CN102313794A - Preparation method of duck simulated intestinal fluid - Google Patents
Preparation method of duck simulated intestinal fluid Download PDFInfo
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- CN102313794A CN102313794A CN201110140044A CN201110140044A CN102313794A CN 102313794 A CN102313794 A CN 102313794A CN 201110140044 A CN201110140044 A CN 201110140044A CN 201110140044 A CN201110140044 A CN 201110140044A CN 102313794 A CN102313794 A CN 102313794A
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Abstract
The invention discloses a preparation method of a duck simulated intestinal fluid. The method comprises the following steps of: putting collected duck intestinal chyme in a reagent bottle to shake uniformly, putting in a centrifuge bottle to perform centrifuging treatment, and collecting a supernatant fluid; filtering the supernatant fluid, freezing and concentrating into a mixed concentrated liquid; after dialyzing the mixed concentrated liquid and removing impurities, freezing and drying into lyophiled powder; measuring the activities of alpha-amylase, lipase, trypsin and chymotrypsin in the lyophiled powder, and measuring the activities of corresponding digestive enzymes in the reagent level alpha-amylase, lipase, trypsin and chymotrypsin; and according to the principal that the gross activities of various digestive enzymes in the duck simulated intestinal fluid and the targeted duck intestinal fluid are equal, adding corresponding capacity reagent level alpha-amylase, corresponding weight reagent level lipase, corresponding weight reagent level trypsin, corresponding weight reagent level chymotrypsin and a corresponding capacity water solution in the lyophiled powder with a set weight. The method can be carried out in a low-cost way, and can also be used for preparing the duck simulated intestinal fluid basically homologous with four digestive enzymes in a real duck body.
Description
Technical field
The present invention relates to a kind of preparation method of animal simulation digestive juice, refer to a kind of preparation method of duck simulation intestinal fluid especially.
Background technology
The duck number of animals raised of China accounts for more than 70% of world's total amount.In the preparation of duck daily ration, the energy of feedstuff and amino acid bio are learned and tired is the parameter that feed formula at first need be understood always.Yet, measure the metabolizable energy of feedstuff and can digest amino acid no with the biology method according to consuming time, consumption power, expensive, become the major technique bottleneck that the feedstuff basic data is upgraded in the production.For a long time, the various countries nutritionist attempts to set up a kind of quantitative determination fodder energy through simulating feed digestion in animal body, amino acid bio is learned the technical system of tiring, to realize the real-time monitoring to the feed nutrient biological value.But this technical system is not really set up on industry all the time, and is at the experimental stage always.
Since the nineties in last century; American-European scholar in the preparation of poultry simulation intestinal fluid be according to certain hour in the percent hydrolysis of feed needed minimum pig pancreatin (enzyme) amount is confirmed when reaching maximum that (document sees reference: Valdes; E.V., S.Leeson.Measurement of metabolizable energy in poultry feeds by an in vitro system, Poultry Science; 1992,71:1493-1503).Simulation intestinal fluid by this method prepares not only is difficult to repeat fully on the digestion activity, but also has broken away from the physiological level of digestive enzyme activity in the interior unit volume intestinal juice of animal body.In addition, this method adopts SILVER REAGENT pig pancreatin on the source of enzyme, thus cause digestive ferment enzymatic property maybe with corresponding digestive ferment in poultry body homology not.
In recent years, some scholars transfer to the breakthrough point again on the basic theory of digestive physiology in the preparation of animal simulation digestive juice, with the research object that is prepared as of poultry intestinal fluid, have obtained respond well simulation poultry intestinal fluid.But; What the preparation method of this simulation poultry intestinal fluid adopted is the single digestive ferment of SILVER REAGENT; And mostly digestive ferment is pig source property or microbe-derived; Therefore exist the not problem of homology of enzymatic property, and implementation cost is very high, is difficult to herding enterprise and carries out the cost accounting that the feed nutrient biological value detects and accept.Therefore, opening up new approach, to prepare poultry simulation intestinal fluid imperative.
Summary of the invention
The object of the present invention is to provide a kind of preparation method of duck simulation intestinal fluid; This method implementation cost is low; Production technology is efficiently quick, can prepare the duck simulation intestinal fluid with the interior intestinal fluid AMS of true duck body, lipase, trypsase, 4 kinds of basic homologies of digestive ferment of chymotrypsin.
To achieve these goals, the present invention has adopted following technical scheme:
A kind of preparation method of duck simulation intestinal fluid, it is characterized in that: this method comprises the steps:
Step 1: the duck small intestine chyme that will come with collection the duck small intestine of duck from the system liquid of setting quantity is put into a reagent bottle and is shaken up; The potpourri that obtains after shaking up is put into a centrifugal bottle; Under design temperature, carry out centrifugal treating through hydro-extractor; After centrifugal treating finishes, collect the supernatant in this centrifugal bottle;
Step 2: behind the supernatant liquid filtering that step 1 is obtained, become mixed concentrated liquid through the freeze drier freeze concentration;
Step 3: the mixed concentrated liquid that step 2 is obtained is after impurity is removed in the bag filter dialysis, and freeze drying becomes freeze-dried powder;
Step 4: the activity of AMS, lipase, trypsase, chymotrypsin in the freeze-dried powder that obtains of determination step three respectively, and measure chymotrypsin and tryptic activity in the activity, SILVER REAGENT chymotrypsin of trypsase and chymotrypsin in the activity, SILVER REAGENT trypsase of lipase in the activity, SILVER REAGENT lipase of AMS in the SILVER REAGENT AMS;
Step 5: the principle that equates according to each digestive ferment gross activity in duck simulation intestinal fluid and the target duck intestinal fluid; The SILVER REAGENT AMS, the SILVER REAGENT lipase of corresponding weight, the SILVER REAGENT trypsase of corresponding weight, the SILVER REAGENT chymotrypsin of corresponding weight and the WS of respective volume that in the freeze-dried powder of setting weight, add respective volume; Preparing duck simulation intestinal fluid, the activity of AMS, lipase, trypsase, chymotrypsin equates with the activity of AMS, lipase, trypsase, chymotrypsin in the target duck intestinal fluid respectively in this duck of preparing simulation intestinal fluid.
