CN101988054B - Method for preparing cyclodextrin lysozyme inclusion complex, prepared feed additive and application of inclusion complex in compound feed - Google Patents
Method for preparing cyclodextrin lysozyme inclusion complex, prepared feed additive and application of inclusion complex in compound feed Download PDFInfo
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- CN101988054B CN101988054B CN2009100558695A CN200910055869A CN101988054B CN 101988054 B CN101988054 B CN 101988054B CN 2009100558695 A CN2009100558695 A CN 2009100558695A CN 200910055869 A CN200910055869 A CN 200910055869A CN 101988054 B CN101988054 B CN 101988054B
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- Fodder In General (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
The invention relates to a method for preparing a cyclodextrin lysozyme inclusion complex, a prepared feed additive and application of the inclusion complex in a compound feed. The preparation method of the inclusion complex comprises the following steps of: mixing 5-90 percent by weight of lysozyme and 10-95 percent by weight of cyclodextrin together and carrying out inclusion at the temperature of 40-90 DEG C for 1-3 hours to obtain the cyclodextrin lysozyme inclusion complex. The heat resistance of the lysozyme coated by the cyclodextrin is obviously improved. Very small amount of water is added in the preparation process of the lysozyme inclusion complex or water is not added at all; any organic solvent does not need to be added, and thus, no solvent residues are generated and the aftertreatment is convenient; the process is simple and convenient, and the inclusion speed is high. Then the inclusion complex of the cyclodextrin and the lysozyme is mixed in the proportion of 1:1-3 to obtain the feed additive; the feed additive containing the cyclodextrin and the lysozyme has better tolerance to pepsase, and moreover, the feed additive is mixed into the compound feed according to the additive amount of 0.05-0.5 percent, and after being evenly stirred, the feed additive has very obvious in vitro bacteriostasis effect.
Description
Technical field
The present invention relates to a kind of cyclodextrin coating technique that utilizes and handle the method that N,O-Diacetylmuramidase improves its tolerance that temperature and enzyme are cut, obtain a kind of product that can be used as fodder additives, belong to technical field of enzyme engineering by this method.
Background technology
N,O-Diacetylmuramidase (Lysozyme; EC3.2.1.17) be the lytic enzyme that acts on microorganism wall specially; claim Lysozyme (Muramidase) again; be a kind of green, pollution-free, nuisanceless, noresidue, high alkali protein that security is very high; the glycosidic linkage that can act on to specificity peptidoglycan makes it fracture, makes bacteria cell wall become loose, loses the provide protection of pair cell; last cytolysis death, and can not have a negative impact to the human body cell that does not have cell walls.Because it has selectivity bacteriostatic characteristics, be that food preservatives or preservation agent use by many countries and tissue approval at present.At present, N,O-Diacetylmuramidase is as additive for farm animal feed ability ground zero, and only some preliminary study have been done by USSR (Union of Soviet Socialist Republics), France, has obtained better effects but the domestic people of having reports the utilization N,O-Diacetylmuramidase in Production of Livestock and Poultry.N,O-Diacetylmuramidase has preventive and therapeutic effect to grice diarrhoea, and it and immunoglobulin (Ig) have closely on function gets in touch, and can go into complement with other life active compounds, strengthens the activity of antibody, thus kill bacteria.Other has report, and N,O-Diacetylmuramidase can be used for preventing and treating diseases such as gastro-enteritis, maldigestion, suppuration, and is verified on livestock and poultry such as the broiler chicken of feeding, calf, piglet; N,O-Diacetylmuramidase is just becoming a kind of " green " fodder enzyme preparation, will have wide application value and huge development prospect in herding at home and abroad and the culture fishery.Yet, assessment for fodder enzyme preparation at present mainly concentrates on the active simple mensuration of product aspect, it mainly is the activity of enzyme, and also rarely found for the effective enzyme that zymin really plays a role in the animal digestive tract assessment of living, because the complex environment in the enteron aisle makes that the research of this part content is very difficult.As everyone knows, before having an effect in the corresponding site of zymin arrival animal digestive tract, its activity can be subjected to the loss of wide range of forms.At first be in the feed course of processing, high temperature and humidity can cause the active reduction of zymin; Enter afterwards in the animal stomach, can be subjected to hydrochloric acid in gastric juice and pepsic cutting; Enter after the small intestine, also can receive the effect of proteolytic enzyme such as pancreatin, cause the active loss of zymin.Therefore, for fodder enzyme preparation, it not only will stand thermal treatment such as hot and humid grade in the feed course of processing, also will stand the influence of interior different pH environment of animal gastrointestinal tract and endogenous proteinase.Report in the past is more to the zymologic property Journal of Sex Research of fodder enzyme preparation, as optimum temperuture, pH and thermostability etc., and less to its stability study in animal intestinal.A kind ofly solve zymin is acidproofly separated, anti-enzyme is cut effective ways and exactly product is wrapped processedly, improve the stability of product in enteron aisle, give full play to its due feature.
