CN103444984A

CN103444984A - Lysine and xylanase fermented and coated feed

Info

Publication number: CN103444984A
Application number: CN2013104037246A
Authority: CN
Inventors: 马吉银; 陈崇安; 孟刚; 曹洪; 程耀东; 刘鑫
Original assignee: NINGXIA EPPEN BIOTECH CO Ltd
Current assignee: NINGXIA EPPEN BIOTECH CO Ltd
Priority date: 2012-06-07
Filing date: 2012-06-07
Publication date: 2013-12-18
Anticipated expiration: 2032-06-07
Also published as: CN103444984B; CN102763771A; CN102763771B

Abstract

The invention relates to lysine and xylanase fermented and a coated feed, and provides a method for preparing an L-lysine product by fermentation. The method comprises the steps: fermenting to produce an L-lysine fermented liquor, mixing the L-lysine fermented liquor with high-temperature-resisting dewatered xylanase obtained through fermentation, and coating by using fat after high temperature drying. In addition, the invention also provides a product produced by using the method, the high-temperature-resisting dewatered xylanase and the like.

Description

The fermentation of lysine, zytase and coated feed

Technical field

The invention belongs to amino acid whose fermentation field, particularly, the present invention relates to the method that fermentation prepares the 1B goods, it comprises that fermentation produces the 1B zymotic fluid, this zymotic fluid can mix with the zytase of the high temperature resistant dehydration that obtains of can fermenting, and after high temperature drying, with fat, is coated with.In addition, the present invention also provides the product of described method production and the zytase of high temperature resistant dehydration etc.

Background technology

Zytase can effectively be degraded containing the material of lignin (as, feed), therefore in feed, adds the absorption rate that zytase can strengthen feed, thereby saves feeding cost.For example, Chinese patent 96196760.9 discloses the enzyme additive of ruminant feed, and it comprises zytase.

1B is important amino acid starting material, can be used as flavouring, food, feed addictive use, also can be used as the effective or adjunct ingredient in health products, medicine, is widely used in grocery trade, feed industry, pharmacy industry and other chemical industry.Current, the production of 1B is mainly by the fermenting and producing of microorganism, as utilized corynebacteria production.

Yet, when lysine, as feed the time, because it belongs to little molecular nutrition material, can be discharged into soon in animal body, especially, when huge uptake, affected the efficiency that animal absorbs.Usually the method solved is feeding animals in multiple times on a small quantity, but this will increase the workload of animal feeding.Therefore, the present invention proposes the direct method for preparing coated lysine goods with the fermenting lysine nutrient solution, can effectively slow down the speed that lysine discharges in animal body.

According to conventional thinking, coated lysine goods can with containing the enzyme additive of zytase in feeding while raising and being mixed together animal feed.Cumbersome in using like this, and easily mix inhomogeneously, corresponding utilization rate is low.

Inventor's imagination, in preparation, in the process of coated lysine goods, zytase and/or cellulase are directly mixed with the fermenting lysine nutrient solution, can be mixedly very even, then be coated with, yet but find to be limited by being coated with high temperature and the dry run that will experience, conventional enzymatic activity will greatly weaken, and even completely lose.Unexpectedly, the inventor has found the heat-resisting and drought-enduring fungal strain of a strain, through arduous intensive research, still can keep high xylanase activity after the zytase experience high temperature drying of wherein separating, therefore be suitable for using easily therein, reduced and fed the trouble of using while raising.

Summary of the invention

The technical problem to be solved in the present invention is that the fermentation that provides new prepares the method for 1B goods, it comprises that fermentation produces the 1B zymotic fluid, this zymotic fluid can mix with the zytase of the high temperature resistant dehydration that obtains of can fermenting, and after high temperature drying, with fat, is coated with.In addition, the present invention also provides product that described method produces and material used etc. wherein.

Particularly, in first aspect, the invention provides the method that fermentation prepares the 1B goods, comprise, fermentation produces the 1B zymotic fluid, and this zymotic fluid can mix with the zytase of high temperature resistant dehydration, and the particles with fat of collecting after high temperature drying is coated.Therefore, the 1B goods of the method for first aspect present invention fermentation preparation divide two-layer, outer wrapping inner layer, and its ectomesoderm is fat, and the zytase that internal layer contains 1B and high temperature resistant dehydration.These 1B goods can slow down the speed that lysine discharges in animal body, and can have xylanase activity.These goods are feed addictive preferably.

More specifically, in first aspect, the invention provides the method that fermentation prepares the 1B goods, it comprises:

(1) under fermentation conditions cultivate the bacterium of the product 1B of the polynucleotides that imported the coding pyridine nucleotide transhydrogenase, obtain zymotic fluid, after filtering, retain filtrate;

(2) under fermentation conditions cultivate the secreting, expressing bacterium of the polynucleotides of the zytase that has imported the high temperature resistant dehydration of encoding, after filtering, retain supernatant;

(3) supernatant that the filtrate that blend step (1) obtains and step (2) obtain, obtain mixed liquor;

(4) mixed liquor high temperature spray-drying step (3) obtained, and optionally sieved and/or fragmentation collecting granules; With

(5) particles with fat of step (4) being collected is coated.

