is: adopt bent room multi-layer solid fermentation mode or the production of solid fermentation machine, spore suspension is sprayed in the solid fermentation culture medium in 4%～5% ratio, stir, in temperature is that 27～30 ℃ and relative humidity are to cultivate 60-72h under 85%～90% the condition, then under 45～50 ℃, low temperature air flow drying to moisture is 8～10%, pulverizes promptly to obtain complex enzyme preparation for feeding of the present invention.
The enzyme that described complex enzyme product comprises has: zytase, acid protease, pectase and cellulase etc.
The useful effect that the present invention has is:
1. a large amount of bacterial strains being screened obtains utilizing the oranges and tangerines skin to produce the Aspergillus usamii of high enzyme complex enzyme alive;
2. be primary raw material with oranges and tangerines process residues such as oranges and tangerines skins, add a spot of dregs of beans, wheat bran and inorganic salts, its raw material sources are wide, and are cheap, and production cost is low;
3. applying solid fermentation process, available bent room multilayer batch fermentation method is produced, or adopts the production of simple and easy fermentation machine, and cultivation cycle is short, has small investment, and operation is simple and feasible, tallies with the national condition;
4. one time fermentation can obtain multienzyme kind, high enzyme leaven material alive, through low temperature air flow drying, pulverizing, can prepare the complex enzyme feed additive that can directly add.Easy and simple to handle, promote easily;
5. the complete and enzyme of product enzyme system is lived highly, and as the green feed additive of the cultivated animals of poultry, fowl and aquatic products, the scope of application is wide;
6. do not produce waste water and dregs in producing, can not cause secondary environmental pollution.
The specific embodiment
One, strain preparation
Bacterial classification is that Zhejiang Microbe Inst. buys Aspergillus usamii (Aspergillus usaanii).Strain preparation is divided into the I and II strain preparation.
First class inoculum: culture medium is fresh potato-glucose-agar (PDA) inclined-plane, the inoculation of Aspergillus usamii preservation strain method of scoring, cultivate 84-120h down for 27～30 ℃, wash spore with sterilized water, be transferred to no cingula bead triangular flask, spore is broken up in vibration, makes spore suspension, regulates spore concentration 10
6～10
7Between individual/mL, stand-by.
Second class inoculum: according to dry citrus peel meal 65～75%, bean cake powder 20～30%, urea 1～2%, ammonium sulfate 2～3%, the bottled 20g siccative of each 500ml triangle, material-water ratio is 1: 1.0～1.3,121 ℃ of 30min that sterilize down, cooling inserts the first order seed spore suspension, each triangle bottle graft 2ml stirs, and cultivates about 60-84h down in 27～30 ℃, then, wash spore with sterilized water, make spore suspension, regulating spore concentration is 10
6～10
7Individual/mL.
Two, fermenting and producing
1. fermentation mode can adopt bent room multilayer fermentation method or the production of solid fermentation machine, and material thickness is 3～5cm, keeps well ventilation, temperature control and control wet.
2. solid fermentation culture medium preparation culture medium siccative component is: dry citrus peel meal 55～75%, and wheat bran 0～15%, bean cake powder 20～35%, urea 1～2%, manganese sulfate 2～3%, sodium hydrogen phosphate 2～3%, material-water ratio are 1: 1.0～1.3; Concrete operation method is: earlier citrus peel meal, bean cake powder and wheat bran are mixed in proportion, urea and sodium hydrogen phosphate are dissolved in the corresponding running water again, evenly spray in the material 121 ℃ of autoclaving 30min.
3. fermented and cultured evenly is sprayed to solid fermentation with the spore suspension of second class inoculum by 4～5% inoculum concentration and produces the enzyme culture medium, ferment after stirring, fermentation temperature is controlled at 27～30 ℃, and relative humidity is controlled at 85%～90%, and fermentation time is 60～72h.
4. drying will have been fermented solid in 45～50 ℃ of following low temperature air flow dryings, pulverize, and be packaged to be enzyme preparation.