Cyclodextrin (Cyclodextrin, be called for short CD) be nonpoisonous and tasteless white powder solid, the general name of a series of cyclic oligosaccharides that to be amylose starch generate under the cyclomaltodextrin glucanotransferase effect that is produced by genus bacillus, because connecting the glycosidic link of glucose unit can not rotate freely, make cyclodextrin form slightly tapered annulus, the primary hydroxyl of cyclodextrin has surrounded the osculum of taper, and its secondary hydroxyl has surrounded big mouthful of taper.Based on the constructional feature of cyclodextrin itself, so it has hydrophobic inner chamber and hydrophilic surface.Wherein (β-CD) as molecular vehicle, at medicine industry, food and other industrial circle have obtained widespread use to beta-cyclodextrin.In recent years, about of common occurrence with the report of cyclodextrin and derivative inclusion volatile oil, VITAMIN and common drug etc., but the inclusion treatment technology of relevant N,O-Diacetylmuramidase yet there are no report.The present invention utilizes the hydrophobic cavity of cyclodextrin to generate the ability of inclusion compound, make N,O-Diacetylmuramidase and cyclodextrin generate inclusion compound, reach the physico-chemical property of stablizing N,O-Diacetylmuramidase, reduce purposes such as oxidation and thermo-sensitivity, for N,O-Diacetylmuramidase applying in fodder industry provides certain theoretical foundation, thereby be that N,O-Diacetylmuramidase is brought into play better economic benefit in feedstuff industry and social benefit lays the foundation.
Summary of the invention
Technical problem to be solved by this invention is to provide a kind of the prepare method of cyclodextrin N,O-Diacetylmuramidase inclusion compound and the application in mixed feed thereof, for N,O-Diacetylmuramidase applying in fodder industry provides certain theoretical foundation, thereby be that N,O-Diacetylmuramidase is brought into play better economic benefit in feedstuff industry and social benefit lays the foundation.
The technical problem that will solve required for the present invention can be achieved through the following technical solutions:
Main contents of the present invention comprise three aspects, mix a certain amount of N,O-Diacetylmuramidase first aspect with cyclodextrin, controlled temperature and time get product, by hot and humid condition in the feed course of processing inclusion is handled the influence that the back N,O-Diacetylmuramidase is lived, estimate the stability that inclusion is handled the back N,O-Diacetylmuramidase.
(1) preparation of N,O-Diacetylmuramidase inclusion compound
Adopt the heated sealed method, N,O-Diacetylmuramidase sample and cyclodextrin are mixed, temperature is controlled at 40-90 ℃, and the inclusion time is 1-3h.
Wherein, the consumption of N,O-Diacetylmuramidase is 5-90%, and the consumption of cyclodextrin is 10-95% (by weight percentage), makes the N,O-Diacetylmuramidase inclusion compound.
(2) enzyme of N,O-Diacetylmuramidase inclusion compound is lived
Precision takes by weighing inclusion compound 0.01g and is dissolved in the buffered soln of PBS (pH6.2), and it is standby fully to shake up 100 times of back dilutions; Organize in contrast with N,O-Diacetylmuramidase and the direct mixed mixture of cyclodextrin simultaneously,, detect the enzyme of inclusion compound and live according to the measuring method that the N,O-Diacetylmuramidase enzyme is lived.