The coated particle of fat can be sprayed on the fat of fusing on particle surface and realize by the fluidized granulation seed-coating machine, thus preferred fat be under room temperature state for solid-state fusing point a little more than room temperature (as, 45-80 ℃, preferably 50-65 ℃) fat.Preferably, in the method for first aspect present invention, fat is fractionation palm fat preferably, is more preferably hundred Canons (BergaFat), and it can obtain easily by commercial sources.In the specific embodiment of the present invention, coated fat used is hundred HTL316 of Canon.

Inventor's discovery, fat deposit coated (spraying) must be thicker, and the anti-degradation capability of the 1B goods that prepare is stronger.Preferably, in the method for first aspect present invention, in step (5), the particle that step (4) obtains and fatty weight ratio are 2 ~ 8:8 ~ 2, are preferably 3 ~ 7:7 ~ 3, more preferably 5 ~ 6.5:5 ~ 3.5.

In step (4), the particle diameter skewness for wherein drying obtains, can be sieved, and collects the particle of certain particle size range; And/or, for the larger particle of particle diameter dry and/or that screening obtains, can be pulverized, then proceed screening, collect the particle of certain particle size range.Preferably, in the method for first aspect present invention, the particle diameter of the particle of collection is less than 0.8mm, preferably is less than 0.5mm.Be greater than the particle of 0.5mm.Preferred in the method for first aspect present invention in addition, high temperature spray-drying can carry out in fluid bed dryer.For the shape that makes final particle is more even, fluid bed dryer inner exhaust gas temperature is unsuitable too high, usually not higher than 100 ℃, preferably, not higher than 85 ℃, more preferably remains on 80 +3 ℃.Therefore, preferably, in the method for first aspect present invention, in step (4), high temperature is 60-100 ℃, is preferably 65-90 ℃, more preferably 75-85 ℃.

Lysine is as micromolecular compound, and the high temperature dehydration drying can not produce too much adverse effect to it; And zytase through high temperature and/or dehydration, likely makes its degraded and/or sex change as macro-molecular protein, thereby cause remarkable reduction or the forfeiture of its enzymatic activity, can't play respective action.Therefore the present invention can adopt the zytase of high temperature resistant dehydration, and wherein high temperature is 60-100 ℃, is preferably 65-90 ℃, more preferably 75-85 ℃.

Preferably when using as feed addictive, because the requirement of zytase is less than lysine, therefore in the 1B goods of the method for first aspect present invention fermentation preparation, preferably lysine content is high, and zytase content is low simultaneously, can be low to moderate the activity that meets the required zytase of feed.Preferably, in the method for first aspect present invention, the volume ratio of the supernatant that the filtrate that step (1) obtains and step (2) obtain is 10:0.01-10, is preferably 10:0.05-5, more preferably 10:0.1 ~ 1.

Polynucleotides can be imported in bacterium by various modes well-known to those skilled in the art, make this bacterium express the enzyme of described polynucleotide encoding, as pyridine nucleotide transhydrogenase and/or zytase.Described polynucleotides can directly be imported into, such as utilizing the transfered cells such as microsome, particle gun; Also can indirectly be imported into, for example can be by being structured in transfered cell on plasmid vector.The described polynucleotides that import can be incorporated on the genome of cell and express, and expression also can dissociate.Preferably in the method for first aspect present invention, the polynucleotides of codase can utilize shuttle plasmid to import in bacterium, as the polynucleotides of the pyridine nucleotide transhydrogenase of encoding can utilize Escherichia coli-corynebacteria shuttle plasmid to import in the bacterium (as corynebacteria) that produces 1B, the polynucleotides of the zytase of and for example encoding can utilize E. coli-Yeast bacterium shuttle plasmid to import in secreting, expressing bacterium (as pichia yeast).

The fermentation of 1B has many prior aries to select for those skilled in the art, for example can utilize wild type pyridine nucleotide transhydrogenase or its variant.The wild type pyridine nucleotide transhydrogenase is that those skilled in the art know, it comprises α subunit and β subunit, and wherein the sequence of α subunit and β subunit is respectively as NCBI(http: //www.ncbi.nlm.nih.gov) as shown in albumen and gene accession number NP416120.1 and AAC74674.1.Preferred variant can be the disclosed pyridine nucleotide transhydrogenase variant of Chinese patent 201110065683.5, increased the output (also can referring to Chinese patent 201110151495.4) of lysine in actual production, it comprises α subunit variant and β subunit variant, wherein α subunit variant is replaced by other natural amino acids on the position of M102, L127 or Q322 with respect to wild type α subunit, and preferably replacing is M102L, L127R and Q322K; β subunit variant is replaced by other natural amino acids on the position of A398 or D400 with respect to the wild-type beta subunit, and preferably replacing is A398S and D400H.In the specific embodiment of the present invention, adopt the pyridine nucleotide transhydrogenase variant, it is nucleotide sequence coded by as shown in SEQ ID No:5.

In the method for first aspect present invention, the zytase that the zytase of the high temperature resistant dehydration bacterial strain that preferably inventor finds unexpectedly produces, this zytase is very low with existing known zytase homology, and there is the advantage of high temperature resistant dehydration, even verified by after the high temperature evaporation drying, still can retain the enzymatic activity more than 80%, thereby make the method for first aspect present invention can be able to smooth enforcement.In the method for preferred first aspect present invention, the amino acid sequence of the zytase of high temperature resistant dehydration:

(1) as shown in SEQ ID No:2; Or

(2) be to SEQ ID No:2 disappearance, add and/or replace one or several (preferably be no more than 7, more preferably no more than 5, as be no more than 3) amino acid residue and the sequence of the reservation SEQ ID No:2 activity that obtains.Those skilled in the art, by recombinant DNA technology, can prepare the misfolded proteins obtained through disappearance, interpolation and/or substituted amino acid residue.In the specific embodiment of the present invention, zytase is nucleotide sequence coded by as shown in SEQ ID No:1.