Advantage of the present invention is:
The activity (hydrolysis ability) of 4 kinds of main digestive ferments (AMS, trypsase, chymotrypsin, lipase) is the same with the duck intestinal fluid of reality in duck that the present invention the prepares simulation intestinal fluid, can simulate the digestive juice that feed digests in the duck small intestine as a kind of.
The present invention is not to use single SILVER REAGENT digestive ferment, and (mostly the SILVER REAGENT digestive ferment is pig source property or microbe-derived; Have the different source problems of enzymatic property) prepare duck simulation intestinal fluid; But mainly use the 4 kinds of main digestive ferments (AMS, trypsase, chymotrypsin, lipase) in the intestinal fluid of true duck to be equipped with SILVER REAGENT digestive ferment (SILVER REAGENT AMS, SILVER REAGENT lipase, SILVER REAGENT trypsase, SILVER REAGENT chymotrypsin; Contained digestive ferment is active minimum in the SILVER REAGENT digestive ferment) prepare duck simulation intestinal fluid, make the duck of preparing simulate the enzymatic property and the interior basic homology of intestinal fluid of actual duck body of intestinal fluid like this.
It is primary raw material that the present invention adopts the duck intestinal fluid freeze-dried powder of purification, greatly reduces production cost.The present invention can control the digestion power of duck simulation intestinal fluid through the activity of 4 kinds of main digestive ferments of control (AMS, trypsase, chymotrypsin, lipase), thereby makes that can simulate intestinal fluid to duck carries out standardized production.Implementation cost of the present invention is low, and production technology is efficient, quick, can carry out the large-scale production of duck simulation intestinal fluid.
Description of drawings
Fig. 1 is realization flow figure of the present invention.
Embodiment
As shown in Figure 1, the preparation method of duck simulation intestinal fluid of the present invention comprises the steps one to five:
Step 1: the duck small intestine chyme that will come with collection the duck small intestine of duck from the system liquid of setting quantity is put into a reagent bottle and is shaken up; The potpourri that obtains after shaking up is put into a centrifugal bottle (as selecting 250 milliliters of centrifugal bottles for use); Under design temperature (as 4 ℃), carry out centrifugal treating through hydro-extractor; After centrifugal treating finishes, collect the supernatant in this centrifugal bottle;
Step 2: behind the supernatant liquid filtering that step 1 is obtained, become mixed concentrated liquid through the freeze drier freeze concentration;
Step 3: the mixed concentrated liquid that step 2 is obtained is after impurity is removed in the bag filter dialysis, and freeze drying becomes freeze-dried powder;
Step 4: the activity (activity of unit weight) of AMS, lipase, trypsase, chymotrypsin in the freeze-dried powder (can be described as the intestinal juice pulvis again) that obtains of determination step three respectively, and measure chymotrypsin and tryptic activity (activity of unit weight) in the activity (activity of unit weight), SILVER REAGENT chymotrypsin of trypsase and chymotrypsin in the activity (activity of unit weight), SILVER REAGENT trypsase of lipase in activity (activity of unit milliliter), SILVER REAGENT lipase of AMS in the employed SILVER REAGENT AMS of this preparation duck simulation intestinal fluid;
Step 5: the principle that equates according to each digestive ferment gross activity in duck simulation intestinal fluid and the target duck intestinal fluid; The SILVER REAGENT AMS that in the freeze-dried powder of setting weight (being generally 3～9 grams gets final product), adds respective volume (can be 0 according to circumstances; Promptly do not add the SILVER REAGENT AMS; Analyze and to state as follows), the SILVER REAGENT lipase (can be 0 according to circumstances, promptly do not add SILVER REAGENT lipase, analyze and state as follows) of corresponding weight, the SILVER REAGENT trypsase of corresponding weight (can be 0 according to circumstances; Promptly do not add SILVER REAGENT trypsase; Analyze and to state as follows), the SILVER REAGENT chymotrypsin (can be 0 according to circumstances, promptly do not add the SILVER REAGENT chymotrypsin, analyze and state as follows) of corresponding weight and the WS (the optional deionized water of using) of respective volume; Preparing duck simulation intestinal fluid, and the activity of AMS, lipase, trypsase, chymotrypsin equates with the activity of AMS, lipase, trypsase, chymotrypsin in the target duck intestinal fluid respectively in this duck of preparing simulation intestinal fluid.
Among the present invention involved duck preferably the age surpasses 6 ages in week and body weight the duck more than 2 kilograms.For example, the duck preferred age of using in the step 1 surpasses 6 ages in week and body weight the duck more than 2 kilograms, if duck is too little, with regard to bad collection duck small intestine chyme.
In step 1; From the duck small intestine, gathering duck small intestine chyme can realize through following method: duck is undergone surgery; Poultry enteron aisle chyme is installed in the small intestine of duck is gathered sleeve pipe (is the Chinese utility model patent " poultry enteron aisle chyme is gathered sleeve pipe " of ZL201020257424.3 referring to the patent No.); After treating surgery recovery tens to tens days (as 20 days), gather sleeve pipe through poultry enteron aisle chyme duck small intestine chyme is collected in the sample bottle, sample bottle preferably is under 4～10 ℃ the cryogenic conditions.In addition, for step 1, in reality was implemented, it was centrifugal to adopt Sorvall RC-5C Plus hydro-extractor that the potpourri that obtains after shaking up is carried out 1250g, and the time of centrifugal treating was controlled at 5～10 minutes.
In step 2, supernatant can filter via 200 order nylon filter clothes, and freeze drier is to the supernatant freeze concentration certain hour (being generally several hours to tens hours) after filtering, so that its freeze concentration becomes mixed concentrated liquid more than 1 times.
