CN101912048B - T4 lysozyme premix for animals and preparation method thereof - Google Patents
T4 lysozyme premix for animals and preparation method thereof Download PDFInfo
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- CN101912048B CN101912048B CN2010102527529A CN201010252752A CN101912048B CN 101912048 B CN101912048 B CN 101912048B CN 2010102527529 A CN2010102527529 A CN 2010102527529A CN 201010252752 A CN201010252752 A CN 201010252752A CN 101912048 B CN101912048 B CN 101912048B
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Abstract
The invention belongs to the field of feed additives, and in particular relates to a pemix containing T4 lysozyme from T4 bacteriophage. The T4 lysozyme from the T4 bacteriophage replaces the traditional animal antibiotics, and the antibacterial growth-promoting feed premix containing the T4 lysozyme is prepared. A method for preparing the premix comprises the following steps of: centrifuging T4 lysozyme basic solution to remove impurities, performing membrane concentration, refining and purifying by using cation exchange resin, preparing protease powder by using a low-temperature spray drying method, screening the protease powder, adding auxiliary materials, and uniformly mixing at a high speed by a mixer to prepare the T4 lysozyme premix. The added auxiliary materials comprise soya bean meal, bran, wheatmeal, wheat middling, bentonite, CaCO3, and zeolite. Experimental results show that the premix containing the T4 lysozyme has the effect of treating piglet diarrhea, which is obviously superior to that of a premix containing lysozyme from chicken egg albumin; and experimental results for feeding meat chickens and carp show that the premix containing the T4 lysozyme has the weight increasing effect and feed utilization rate which are slightly superior to those of the premix containing lysozyme from chicken egg albumin.
Description
Technical field
The invention belongs to feed additive field, be specifically related to prepare a kind of T4 Lysozyme pre-mixing agent that derives from the T4 bacteriophage that contains.
Background technology
China is vast in territory, populous, economic development is rapid in recent years, living standards of the people progressively improve, high-quality animal protein product consumption is constantly risen, driven thus the fast development of domestic animal health-care product industry, domestic food animal health-care product market has been the second largest market after the U.S..
Along with China puts into effect various Food Securities successively, the safe and efficient nontoxic animal feed additive series products of corresponding exploitation has become the developing direction of animal health-care product industry.Lysozyme is that the selective object microorganism wall that makes of a kind of energy dissolves, and organism itself is had the enzyme of protective effect, and has the characteristic of noresidue in broad-spectrum high efficacy sterilization, body, environmental nonpollution, is widely studied in recent years.Lysozyme is used for animal feed additive to substitute traditional veterinary drug antibiotic product, has higher economic and social benefit.
People's early start research lysozyme is from the report that Nicoile delivered the bacillus subtilis dissolution factor in 1907; After 2 years, Laschtschenko points out: the white strong bacteriostasis of ovum gallinaceum is the result of enzyme; There is the enzyme of energy dissolution of bacteria cell membrane in nineteen twenty-two Britain bacteriologist Fleming finder's saliva, tears, because it has bacteriolysis, so the called after lysozyme.People were gradually in Various Tissues, secretion and the plant of humans and animals, the existence that microorganism has also been found lysozyme afterwards.Along with the constantly deeply development of research to lysozyme, people have had further understanding to its characteristic.
Lysozyme is a kind of enzyme that can dissolve microorganism wall, be a class material general name.Can be divided into the animal sources lysozyme according to its source and mechanism of action difference: comprise C type (chicken-lysozyme type), G type (goose-type), I type (intertebrate-type); The plant source lysozyme; Microbial source lysozyme: comprise N-acetylmuramic glycanchydrolase, amidase, endopeptidase, β-1,3,1,6 dextranase and mannonase chitinase; The bacteriophage T4 Lysozyme.Different its action and efficacy in lysozyme source are different.
Phage lysozyme is coded by various bacteriophages self that can the bacterial infection cell, is usually located in the protein coat of bacteriophage, and in the process of host cells infected, they are playing an important role aspect the dissolution of bacteria cell membrane.T4 Lysozyme is to be produced when the ehec infection E.coli by the T4 bacteriophage, and it is the highest a kind of lysozyme of bactericidal activity that it is found that up to now.