In step (2), remove by filter thalline and retain supernatant, wherein preferably use 0.22 μ m filter membrane to be filtered.Supernatant can be purified and/or be concentrated, also can be directly used in the method for first aspect present invention, preferably the latter.

Preferably, in the method for first aspect present invention, in step (2), the xylosidase enzymatic activity of supernatant is not less than 1000IU/L, preferably is not less than 5000IU/L, more preferably is not less than 10000IU/L, for example is not less than 15000IU/L.The zytase of high temperature resistant dehydration of the present invention, through the high temperature dehydration drying, still can retain original enzyme activity substantially.The method of under fermentation conditions cultivating in preferred step (2) comprises:

(i) by the bacterium of the zytase of the high temperature resistant dehydration of secreting, expressing (as, saccharomycete) access fluid nutrient medium, be cultured to OD600 in 28-33 ℃ and reach 3.0-8.0;

The nutrient solution (ii) step (i) obtained is with the 3-7%(volume) inoculum concentration be inoculated in fluid nutrient medium, cultivate 20-28 hour in 28-33 ℃;

(iii) by methyl alcohol with the 0.3-0.7%(volume) the access amount add step (ii) to obtain nutrient solution in, induce and cultivate 6-12 days, within every 24 hours during this time, add the 0.3-0.7%(volume) methyl alcohol of access amount, ventilation is controlled dissolved oxygen and is greater than 20%, and to control pH be 5.8-6.4.

In this article, inoculum concentration or access measurer capable field technique personnel institute can conventional understanding implication, specifically, when meaning with percent by volume, refer to the percentage of other liquid volumes of bacterial culture fluid (the bacterium liquid of access) or access with respect to the culture volume be access in.

Wherein, fluid nutrient medium is the Liquid Culture agent of the bacterium that can make the zytase of the high temperature resistant dehydration of secreting, expressing (as, saccharomycete) growth.Step (i) and the step fluid nutrient medium in (ii) can be identical, also can make difference, preferably identical, as be the BMGY fluid nutrient medium, its formula is in 100mM kaliumphosphate buffer (pH6.0), containing 1%(weight) yeast extract, 2%(weight) peptone, yeast nitrogenous base 1.34%(weight), biotin 4*10 ^-5%(weight), glucose 2%(weight), glycerine 1%(volume).

In step (1), remove by filter thalline and retain filtrate, wherein preferably use milipore filter to be filtered.Filtrate can be purified and/or is concentrated, also can be directly used in the method for first aspect present invention, preferably the latter.

Preferably, in the method for first aspect present invention, in step (1), in filtrate, the content of 1B is higher than 50g/L, preferably higher than 70g/L, more preferably higher than 90g/L.This can utilize many prior aries to realize, comprises the improvement of bacterial classification and/or the improvement of fermentation flow process.The method of under fermentation conditions cultivating in exemplary step (1) comprises:

(a) bacterium that will produce 1B is accessed the first fermentation tank in 31-38 ℃ of cultivation 10-20 hour;

(b) nutrient solution step (a) obtained is with the 3-7%(volume) inoculum concentration be inoculated in the second fermentation tank, cultivate 3-8 hour in 36-40 ℃;

(c), to the second fermentation tank Continuous-flow sugaring, wherein the dosage that per hour flows of sugar is to cultivate the 0.2-0.35%(weight of liquid measure in the second fermentation tank), carry out 10-18 hour;

(d) to the second sugaring of fermentation tank Continuous-flow and nitrogenous source, wherein the dosage that per hour flows of sugar is to cultivate the 0.3-0.5%(weight of liquid measure in the second fermentation tank), and the dosage that per hour flows of nitrogenous source is to cultivate the 0.1-0.25%(weight of liquid measure in the second fermentation tank), carry out 30-65 hour, preferably 50-55 hour.

In this article, " first " and " second ", when modifying fermentation tank, is the fermentation tank of modifying for district office, and the first fermentation tank and the second fermentation tank are different.Wherein, preferably the culture medium prescription in the first fermentation tank is: contain glucose 500-750 kilogram, cane molasses 50-300 kilogram, corn steep liquor 400-600 kilogram, KH in every 18 cubic metres of culture mediums ₂pO ₄the 30-80 kilogram, MgSO ₄7H ₂o 3-15 kilogram, FeSO ₄7H ₂o 0.1-1 kilogram, MnSO ₄7H ₂o 0.1-1 kilogram, biotin 5-30 gram, and folic acid 3-10 gram, and/or preferably the culture medium prescription of the second fermentation tank is: contain glucose 10000-15000 kilogram in every 315 cubic metres of culture mediums, cane molasses 50-3000 kilogram, corn steep liquor 10000-15000 kilogram, KH ₂pO ₄the 700-1200 kilogram, MgSO ₄7H ₂o 5-220 kilogram, FeSO ₄7H ₂o 5-25 kilogram, MnSO ₄7H ₂o 5-220 kilogram, biotin 150-300 gram, and folic acid 50-120 gram.

