CN105767534A - Grass carp bionic digestive reagent enzyme recipe preparation and use method thereof - Google Patents
Grass carp bionic digestive reagent enzyme recipe preparation and use method thereof Download PDFInfo
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- CN105767534A CN105767534A CN201610131940.3A CN201610131940A CN105767534A CN 105767534 A CN105767534 A CN 105767534A CN 201610131940 A CN201610131940 A CN 201610131940A CN 105767534 A CN105767534 A CN 105767534A
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Abstract
The present invention discloses a grass carp bionic digestive reagent enzyme recipe preparation and a use method thereof. The recipe preparation is prepared by using trypsin, alpha-chymotrypsin, amylase and lipase as raw materials, mixing the raw materials, and adding a dialysate into the mixture to dissolve. The dialysate comprises Na<2>HPO<4>12H<2>O, NaH<2>PO<4>2H<2>O, Nacl and sodium ampicillin, and the mixed liquid has a pH of 6.8-7.2. The recipe preparation is combinedly used with a dialysis bag and a digestive tract, a digestive device is used to conduct a bionic digestion to obtain a digested residue dry matter. Then indexes of protein content, amino acid content, crude fat content and crude fiber content of the dry matter are measured. The grass carp bionic digestive reagent enzyme recipe preparation has characteristics of being modest in cost and stable in digestive effects.
Description
Technical field
The present invention is a kind of Ctenopharyngodon idellus Bionic digestion reagent enzyme pharmaceutical formulation and using method thereof, is specifically related to Ctenopharyngodon idellus external imitative
Raw digestive enzyme composes the using method of agent and said preparation, belongs to Feed Manufacturing research field.
Background technology
Current domestic cultural technique research is mainly carried out by animal experiment, but animal experiment cost is the highest, typically
In the case of make effect and need to expend long time, and in process of the test, uncontrollable factor is too many, to result accuracy and
Stability influence is very big, and the technique study of the most in-vitro simulated animal digestion process is paid close attention to.Imitating at livestock and poultry animal
Raw digestion research field, domestic experts and scholars have been carried out substantial amounts of research, defined can ripe use pig,
Chicken, duck Bionic digestion rule of operation.In patent of invention CN201210352892.2 " a bionical rating method of boar food available phosphorus "
Provide a kind of Bionic digestion method evaluating pig feed available phosphorus.But stomach-large intestine-small intestinal three step digestion that the method uses
Method is directed to the digestive physiology process of pig, does not try out in other cultivated animals.
And Ctenopharyngodon idellus is as one of important freshwater aquiculture economic fish of China, its cultivation amount in the whole nation and consumption figure are huge
Greatly.Simultaneously as Growth of Grass Carps Ctenopharyngodon Idellus is in water body, be doomed for Ctenopharyngodon idellus digest research be the most difficult operation, therefore for
The external Bionic digestion research of Ctenopharyngodon idellus just seems particularly necessary.State Intellectual Property Office CN201510055502.9 is entitled " a kind of
Ctenopharyngodon idellus simulates endogenous Digestive system " patent of invention provide and a kind of simulate the formula of Digestive system in Ctenopharyngodon idellus body.Intestinal in this formula
The active concentration of amylase, trypsin and Chymotrypsin is respectively 608.1 U/ml, 183.8 U/ml and 51.6 U/ml, pH
Value is set to 6.95 and 6.92 according to ideocolon, and the concentration of ideocolon sodium dihydrogen phosphate and disodium hydrogen phosphate is respectively 0.187
Mol/L, 0.357 mol/L and 0.199 mol/L, 0.352 mol/L.But, just for digestion carbon in this patent zymogram formula
Hydrate and protein-based, it is impossible to avoid well in evaluation object the impact of fatty offseting result, and this
The bright one zymogram that is provided solely for forms, and under identical enzyme activity concentration levels, uses different reagent grade, different manufacturer
The Digestive system product allotted of enzyme preparation all have different digestion effects.In this case, need a kind of moderate cost badly, disappear
Change the Ctenopharyngodon idellus Bionic digestion reagent enzyme product mix of effect stability, and for ensureing application effect stability, in addition it is also necessary to determine that one is joined
Close the operational approach of the Ctenopharyngodon idellus Bionic digestion that this enzyme product uses.Therefore, the present invention arises at the historic moment.