The lysozyme commonly used that uses in feed addictive at present is mostly to adopt chemical method to extract preparation from egg, but due to production scale and expensive restriction, makes it be difficult to large-scale promotion in feedstuff industry.The patent 200610137767 of Liu Dehu researcher's invention of the Chinese Academy of Agricultural Sciences discloses a kind of method of construction expression high disinfection vitality T 4 lysozyme recombinant protein gene engineering microzyme.T4 Lysozyme set forth in the present invention adopts the genetic engineering bacterium large scale fermentation that Liu Dehu researcher builds to produce, decrease product cost.And because its bacteriostatic activity is high, using dosage is low, has a extensive future in feed additive industry.Before the present invention, yet there are no scale and prepare the method for the T4 Lysozyme that derives from the T4 bacteriophage and the report of corresponding pre-mixing agent product.
Summary of the invention
The present invention describes be a kind of contain the T4 Lysozyme pre-mixing agent that derives from the T4 bacteriophage and the preparation method.
The present invention includes following two aspects:
1. extract T4 Lysozyme from zymotic fluid, zymotic fluid is through plate-frame filtering, the filtered fluid supernatant is through ceramic membrane ultrafitration, ceramic membrane filter liquid is through the filter of doughnut column chromatography essence, then according to the molecular weight of T4 Lysozyme product, the organic nanofiltration membrane nanofiltration of preferred suitable pore size is concentrated, and nanofiltration liquid is analysed refining purifying with cation exchange resin layer, preferred CM-Sephadex-C25 (experiment is used) or AmberliteIRC-50 (commercial scale, 50～100 orders) cationic ion-exchange resin.The ion exchange liquid spray-drying makes the enzyme powder, and the initial solid content of its spray-drying mother liquor is 2-10%, and EAT is 80-180 ℃, and leaving air temp is 40-80 ℃, and enzyme dry powder albumen bactericidal activity can reach 95% of the front solid content activity of spraying.
2. the enzyme powder of spray-drying preparation is crossed the 60-120 mesh sieve, adds 80-120 mesh sieve zein, the defatted soybean meal powder, and wheat bran, wheat time powder adds bentonite, zeolite powder and CaCO simultaneously
3, mixed at high speed is even, and preparation content is the T4 Lysozyme pre-mixing agent of 1-10%.
Advantage of the present invention is that the lysozyme that adopts derives from the T4 bacteriophage; the mode of production is the fermentation method large-scale production; the T4 Lysozyme purity high activity of selecting the extraction process of optimization to extract is strong; cost is low simultaneously; with respect to the existing lysozyme product that comes from chicken egg white, have larger actual application value and economic benefit.
The specific embodiment
Embodiment 1
The T4 Lysozyme zymotic fluid, plate-frame filtering is removed mycelium, the frame filtered fluid is with 0.1 micrometer ceramics film ultrafiltration, preferred aperture is 50,000 daltonian doughnut column chromatography essence filter, according to the molecular weight of T4 Lysozyme product, preferred aperture is that 2500 daltonian organic nanofiltration membrane nanofiltrations are concentrated, nanofiltration liquid is analysed refining purifying with cation exchange resin layer, preferred CM-Sephadex-C25 cationic ion-exchange resin.The ion exchange liquid spray-drying makes the enzyme powder, and the initial solid content of its spray-drying mother liquor is 4%, and EAT is 90 ℃, and leaving air temp is 60 ℃.The enzyme powder is crossed the 60-120 mesh sieve, takes the 62.5g sieving, adds zein 350g and the wheat bran 250g of 80-120 mesh sieve, add simultaneously bentonite 50g, CaCO3 200g, zeolite powder 87.5g, high-speed mixer is evenly mixed, makes T4 Lysozyme pre-mixing agent (5%).
Embodiment 2
The T4 Lysozyme zymotic fluid, plate-frame filtering is removed mycelium, the frame filtered fluid is with 0.2 micrometer ceramics film ultrafiltration, preferred aperture is 25,000 daltonian doughnut column chromatography essence filter, according to the molecular weight of T4 Lysozyme product, preferred aperture is that 2500 daltonian organic nanofiltration membrane nanofiltrations are concentrated, nanofiltration liquid is analysed refining purifying with cation exchange resin layer, preferred AmberliteIRC-50 cationic ion-exchange resin.The ion exchange liquid spray-drying makes the enzyme powder, and the initial solid content of its spray-drying mother liquor is 8%, and EAT is 100 ℃, and leaving air temp is 80 ℃.The enzyme powder is crossed the 60-120 mesh sieve, takes the 118g sieving, adds zein 350g and the wheat bran 200g of 80-120 mesh sieve, adds simultaneously bentonite 100g, CaCO
3150g, zeolite powder 82g, high-speed mixer is evenly mixed, makes T4 Lysozyme pre-mixing agent (10%).