In step 3; The molecular cut off of bag filter is below 20000 dalton; Mixed concentrated liquid is successively via pH value dialyse at the phosphate buffer between 7.0～8.4, deionized water (certain hour of in phosphate buffer, deionized water, dialysing respectively); Perhaps, mixed concentrated liquid is successively via pH value dialyse at the phosphate buffer between 7.0～8.4, distilled water (certain hour of in phosphate buffer, distilled water, dialysing respectively).Mixed concentrated liquid just can be removed the impurity in it after dialysis, like impurity such as small molecular proteins, then, behind the mixed concentrated liquid freeze drying certain hour with dialysis removal impurity, just can obtain freeze-dried powder.
For example, the molecular cut off of bag filter can be 20000 dalton, and the pH value of phosphate buffer can be 7.0.Again for example, the molecular cut off of bag filter can be 14400 dalton, and the pH value of phosphate buffer can be 8.4.Again for example, the molecular cut off of bag filter can be 100 dalton, and the pH value of phosphate buffer can be 8.0.It should be noted that; The molecular cut off of bag filter is controlled at can reach the purpose of removing impurity below 20000 dalton; If the molecular cut off of bag filter greater than 20000 dalton, then can cause a large amount of losses of digestive ferment when removing impurity, be worthless.
For step 5; The principle that each digestive ferment gross activity equates in duck simulation intestinal fluid and the target duck intestinal fluid is meant that the duck of preparing simulates the activity (in every ml soln) of AMS, lipase, trypsase, chymotrypsin in the intestinal fluid and should be respectively equate with the activity (in every ml soln) of AMS, lipase, trypsase, chymotrypsin in the target duck intestinal fluid, could simulate the digestion situation that intestinal fluid comes the simulated target duck with duck like this.
The activity of AMS, lipase, trypsase, chymotrypsin can use duck to draw (the active mean value that draws above-mentioned 4 kinds of digestive ferments) as the determination of activity that object carries out AMS, lipase, trypsase, chymotrypsin respectively with the ownership system liquid of using in the step 1 in the above-mentioned target duck intestinal fluid; The target duck that the duck of preparing like this, simulation intestinal fluid is simulated just is meant that the system liquid of using in the step 1 uses duck.
In addition; The activity of AMS, lipase, trypsase, chymotrypsin can be that the determination of activity that object carries out AMS, lipase, trypsase, chymotrypsin respectively draws (the active mean value that draws above-mentioned 4 kinds of digestive ferments through a large amount of experiments) with a large amount of ducks also in the above-mentioned target duck intestinal fluid; Like this, the target duck that the duck simulation intestinal fluid of preparing is simulated just can refer to any duck.Through being that the average activity of AMS, lipase, trypsase, chymotrypsin in the target duck intestinal fluid that determines of object is respectively 401.56U/ml (active unit/milliliter), 1.10U/ml, 108.75U/ml, 39.01U/ml with a large amount of ducks.
No matter be duck intestinal fluid, freeze-dried powder are carried out the determination of activity of AMS, lipase, trypsase, chymotrypsin; Still chymotrypsin and trypsase in trypsase and chymotrypsin, the SILVER REAGENT chymotrypsin in lipase, the SILVER REAGENT trypsase in AMS, the SILVER REAGENT lipase in the SILVER REAGENT AMS are carried out determination of activity; In a word; Determination of activity to AMS, lipase, trypsase, chymotrypsin is known technology; Wherein: but the associated description in the determination of activity list of references of AMS " Dahlqvist A.A method for the determination of amylase in intestinal content [J] .Scandinavian Journal of Clinical and Laboratory Investigation; 1962,14:145-151 "; The determination of activity of lipase can be adopted the lipase kit measurement method of German DiaSys company; But the associated description in the tryptic determination of activity list of references " Wirnt R.Trypsin, measurement with n α-p-toluenesulfonyl-l-arginine methyl ester as substrate [A] .In:Bergmeyer H U.Methods of enzymatic analysis [M] .Weinheinm:Verlag chemie.1974 "; But the associated description in the determination of activity list of references of chymotrypsin " Wirnt R.Chymotrypsin, measurements with n-benzoyl-l-tyrosin ethyl ester as substrate [A] .In:Bergmeyer H U.Methods of enzymatic analysis [M] .Weinheinm:Verlag chemie.1974 ".
In duck simulation intestinal fluid; The activity of the AMS that freeze-dried powder provides, lipase, trypsase, chymotrypsin is main; The activity of the corresponding digestive ferment that SILVER REAGENT AMS, SILVER REAGENT lipase, SILVER REAGENT trypsase and SILVER REAGENT chymotrypsin provide respectively is very little, is auxilliary.And,, just need not add reagent corresponding level digestive ferment for active satisfactory digestive ferment in the freeze-dried powder.For example; In the freeze-dried powder AMS, chymotrypsin and tryptic activity respectively with target duck intestinal fluid in AMS, chymotrypsin, tryptic activity equate; Just the activity of lipase does not reach the words of the activity of lipase in the target duck intestinal fluid; Only need to add an amount of SILVER REAGENT lipase in addition, the activity that the activity that makes the duck that makes simulate lipase in the intestinal fluid equals lipase in the target duck intestinal fluid gets final product.That is to say; Duck that the present invention prepares simulation intestinal fluid is to replenish an amount of corresponding reagent level digestive ferment (any in SILVER REAGENT AMS, SILVER REAGENT lipase, SILVER REAGENT trypsase or the SILVER REAGENT chymotrypsin or appoint several kinds, and the digestive enzyme activity value in the corresponding reagent level digestive ferment that replenishes is very little) on the basis of the 4 kinds of main digestive ferments (AMS, trypsase, chymotrypsin, lipase) in the intestinal fluid of true duck to prepare duck simulation intestinal fluid.