Step (c) and part condition of culture (d) (as, temperature, pH etc.) with step (b) can be identical, also can be different.In step (b), do not flowed add operation; And, in step (c) with (d), pH maintains between 6.5 to 7.8, this can add alkali by stream simply or acid realizes.

In this article, the stream dosage has the implication that those skilled in the art institute can conventional understanding, specifically the time as expressed in weight percent, refers to the percentage that the weight that adds material accounts for the weight that is added into material (as, nutrient solution).Preferred steps (c) and (d) in sugar be glucose.In step (c), the dosage that per hour flows of glucose is to cultivate the 0.2-0.35%(weight of liquid measure in the second fermentation tank), be preferably 0.27-0.33%(weight).In step (d), the dosage that per hour flows of glucose is to cultivate the 0.3-0.5%(weight of liquid measure in the second fermentation tank), be preferably 0.42-0.48%(weight).

Nitrogenous source in preferred steps (d) is inorganic nitrogen-sourced, and preferably sulfate of ammoniac or ammonium chloride, as sulfate of ammoniac.In step (d), the dosage that per hour flows of sulfate of ammoniac is to cultivate the 0.1-0.25%(weight of liquid measure in the second fermentation tank), be preferably 0.17-0.23%(weight).

In second aspect, the invention provides the 1B goods (as, feed addictive), it comprises the particle with the fat parcel, the zytase that wherein particle comprises 1B and high temperature resistant dehydration, the zytase of preferred high temperature resistant dehydration is the zytase that the bacterial strain found unexpectedly of the inventor produces.Preferably the goods in second aspect present invention are to be fermented and prepared by the method for first aspect present invention.

In the third aspect, the invention provides the zytase of high temperature resistant dehydration or its encoding gene, its carrier, bacterial classification.Particularly, the present invention:

Providing the zytase of high temperature resistant dehydration aspect I, its amino acid sequence:

(1) as shown in SEQ ID No:2; Or

(2) be to SEQ ID No:2 disappearance, add and/or replace one or several (preferably be no more than 7, more preferably no more than 5, as be no more than 3) amino acid residue and the sequence of the reservation SEQ ID No:2 activity that obtains.

The gene of the zytase of the I aspect of encoding is being provided aspect II, and preferably its nucleotide sequence is as shown in SEQ ID No:1.

The carrier of the gene that comprises the II aspect is being provided aspect III.

Bacterial classification is being provided aspect IV, secreting, expressing bacterium (as, yeast) especially, the gene that it comprises the II aspect, or transform or transfection with the carrier of III aspect.

In the method that the fermenting and producing zytase is provided aspect V, it comprises the bacterial classification of under fermentation conditions cultivating the IV aspect, isolates the zytase of I aspect from culture.

Purposes in the zytase that the I aspect is provided aspect VI is preparing feed or feed addictive.

The present invention has following beneficial effect: goods of the present invention are safe and reliable, can effectively reduce the rate of release of lysine in animal body, have xylanase activity, can further improve the utilization rate of feed; Zytase of the present invention has the advantage of high temperature resistant dehydration.

For the ease of understanding, below will to the present invention, be described in detail by specific embodiment.It needs to be noted, these descriptions are only exemplary descriptions, do not form limitation of the scope of the invention.According to the discussion of this specification, many variations of the present invention, change are all apparent concerning one of ordinary skill in the art.

In addition, the present invention has quoted open source literature, and these documents are in order more clearly to describe the present invention, and their full text content is all included this paper in and carried out reference, and the full text that just looks like them repeats in this article and clearly put down in writing the same.

The specific embodiment

Further illustrate by the following examples content of the present invention.As do not specialize, the conventional means that in embodiment, technological means used is well known to those skilled in the art and commercially available common instrument, reagent, can be referring to the references such as manufacturers instruction of " molecular cloning experiment guide (the 3rd edition) " (Science Press), " Microbiology Experiment (the 4th edition) " (Higher Education Publishing House) and corresponding instrument and reagent.

the preparation of the expression construct of the xylanase gene of the heat-resisting and anti-dehydration of embodiment 1

The bacterial strain of finding according to us, entrust Institute of Microorganism, Academia Sinica, carry out sequence analysis, found new xylanase gene, its nucleotide sequence is as shown in the SEQ ID No:1 of sequence table, and the amino acid sequence of coded zytase is as shown in SEQ ID No:2.Then, build the yeast of secreting, expressing according to the operating guidance of " molecular cloning experiment guide " and commercialization reagent used, in brief, the gene of this zytase of being about to encode (has been introduced EcoR I restriction enzyme site) with forward primer as shown in the SEQ ID No:3 of sequence table, use EcoR I and Not I double digestion after amplification that reverse primer (has been introduced Not I restriction enzyme site) as shown in the SEQ ID No:4 of sequence table, and be connected with the T4 DNA ligase with pPIC3.5 plasmid through these two endonuclease digestions (can purchased from Invitrogen company), be transformed in bacillus coli DH 5 alpha.Choose positive colony, extract plasmid wherein, after sequence verification is correct, positive plasmid, with after Sal I linearization for enzyme restriction, is transformed into to pichia yeast GS115 strain by electrotransformation, containing on the flat board of G418, choosing positive Yeast transformant, return.

fermentation preparation and the determination of activity of the zytase of the heat-resisting and anti-dehydration of embodiment 2

The positive Yeast transformant that embodiment 1 is obtained is inoculated into 50ml BMGY fluid nutrient medium, and (100mM kaliumphosphate buffer (pH6.0), wherein containing 1% yeast extract, 2% peptone, yeast nitrogenous base 1.34%, biotin 4*10 ^-5%, glucose 2%, glycerine 1%) in, be cultured to OD600 and reach 5.0 in 30 ℃, 200rpm.