Embodiment 3
The T4 Lysozyme zymotic fluid, plate-frame filtering is removed mycelium, the frame filtered fluid is with 0.1 micrometer ceramics film ultrafiltration, preferred aperture is 25,000 daltonian doughnut column chromatography essence filter, according to the molecular weight of T4 Lysozyme product, preferred aperture is that 2500 daltonian organic nanofiltration membrane nanofiltrations are concentrated, nanofiltration liquid is analysed refining purifying with cation exchange resin layer, preferred CM-Sephadex-C25 cationic ion-exchange resin.The ion exchange liquid spray-drying makes the enzyme powder, and the initial solid content of its spray-drying mother liquor is 6%, and EAT is 95 ℃, and leaving air temp is 70 ℃.The enzyme powder is crossed the 60-120 mesh sieve, takes the 56g sieving, adds defatted soybean meal 350g and the wheat time powder 250g of 80-120 mesh sieve, adds simultaneously bentonite 80g, CaCO
3200g, zeolite powder 64g, high-speed mixer is evenly mixed, makes T4 Lysozyme pre-mixing agent (5%).
Embodiment 4
The T4 Lysozyme zymotic fluid, plate-frame filtering is removed mycelium, the frame filtered fluid is with 0.2 micrometer ceramics film ultrafiltration, preferred aperture is 50,000 daltonian doughnut column chromatography essence filter, according to the molecular weight of T4 Lysozyme product, preferred aperture is that 2500 daltonian organic nanofiltration membrane nanofiltrations are concentrated, nanofiltration liquid is analysed refining purifying with cation exchange resin layer, preferred AmberliteIRC-50 cationic ion-exchange resin.The ion exchange liquid spray-drying makes the enzyme powder, and the initial solid content of its spray-drying mother liquor is 5%, and EAT is 95 ℃, and leaving air temp is 75 ℃.The enzyme powder is crossed the 60-120 mesh sieve, takes the 89g sieving, adds defatted soybean meal 400g and the wheat time powder 250g of 80-120 mesh sieve, adds simultaneously bentonite 50g, CaCO
3150g, zeolite powder 61g, high-speed mixer is evenly mixed, makes T4 Lysozyme pre-mixing agent (8%).
Embodiment 5
Application Example
Test for piglet:
(1) experimental design
180 30 age in days piglets (body weight 8.5kg left and right), take random single-factor design, be divided into three groups, be respectively control group (not adding lysozyme), test 1 group of (the per kilogram feed adds 180mg chicken egg white lysozyme), 2 groups of tests (the per kilogram feed adds the 180mgT4 lysozyme), every group of three repetitions, each repeats 20 piglets.The chicken egg white lysozyme formulation and the T4 Lysozyme formulation content that add are 500,000 U/g, every group of 3 repetitions, 6 weeks of experimental period.Between group, between repeating groups, body weight is no more than 5% of average weight.Each group diarrhoea number of times of observation, and note down.Diarrhoea treatment at once appears.
(2) results and analysis
Table 1 grice diarrhoea situation
| Group | Control group | Test 1 group | Test 2 groups |
| Average diarrhea rate (%) | 57.5 | 25 | 10 |
As can be seen from Table 1, front two groups of the average diarrhea rate of 2 groups of tests has reduced respectively 83% and 60%.As can be seen from the above results, do not add any antibiotic or other anti-diarrhea additives in weanling pig, diarrhoea is serious, add lysozyme and can effectively control diarrhoea than the blank group in the weanling pig daily ration, but in the situation that the Antidiarrheic effect that same addition adds T4 Lysozyme is more excellent than the chicken egg white lysozyme.