Embodiment one
20 8 week age, average weight poultry enteron aisle chyme collection sleeve pipe is installed at the system liquid more than 3.0 kilograms in the duck small intestine of duck; Treat that surgery recovery is after 20 days; Gather sleeve pipe through poultry enteron aisle chyme and gather duck small intestine chyme, 3 liters duck small intestine chyme is collected into and is in 4 ℃ of sample bottles under the cryogenic conditions.
3 liters of duck small intestine chymes collecting are put into reagent bottle to be shaken up; The potpourri that obtains after shaking up is put into 250 milliliters of centrifugal bottles; Under 4 ℃ of conditions, carry out 10 minutes 1250g centrifugal treating through hydro-extractor, centrifugal treating finishes the back and collects the supernatant in the centrifugal bottle.
With the supernatant that obtains after 200 order nylon filter clothes filter, again through freeze drier freeze concentration 12 hours, so that 1.5 times of supernatant freeze concentration become required mixed concentrated liquid.
It is in the 14400 daltonian bag filters that the mixed concentrated liquid that obtains is transferred to molecular cut off; In the phosphate buffer of pH7.0, dialysed 2 hours earlier; In deionized water, dialysed 2 hours then; Behind the impurity such as removing small molecular protein of dialysing, freeze drying is 48 hours again, obtains freeze-dried powder.
According to the activity determination method of above-mentioned AMS, lipase, trypsase, 4 kinds of digestive ferments of chymotrypsin, the activity of measuring 4 kinds of main digestive ferments in the above-mentioned freeze-dried powder that obtains is: AMS, 9.93U/mg (active unit/milligram); Lipase, 0.012U/mg; Trypsase, 3.51U/mg; Chymotrypsin, 0.63U/mg.The activity of measuring the AMS that contains in the employed SILVER REAGENT AMS of this preparation duck simulation intestinal fluid is 22947.41U/ml; The activity of the lipase that contains in the SILVER REAGENT lipase is 3.27U/mg; The tryptic activity that contains in the SILVER REAGENT trypsase is 170.87U/mg; And the activity of the chymotrypsin that contains is 0.67U/mg, and the activity of the chymotrypsin that contains in the SILVER REAGENT chymotrypsin is 63.31U/mg, and the tryptic activity that contains is 2.34U/mg.
This used 20 of preparation duck simulation intestinal fluid is made liquid with the determination of activity object of duck as AMS, lipase, trypsase, chymotrypsin in the target duck intestinal fluid; Measuring these 20 system liquid uses the active mean value of 4 kinds of main digestive ferments in the intestinal fluid in the duck body to be: AMS, 460.92U/ml; Lipase, 0.18U/ml; Trypsase, 133.44U/ml; Chymotrypsin, 35.01U/ml.The principle that equates according to each digestive ferment gross activity in duck simulation intestinal fluid and the target duck intestinal fluid; The deionized water that in 3.78 gram freeze-dried powders, adds 3.39 milliliters SILVER REAGENT AMS, 0.12 gram SILVER REAGENT trypsase, 0.10 gram SILVER REAGENT chymotrypsin and respective volume; Finally obtain 250 milliliters of duck simulation intestinal fluids and (simulate in intestinal fluid AMS 460.92U/ml, lipase 0.18U/ml this duck; Trypsase 133.44U/ml, chymotrypsin 35.01U/ml.); The activity of AMS, lipase, trypsase, chymotrypsin equates with the active mean value of used 20 the system liquid of this preparation duck simulation intestinal fluid with the AMS in the duck body, lipase, trypsase, chymotrypsin respectively in this duck simulation intestinal fluid, can be used to simulate the digestion power of these 20 system liquid with duck.
Checking: in monogastric animal bionic digestive system (being the associated description in 200910078147.1 the Chinese invention patent application " monogastric animal bionic digestive system and based on the method for this system simulation nonruminant digestion " referring to number of patent application); With 100 milliliters of intestinal fluids in the duck body 10 gram corns are carried out 15 hours digestion with 100 milliliters of duck simulation intestinal fluids that make, used 20 the system liquid of this duck simulation intestinal fluid of this preparation respectively; The digestion experiment result is merely 0.07% with the intestinal fluid in the duck body to the digestibility difference of the dry of corn for this duck simulation intestinal fluid and 20 system liquid, and this has just confirmed that the duck simulation intestinal fluid of preparing can substitute these 20 fully and make liquid and simulate these 20 digestion powers of making liquid with duck with duck.
Embodiment two
30 12 week age, average weight poultry enteron aisle chyme collection sleeve pipe is installed at the system liquid more than 3.2 kilograms in the duck small intestine of duck; Treat that surgery recovery is after 25 days; Gather sleeve pipe through poultry enteron aisle chyme and gather duck small intestine chyme, 5 liters duck small intestine chyme is collected into and is in 8 ℃ of sample bottles under the cryogenic conditions.
5 liters of duck small intestine chymes collecting are put into reagent bottle to be shaken up; The potpourri that obtains after shaking up is put into 250 milliliters of centrifugal bottles; Under 4 ℃ of conditions, carry out 8 minutes 1250g centrifugal treating through hydro-extractor, centrifugal treating finishes the back and collects the supernatant in the centrifugal bottle.
With the supernatant that obtains after 200 order nylon filter clothes filter, again through freeze drier freeze concentration 36 hours, so that 10 times of supernatant freeze concentration become required mixed concentrated liquid.
It is in the 12400 daltonian bag filters that the mixed concentrated liquid that obtains is transferred to molecular cut off; In the phosphate buffer of pH7.5, dialysed 2 hours earlier; In deionized water, dialysed 2 hours then; Behind the impurity such as removing small molecular protein of dialysing, freeze drying is 48 hours again, obtains freeze-dried powder.