Embodiment 1
The Aspergillus usamii slant strains of low-temperature preservation is transferred on the fresh PDA inclined-plane, place constant incubator, temperature is controlled at 28 ℃, cultivated through 84 hours, after treating that mycelia is covered with the inclined-plane and covers with the grey black spore, add an amount of sterilized water and wash spore, make spore suspension, regulate spore concentration to 10
7Individual/ml.In the 500mL triangular flask, the 40g oranges and tangerines skin solid fermentation culture medium of packing into, comprising: citrus peel meal 15g, dregs of beans 5g, urea 0.2g, ammonium sulfate 0.4g, water 20ml, 121 ℃ of 20min that sterilize down, the 2mL spore suspension is inserted in the cooling back, places 28 ℃ of biochemical incubator fermented and cultured, after process 72h treats that the fermentation solid surface is covered with the grey black spore, add sterilized water, wash spore, regulating spore concentration is 10
7Individual/mL, standby.Above-mentioned cultured spore suspension 4L evenly is sprayed in the multilayer koji tray that 100 kilograms of fermentation mediums are housed, every layer of material thickness is 3～4cm, 100 kilograms of fermentation mediums comprise: 30 kilograms of dry citrus peel meals, 10 kilograms of dregs of beans, 1 kilogram of urea, 2 kilograms of manganese sulfates, 2 kilograms of sodium hydrogen phosphates, 55 kilograms of running water, 121 ℃ of sterilization 30min, under 27～28 ℃, humidity is to cultivate 24 hours under 90% condition,, turn over song for the first time, continuation is at 27～28 ℃, humidity is to cultivate 24 hours under 85% the condition, turn over for the second time song, after continuing to cultivate 18 hours under the same conditions, the yellow mycelia on the culture medium changes into the grey black spore, then it is sent into pneumatic drier, being dried to water content under 50 ℃ is 8～9%, is ground into 40 order powders in the input pulverizer, and 100 bags in 1 kilogram of complex enzyme for feed goods of the present invention are dressed up with double-layer plastic bag vacuum packet in the qualified back of quality inspection.
After measured, zytase is that 1973U/g, beta-glucosidase are that 87U/g, acid protease are that 7290U/g, pectase 1573U/g and CMC enzyme are that 23.6U/g, filter paper enzyme activity are 11.3U/g etc.
Embodiment 2
The Aspergillus usamii slant strains of low-temperature preservation is transferred on the fresh PDA inclined-plane, place constant incubator, temperature is controlled at 28 ℃, cultivated through 84 hours, after treating that mycelia is covered with the inclined-plane and covers with the grey black spore, add an amount of sterilized water and wash spore, make spore suspension, regulate spore concentration to 10
7Individual/ml.In each 500mL triangular flask, the 40g oranges and tangerines skin solid fermentation culture medium of packing into, comprising: citrus peel meal 16g, dregs of beans 4g, urea 0.3g, ammonium sulfate 0.3g, water 20ml, 121 ℃ of 20min that sterilize down, the 2mL spore suspension is inserted in the cooling back, place 28 ℃ of biochemical incubator fermented and cultured, cultivate through 84h, treat that the fermentation solid surface is covered with the grey black spore after, add sterilized water, wash spore, regulate spore concentration and be 107/mL, standby.
Above-mentioned cultured spore suspension 5L evenly is sprayed in the multilayer koji tray that 100 kilograms of fermentation mediums are housed, every layer of material thickness is 4～5cm, 100 kilograms of solid fermentation culture mediums comprise: 25 kilograms of dry citrus peel meals, 5 kilograms of wheat brans, 12 kilograms of dregs of beans, 1.5 kilogram urea, 2.5 kilogram manganese sulfate, 2 kilograms of sodium hydrogen phosphates, 52 kilograms of running water, 121 ℃ of sterilization 30min are at 29～30 ℃, humidity is to cultivate 24 hours under 90% condition, turns over song for the first time, continuation is at 29～30 ℃, humidity is to cultivate 24 hours under 85% condition, turn over for the second time song, after continuing to cultivate 12 hours under the same conditions, the yellow mycelia on the culture medium changes into the grey black spore, then it is sent into pneumatic drier, being dried to water content under 45 ℃ is 9～10%, is ground into 40 order powders in the input pulverizer, and 100 bags in 1 kilogram of complex enzyme for feed goods of the present invention are dressed up with double-layer plastic bag vacuum packet in the qualified back of quality inspection.