Test for broiler chicken:
(1) experimental design
To test the meat chick and be divided at random three groups, every group of 4 repetitions, each repeats 200 plumages.Control group is daily diet group, and testing 1 group is to add the chicken egg white lysozyme on the basis of daily diet group, and testing 2 groups is to add T4 Lysozyme on the basis of daily diet group.Test period was 8 weeks, and front surrounding is added corresponding lysozyme amount in testing 1 group and 2 groups be 60g; Rear surrounding is added corresponding lysozyme amount in testing 1 group and 2 groups be 30g.The chicken egg white lysozyme formulation and the T4 Lysozyme formulation content that add are 500,000 U/g.Daily gain, daily ingestion amount, the death rate are respectively organized in observation, and note down, and between group, between repeating groups, body weight is no more than 5% of average weight.
(2) results and analysis
The impact (1-4 week) of table 2 on the broiler growth performance
| Project | Average daily gain (g) | Daily ingestion amount (g) | The material anharmonic ratio | The death rate (%) |
| Control group | 75.96 | 95.71 | 1.26 | 1.83 |
| Test 1 group | 76.31 | 95.03 | 1.246 | 1.49 |
| Test 2 groups | 80.01 | 95.11 | 1.189 | 0.79 |
1-4 week is added the production performance that lysozyme can improve the meat chick in the feed of meat chick as can be seen from Table 2, add the T4 Lysozyme average daily gain and improved respectively 5.3% and 4.8% than the above two, the material anharmonic ratio has reduced respectively 5.6% and 4.6%, the death rate has also reduced by 56.8% and 46.9%, and effect is better than the chicken egg white lysozyme.
The impact (5-8 week) of table 3 on the broiler growth performance
| Project | Average daily gain (g) | Daily ingestion amount (g) | The material anharmonic ratio | The death rate (%) |
| Control group | 171.53 | 349.21 | 2.036 | 1.71 |
| Test 1 group | 173.22 | 351.08 | 2.027 | 1.32 |
| Test 2 groups | 176.94 | 354.86 | 2.006 | 0.68 |
Add as can be seen from Table 3 T4 Lysozyme 5-8 week and can reach good production performance in the meat chick fodder, material anharmonic ratio and the death rate of meat chick all improve significantly, and effect is better than the chicken egg white lysozyme.
Test for aquaculture
(1) experimental design
Choosing average weight is the test fish of 80g left and right, be divided into 3 groups, be respectively control group (not adding lysozyme), test 1 group of (the per kilogram feed adds 75mg chicken egg white lysozyme), 2 groups of tests (the per kilogram feed adds the 75mgT4 lysozyme), the chicken egg white lysozyme formulation and the T4 Lysozyme formulation content that add are 500,000 U/g, establish three repeating groups for every group.Between group, between repeating groups, body weight is no more than 5% of average weight.30 days experimental periods, carry out in indoor water tank, control water temperature at 25-30 ℃, between pH6.8-7.2.
(2) results and analysis
The impact of table 4 on test fish (carp) growth performance
| Project | Average weight gain (g) | Average food ration (g) | Feed coefficient |
| Control group | 6.68 | 25.12 | 3.761 |
| Test 1 group | 8.78 | 26.54 | 3.023 |
| Test 2 groups | 9.81 | 26.79 | 2.731 |
As shown in Table 4, the rate of body weight gain and the feed coefficient that add T4 Lysozyme group test fish significantly are better than control group and add the egg white powder lysozyme, have descended respectively 27.4% and 9.7% than control group and egg white powder lysozyme group feed coefficient.
Claims (13)
1. method for preparing the T4 Lysozyme pre-mixing agent is characterized in that comprising the following steps:
A. separating-purifying T4 Lysozyme from zymotic fluid, zymotic fluid are through plate-frame filtering, and film is concentrated, and cationic ion-exchange resin is made with extra care purifying, and spray-drying makes the protoenzyme powder; The initial solid content of its spray-drying mother liquor is 2-10%; EAT is 80-180 ℃; Leaving air temp is 40-80 ℃; Spray-drying lysozyme powder content is 70%-90%, and crosses the 60-120 mesh sieve;
B. added the auxiliary material of 80-120 mesh sieve in above-mentioned T4 Lysozyme enzyme powder, the mixer mixed at high speed is even, is prepared into the T4 Lysozyme pre-mixing agent.
2. by the method for preparing the T4 Lysozyme pre-mixing agent claimed in claim 1, it is characterized in that the initial solid content of described spray-drying mother liquor is 8%.