According to the activity determination method of above-mentioned AMS, lipase, trypsase, 4 kinds of digestive ferments of chymotrypsin, the activity of measuring 4 kinds of main digestive ferments in the above-mentioned freeze-dried powder that obtains is: AMS, 10.25U/mg; Lipase, 0.015U/mg; Trypsase, 4.12U/mg; Chymotrypsin, 0.84U/mg.The activity of measuring the AMS that contains in the employed SILVER REAGENT AMS of this preparation duck simulation intestinal fluid is 22947.41U/ml; The activity of the lipase that contains in the SILVER REAGENT lipase is 3.27U/mg; The tryptic activity that contains in the SILVER REAGENT trypsase is 170.87U/mg; And the activity of the chymotrypsin that contains is 0.67U/mg, and the activity of the chymotrypsin that contains in the SILVER REAGENT chymotrypsin is 63.31U/mg, and the tryptic activity that contains is 2.34U/mg.
This used 30 of preparation duck simulation intestinal fluid is made liquid with the determination of activity object of duck as AMS, lipase, trypsase, chymotrypsin in the target duck intestinal fluid; Measuring these 30 system liquid uses the active mean value of 4 kinds of main digestive ferments in the intestinal fluid in the duck body to be: AMS, 420.53U/ml; Lipase, 0.20U/ml; Trypsase, 130.24U/ml; Chymotrypsin, 41.50U/ml.The principle that equates according to each digestive ferment gross activity in duck simulation intestinal fluid and the target duck intestinal fluid; The deionized water that in 3.33 gram freeze-dried powders, adds 3.09 milliliters SILVER REAGENT AMS, 0.11 gram SILVER REAGENT trypsase, 0.12 gram SILVER REAGENT chymotrypsin and respective volume; Finally obtain 250 milliliters of duck simulation intestinal fluids and (simulate in intestinal fluid AMS 420.53U/ml, lipase 0.20U/ml this duck; Trypsase 130.24U/ml, chymotrypsin 41.50U/ml.); The activity of AMS, lipase, trypsase, chymotrypsin equates with the active mean value of used 30 the system liquid of this preparation duck simulation intestinal fluid with the AMS in the duck body, lipase, trypsase, chymotrypsin respectively in this duck simulation intestinal fluid, can be used to simulate the digestion power of these 30 system liquid with duck.
Checking: in the monogastric animal bionic digestive system; With 100 milliliters of intestinal fluids in the duck body 5 gram dregs of beans are carried out 15 hours digestion with 100 milliliters of duck simulation intestinal fluids that make, used 30 the system liquid of this duck simulation intestinal fluid of this preparation respectively; The digestion experiment result is merely 1.02% with the intestinal fluid in the duck body to the digestibility difference of the dry of dregs of beans for this duck simulation intestinal fluid and 30 system liquid, and this has just confirmed that the duck simulation intestinal fluid of preparing can substitute these 30 fully and make liquid and simulate these 30 digestion powers of making liquid with duck with duck.
Embodiment three
60 18 week age, average weight poultry enteron aisle chyme collection sleeve pipe is installed at the system liquid more than 3.2 kilograms in the duck small intestine of duck; Treat that surgery recovery is after 30 days; Gather sleeve pipe through poultry enteron aisle chyme and gather duck small intestine chyme, 5 liters duck small intestine chyme is collected into and is in 5 ℃ of sample bottles under the cryogenic conditions.
5 liters of duck small intestine chymes collecting are put into reagent bottle to be shaken up; The potpourri that obtains after shaking up is put into 250 milliliters of centrifugal bottles; Under 4 ℃ of conditions, carry out 5 minutes 1250g centrifugal treating through hydro-extractor, centrifugal treating finishes the back and collects the supernatant in the centrifugal bottle.
With the supernatant that obtains after 200 order nylon filter clothes filter, again through freeze drier freeze concentration 48 hours, so that 20 times of supernatant freeze concentration become required mixed concentrated liquid.
It is in the 14400 daltonian bag filters that the mixed concentrated liquid that obtains is transferred to molecular cut off; In the phosphate buffer of pH7.8, dialysed 2 hours earlier, in distilled water, dialysed 2 hours then, behind the impurity such as removing small molecular protein of dialysing; Freeze drying is 48 hours again, obtains freeze-dried powder.
According to the activity determination method of above-mentioned AMS, lipase, trypsase, 4 kinds of digestive ferments of chymotrypsin, the activity of measuring 4 kinds of main digestive ferments in the above-mentioned freeze-dried powder that obtains is: AMS, 12.04U/mg; Lipase, 0.015U/mg; Trypsase, 3.85U/mg; Chymotrypsin, 0.78U/mg.The activity of measuring the AMS that contains in the employed SILVER REAGENT AMS of this preparation duck simulation intestinal fluid is 22947.41U/ml; The activity of the lipase that contains in the SILVER REAGENT lipase is 3.27U/mg; The tryptic activity that contains in the SILVER REAGENT trypsase is 170.87U/mg; And the activity of the chymotrypsin that contains is 0.67U/mg, and the activity of the chymotrypsin that contains in the SILVER REAGENT chymotrypsin is 63.31U/mg, and the tryptic activity that contains is 2.34U/mg.
This used 60 of preparation duck simulation intestinal fluid is made liquid with the determination of activity object of duck as AMS, lipase, trypsase, chymotrypsin in the target duck intestinal fluid; Measuring these 60 system liquid uses the active mean value of 4 kinds of main digestive ferments in the intestinal fluid in the duck body to be: AMS, 410.25U/ml; Lipase, 0.19U/ml; Trypsase, 125.50U/ml; Chymotrypsin, 36.47U/ml.The principle that equates according to each digestive ferment gross activity in duck simulation intestinal fluid and the target duck intestinal fluid; The deionized water that in 3.15 gram freeze-dried powders, adds 2.88 milliliters SILVER REAGENT AMS, 0.13 gram SILVER REAGENT trypsase, 0.08 gram SILVER REAGENT lipase and respective volume; Finally obtain 250 milliliters of duck simulation intestinal fluids and (simulate in intestinal fluid AMS 410.25U/ml, lipase 0.19U/ml this duck; Trypsase 125.50U/ml, chymotrypsin 36.47U/ml.); The activity of AMS, lipase, trypsase, chymotrypsin equates with the active mean value of used 60 the system liquid of this preparation duck simulation intestinal fluid with the AMS in the duck body, lipase, trypsase, chymotrypsin respectively in this duck simulation intestinal fluid, can be used to simulate the digestion power of these 60 system liquid with duck.