After measured, zytase is that 1873U/g, beta-glucosidase are that 104U/g, acid protease are that 6290U/g, pectase 1453U/g and CMC enzyme are that 24.5U/g, filter paper enzyme activity are 9.7U/g etc.
Embodiment 3
The Aspergillus usamii slant strains of low-temperature preservation is transferred on the fresh PDA inclined-plane, place constant incubator, temperature is controlled at 28 ℃, cultivated through 72 hours, after treating that mycelia is covered with the inclined-plane and covers with the grey black spore, add an amount of sterilized water and wash spore, make spore suspension, regulate spore concentration to 10
7Individual/ml.
In the 500mL triangular flask, the 45g oranges and tangerines skin solid fermentation culture medium of packing into, comprising: citrus peel meal 16g, dregs of beans 4g, urea 0.3g, ammonium sulfate 0.3g, water 25ml, 121 ℃ of 20min that sterilize down, the 2mL spore suspension is inserted in the cooling back, place 28 ℃ of biochemical incubator fermented and cultured, after process 72h treats that the fermentation solid surface is covered with the grey black spore, add sterilized water, wash spore, regulate spore concentration and be 107/mL, standby.
Above-mentioned cultured spore suspension 5L evenly is sprayed in the multilayer koji tray that 100 kilograms of fermentation mediums are housed, every layer of material thickness is 3～4cm, 100 kilograms of fermentation mediums comprise: 24 kilograms of dry citrus peel meals, 8 kilograms of wheat brans, 8 kilograms of dregs of beans, 2 kilograms of urea, 1.5 kilogram manganese sulfate, 1.5 kilogram sodium hydrogen phosphate, 55 kilograms of running water, 121 ℃ of sterilization 30min are under 27～28 ℃, humidity is to cultivate 24 hours under 90% condition, turn over for the first time song, continue at 27～28 ℃, humidity is to cultivate 24 hours under 85% the condition, turns over song for the second time, after continuing to cultivate 18 hours under the same conditions, yellow mycelia on the culture medium changes into the grey black spore, then it is sent into pneumatic drier, and being dried to water content under 50 ℃ is 8～9%, be ground into 40 order powders in the input pulverizer, 100 bags in 1 kilogram of complex enzyme for feed goods of the present invention are dressed up with double-layer plastic bag vacuum packet in the qualified back of quality inspection.
After measured, zytase is that 1873U/g, beta-glucosidase are that 104U/g, acid protease are that 6890U/g, pectase 1486U/g and CMC enzyme are that 25.6U/g, filter paper enzyme activity are 8.3U/g etc.
Attached enzyme extracts and activity determination method is:
The 0.1M, the pH5.0 acetic acid-sodium-acetate buffer that add 10 times of fermentation solid quality in triangular flask intermittently stir 2h, filter paper filter crude enzyme liquid, make enzymatic determination with the cushioning liquid dilution and use.
The determination of activity of zytase:
Get the suitably crude enzyme liquid of dilution of 0.2mL, be added to 1% the xylan solution of 1mL with the acetic acid of 0.2M, Ph4.8-sodium-acetate buffer preparation, 50 ℃ of enzymolysis 30 minutes, add 2mL with distilled water, add DNS reagent 2mL, in boiling water, boiled 15 minutes, after the cooling, moisturizing is measured the reduced sugar amount that forms to 10mL at the 550nm place, calculates enzyme and lives.
The CMC enzyme testing method:
What add 1mL earlier is 1% CMC-Na solution of 4.8 acetic acid-sodium acetate buffer solution preparation with the pH value, adds 0.5mL dilution enzyme liquid again, at 50 ℃ of following isothermal reaction 30min, add 1.5mLDNS reagent then, boiling water bath reaction 5min, cooling, be settled to 25mL, under 520nm, measure light absorption value.
The beta-glucosidase enzymatic determination: the CMC-Na solution in the CMC method is replaced with 1% salicin solution, all the other with.