Summary of the invention
It is an object of the invention to provide a kind of Ctenopharyngodon idellus Bionic digestion reagent enzyme pharmaceutical formulation, this pharmaceutical formulation is with Ctenopharyngodon idellus body
Based on interior four kinds of digestive enzyme (trypsin, Chymotrypsin, amylase and lipase) activity levels and proportion of composing, by common
Domestic enzyme preparation proportioning by a certain percentage makes Ctenopharyngodon idellus external Bionic digestion zymogram formulation products, and this product is at simulation grass carp intestinal
Must the factor that affect result stability be evaded minimum as far as possible on the basis of digestion situation, there is moderate cost, digestion effect
The most stable feature.
Another object of the present invention is to provide the using method of Ctenopharyngodon idellus Bionic digestion reagent enzyme pharmaceutical formulation, the method relates to
And to digestion parameter there is the feature of digestion situation in simulation Ctenopharyngodon idellus body, and more steady than the result that internal true digestion obtains
Fixed, repeatability is higher.
The present invention is achieved through the following technical solutions: a kind of Ctenopharyngodon idellus Bionic digestion reagent enzyme pharmaceutical formulation, it is characterised in that:
This pharmaceutical formulation is with trypsin, Chymetin, amylase, lipase as raw material, after mixing add dialysis solution dissolve and
Preparing, described dialysis solution includes Na2HPO4·12H2O、NaH2PO4·2H2The pH value of O, Nacl, sodium ampicillin composition is
The mixed solution of 6.8～7.2, in described pharmaceutical formulation, the final concentration of each component is as follows:
Trypsin: 5.4 × 10-3～6.6 × 10-3G/ml,
Chymetin: 2.25 × 10-4～2.75 × 10-4G/ml,
Amylase: 4.19 × 10-2～5.12 × 10-2G/ml,
Lipase: 2.16 × 10-2～2.64 × 10-2G/ml,
Na2HPO4·12H2O:4.37 × 10-2G/ml,
NaH2PO4·2H2O:1.22 × 10-2G/ml,
Nacl:6.5 × 10-3G/ml,
Sodium ampicillin: 5.0 × 10-4～5.5 × 10-4g/ml。
Based on the activity level of present invention digestive enzyme in Ctenopharyngodon idellus body and proportion of composing, meeting pH value at dialysis solution is
6.8～7.2, Nacl:6.5 × 10-3Under the conditions of g/ml etc., trypsin, Chymetin, amylase and lipase are being limited
In above-mentioned scope of data, formulated pharmaceutical formulation is used for such as the external Bionic digestion of the fresh-water fishes such as Ctenopharyngodon idellus, can simulate fish
The digestion situation of the multiple nutrients such as vivo protein class, lipid, carbohydrate, has digestion effect and stablizes, digests result reproduction
The advantages such as rate is high, preparation cost is moderate.
The invention allows for the using method of Ctenopharyngodon idellus Bionic digestion reagent enzyme pharmaceutical formulation, it is characterised in that: include with
Lower step:
A, take cleaning after bag filter be inserted in digestive tube, bag filter two ends are fixed on digestive tube termination after outwards overturning, take 1g
Pharmaceutical formulation described in sample digestion and 20ml puts into bag filter, is sealed by digestive tube with rubber closure；
B, will seal after digestive tube load digestion facility carry out Bionic digestion, the setup parameter of described digestion facility is as follows: disappear
Change time ＞ 16h, digestion temperature 28～29 DEG C, shaking speed 175～185r/m, flow 400～450ml/min of dialysing.
After C, Bionic digestion terminate, with distilled water, slaking residue remaining in bag filter is flushed in container, through repeatedly
After drying, dry cooling, it is thus achieved that slaking residue dry；
D, take out step C obtain slaking residue dry, carry out respectively protein content, amino acid content, crude fat content,
The index determining of crude fiber content.
In described step C, repeatedly dry, be dried cooling include: more than 50～65 DEG C of oven for drying 12h, room temperature is put
Put back into tide more than 6h, then by 101～105 DEG C of oven for drying 4～6h, put into and exsiccator is cooled to room temperature.