N,O-Diacetylmuramidase directly and the mixed enzyme work of cyclodextrin descend gradually along with the rising of temperature, in the time of 100 ℃, residual enzyme work is about 20%, illustrates that the N,O-Diacetylmuramidase structure of inclusion has not been subjected to destruction; And cyclodextrin encapsulated N,O-Diacetylmuramidase is at high temperature still keeping higher enzyme to live, and behind the reservation 20min, residual enzyme work is more than 83% in the time of 100 ℃.Presentation of results uses the stability of cyclodextrin encapsulated N,O-Diacetylmuramidase obviously to improve.
(3) the resistance toheat experiment of N,O-Diacetylmuramidase inclusion compound in the feed granulating process
Enzyme is the protein of biologically active, and most of zymins do not have tolerance pyritous character more than 90 ℃.Feed in process of production, processes such as pulverizing, premix, granulation all can cause the zymin loss of activity even not reversibly inactivated.Thermostability for the analysis package compound, accurately take by weighing a certain amount of N,O-Diacetylmuramidase sample in test tube, place the steam (being wet method) of differing temps and warm air (being dry method) after the insulation regular hour respectively, the detection method of living according to the N,O-Diacetylmuramidase enzyme detects separately enzyme and lives.
The N,O-Diacetylmuramidase sample is incubated the decline alive of back enzyme under the condition of different temperatures of dry method not obvious.Behind the insulation 3min, enzyme is lived all more than 92% relatively; Behind the insulation 5min, enzyme is lived all more than 91% relatively; The wet method insulation is more obvious than dry method to the influence alive of the enzyme of N,O-Diacetylmuramidase.Behind the insulation 3min, enzyme is lived all more than 90% relatively, and enzyme loss alive is less, and this result to the dry method insulation is similar; Relative enzyme work is between 88%-93% behind the 5min.Comprehensive dry method insulation and wet method insulation influence the result as can be known to what inclusion compound N,O-Diacetylmuramidase enzyme was lived, no matter dry method or wet method, after keeping 3min under 75 ℃ of-90 ℃ of high temperature, the loss alive of the enzyme of N,O-Diacetylmuramidase is all less, these conditions are enough to satisfy the temperature and time that feed is stood in pelletization, illustrate that high temperature in the feed granulating process can significantly not have influence on behind the inclusion enzyme of N,O-Diacetylmuramidase and live, can bear high temperature and high humidity treatment process in the general granulating process after promptly the N,O-Diacetylmuramidase inclusion is handled fully.
A second aspect of the present invention is with the N,O-Diacetylmuramidase and the N,O-Diacetylmuramidase inclusion compound of inclusion are not formed a kind of additive agent for feeding according to certain mixed.
In the body of livestock and poultry, because the chyme residence time under one's belt is about two hours, its strong acid environment (pH2-3) adds pepsic effect etc. can make a lot of zymin sex change, thereby reduces the bioavailability of enzyme.The content of this part is improved at these problems and shortcoming exactly, the application method of a kind of N,O-Diacetylmuramidase in mixed feed is provided, make its can be smoothly strong acid environment by stomach, raising is to pepsic tolerance, thereby reduce the loss that enzyme is lived, and can play a role faster after reaching small intestine, improve the utilization ratio of zymin to greatest extent.
N,O-Diacetylmuramidase sample and N,O-Diacetylmuramidase inclusion compound is even with 1: 1～3 mixed, promptly get a kind of additive agent for feeding sample,, stir.
The weight ratio of described N,O-Diacetylmuramidase and N,O-Diacetylmuramidase inclusion compound is 1: 2.
A third aspect of the present invention, the application of a kind of cyclodextrin N,O-Diacetylmuramidase inclusion compound in mixed feed, it is characterized in that, bacteriolyze bacterium enzyme and N,O-Diacetylmuramidase inclusion compound is even with the mixed of weight ratio 1: 1-3, promptly get a kind of additive agent for feeding, addition according to 0.05%-0.5% is blended in the mixed feed respectively, stirs.