3. by the method for preparing the T4 Lysozyme pre-mixing agent claimed in claim 1, it is characterized in that described EAT is 90 ℃.
4. by the method for preparing the T4 Lysozyme pre-mixing agent claimed in claim 1, it is characterized in that described leaving air temp is 60 ℃.
5. by the method for preparing the T4 Lysozyme pre-mixing agent claimed in claim 1, it is characterized in that described auxiliary material comprises that addition is the zein of 200-500g/kg pre-mixing agent, addition is the defatted soybean meal powder of 200-500g/kg pre-mixing agent, addition is the wheat bran of 200-500g/kg pre-mixing agent, addition is the wheat time powder of 200-500g/kg pre-mixing agent, addition is the bentonite of l0-l00g/kg pre-mixing agent, and addition is the CaCO of l00-300g/kg pre-mixing agent
3, addition is the zeolite powder of 50-200g/kg pre-mixing agent.
6. the T4 Lysozyme pre-mixing agent of the described method preparation of any one according to claim 1-5, is characterized in that preparing the T4 Lysozyme pre-mixing agent that content is 1-10%.
7. T4 Lysozyme pre-mixing agent according to claim 6, is characterized in that preparing content and be preferably 5% T4 Lysozyme pre-mixing agent.
8. the T4 Lysozyme pre-mixing agent of the described method preparation of any one according to claim 1-5, the object that it is characterized in that its use is various fowl poultry kinds and aquaculture class.
9. T4 Lysozyme pre-mixing agent according to claim 8, is characterized in that the object of its use is preferably piglet, broiler chicken.
10. T4 Lysozyme pre-mixing agent according to claim 9, is characterized in that the addition of T4 Lysozyme in feed is the 50-500mg/kg feed.
11. T4 Lysozyme pre-mixing agent according to claim 10 is characterized in that T4 Lysozyme addition in pig starter feed is the 180mg/kg feed.
12. T4 Lysozyme pre-mixing agent according to claim 10 is characterized in that T4 Lysozyme addition in broiler fodder is the 60mg/kg feed.
13. T4 Lysozyme pre-mixing agent according to claim 8 is characterized in that the addition of T4 Lysozyme in the freshwater fish feed is the 75mg/kg feed.
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Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102296056B (en) * | 2011-08-30 | 2013-06-05 | 中国人民解放军军事医学科学院生物工程研究所 | Mycobacteriophage D29 particles and preparation method and use thereof |
| CN103461676B (en) * | 2013-09-18 | 2014-08-13 | 杭州康源饲料科技有限公司 | Feed additive comprising lysozyme, pesticide composition and application of feed additive |
| AU2016286612B2 (en) * | 2015-07-02 | 2021-01-28 | Novozymes A/S | Animal feed compositions and uses thereof |
| CN108719607B (en) * | 2017-04-14 | 2021-07-06 | 浙江伊杰斯生物科技有限公司 | Feed additive containing lysozyme |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101173260A (en) * | 2006-10-31 | 2008-05-07 | 刘德虎 | Representation of high disinfection vitality T4 lysozyme in yeast and producing method thereof |
| CN101530161A (en) * | 2008-03-10 | 2009-09-16 | 北京大北农科技集团股份有限公司 | Novel feed additive and preparation method thereof |
| CN101611765A (en) * | 2009-06-19 | 2009-12-30 | 杭州康源饲料科技有限公司 | Microbic muramidase substitutes antibiotic application process in feed |
| CN101619308A (en) * | 2009-08-14 | 2010-01-06 | 湖北神地农业科贸有限公司 | Preparation method for extracting lysozyme from egg white |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101173260A (en) * | 2006-10-31 | 2008-05-07 | 刘德虎 | Representation of high disinfection vitality T4 lysozyme in yeast and producing method thereof |
| CN101530161A (en) * | 2008-03-10 | 2009-09-16 | 北京大北农科技集团股份有限公司 | Novel feed additive and preparation method thereof |
| CN101611765A (en) * | 2009-06-19 | 2009-12-30 | 杭州康源饲料科技有限公司 | Microbic muramidase substitutes antibiotic application process in feed |
| CN101619308A (en) * | 2009-08-14 | 2010-01-06 | 湖北神地农业科贸有限公司 | Preparation method for extracting lysozyme from egg white |
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