Above-mentioned culture is transferred in 1L BMGY fluid nutrient medium, in 30 ℃, 200rpm, cultivate 24 hours, add 5ml methyl alcohol, then continuing at 30 ℃, 200rpm cultivates 8 days, within every 24 hours during this time, add 5ml methyl alcohol, ventilation is controlled dissolved oxygen (DO) and is greater than 20%, and control pH is that 6.0(regulates with ammoniacal liquor).After cultivation completes, with 0.22 μ m membrane filtration and retain supernatant, this supernatant detects and wherein is rich in the protein that zytase is corresponding through SDS-PAGE, and fermentation obtains the zytase supernatant thus.

Get fresh supernatant, be divided into three parts, a refrigeration stand-by (A), a freeze drying is again dissolved (B) with kaliumphosphate buffer (pH6.0) to solid, and portion evaporation drying in 80 ℃ of baking ovens is again dissolved (C) with kaliumphosphate buffer (pH6.0) to solid; Contrast adopts the commercially available zytase (can purchased from Jiangsu Province's paddy bio tech ltd difficult to understand) of 5000IU/g specification, be divided into three parts after dissolving with kaliumphosphate buffer (pH6.0), a refrigeration stand-by (CA), the freeze drying (CB) of another two parts of difference and 80 ℃ of baking oven evaporation dryings (CC), then heavily be dissolved in kaliumphosphate buffer (pH6.0).Measure respectively these xylanase activities, the enzyme that is converted into original unit's volume supernatant or Unit Weight is lived, the enzyme loss late alive that calculating causes due to high temperature dehydration, result is as shown in table 1, show almost to have lost with respect to commercially available prod the situation that enzyme is lived, the high temperature resistant dehydration of zytase of the present invention, even if through high temperature dehydration, still can retain the activity more than 80%, substantially retain protoenzyme function alive.

The high temperature dehydration enzyme of table 1 the present invention and the commercially available zytase loss late of living

Sample	Enzyme is lived	Loss late	Sample	Enzyme is lived	Loss late
						A	2.38*10 ⁴IU/L		CA	5.21*10 ³IU/g
B	2.20*10 ⁴IU/L	6.8%	CB	5.05*10 ³IU/g	3.1%
						C	1.96*10 ⁴IU/L	17.6%	CC	0.87*10 ³IU/g	83.3%

the fermenting and producing of embodiment 3 1Bs

As described in Chinese patent 201110065683.5, pyridine nucleotide transhydrogenase variant gene (its nucleotide sequence is as shown in the SEQ ID No:5 of sequence table) is cloned into to Escherichia coli-corynebacteria shuttle plasmid pMS2(according to a conventional method can be purchased from U.S.'s representative microbial preservation center (ATCC), goods number ATCC 67189) between EcoR I and Xba I restriction enzyme site, electricity is transformed into the corynebacteria engineering bacteria of 1B fermentation (can be purchased from U.S.'s representative microbial preservation center (ATCC), goods number ATCC 31269), in, obtain the corynebacteria engineering bacteria.

This corynebacteria engineering bacteria be take to 0.5% inoculum concentration access 20 cubic metres of fermentation tanks, and (culture medium prescription wherein is 600 kilograms of glucose, 200 kilograms of cane molasses, 520 kilograms of corn steep liquors, KH ₂pO ₄45 kilograms, MgSO ₄7H ₂7 kilograms of O, FeSO ₄7H ₂0.5 kilogram of O, MnSO ₄7H ₂0.5 kilogram of O, biotin 12 grams, and folic acid 5 grams, water is settled to 18 cubic metres), in 35 ℃ of saturated ventilations, cultivate 15 hours, improve cell density.

Then, (culture medium prescription wherein is: 12000 kilograms of glucose, 1000 kilograms of cane molasses, 12000 kilograms of corn steep liquors, KH the nutrient solution of 20 cubic metres of fermentation tanks directly to be injected to 350 cubic metres of fermentation tanks ₂pO ₄1000 kilograms, MgSO ₄7H ₂100 kilograms of O, FeSO ₄7H ₂12 kilograms of O, MnSO ₄7H ₂the O 120 kg, biotin 200 grams, and folic acid 85 grams, water is settled to 315 cubic metres), in 38 ℃ of saturated ventilations, cultivate 5 hours.Then, per hour stream adds 1000 kilograms of glucose, continues 15 hours, during exhaust steam to keep volume; Afterwards, per hour stream adds 1500 kilograms of glucose and 650 kilograms of ammonium sulfate, continuing fermentation 50 hours, during exhaust steam to keep volume.During stream adds, add NaOH and concentrated hydrochloric acid that pH is maintained between 6.5 to 7.8, add alkali during lower than lower bound, higher than the high acid adding of prescribing a time limit.Ferment complete, thin-layer chromatography detects and wherein produces 1B 97g/L, reaches the standard of commercial Application.

being coated with of embodiment 4 1B feeds

The zymotic fluid of embodiment 3 preparation is carried out to film separation, filtering solid insoluble by Suntar-III type milipore filter (can purchased from SanDa film Science Co., Ltd).Then, filtrate is mixed with the volume ratio of 10:0.5 with the zytase supernatant that embodiment 2 fermentations obtain, directly with vaporific, spray in fluid bed dryer and keep exhaust temperature 80 +3 ℃ constant, collects the particle of discharging.Particle to discharging is sieved, and the particle that is more than or equal to 0.5mm for particle diameter is sent into crusher in crushing, and the particle that the particle diameter then particle after fragmentation obtained with screening is less than 0.5mm mixes and again sprays in fluid bed dryer and keep exhaust temperature 80 +3 ℃ constant, collects the particle of discharging and sieve out the particle that particle diameter is less than 0.5mm, is the 1B feed granules agent with xylanase activity.The particle that particle diameter is greater than 0.5mm is again after fragmentation, and the particle of the discharging obtained with the filtrate drying of next batch mixes.