Checking: in the monogastric animal bionic digestive system; With 100 milliliters of intestinal fluids in the duck body 10 gram corn-dregs of beans daily rations are carried out 15 hours digestion with 100 milliliters of duck simulation intestinal fluids that make, used 60 the system liquid of this duck simulation intestinal fluid of this preparation respectively; The digestion experiment result is merely 1.07% with the intestinal fluid in the duck body to the dry digestibility difference of corn-dregs of beans daily ration for this duck simulation intestinal fluid and 60 system liquid, and this has just confirmed that the duck simulation intestinal fluid of preparing can substitute these 60 fully and make liquid and simulate these 60 digestion powers of making liquid with duck with duck.
Embodiment four
25 15 week age, average weight poultry enteron aisle chyme collection sleeve pipe is installed at the system liquid more than 3.0 kilograms in the duck small intestine of duck; Treat that surgery recovery is after 30 days; Gather sleeve pipe through poultry enteron aisle chyme and gather duck small intestine chyme, 4 liters duck small intestine chyme is collected into and is in 4 ℃ of sample bottles under the cryogenic conditions.
4 liters of duck small intestine chymes collecting are put into reagent bottle to be shaken up; The potpourri that obtains after shaking up is put into 250 milliliters of centrifugal bottles; Under 4 ℃ of conditions, carry out 8 minutes 1250g centrifugal treating through hydro-extractor, centrifugal treating finishes the back and collects the supernatant in the centrifugal bottle.
With the supernatant that obtains after 200 order nylon filter clothes filter, again through freeze drier freeze concentration 30 hours, so that 9 times of supernatant freeze concentration become required mixed concentrated liquid.
It is in the 14400 daltonian bag filters that the mixed concentrated liquid that obtains is transferred to molecular cut off; In the phosphate buffer of pH7.9, dialysed 2 hours earlier; In deionized water, dialysed 2 hours then; Behind the impurity such as removing small molecular protein of dialysing, freeze drying is 48 hours again, obtains freeze-dried powder.
According to the activity determination method of above-mentioned AMS, lipase, trypsase, 4 kinds of digestive ferments of chymotrypsin, the activity of measuring 4 kinds of main digestive ferments in the above-mentioned freeze-dried powder that obtains is: AMS, 11.02U/mg; Lipase, 0.016U/mg; Trypsase, 4.23U/mg; Chymotrypsin, 0.90U/mg.The activity of measuring the AMS that contains in the employed SILVER REAGENT AMS of this preparation duck simulation intestinal fluid is 22947.41U/ml; The activity of the lipase that contains in the SILVER REAGENT lipase is 3.27U/mg; The tryptic activity that contains in the SILVER REAGENT trypsase is 170.87U/mg; And the activity of the chymotrypsin that contains is 0.67U/mg, and the activity of the chymotrypsin that contains in the SILVER REAGENT chymotrypsin is 63.31U/mg, and the tryptic activity that contains is 2.34U/mg.
This used 25 of preparation duck simulation intestinal fluid is made liquid with the determination of activity object of duck as AMS, lipase, trypsase, chymotrypsin in the target duck intestinal fluid; Measuring these 25 system liquid uses the active mean value of 4 kinds of main digestive ferments in the intestinal fluid in the duck body to be: AMS, 440.50U/ml; Lipase, 0.205U/ml; Trypsase, 130.29U/ml; Chymotrypsin, 35.08U/ml.The principle that equates according to each digestive ferment gross activity in duck simulation intestinal fluid and the target duck intestinal fluid; The deionized water that in 3.20 gram freeze-dried powders, adds 3.26 milliliters SILVER REAGENT AMS, 0.11 gram SILVER REAGENT trypsase, 0.09 gram SILVER REAGENT chymotrypsin and respective volume; Finally obtain 250 milliliters of duck simulation intestinal fluids and (simulate in intestinal fluid AMS 440.50U/ml, lipase 0.205U/ml this duck; Trypsase 130.29U/ml, chymotrypsin 35.08U/ml.); The activity of AMS, lipase, trypsase, chymotrypsin equates with the active mean value of used 25 the system liquid of this preparation duck simulation intestinal fluid with the AMS in the duck body, lipase, trypsase, chymotrypsin respectively in this duck simulation intestinal fluid, can be used to simulate the digestion power of these 25 system liquid with duck.
Checking: in the monogastric animal bionic digestive system; With 100 milliliters of intestinal fluids in the duck body 5 gram cotton dregs are carried out 15 hours digestion with 100 milliliters of duck simulation intestinal fluids that make, used 25 the system liquid of this duck simulation intestinal fluid of this preparation respectively; The digestion experiment result is merely 0.53% with the intestinal fluid in the duck body to the digestibility difference of the dry of cotton dregs for this duck simulation intestinal fluid and 25 system liquid, and this has just confirmed that the duck simulation intestinal fluid of preparing can substitute these 25 fully and make liquid and simulate these 25 digestion powers of making liquid with duck with duck.
Embodiment five
40 10 week age, average weight poultry enteron aisle chyme collection sleeve pipe is installed at the system liquid more than 3.0 kilograms in the duck small intestine of duck; Treat that surgery recovery is after 40 days; Gather sleeve pipe through poultry enteron aisle chyme and gather duck small intestine chyme, 8 liters duck small intestine chyme is collected into and is in 10 ℃ of sample bottles under the cryogenic conditions.
8 liters of duck small intestine chymes collecting are put into reagent bottle to be shaken up; The potpourri that obtains after shaking up is put into 250 milliliters of centrifugal bottles; Under 4 ℃ of conditions, carry out 10 minutes 1250g centrifugal treating through hydro-extractor, centrifugal treating finishes the back and collects the supernatant in the centrifugal bottle.