Also include step E, after Bionic digestion terminates, use by 1.6 g KOH/600 ml H2The cleaning of O proportions
Liquid, pours in the cleaning cylinder of digestion facility, and standby after running cleaning procedure, the cleanout fluid of this ratio can remove digestion facility pipeline
In be attached to inner-walls of duct residual dialysis solution, it is to avoid antibacterial grow to next time measure impact.
The present invention compared with prior art, has the following advantages and beneficial effect:
(1) present invention is with the Ctenopharyngodon idellus in aquatic animal as simulated object, with the actually detected normal cultivation full intestinal of Ctenopharyngodon idellus four kinds
Digestive enzyme activity is zymogram proportioning foundation, protide, lipid, carbohydrate in preparation enzyme liquid energy altitude simulation grass carp intestinal
The digestion situation of nutrient.
(2) in the present invention, the preparation four kinds of enzyme preparations used by digestive enzyme liquid are domestic biochemical reagents, with additive method institute
Import enzyme preparation compare there is fast, the moderate advantage of the supply of material, and each stock amount can shorten, it is ensured that enzyme system
The activity of agent is at effect level, it is ensured that stablizing of digestion result.
(3) present invention coordinates Bionic digestion enzyme liquid to provide standard set using method, and relate in the method disappears
Change parameter and there is the feature of digestion situation in simulation Ctenopharyngodon idellus body, and more more stable than the result that internal true digestion obtains, repeatability
Higher.
(4) for controlling product cost, in dialysis solution of the present invention, the concentration of sodium ampicillin is limited to 5.0 × 10-4～5.5
×10-4G/ml, primarily serves the effect preventing microorganism from disturbing.
(5) slaking residue is dried and is dried by the present invention repeatedly, including more than 50～65 DEG C of oven for drying 12h,
Place moisture regain more than 6h, most free moisture in energy solid residue by room temperature, make air-dry sample, during practical operation,
When rinsing generally according to environment and residue moisture number control concrete duration, it is ensured that sample parches.
Accompanying drawing explanation
Fig. 1 is the connection diagram of digestive tube of the present invention.
Wherein, 1 digestive tube, 2 plastic bottles.
Detailed description of the invention
Below the goal of the invention of the present invention, technical scheme and beneficial effect are described in further detail.
It it is noted that described further below is all exemplary, it is intended to the required present invention is provided further
Bright, except as otherwise noted, all technology used herein and scientific terminology have the common skill with the technical field of the invention
The identical meanings that art personnel are generally understood that.
The patent of invention of State Intellectual Property Office CN201510055502.9 entitled " a kind of Ctenopharyngodon idellus simulates endogenous Digestive system "
Provide and a kind of simulate the formula of Digestive system, the defect existed for this patent:, zymogram formula is just for digestion in Ctenopharyngodon idellus body
Carbohydrate and protein-based, it is impossible to avoid well in evaluation object the impact of fatty offseting result；Two, should
Zymogram formula provide only a kind of zymogram composition, under identical enzyme activity concentration levels, uses different reagent grade, not syngenesis
The Digestive system product that the enzyme preparation of product producer is allotted all has different digestion effects.Based on above-mentioned situation, the invention provides
A kind of Ctenopharyngodon idellus Bionic digestion reagent enzyme pharmaceutical formulation and using method thereof.