N,O-Diacetylmuramidase sample and N,O-Diacetylmuramidase inclusion compound is even with 1: 1～3 mixed, promptly get a kind of additive agent for feeding sample, be blended in the mixed feed (not adding any microbiotic) according to 0.5%, 0.25% and 0.05% addition respectively, stir.
Respectively with 1: 1,1: 2,1: 3 mixed was even, promptly got a kind of additive agent for feeding sample with N,O-Diacetylmuramidase sample and N,O-Diacetylmuramidase inclusion compound, by to pepsic tolerance experiment, determined N,O-Diacetylmuramidase and the suitable proportion of composing of N,O-Diacetylmuramidase inclusion compound.
Accurately take by weighing a certain amount of stomach en-, be dissolved in (1.0mg/mL in 5mL citric acid-phosphate buffer soln, pH2.0), get the additive agent for feeding sample that N,O-Diacetylmuramidase and N,O-Diacetylmuramidase inclusion compound different ratios are formed then respectively, in 37 ℃, behind the 100rpm vibration dissolving 2h, get a certain amount of supernatant liquor, after 100 times of buffered soln dilutions, according to the measuring method of antalzyme activity, detect the enzyme of each sample and live, set up control group simultaneously, be 100% with the enzyme work without the additive of pepsin promptly, the per-cent that all the other treated enzyme activities obtain by comparison is the relative surplus enzyme of this sample and lives.
In the acid range of pH 2.0～5.0, behind pepsin 2h, the additive agent for feeding that N,O-Diacetylmuramidase sample and N,O-Diacetylmuramidase inclusion compound are formed according to different ratios shows tolerance preferably to stomach en-, and the relative surplus enzyme is lived and all remained at more than 80%;
Especially according to the additive agent for feeding of 1: 2 ratio composition, the work of relative surplus enzyme is 98% behind pH 2.0 insulation 2h, illustrates that the N,O-Diacetylmuramidase inclusion compound has stronger tolerance to stomach en-, and stability is significantly improved.
Described N,O-Diacetylmuramidase sample and N,O-Diacetylmuramidase inclusion compound are according to the additive agent for feeding of 1: 2 ratio composition, and the addition according to 0.05%-0.5% is blended in the mixed feed respectively, stirs.
Illustrate that the additive agent for feeding that this ratio is formed can be smoothly by the strong acid environment of stomach, enzyme is lived in losing and is reduced, and obtains the action time of enzyme prolonging, and can play a role in enteron aisle, improves bioavailability.
Embodiment
In order to make technique means of the present invention, creation characteristic, to reach purpose and effect is easy to understand,, further set forth the present invention below in conjunction with specific embodiment.
(1) preparation of N,O-Diacetylmuramidase inclusion compound and stability
(1) preparation of N,O-Diacetylmuramidase inclusion compound
Adopt the heated sealed method, N,O-Diacetylmuramidase sample and cyclodextrin are mixed, temperature is controlled at 40-90 ℃, and the inclusion time is 1-3h.Wherein, the consumption of N,O-Diacetylmuramidase is 5-90%, and the consumption of cyclodextrin is 10-95% (by weight), and inclusion compound gets product.
(2) enzyme of N,O-Diacetylmuramidase inclusion compound is lived
Precision takes by weighing inclusion compound 0.01g and is dissolved in the buffered soln of PBS (pH6.2), and after fully shaking up, it is standby with 100 times of PBS damping fluid dilutions to get a certain amount of solution; Organize in contrast with N,O-Diacetylmuramidase and the direct mixed mixture of cyclodextrin simultaneously,, detect the enzyme of inclusion compound and live according to the measuring method that the N,O-Diacetylmuramidase enzyme is lived.
The experimental result of table 1 N,O-Diacetylmuramidase inclusion compound stability
As can be seen from Table 1, N,O-Diacetylmuramidase directly and the mixed enzyme work of cyclodextrin descend gradually along with the rising of temperature, in the time of 100 ℃, residual enzyme work is about 20%, illustrates that the N,O-Diacetylmuramidase structure of inclusion has not been subjected to destruction; And cyclodextrin encapsulated N,O-Diacetylmuramidase is at high temperature still keeping higher enzyme to live, and behind the reservation 20min, residual enzyme work is more than 83% in the time of 100 ℃.Presentation of results uses the stability of cyclodextrin encapsulated N,O-Diacetylmuramidase obviously to improve.