With the supernatant that obtains after 200 order nylon filter clothes filter, again through freeze drier freeze concentration 24 hours, so that 4 times of supernatant freeze concentration become required mixed concentrated liquid.
It is in the 12400 daltonian bag filters that the mixed concentrated liquid that obtains is transferred to molecular cut off; In the phosphate buffer of pH8.0, dialysed 2 hours earlier, in distilled water, dialysed 2 hours then, behind the impurity such as removing small molecular protein of dialysing; Freeze drying is 48 hours again, obtains freeze-dried powder.
According to the activity determination method of above-mentioned AMS, lipase, trypsase, 4 kinds of digestive ferments of chymotrypsin, the activity of measuring 4 kinds of main digestive ferments in the above-mentioned freeze-dried powder that obtains is: AMS, 15.18U/mg; Lipase, 0.012U/mg; Trypsase, 5.14U/mg; Chymotrypsin, 0.75U/mg.The activity of measuring the AMS that contains in the employed SILVER REAGENT AMS of this preparation duck simulation intestinal fluid is 22947.41U/ml; The activity of the lipase that contains in the SILVER REAGENT lipase is 3.27U/mg; The tryptic activity that contains in the SILVER REAGENT trypsase is 170.87U/mg; And the activity of the chymotrypsin that contains is 0.67U/mg, and the activity of the chymotrypsin that contains in the SILVER REAGENT chymotrypsin is 63.31U/mg, and the tryptic activity that contains is 2.34U/mg.
This used 40 of preparation duck simulation intestinal fluid is made liquid with the determination of activity object of duck as AMS, lipase, trypsase, chymotrypsin in the target duck intestinal fluid; Measuring these 40 system liquid uses the active mean value of 4 kinds of main digestive ferments in the intestinal fluid in the duck body to be: AMS, 455.20U/ml; Lipase, 0.204U/ml; Trypsase, 148.40U/ml; Chymotrypsin, 42.52U/ml.The principle that equates according to each digestive ferment gross activity in duck simulation intestinal fluid and the target duck intestinal fluid; The deionized water that in 4.25 gram freeze-dried powders, adds 2.15 milliliters SILVER REAGENT AMS, 0.09 gram SILVER REAGENT trypsase, 0.12 gram SILVER REAGENT chymotrypsin and respective volume; Finally obtain 250 milliliters of duck simulation intestinal fluids (in this duck simulation intestinal fluid; AMS, 455.20U/ml; Lipase, 0.204U/ml; Trypsase, 148.40U/ml; Chymotrypsin, 42.52U/ml.); The activity of AMS, lipase, trypsase, chymotrypsin equates with the active mean value of used 40 the system liquid of this preparation duck simulation intestinal fluid with the AMS in the duck body, lipase, trypsase, chymotrypsin respectively in this duck simulation intestinal fluid, can be used to simulate the digestion power of these 40 system liquid with duck.
Checking: in the monogastric animal bionic digestive system; With 100 milliliters of intestinal fluids in the duck body 10 gram corn-dregs of beans daily rations are carried out 15 hours digestion with 100 milliliters of duck simulation intestinal fluids that make, used 40 the system liquid of this duck simulation intestinal fluid of this preparation respectively; The digestion experiment result is merely 0.85% with the intestinal fluid in the duck body to the dry digestibility difference of corn-dregs of beans daily ration for this duck simulation intestinal fluid and 40 system liquid, and this has just confirmed that the duck simulation intestinal fluid of preparing can substitute these 40 fully and make liquid and simulate these 40 digestion powers of making liquid with duck with duck.
At present; In the poultry small intestine, can detect and understand about 19 kinds of the digestive ferment of its zymetology fundamental characteristics through effective ways, AMS wherein, trypsase, chymotrypsin proportion just account for 60% (referring to the description in the document " Pubols; M.H.Ratio of enzymes in the chick pancreas [J]; Poultry Science, 1991,70:337-342 ") of whole protein content.And; From secretory volume and hydrolysis ability; The per hour secretory volume of AMS, trypsase, chymotrypsin, lipase is respectively 7235.74U, 1780.43U, 811.43U, 20.30U in the duck intestinal fluid, hydrolyzable 140g starch, 19.9g protein, 0.3g fat.And the summation of starch, protein and fatty three's content accounts for more than 80% of total amount of organic in the daily ration.Can see according to above data; The secretion of the enteral AMS of duck, trypsase, chymotrypsin, these four kinds of digestive ferments of lipase and hydrolysing activity fully can hydrolysate feed in most organism; Therefore; As long as in the duck of the preparation simulation intestinal fluid AMS, trypsase, chymotrypsin, these four kinds of digestive ferments of lipase are arranged; And the activity that makes AMS in the duck simulation intestinal fluid, trypsase, chymotrypsin, these four kinds of digestive ferments of lipase respectively with the duck intestinal fluid of reality (the system liquid that refers to set quantity is with duck or duck arbitrarily) in AMS, trypsase, chymotrypsin, lipase active the same, just can simulate intestinal fluid and simulate the digestion power of duck (the system liquid that refers to set quantity is with duck or any duck) various daily rations with duck.And; Can see from above-mentioned each embodiment; The duck simulation intestinal fluid that the present invention prepares is to the very approaching actual duck intestinal fluid of the digestibility of various daily rations; Digestibility difference is minimum, that is to say, the duck simulation intestinal fluid that the present invention prepares can substitute actual duck intestinal fluid fully and come the digestion power of various daily rations is experimentized.
In the present invention, hydro-extractor, freeze drier, bag filter etc. are known device, and the computing method of processing such as centrifugal, the freeze drying that relates to, dialysis and solid-liquid conversion are known technology.
Advantage of the present invention is:
The activity (hydrolysis ability) of 4 kinds of main digestive ferments (AMS, trypsase, chymotrypsin, lipase) is the same with the duck intestinal fluid of reality in duck that the present invention the prepares simulation intestinal fluid, can simulate the digestive juice that feed digests in the duck small intestine as a kind of.