FPA (filter paper enzyme activity mensuration):
Add one of the filter paper bar (roll and put into) of 6cm * 1cm in each test tube, respectively add 1mL acetic acid-sodium acetate buffer solution again, 50 ℃ of reaction 1h add DNS reagent 1.5mL then, boiling water bath reaction 5min, and cooling is settled to 25mL, measures OD
520
Acid protease activity is measured (forint phenol method):
Crude enzyme liquid dilution 1mL, preheating 2min in 40 ℃ of water-baths, 2% of the same preheating of adding warp casein solution (with lactic acid-sodium lactate buffer preparation of pH2.5) 1mL accurately is incubated 10min, after the time arrives again, add 0.4mol trichloroacetic acid 2mL immediately again, with cessation reaction, continue to be incubated 20min in the water-bath, make the centrifugal or filtration of residual protein post precipitation, get filtrate 1mL, add 0.4molNa again
2CO
3The Folin reagent 1mL of 5mL and dilution shakes up, and 40 ℃ of insulation color development 20min measure OD
660
Blank: basic step is the same, adds 0.4mol trichloroacetic acid 2mL in preheating enzyme liquid 1mL, adds 2% casein 1mL of preheating behind the effect 10min again, filters, and filtrate is handled same sample.
The pectase assay method:
Get the suitable enzyme liquid of 2ml dilution and 2ml and mix, spend in the waters bath with thermostatic control 45 and react 30min, add 1.5mlDNS reagent then, in boiling water, boiled 5 minutes, be settled to 25ml, mensuration OD with 1% pectin solution of acetic acid-sodium-acetate buffer preparation
520
The expression of enzyme activity
The xylanase activity definition: at Ph4.8, under 50 ℃ of conditions, the per minute hydrolyzed xylan produces the enzyme amount of 1 μ mol wood sugar as a unit (is standard with the wood sugar).
The pectinase activity international unit is defined as: at pH4.8, under 45 ℃ of conditions, per hour the required enzyme amount of hydrolysis of pectin matter generation 1mg galacturonic acid is an enzyme unit alive.
The CMCase enzyme is lived and defined: at Ph4.8, under 50 ℃ of conditions, it is enzyme unit alive that per minute hydrolyzed carboxymethylcellulo, e sodium (CMC) produces the required enzyme amount of 1 μ mol glucose.
The definition of beta-glucosidase enzyme activity: with 1% salicin is substrate, and under reaction condition, the enzyme amount that forms 1 μ mol glucose with per minute catalysis salicin in the hydrolysis is an enzyme unit (U) alive.
The filter paper enzyme activity definition: with filter paper is substrate, and under reaction condition, the enzyme amount that forms 1 μ mol glucose with per minute catalyzing cellulose hydrolysis in the hydrolysis is an enzyme unit (U) alive.
The acid protease enzyme is lived and is defined: produce 1 μ g tyrosine at 40 ℃ of following per minute caseinhydrolysates, be defined as 1 protease activity unit of force.
Above enzyme is lived and is all represented with amount of dry matter, and the extract enzyme work that records is got through conversion.
Claims (6)
1. one kind is the method that raw material by solid fermentation is produced complex enzyme for feed with the oranges and tangerines skin, it is characterized in that the oranges and tangerines skin is mixed with dregs of beans, wheat bran, add small amounts of inorganic salt and make the solid fermentation culture medium, add the water mixing, through sterilization, after the cooling, insert the Aspergillus usamii bacterial classification, through a solid fermentation, promptly obtain the product that enzyme of several kinds of enzyme and with high is lived after the drying.
2. according to claim 1 is the method that raw material by solid fermentation is produced complex enzyme for feed with the oranges and tangerines skin, it is characterized in that: the preparation method of described solid fermentation culture medium is: dry citrus peel meal 55～75%, wheat bran 0～15%, bean cake powder 20～35%, urea 1～2%, sodium hydrogen phosphate 3～4%, material-water ratio are 1: 1.0～1.3,121 ℃ of sterilization 30min cool off standby.