Get 370 HTL316(of gram hundred Canons (BergaFat) and can converge new peak herding Co., Ltd purchased from Nanning) in 60 ℃ of baking ovens, melt, then with the fluidized granulation seed-coating machine, it is all evenly sprayed on the surface of 1B feed granules of the above-mentioned preparation of 630 gram and forms coated layer, obtain the coated 1B feed addictive with xylanase activity.

the degraded test of the 1B feed addictive with xylanase activity that embodiment 5 is coated

According to Zhao He etc. (research of different proportion sheep's hay and ensilage dry matter degradability in cow rumen. Chinese Cattle industry science, 34 (2): Nylon Bag in the body of 13-16) describing, to coated feed addictive (the coated 1B feed granules agent with xylanase activity of take the is contrast) test of degrading of embodiment 4 preparation, using wherein 1B as testing index in adult dairy cattle.Degradation rate after impinging upon 2 hours is reached to (being 1B conservation rate less than 5%) more than 95%, and the conservation rate of coated feed addictive is as shown in the table:

The time of experience (hour)	Conservation rate (%)	The time of experience (hour)	Conservation rate (%)
				2	67.0	16	18.7
4	43.6	24	14.1
				6	30.5	36	12.9
8	25.2	48	11.6

Reach one week thereafter during animal experiment, animal subject, with respect to common feeding animals, is not observed bad reaction and is occurred.As can be seen here, coating technique of the present invention can the remaining time of significant prolongation 1B in the animal stomach, in 24 hours, can in the ox stomach, discharge more equably, and simultaneously can be so that feed addictive has xylanase activity, thereby significantly improved the utilization rate of feed, and animal has been had no adverse effect simultaneously.

<110 > Ningxia Yi Pin biotech inc

<120 > fermentation of lysine, zytase and coated feed

<130 > electronic application

<160> 5

<170> PatentIn version 3.3

<210> 1

<211> 1428

<212> DNA

<213 > mould

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accccgctgg aaaccagcct gccggtgtgg atgaccgaat gggcggatct gaacgataac 900