The following is and the further of the present invention is summarized:
The digestion reagent enzyme pharmaceutical formulation that the present invention relates to is to be former with trypsin, Chymetin, amylase, lipase
Material, adds dialysis solution and dissolves and prepare after mixing, dialysis solution includes Na2HPO4·12H2O、NaH2PO4·2H2O, Nacl, ammonia benzyl
The mixed solution that pH value is 6.8～7.2 of penicillin sodium composition, in above-mentioned pharmaceutical formulation, the final concentration of each component is as follows:
Trypsin: 5.4 × 10-3～6.6 × 10-3G/ml,
Chymetin: 2.25 × 10-4～2.75 × 10-4G/ml,
Amylase: 4.19 × 10-2～5.12 × 10-2G/ml,
Lipase: 2.16 × 10-2～2.64 × 10-2G/ml,
Na2HPO4·12H2O:4.37 × 10-2G/ml,
NaH2PO4·2H2O:1.22 × 10-2G/ml,
Nacl:6.5 × 10-3G/ml,
Sodium ampicillin: 5.0 × 10-4～5.5 × 10-4g/ml。
The using method of digestion reagent enzyme pharmaceutical formulation, comprises the following steps:
A, take cleaning after bag filter be inserted in digestive tube 1, bag filter two ends are fixed on digestive tube 1 termination after outwards overturning, take
Pharmaceutical formulation described in 1g sample digestion and 20ml puts into bag filter, is sealed by digestive tube 1 with rubber closure；
B, will seal after digestive tube 1 load digestion facility and carry out Bionic digestion, the setup parameter of described digestion facility is as follows: disappear
Change time ＞ 16h, digestion temperature 28～29 DEG C, shaking speed 175～185r/m, flow 400～450ml/min of dialysing.
After C, Bionic digestion terminate, with distilled water, slaking residue remaining in bag filter is flushed in container, with 50～
More than 65 DEG C of oven for drying 12h, room temperature places moisture regain more than 6h, then by 101～105 DEG C of oven for drying 4～6h, puts into exsiccator
In be cooled to room temperature after, it is thus achieved that slaking residue dry；
D, take out step C obtain slaking residue dry, carry out respectively protein content, amino acid content, crude fat content,
The index determining of crude fiber content, assay method is all measured with corresponding feedstuff national standard method；
E, after Bionic digestion terminates, use by 1.6 g KOH/600 ml H2The cleanout fluid of O proportions, pours digestion into and sets
In standby cleaning cylinder, standby after running cleaning procedure.
The digestion facility that the present invention uses can use (in Changsha, this intelligence company limited produces) nonruminant to imitate
Raw digestive system SDS-II, this equipment is as it is shown in figure 1, at most can arrange 10 digestive tubes 1, and every 5 digestive tubes 1 are depended on by flexible pipe
Secondary being connected in a plastic bottle 2 filling dialysis solution, and form a closed circulation passage, plastic bottle 2 is saturating built with 800ml
Analysis liquid, and is placed on the water-bath inside holding that digestion facility is supporting, it is ensured that dialysate temperature simulation Ctenopharyngodon idellus body temperature (28～
29 DEG C) in, during bionical effect, by equipment peristaltic pump by dialysis solution plastic bottle 2 from water-bath is extracted out, pass sequentially through
The digestive tube 1 of 5 series connection, takes away the little molecule digestion product through bag filter, then returns in plastic bottle 2 by flexible pipe,
In bionic process, keep the persistent loop of dialysis solution.
When practical operation, each closed circulation passage can arrange 1～5 digestive tube 1, when the digestive tube 1 arranged
When quantity is 5, after Bionic digestion, usually leading to 4～5 Duplicate Samples results, the difference according to Duplicate Samples result is permissible
React the stability of this Bionic digestion result.
The detailed description of the invention of the explanation present invention, certainly, the protection of the present invention is enumerated below with several exemplary embodiments
Scope is not limited to following example.