The N,O-Diacetylmuramidase enzyme activity determination: adopt the enzyme of turbidimetry for Determination N,O-Diacetylmuramidase inclusion compound to live, promptly with micrococcus lysodeikticus suspension as substrate (25 ℃), measure OD at different time respectively at 450nm wavelength place
450Numerical value.Enzyme is lived and is defined: under this experiment condition, and per minute OD
450Numerical value decline 0.001 is a unit of activity;
(3) the resistance toheat experiment of N,O-Diacetylmuramidase inclusion compound in the feed granulating process
Enzyme is the protein of biologically active, and most of zymins do not have tolerance pyritous character more than 90 ℃.Feed in process of production, processes such as pulverizing, premix, granulation all can cause the zymin loss of activity even not reversibly inactivated.For analyzing the thermostability of N,O-Diacetylmuramidase inclusion compound, accurately take by weighing a certain amount of N,O-Diacetylmuramidase inclusion compound sample in test tube, place the steam (being wet method) of differing temps and warm air (being dry method) after the insulation regular hour respectively, the detection method of living according to the N,O-Diacetylmuramidase enzyme detects separately enzyme and lives.
The resistance toheat experimental result (%) of table 2 N,O-Diacetylmuramidase inclusion compound
Table 2 shows that the N,O-Diacetylmuramidase sample is incubated the decline alive of back enzyme under the condition of different temperatures of dry method not obvious.Behind the insulation 3min, enzyme is lived all more than 92% relatively; Behind the insulation 5min, enzyme is lived all more than 91% relatively; The wet method insulation is more obvious than dry method to the influence alive of the enzyme of N,O-Diacetylmuramidase.Behind the insulation 3min, enzyme is lived all more than 90% relatively, and enzyme loss alive is less, and this result to the dry method insulation is similar; Relative enzyme work is between 88%-93% behind the 5min.Comprehensive dry method insulation and wet method insulation influence the result as can be known to what inclusion compound N,O-Diacetylmuramidase enzyme was lived, no matter dry method or wet method, after keeping 3min under 75 ℃～90 ℃ high temperature, the loss alive of the enzyme of N,O-Diacetylmuramidase is all less, these conditions are enough to satisfy the temperature and time that feed is stood in pelletization, illustrate that high temperature in the feed granulating process can significantly not have influence on behind the inclusion enzyme of N,O-Diacetylmuramidase and live, can bear high temperature and high humidity treatment process in the general granulating process after promptly the N,O-Diacetylmuramidase inclusion is handled fully.
(2) additive agent for feeding of N,O-Diacetylmuramidase and N,O-Diacetylmuramidase inclusion compound composition
In the body of livestock and poultry, because the chyme residence time under one's belt is about two hours, its strong acid environment (pH2-3) adds pepsic effect etc. can make a lot of zymin sex change, thereby reduces the bioavailability of enzyme.The content of this part is improved at these problems and shortcoming exactly, the application method of a kind of N,O-Diacetylmuramidase in mixed feed is provided, make its can be smoothly strong acid environment by stomach, raising is to pepsic tolerance, thereby reduce the loss that enzyme is lived, and can play a role faster after reaching small intestine, improve the utilization ratio of zymin to greatest extent.
With 1: 1,1: 2,1: 3 mixed was even, promptly got a kind of additive agent for feeding sample with N,O-Diacetylmuramidase sample and N,O-Diacetylmuramidase inclusion compound, by to pepsic tolerance experiment, determined suitable proportion of composing; The additive agent for feeding that proper ratio is formed is blended in the mixed feed according to 0.05%, 0.25% and 0.5% addition respectively then, stirs, and studies the fungistatic effect of enzyme-added back mixed feed respectively.