tataccgcgg cgtggtatga aaacggcgcg ccgggcgaag gcctgacctg ggcgaaccgc 960

attcaggatg cgtttacccg cagcaacgtg agcgcgtttc tgcattggat tggcgcggaa 1020

aacggcacca gcaacagccc gctgattaac ctgaacggcg atagctatgt ggcgagcaaa 1080

cgcctgtggg cgtttggcca gtttagccgc tttgtgcgcc cgggcgcggt gcgcattgat 1140

gcggcgagca gcgatccgct ggtgaccgtg agcgcgtttc agaacaaaga aaacggcgtg 1200

gtggcgaccc aggtgattaa caacgcggat accgattatg aagtggaagt ggatctgacc 1260

ggccgcggcc atctggtgag cgtggtgcag ccgtatctga ccaacaacga acatgatctg 1320

gaagcgagca gcccgattat taccctggcg ctgggcgcgg cgaccaaatt taccagcatt 1380

gtgccggcgc gcagcctggt gagctttgtg agcaaaaccg cgctgtaa 1428

<210> 2

<211> 475

<212> PRT

<213 > mould

<400> 2

Met Ile Ser Leu Ala Ala Ser Leu Pro Ile Pro Leu Val Ser Ala Leu

1 5 10 15

Pro His Ala His Ile Ser Val Asn Ala His Thr Arg Phe Gln Gln Val

20 25 30

Asp Gly Phe Gly Phe Ser Glu Ala Phe Gln Arg Ala Arg Asp Ile Tyr

35 40 45

Gly Lys Asp Gly Leu Ser Pro Glu Asp Arg Thr Arg Val Leu Asp Leu

50 55 60

Leu Phe Ser Asp Thr Arg Gly Ala Gly Leu Thr Ile Val Arg Asn Gly

65 70 75 80

Ile Gly Ser Asn Ser Thr Ile Lys Asp Phe Met Asn Ser Ile Glu Pro

85 90 95

Phe Ser Pro Gly Ser Pro Ser Ala Pro Pro His Tyr Val Trp Asp Arg

100 105 110

Asn Asp Ser Gly Gln Val Trp Leu Ser His Glu Ala Ala Ser Tyr Gly

115 120 125

Val Asn Thr Phe Tyr Ala Asp Ala Trp Ser Ala Pro Gly Tyr Met Lys

130 135 140

Thr Asn Asp Asp Asp Ser Asn Gly Gly Tyr Leu Cys Gly Val Ser Asn

145 150 155 160

Thr Ser Cys Ala Ser Gly Asp Trp Lys Gln Ala Tyr Ala Asn Tyr Leu

165 170 175

Val Gln Tyr Ile His Phe Tyr Gln Gln Val Gly Ile Lys Val Thr His

180 185 190

Leu Gly Phe Leu Asn Glu Pro Gln Glu Asp Val Thr Tyr Ala Ser Met

195 200 205

Leu Ser Asp Gly Thr Gln Ala Ala Ala Asp Phe Ile Lys Val Leu Ala

210 215 220

Pro Thr Val Lys Ala Ala Gly Leu Asp Val Lys Leu Thr Cys Cys Asp

225 230 235 240

Gly Val Gly Trp Glu Glu Gln Arg Ala Met Leu Pro Gly Leu Gln Ala

245 250 255

Gly Gly Pro Glu His Ser Ala Glu Ser Tyr Leu Ser Val Ile Thr Ala

260 265 270

His Gly Tyr Asn Ser Pro Pro Thr Thr Pro Leu Glu Thr Ser Leu Pro

275 280 285

Val Trp Met Thr Glu Trp Ala Asp Leu Asn Asp Asn Tyr Thr Ala Ala

290 295 300

Trp Tyr Glu Asn Gly Ala Pro Gly Glu Gly Leu Thr Trp Ala Asn Arg

305 310 315 320

Ile Gln Asp Ala Phe Thr Arg Ser Asn Val Ser Ala Phe Leu His Trp

325 330 335

Ile Gly Ala Glu Asn Gly Thr Ser Asn Ser Pro Leu Ile Asn Leu Asn

340 345 350

Gly Asp Ser Tyr Val Ala Ser Lys Arg Leu Trp Ala Phe Gly Gln Phe

355 360 365

Ser Arg Phe Val Arg Pro Gly Ala Val Arg Ile Asp Ala Ala Ser Ser

370 375 380

Asp Pro Leu Val Thr Val Ser Ala Phe Gln Asn Lys Glu Asn Gly Val

385 390 395 400

Val Ala Thr Gln Val Ile Asn Asn Ala Asp Thr Asp Tyr Glu Val Glu

405 410 415

Val Asp Leu Thr Gly Arg Gly His Leu Val Ser Val Val Gln Pro Tyr

420 425 430

Leu Thr Asn Asn Glu His Asp Leu Glu Ala Ser Ser Pro Ile Ile Thr

435 440 445

Leu Ala Leu Gly Ala Ala Thr Lys Phe Thr Ser Ile Val Pro Ala Arg

450 455 460

Ser Leu Val Ser Phe Val Ser Lys Thr Ala Leu

465 470 475

<210> 3

<211> 22

<212> DNA

<213> Artificial

<220>

<223 > forward primer

<400> 3

cgaattcgat gattagcctg gc 22

<210> 4

<211> 25

<212> DNA

<213> Artificial

<220>

<223 > reverse primer

<400> 4

agcggccgca ttacagcgcg gtttt 25

<210> 5

<211> 2922

<212> DNA

<213 > Escherichia coli

<400> 5

atgcgaattg gcataccaag agaacggtta accaatgaaa cccgtgttgc agcaacgcca 60

aaaacagtgg aacagctgct gaaactgggt tttaccgtcg cggtagagag cggcgcgggt 120

caactggcaa gttttgacga taaagcgttt gtgcaagcgg gcgctgaaat tgtagaaggg 180

aatagcgtct ggcagtcaga gatcattctg aaggtcaatg cgccgttaga tgatgaaatt 240

gcgttactga atcctgggac aacgctggtg agttttatct ggcctgcgca gaatccggaa 300

ttactgcaaa aacttgcgga acgtaacgtg accgtgatgg cgatggactc tgtgccgcgt 360

atctcacgcg cacaatcgcg ggacgcacta agctcgatgg cgaacatcgc cggttatcgc 420

gccattgttg aagcggcaca tgaatttggg cgcttcttta ccgggcaaat tactgcggcc 480

gggaaagtgc caccggcaaa agtgatggtg attggtgcgg gtgttgcagg tctggccgcc 540

attggcgcag caaacagtct cggcgcgatt gtgcgtgcat tcgacacccg cccggaagtg 600

aaagaacaag ttcaaagtat gggcgcggaa ttcctcgagc tggattttaa agaggaagct 660

ggcagcggcg atggctatgc caaagtgatg tcggacgcgt tcatcaaagc ggaaatggaa 720

ctctttgccg cccaggcaaa agaggtcgat atcattgtca ccaccgcgct tattccaggc 780

aaaccagcgc cgaagctaat tacccgtgaa atggttgact ccatgaaggc gggcagtgtg 840

attgtcgacc tggcagccca aaacggcggc aactgtgaat acaccgtgcc gggtgaaatc 900

ttcactacgg aaaatggtgt caaagtgatt ggttataccg atcttccggg ccgtctgccg 960

acgaaatcct cacagcttta cggcacaaac ctcgttaatc tgctgaaact gttgtgcaaa 1020