Embodiment 1:
The present embodiment relates to a kind of Ctenopharyngodon idellus Bionic digestion reagent enzyme pharmaceutical formulation, and this pharmaceutical formulation is with trypsin, α-gruel egg
White enzyme, amylase, lipase are raw material, add dialysis solution and dissolve and prepare after mixing, and described dialysis solution includes Na2HPO4·
12H2O、NaH2PO4·2H2The mixed solution that pH value is 6.8～7.2 of O, Nacl, sodium ampicillin composition, described formula system
In agent, the final concentration of each component is as follows:
Trypsin: 5.4 × 10-3G/ml,
Chymetin: 2.25 × 10-4G/ml,
Amylase: 4.19 × 10-2G/ml,
Lipase: 2.16 × 10-2G/ml,
Na2HPO4·12H2O:4.37 × 10-2G/ml,
NaH2PO4·2H2O:1.22 × 10-2G/ml,
Nacl:6.5 × 10-3G/ml,
Sodium ampicillin: 5.0 × 10-4g/ml。
Embodiment 2:
The present embodiment relates to a kind of Ctenopharyngodon idellus Bionic digestion reagent enzyme pharmaceutical formulation, and this pharmaceutical formulation is as follows with the difference of embodiment 1:
In pharmaceutical formulation, the final concentration of each component is as follows:
Trypsin: 6.6 × 10-3G/ml,
Chymetin: 2.75 × 10-4G/ml,
Amylase: 5.12 × 10-2G/ml,
Lipase: 2.64 × 10-2G/ml,
Na2HPO4·12H2O:4.37 × 10-2G/ml,
NaH2PO4·2H2O:1.22 × 10-2G/ml,
Nacl:6.5 × 10-3G/ml,
Sodium ampicillin: 5.5 × 10-4g/ml。
Embodiment 3:
The present embodiment relates to a kind of Ctenopharyngodon idellus Bionic digestion reagent enzyme pharmaceutical formulation, and this pharmaceutical formulation is as follows with the difference of embodiment 1:
In pharmaceutical formulation, the final concentration of each component is as follows:
Trypsin: 6.2 × 10-3G/ml,
Chymetin: 2.5 × 10-4G/ml,
Amylase: 4.82 × 10-2G/ml,
Lipase: 2.41 × 10-2G/ml,
Na2HPO4·12H2O:4.37 × 10-2G/ml,
NaH2PO4·2H2O:1.22 × 10-2G/ml,
Nacl:6.5 × 10-3G/ml,
Sodium ampicillin: 5.25 × 10-4g/ml。
Embodiment 4:
The present embodiment relates to a kind of Ctenopharyngodon idellus Bionic digestion reagent enzyme pharmaceutical formulation, and this pharmaceutical formulation is as follows with the difference of embodiment 1:
In pharmaceutical formulation, the final concentration of each component is as follows:
Trypsin: 5.89 × 10-3G/ml,
Chymetin: 2.66 × 10-4G/ml,
Amylase: 4.68 × 10-2G/ml,
Lipase: 2.45 × 10-2G/ml,
Na2HPO4·12H2O:4.37 × 10-2G/ml,
NaH2PO4·2H2O:1.22 × 10-2G/ml,
Nacl:6.5 × 10-3G/ml,
Sodium ampicillin: 5.30 × 10-4g/ml。
Embodiment 5:
The present embodiment is the using method of Ctenopharyngodon idellus Bionic digestion reagent enzyme pharmaceutical formulation described in embodiment 1, comprises the following steps:
A, take cleaning after bag filter be inserted in digestive tube 1, bag filter two ends are fixed on digestive tube 1 termination after outwards overturning, take
Pharmaceutical formulation described in 1g sample digestion and 20ml puts into bag filter, is sealed by digestive tube 1 with rubber closure；
B, will seal after digestive tube 1 load digestion facility and carry out Bionic digestion, the setup parameter of described digestion facility is as follows: disappear
Change time 18h, digests temperature 28 DEG C, shaking speed 175r/m, and dialyse flow 400ml/min.
After C, Bionic digestion terminate, with distilled water, slaking residue remaining in bag filter is flushed in container, through repeatedly
After drying, dry cooling, it is thus achieved that slaking residue dry；
D, take out step C obtain slaking residue dry, carry out respectively protein content, amino acid content, crude fat content,
The index determining of crude fiber content.
Embodiment 6:
The present embodiment is the using method of Ctenopharyngodon idellus Bionic digestion reagent enzyme pharmaceutical formulation described in embodiment 2, comprises the following steps:
A, take cleaning after bag filter be inserted in digestive tube 1, bag filter two ends are fixed on digestive tube 1 termination after outwards overturning, take
Pharmaceutical formulation described in 1g sample digestion and 20ml puts into bag filter, is sealed by digestive tube 1 with rubber closure；
B, will seal after digestive tube 1 load digestion facility and carry out Bionic digestion, the setup parameter of described digestion facility is as follows: disappear
Change time 16.5h, digests temperature 29 DEG C, shaking speed 185r/m, and dialyse flow 450ml/min.
After C, Bionic digestion terminate, with distilled water, slaking residue remaining in bag filter is flushed in container, through repeatedly
After drying, dry cooling, it is thus achieved that slaking residue dry；
D, take out step C obtain slaking residue dry, carry out respectively protein content, amino acid content, crude fat content,
The index determining of crude fiber content.