Accurately take by weighing a certain amount of stomach en-, being dissolved in pH is (1.0mg/mL) in 2.05mL citric acid-phosphate buffer soln, get the additive agent for feeding sample that N,O-Diacetylmuramidase and N,O-Diacetylmuramidase inclusion compound different ratios are formed then respectively, in 37 ℃, behind the 100rpm vibration dissolving 2h, get supernatant liquor and do suitably dilution, measuring method according to antalzyme activity, detecting the enzyme of each sample lives, set up control group simultaneously, be 100% with the enzyme work without the additive of pepsin promptly, the per-cent that all the other treated enzyme activities obtain by comparison is the relative surplus enzyme of this sample and lives.
The anti-pepsic experimental result of additive agent for feeding that table 3 N,O-Diacetylmuramidase and N,O-Diacetylmuramidase inclusion compound are formed
| N,O-Diacetylmuramidase: N,O-Diacetylmuramidase inclusion compound | 1∶1 | 1∶2 | 1∶3 |
| Relative enzyme (%) alive | 90 | 98 | 87 |
Table 3 shows, in the sour environment of pH 2.0, behind pepsin 2h, the additive agent for feeding that N,O-Diacetylmuramidase sample and N,O-Diacetylmuramidase inclusion compound are formed according to different ratios shows tolerance preferably to stomach en-, and the relative surplus enzyme is lived and all remained at more than 85%; Especially according to the additive agent for feeding of 1: 2 ratio composition, the work of relative surplus enzyme is 98% behind pH 2.0 insulation 2h, and stomach en-is had stronger tolerance, and stability is significantly improved.Therefore, can be according to the additive agent for feeding of 1: 2 ratio composition smoothly by the strong acid environment of stomach, enzyme loss alive is minimum, obtains the action time of enzyme prolonging, and can play a role in enteron aisle, improves bioavailability.
N,O-Diacetylmuramidase sample and N,O-Diacetylmuramidase inclusion compound is even with 1: 2 mixed, promptly get a kind of additive agent for feeding sample, be blended in the mixed feed according to 0.05%, 0.25% and 0.5% addition respectively, stir, study the fungistatic effect of enzyme-added back mixed feed respectively.
Embodiment 1:
Get the additive agent for feeding sample 1000g that N,O-Diacetylmuramidase sample and N,O-Diacetylmuramidase inclusion compound are formed with 1: 2 the even back of mixed, add additive agent for feeding 0.5g (being addition 0.05%), thorough mixing is even, accurately gets biased sample 1.0g then, places the triangular flask that the 50mL tap water is housed; Set up control group simultaneously, promptly directly accurately take by weighing mixed feed 1.0g and join in the 50mL tap water, in 37 ℃, behind the 200rpm shaking culture 18h, visual inspection has or not thalli growth, and the kind of microscopic examination thalline adopts dilution spread plate method to carry out enumeration then.
Table 4 addition is 0.05% the external bacteriostatic experiment result of additive agent for feeding in mixed feed
Embodiment 2:
Get the additive agent for feeding sample 400g that N,O-Diacetylmuramidase sample and N,O-Diacetylmuramidase inclusion compound are formed with 1: 2 the even back of mixed, add additive agent for feeding 1.0g (being addition 0.25%), thorough mixing is even, accurately gets biased sample 1.0g then, places the triangular flask that the 50mL tap water is housed; Set up control group simultaneously, promptly directly accurately take by weighing mixed feed 1.0g and join in the 50mL tap water, in 37 ℃, behind the 200rpm shaking culture 18h, visual inspection has or not thalli growth, and the kind of microscopic examination thalline adopts dilution spread plate method to carry out enumeration then.
Table 5 addition is 0.25% the external bacteriostatic experiment result of additive agent for feeding in mixed feed
Embodiment 3:
Get the additive agent for feeding sample 200g that N,O-Diacetylmuramidase sample and N,O-Diacetylmuramidase inclusion compound are formed with 1: 2 the even back of mixed, add additive agent for feeding 1.0g (being addition 0.5%), thorough mixing is even, accurately gets biased sample 1.0g then, places the triangular flask that the 50mL tap water is housed; Set up control group simultaneously, promptly directly accurately take by weighing mixed feed 1.0g and join in the 50mL tap water, in 37 ℃, behind the 200rpm shaking culture 18h, visual inspection has or not thalli growth, and the kind of microscopic examination thalline adopts dilution spread plate method to carry out enumeration then.