gagaaagacg gcaatatcac tgttgatttt gatgatgtgg tgattcgcgg cgtgaccgtg 1080

atccgtgcgg gcgaaattac ctggccggca ccgccgattc aggtatcagc tcagccgcag 1140

gcggcacaaa aagcggcacc ggaagtgaaa actgaggaaa aatgtacctg ctcaccgtgg 1200

cgtaaatacg cgttgatggc gctggcaatc attctttttg gctggatggc aagcgttgcg 1260

ccgaaagaat tccttgggca cttcaccgtt ttcgcgctgg cctgcgttgt cggttattac 1320

gtggtgtgga atgtatcgca cgcgctgcat acaccgttga tgtcggtcac caacgcgatt 1380

tcagggatta ttgttgtcgg agcactgttg cagattggcc agggcggctg ggttagcttc 1440

cttagtttta tcgcggtgct tatagccagc attaatattt tcggtggctt caccgtgact 1500

cagcgcatgc tgaaaatgtt ccgcaaaaat taaatgtctg gaggattagt tacagctgca 1560

tacattgttg ccgcgatcct gtttatcttc agtctggccg gtctttcgaa acatgaaacg 1620

tctcgccagg gtaacaactt cggtatcgcc gggatggcga ttgcgttaat cgcaaccatt 1680

tttggaccgg atacgggtaa tgttggctgg atcttgctgg cgatggtcat tggtggggca 1740

attggtatcc gtctggcgaa gaaagttgaa atgaccgaaa tgccagaact ggtggcgatc 1800

ctgcatagct tcgtgggtct ggcggcagtg ctggttggct ttaacagcta tctgcatcat 1860

gacgcgggaa tggcaccgat tctggtcaat attcacctga cggaagtgtt cctcggtatc 1920

ttcatcgggg cggtaacgtt cacgggttcg gtggtggcgt tcggcaaact gtgtggcaag 1980

atttcgtcta aaccattgat gctgccaaac cgtcacaaaa tgaacctggc ggctctggtc 2040

gtttccttcc tgctgctgat tgtatttgtt cgcacggaca gcgtcggcct gcaagtgctg 2100

gcattgctga taatgaccgc aattgcgctg gtattcggct ggcatttagt cgcctccatc 2160

ggtggtgcag atatgccagt ggtggtgtcg atgctgaact cgtactccgg ctgggcggct 2220

gcggctgcgg gctttatgct cagcaacgac ctgctgattg tgaccggtgc gctggtcggt 2280

tcttcggggg ctatcctttc ttacattatg tgtaaggcga tgaaccgttc ctttatcagc 2340

gttattgcgg gtggtttcgg caccgacggc tcttctactg gcgatgatca ggaagtgggt 2400

gagcaccgcg aaatcaccgc agaagagaca gcggaactgc tgaaaaactc ccattcagtg 2460

atcattactc cggggtacgg catggcagtc gcgcaggcgc aatatcctgt cgctgaaatt 2520

actgagaaat tgcgcgctcg tggtattaat gtgcgtttcg gtatccaccc ggtcgcgggg 2580

cgtttgcctg gacatatgaa cgtattgctg gctgaagcaa aagtaccgta tgacatcgtg 2640

ctggaaatgg acgagatcaa tgatgacttt gctgataccg ataccgtact ggtgattggt 2700

gctaacgata cggttaaccc ggcgtcgcag catgatccga agagtccgat tgctggtatg 2760

cctgtgctgg aagtgtggaa agcgcagaac gtgattgtct ttaaacgttc gatgaacact 2820

ggctatgctg gtgtgcaaaa cccgctgttc ttcaaggaaa acacccacat gctgtttggt 2880

gacgccaaag ccagcgtgga tgcaatcctg aaagctctgt aa 2922

Claims

1. fermentation prepares the method for 1B goods, comprises, fermentation produces the 1B zymotic fluid, and this zymotic fluid mixes with the zytase of high temperature resistant dehydration, and the particles with fat of collecting after high temperature drying is coated.

2. method claimed in claim 1, it comprises:

(5) particles with fat of step (4) being collected is coated.

3. the described method of claim 1 or 2, the particle of wherein collecting and fatty weight ratio are 2-8:8-2, are preferably 3-7:7-3, more preferably 5-6.5:5-3.5; And/or fat is fractionation palm fat, is more preferably hundred Canons, be most preferably hundred HTL316 of Canon; And/or high temperature is 60-100 ℃, is preferably 65-90 ℃, more preferably 75-85 ℃; And/or the particle diameter of the particle of collection is less than 0.8mm, preferably be less than 0.5mm.

4. method claimed in claim 2, wherein, in step (3), the volume ratio of the supernatant that the filtrate that step (1) obtains and step (2) obtain is 10:0.01-10, is preferably 10:0.05-5, more preferably 10:0.1 ~ 1.

5. the described method of claim 1 or 2, the wherein amino acid sequence of the zytase of high temperature resistant dehydration:

(1) as shown in SEQ ID No:2; Or

(2) be to SEQ ID No:2 disappearance, add and/or replace one or several amino acid residue (preferably be no more than 7, more preferably no more than 5, as be no more than 3) and the sequence of the reservation SEQ ID No:2 activity that obtains.

6. method claimed in claim 2, wherein, in step (1), in filtrate, the content of 1B is higher than 50g/L, preferably higher than 70g/L, more preferably higher than 90g/L.

7. the described method of claim 1 or 2, wherein goods are feed addictives.

8. goods, feed addictive preferably, it comprises the particle with the fat parcel, the zytase that wherein particle comprises 1B and high temperature resistant dehydration, preferably prepared by its arbitrary described method by claim 1-7.

9. the zytase of high temperature resistant dehydration, its amino acid sequence:

(1) as shown in SEQ ID No:2; Or

10. II of the present invention, III, IV, V or VI aspect.