Embodiment 7:
The present embodiment is the using method of Ctenopharyngodon idellus Bionic digestion reagent enzyme pharmaceutical formulation described in embodiment 3, comprises the following steps:
A, take cleaning after bag filter be inserted in digestive tube 1, bag filter two ends are fixed on digestive tube 1 termination after outwards overturning, take
Pharmaceutical formulation described in 1g sample digestion and 20ml puts into bag filter, is sealed by digestive tube 1 with rubber closure；
B, will seal after digestive tube 1 load digestion facility and carry out Bionic digestion, the setup parameter of described digestion facility is as follows: disappear
Change time 17h, digests temperature 28.5 DEG C, shaking speed 180r/m, and dialyse flow 425ml/min.
After C, Bionic digestion terminate, with distilled water, slaking residue remaining in bag filter is flushed in container, with 50 DEG C
More than oven for drying 12h, room temperature places moisture regain more than 6h, then by 101 DEG C of oven for drying 4h, puts into and be cooled to room temperature in exsiccator
After, it is thus achieved that slaking residue dry；
D, take out step C obtain slaking residue dry, carry out respectively protein content, amino acid content, crude fat content,
The index determining of crude fiber content.
Embodiment 8:
The present embodiment is the using method of Ctenopharyngodon idellus Bionic digestion reagent enzyme pharmaceutical formulation described in embodiment 4, comprises the following steps:
A, take cleaning after bag filter be inserted in digestive tube 1, bag filter two ends are fixed on digestive tube 1 termination after outwards overturning, take
Pharmaceutical formulation described in 1g sample digestion and 20ml puts into bag filter, is sealed by digestive tube 1 with rubber closure；
B, will seal after digestive tube 1 load digestion facility and carry out Bionic digestion, the setup parameter of described digestion facility is as follows: disappear
Change time 17.5h, digests temperature 28.5 DEG C, shaking speed 182r/m, and dialyse flow 420ml/min.
After C, Bionic digestion terminate, with distilled water, slaking residue remaining in bag filter is flushed in container, with 65 DEG C
More than oven for drying 12h, room temperature places moisture regain more than 6h, then by 105 DEG C of oven for drying 6h, puts into and be cooled to room temperature in exsiccator
After, it is thus achieved that slaking residue dry；
D, take out step C obtain slaking residue dry, carry out respectively protein content, amino acid content, crude fat content,
The index determining of crude fiber content.
E, after Bionic digestion terminates, use by 1.6 g KOH/600 ml H2The cleanout fluid of O proportions, pours into and disappears
In the cleaning cylinder of change equipment, standby after running cleaning procedure.
Embodiment 9:
The present embodiment relates to a kind of Ctenopharyngodon idellus Bionic digestion reagent enzyme pharmaceutical formulation, and this pharmaceutical formulation is prepared by the following method
Arrive:
Step 1: preparation dialysis solution: digesting a sample needs to prepare dialysis solution 1000 ml, and wherein 800 ml dialysis solution are poured into
Analysis liquid bottle in water-bath preheating (preheating temperature be parameter arrange 28.5 DEG C), separately measure 120 ml for preparing Bionic digestion reagent
Enzyme hemodialysis liquid.Weigh analytical pure Na2HPO4·12H2O 43.7 g, analytical pure NaH2PO4·2H2O 12.2 g, analytical pure
Nacl 7.5 g, sodium ampicillin (purity >=95%) 0.5 g that biochemical reagents are pure, add 800 ml distilled water dissolve after with
NaOH solution or Hcl solution regulation pH value are 7.0 ± 0.2, proceed to constant volume in 1000 ml volumetric flasks, shake up standby.
Step 2: preparation Bionic digestion reagent enzyme liquid: digest a sample preparation 120 ml Bionic digestion reagent enzyme liquid (every
Root digestive tube 20 ml, sample do 5 parallel).First four kinds of enzyme preparations are taken from-4 DEG C of refrigerators room temperature and rises again 30
Min, weighs trypsin BR respectively with ten thousand/electronic balance, and Shanghai is tried, production code member 64008860) 0.720 g, α-gruel
Protease (BR, Shanghai examination, production code member 64001631) 0.030 g, amylase (BR, Shanghai examination, production code member 64003436) 5.580
G, lipase (BR, Wo Kai, production code member 64005761) 2.880 g, put in 250 ml beakers, and add that 120 ml prepare is saturating
Analysis liquid, ultrasonic 15 min put in-4 DEG C of refrigerators temporary after dissolving.