3. according to claim 1 is the method that raw material by solid fermentation is produced complex enzyme for feed with the oranges and tangerines skin, it is characterized in that: the preparation method of the spore suspension of described Aspergillus usamii bacterial classification is: Aspergillus usamii is inoculated in the potato dextrose agar, cultivate 84-120h down at 27～30 ℃, make 10 with aseptic
6～10
7The spore suspension of individual/mL, this spore suspension are first class inoculum, and the spore suspension with 3～5% is inoculated in seed culture medium, stir, under 27～30 ℃, cultivate about 60-84h, then, wash spore with sterilized water, make spore suspension, regulating spore concentration is 10
6～10
7Individual/mL, this spore suspension is a second class inoculum.
4. according to claim 3 is the method that raw material by solid fermentation is produced complex enzyme for feed with the oranges and tangerines skin, it is characterized in that: the preparation method of described seed culture medium is: according to dry citrus peel meal 65～75%, bean cake powder 20～30%, urea 1～2%, ammonium sulfate 2～3%, material-water ratio is 1: 1.0～1.3,121 ℃ sterilization 30min, cools off standby.
5. according to claim 1 is the method that raw material by solid fermentation is produced complex enzyme for feed with the oranges and tangerines skin, it is characterized in that: described solid fermentation process is: adopt bent room multi-layer solid fermentation mode or the production of solid fermentation machine, spore suspension is sprayed in the solid fermentation culture medium in 4%～5% ratio, stir, in temperature is that 27～30 ℃ and relative humidity are to cultivate 60-72h under 85%～90% the condition, then under 45～50 ℃, low temperature air flow drying to moisture is 8～10%, pulverizes promptly to obtain complex enzyme preparation for feeding of the present invention.
6. according to claim 1 is the method that raw material by solid fermentation is produced complex enzyme for feed with the oranges and tangerines skin, and it is characterized in that: the enzyme that described complex enzyme product comprises has: zytase, acid protease, pectase and cellulase etc.
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| CNA2005100502865A CN1685924A (en) | 2005-04-18 | 2005-04-18 | Method of producing feed composite enzyme using orange peel as raw material by solid fermentation |
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|---|---|---|---|
| CNA2005100502865A CN1685924A (en) | 2005-04-18 | 2005-04-18 | Method of producing feed composite enzyme using orange peel as raw material by solid fermentation |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102726597A (en) * | 2012-06-22 | 2012-10-17 | 湖南省天金科技有限公司 | Method for preparing feed additive by using citrus waste residues |
| CN104054907A (en) * | 2014-06-10 | 2014-09-24 | 湖南省农产品加工研究所 | Preparation method of protein feed |
| CN105053537A (en) * | 2015-08-10 | 2015-11-18 | 中国科学院重庆绿色智能技术研究院 | High protein feed producing method using orange peel residues as raw materials and feed |
| CN107094993A (en) * | 2017-05-11 | 2017-08-29 | 李平作 | A kind of manufacture method of feature glossy ganoderma fermentation feed |
| CN109770056A (en) * | 2018-11-29 | 2019-05-21 | 北京生泰尔科技股份有限公司 | A kind of Chinese medicine residue degradation bacteria and its preparing the application in poultry fodder additive |
-
2005
- 2005-04-18 CN CNA2005100502865A patent/CN1685924A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102726597A (en) * | 2012-06-22 | 2012-10-17 | 湖南省天金科技有限公司 | Method for preparing feed additive by using citrus waste residues |
| CN104054907A (en) * | 2014-06-10 | 2014-09-24 | 湖南省农产品加工研究所 | Preparation method of protein feed |
| CN105053537A (en) * | 2015-08-10 | 2015-11-18 | 中国科学院重庆绿色智能技术研究院 | High protein feed producing method using orange peel residues as raw materials and feed |
| CN105053537B (en) * | 2015-08-10 | 2019-01-01 | 中国科学院重庆绿色智能技术研究院 | It is a kind of using orange peel slag as the production method of the high protein feed of raw material and feed |
| CN107094993A (en) * | 2017-05-11 | 2017-08-29 | 李平作 | A kind of manufacture method of feature glossy ganoderma fermentation feed |
| CN109770056A (en) * | 2018-11-29 | 2019-05-21 | 北京生泰尔科技股份有限公司 | A kind of Chinese medicine residue degradation bacteria and its preparing the application in poultry fodder additive |
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