The present invention is not to use single SILVER REAGENT digestive ferment, and (mostly the SILVER REAGENT digestive ferment is pig source property or microbe-derived; Have the different source problems of enzymatic property) prepare duck simulation intestinal fluid; But mainly use the 4 kinds of main digestive ferments (AMS, trypsase, chymotrypsin, lipase) in the intestinal fluid of true duck to be equipped with SILVER REAGENT digestive ferment (SILVER REAGENT AMS, SILVER REAGENT lipase, SILVER REAGENT trypsase, SILVER REAGENT chymotrypsin; Contained digestive ferment is active minimum in the SILVER REAGENT digestive ferment) prepare duck simulation intestinal fluid, make the duck of preparing simulate the enzymatic property and the interior basic homology of intestinal fluid of actual duck body of intestinal fluid like this.
It is primary raw material that the present invention adopts the duck intestinal fluid freeze-dried powder of purification, greatly reduces production cost.
The present invention can control the digestion power of duck simulation intestinal fluid through the activity of 4 kinds of main digestive ferments of control (AMS, trypsase, chymotrypsin, lipase), thereby makes that can simulate intestinal fluid to duck carries out standardized production.
Implementation cost of the present invention is low, and production technology is efficient, quick, can carry out the large-scale production of duck simulation intestinal fluid.
Above-mentioned is preferred embodiment of the present invention and the know-why used thereof; For a person skilled in the art; Under the situation that does not deviate from the spirit and scope of the present invention; Any based on conspicuous changes such as the equivalent transformation on the technical scheme of the present invention basis, simple replacements, all belong within the protection domain of the present invention.
Claims (8)
1. a duck is simulated the preparation method of intestinal fluid, and it is characterized in that: this method comprises the steps:
Step 1: the duck small intestine chyme that will come with collection the duck small intestine of duck from the system liquid of setting quantity is put into a reagent bottle and is shaken up; The potpourri that obtains after shaking up is put into a centrifugal bottle; Under design temperature, carry out centrifugal treating through hydro-extractor; After centrifugal treating finishes, collect the supernatant in this centrifugal bottle;
Step 2: behind the supernatant liquid filtering that step 1 is obtained, become mixed concentrated liquid through the freeze drier freeze concentration;
Step 3: the mixed concentrated liquid that step 2 is obtained is after impurity is removed in the bag filter dialysis, and freeze drying becomes freeze-dried powder;
Step 4: the activity of AMS, lipase, trypsase, chymotrypsin in the freeze-dried powder that obtains of determination step three respectively, and measure chymotrypsin and tryptic activity in the activity, SILVER REAGENT chymotrypsin of trypsase and chymotrypsin in the activity, SILVER REAGENT trypsase of lipase in the activity, SILVER REAGENT lipase of AMS in the SILVER REAGENT AMS;
Step 5: the principle that equates according to each digestive ferment gross activity in duck simulation intestinal fluid and the target duck intestinal fluid; The SILVER REAGENT AMS, the SILVER REAGENT lipase of corresponding weight, the SILVER REAGENT trypsase of corresponding weight, the SILVER REAGENT chymotrypsin of corresponding weight and the WS of respective volume that in the freeze-dried powder of setting weight, add respective volume; Preparing duck simulation intestinal fluid, the activity of AMS, lipase, trypsase, chymotrypsin equates with the activity of AMS, lipase, trypsase, chymotrypsin in the target duck intestinal fluid respectively in this duck of preparing simulation intestinal fluid.
2. the preparation method of duck simulation intestinal fluid as claimed in claim 1 is characterized in that:
In said step 2, said supernatant filters via 200 order nylon filter clothes.
3. the preparation method of duck simulation intestinal fluid as claimed in claim 1 is characterized in that:
In said step 3, the molecular cut off of said bag filter is below 20000 dalton, and said mixed concentrated liquid is dialysed via phosphate buffer, the deionized water of pH value between 7.0～8.4 successively.
4. the preparation method of duck simulation intestinal fluid as claimed in claim 1 is characterized in that:
In said step 3, the molecular cut off of said bag filter is below 20000 dalton, and said mixed concentrated liquid is dialysed via phosphate buffer, the distilled water of pH value between 7.0～8.4 successively.
5. the preparation method of duck simulation intestinal fluid as claimed in claim 1 is characterized in that:
In said step 5, the activity of AMS, lipase, trypsase, chymotrypsin is to use duck to carry out the mean value that the determination of activity of AMS, lipase, trypsase, chymotrypsin draws respectively as object with the ownership system liquid of using in the said step 1 in the said target duck intestinal fluid.
6. the preparation method of duck simulation intestinal fluid as claimed in claim 1 is characterized in that:
In said step 5; The activity of AMS, lipase, trypsase, chymotrypsin is to be that object carries out the mean value that the determination of activity of AMS, lipase, trypsase, chymotrypsin draws respectively with a large amount of ducks in the said target duck intestinal fluid, and the average activity of AMS, lipase, trypsase, chymotrypsin is respectively 401.56U/ml, 1.10U/ml, 108.75U/ml, 39.01U/ml in the said target duck intestinal fluid.
7. the preparation method of duck simulation intestinal fluid as claimed in claim 1 is characterized in that:
The said WS in the said step 5 is deionized water.
8. like the preparation method of claim 1 or 5 or 6 described duck simulation intestinal fluids, it is characterized in that:
The age of said duck surpasses 6 ages in week and body weight more than 2 kilograms.
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| CN109596837A (en) * | 2018-12-10 | 2019-04-09 | 中国农业科学院北京畜牧兽医研究所 | The Bionic digestion measuring method of one boar food protein digestibility |
| CN109596837B (en) * | 2018-12-10 | 2022-02-08 | 中国农业科学院北京畜牧兽医研究所 | Bionic digestion determination method for protein digestibility of pig feed |
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