Embodiment 10:
The present embodiment is the using method of Ctenopharyngodon idellus Bionic digestion reagent enzyme pharmaceutical formulation described in embodiment 9, as follows:
Measure under application claims, rapeseed cake sample dry matter and the operational approach of thick protein Bionic digestion rate:
First the culture dish numbering cleaned up 10 is put in 105 ± 2 DEG C of baking ovens and is carried out drying and processing, subsequently by rapeseed cake sample
Product pulverized 60 mesh sieves.Next the bag filter of preservation is soaked after taking out digestive enzyme preparation and EDTA process from-4 DEG C of refrigerators,
Digestive enzyme preparation is put in room temperature about 30 min that rise again, and requires according to Bionic digestion enzyme formula of liquid with ten thousand/balance subsequently
Weigh and put in 250 ml beakers, add ultrasonic 30 min hydrotropies after the dialysis solution of prescribed volume, put in-4 DEG C of refrigerators temporary
Deposit standby；Bag filter is put in the beaker filling 600 ml ultra-pure waters and is repeatedly cleaned, and cleans at librarian use disposable PE glove
Reason, penetrates digestive tube 1 by bag filter after cleaning, be locked under sack two ends are turned-out digestive tube 1 two ends, tightens with rubber band,
One end rubber closure stoppers fixing, and the other end is upwards placed on pipe support.Ten thousand/electronic balance weighs rapeseed cake sample
1.0000 g, (once go up machine digestion and can at most do 10 digestive tubes 1,5 digestion in the lossless bag filter put on digestive tube 1
Pipe 1 is one group, and 1 group is sample cell, 1 group of blank tube being to be not added with sample), each sample adds Bionic digestion liquid 20 ml, covers and disappears
Changing the stopper of pipe 1 other end, concussion makes sample be sufficiently mixed with Digestive system, by the flexible pipe between digestive tube 1 according to instrument up and down
Explanation connects, and is placed on Bionic digestion instrument and preheats, and puts into 2 in the lump equipped with the dialysis bottle of 800 ml dialysis solution simultaneously
Water-bath below instrument, starts preheating after connecting flexible pipe.After digestion temperature reaches requirement, start digestion program.
Ctenopharyngodon idellus Bionic digestion program parameter is provided that
After digestion starts, put in advance in baking oven 10 culture dishs are taken out (need to ensure to dry more than 4h), puts into exsiccator
In be cooled to room temperature after weigh weight record with ten thousand/balance.
After 16 h digestion terminate, instrument shuts down, and is connected by digestive tube 1 immediately and disconnects from instrument, takes off.Instrument is pressed
Being attached according to equipment specification, preparation 1.6g/600 ml KOH solution pours equipment cleaning device into, runs cleaning procedure；With
Slaking residue in bag filter in digestive tube 1 and residue digestive fluid are rinsed into reference numeral by wash bottle equipped with ultra-pure water in the lump
Culture dish in, culture dish is put into together with sample in 65 DEG C of baking ovens and dries 12 h, after treating in culture dish that sample is dry, then
Close baking oven to get damp again at room temperature 12 h, culture dish is put in 105 DEG C of baking ovens the most again and dry to constant weight, weigh weight and remember
Record.
Carefully being scraped by sample sample spoon in culture dish, after pulverizing in culture dish, mixing weighs sample about 0.5g
(recording concrete data for result of calculation), is used for measuring gross protein value.
Embodiment 11:
The present embodiment is for comparing bean cake, the Application Example of rapeseed cake thick protein Bionic digestion rate:
The Bionic digestion enzyme liquid, the formula of dialysis solution that use in the present embodiment are consistent with embodiment 9 with compound method, and use
Operating procedure is consistent with embodiment 10 with Bionic digestion parameter.