Table 6 addition is 0.5% the external bacteriostatic experiment result of additive agent for feeding in mixed feed
As can be seen from the above results, behind the adding N,O-Diacetylmuramidase sample, tangible minimizing has all taken place in kind of microorganism and quantity in the mixed feed.Addition is to have only the sporeformer growth in 0.5% the experimental group, and the order of magnitude obviously reduces, and the growth of a spot of bacillus and coccus is arranged in 0.25% and 0.05% experimental group.According to result in this breadboard early stage, N,O-Diacetylmuramidase can effectively suppress pathogenic bacterium common in the livestock and poultry, as colon bacillus, Salmonellas, clostridieum welchii and streptococcus aureus etc., the N,O-Diacetylmuramidase of same concentrations to the restraining effect of sporeformer a little less than.Based on the above results, as can be seen, the bacteriostatic action of enzyme-added back mixed feed is very obvious, can effectively suppress microbial growths such as common enterobacteria and coccus.
More than show and described ultimate principle of the present invention, principal character and advantage of the present invention.The technician of the industry should understand; the present invention is not restricted to the described embodiments; that describes in the foregoing description and the specification sheets just illustrates principle of the present invention; the present invention also has various changes and modifications without departing from the spirit and scope of the present invention, and these changes and improvements all fall in the claimed scope of the invention.The claimed scope of the present invention is defined by appending claims and equivalent thereof.
Claims (1)
1. a method for preparing cyclodextrin N,O-Diacetylmuramidase inclusion compound is characterized in that, N,O-Diacetylmuramidase is mixed with cyclodextrin, adopts the heated sealed method, and N,O-Diacetylmuramidase and cyclodextrin are mixed, and temperature is controlled at 40-90 ℃, and the inclusion time is 1-3h, makes the N,O-Diacetylmuramidase inclusion compound;
By weight percentage, the consumption of described N,O-Diacetylmuramidase is 5-90%, and the consumption of cyclodextrin is 10-95%, makes the N,O-Diacetylmuramidase inclusion compound.
2, a kind of method for preparing cyclodextrin N,O-Diacetylmuramidase inclusion compound according to claim 1, it is characterized in that described N,O-Diacetylmuramidase inclusion compound is dissolved in the PBS buffered soln of pH6.2, still keeping higher enzyme to live under the high temperature, keep 20min in the time of 100 ℃ after, residual enzyme work is more than 83%.
3, a kind of fodder additives of the cyclodextrin N,O-Diacetylmuramidase inclusion compound preparation by the described method preparation for preparing cyclodextrin N,O-Diacetylmuramidase inclusion compound of claim 1 is characterized in that, N,O-Diacetylmuramidase and the N,O-Diacetylmuramidase inclusion compound mixed with weight ratio 1 ︰ 2 is evenly made.
4, a kind of by the described application of cyclodextrin N,O-Diacetylmuramidase inclusion compound in mixed feed for preparing the method preparation of cyclodextrin N,O-Diacetylmuramidase inclusion compound of claim 1, it is characterized in that, bacteriolyze bacterium enzyme and N,O-Diacetylmuramidase inclusion compound is even with the mixed of weight ratio 1 ︰ 2, promptly get a kind of additive agent for feeding, addition according to 0.05%-0.5% is blended in the mixed feed respectively, stirs.
5, application according to claim 4 is characterized in that, described additive agent for feeding is at 37 ℃, and under the situation of pH 2.0, the work of relative surplus enzyme is 98% behind the insulation 2h, and stomach en-is had stronger tolerance.
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| 肖怀秋等.溶菌酶及其在食品工业中的应用.《中国食物与营养》.2005,(第2期),第32-34页. * |
| 赵娥等.β—环糊精包合技术应用研究进展.《山东医药工业》.2002,第21卷(第1期),第32-33. * |
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