In the present embodiment EXPERIMENTAL DESIGN, go up machine test at twice.Test packet see table:
Note: above bean cake raw material and rapeseed cake raw material were all pulverized 60 mesh and processed.
The result of the test of the present embodiment is as follows: as can be known from the results, and the inventive method measurement result is more stable, at different time
Twice mensuration carried out, basically identical (respectively 87.13% He of the Bionic digestion rate data of thick protein in bean cake raw material
85.71%), the collimation of each digestive tube 1 is preferable.On the other hand, with the protein digestibility of rapeseed cake in a digestion process
Slightly below in bean cake, with actual culture experiment, the protein digestibility result rule of two kinds of raw materials is the most surely closed.
The bionical protein digestibility of Ctenopharyngodon idellus of table 1 bean cake and rapeseed cake compares
Note: each group result processes with sample blank group data normalization, and data represent with means standard deviation (N=4).
The above, be only presently preferred embodiments of the present invention, and the present invention not does any pro forma restriction, every depends on
Any simple modification of being made above example according to the technical spirit of the present invention, equivalent variations, each fall within the protection of the present invention
Within the scope of.
Claims (4)
1. a Ctenopharyngodon idellus Bionic digestion reagent enzyme pharmaceutical formulation, it is characterised in that: this pharmaceutical formulation is with trypsin, α-gruel egg
White enzyme, amylase, lipase are raw material, add dialysis solution and dissolve and prepare after mixing, and described dialysis solution includes Na2HPO4·
12H2O、NaH2PO4·2H2The mixed solution that pH value is 6.8～7.2 of O, Nacl, sodium ampicillin composition, described formula system
In agent, the final concentration of each component is as follows:
Trypsin: 5.4 × 10-3～6.6 × 10-3G/ml,
Chymetin: 2.25 × 10-4～2.75 × 10-4G/ml,
Amylase: 4.19 × 10-2～5.12 × 10-2G/ml,
Lipase: 2.16 × 10-2～2.64 × 10-2G/ml,
Na2HPO4·12H2O:4.37 × 10-2G/ml,
NaH2PO4·2H2O:1.22 × 10-2G/ml,
Nacl:6.5 × 10-3G/ml,
Sodium ampicillin: 5.0 × 10-4～5.5 × 10-4g/ml。
The using method of Ctenopharyngodon idellus Bionic digestion reagent enzyme pharmaceutical formulation the most according to claim 1, it is characterised in that: include
Following steps:
A, take cleaning after bag filter be inserted in digestive tube (1) in, bag filter two ends are fixed on digestive tube (1) end after outwards overturning
Head, takes pharmaceutical formulation described in 1g sample digestion and 20ml and puts into bag filter, is sealed by digestive tube (1) with rubber closure；
B, will seal after digestive tube (1) load digestion facility carry out Bionic digestion, the setup parameter of described digestion facility is as follows:
Digestion time ＞ 16h, digestion temperature 28～29 DEG C, shaking speed 175～185r/m, flow 400～450ml/min of dialysing；
After C, Bionic digestion terminate, with distilled water, slaking residue remaining in bag filter is flushed in container, through repeatedly drying,
After being dried cooling, it is thus achieved that slaking residue dry；
D, take out step C obtain slaking residue dry, carry out respectively protein content, amino acid content, crude fat content,
The index determining of crude fiber content.
The using method of Ctenopharyngodon idellus Bionic digestion reagent enzyme pharmaceutical formulation the most according to claim 2, it is characterised in that: in institute
State in step C, repeatedly dry, be dried cooling include: with 50～65 DEG C of oven for drying 12h more than, room temperature place get damp again more than 6h,
Again by 101～105 DEG C of oven for drying 4～6h, put into and exsiccator is cooled to room temperature.
The using method of Ctenopharyngodon idellus Bionic digestion reagent enzyme pharmaceutical formulation the most according to claim 2, it is characterised in that: also wrap
Include step E, after Bionic digestion terminates, use by 1.6 g KOH/600 ml H2The cleanout fluid of O proportions, pours digestion into
In the cleaning cylinder of equipment, standby after running cleaning procedure.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102313794A (en) * | 2011-05-27 | 2012-01-11 | 中国农业科学院北京畜牧兽医研究所 | Preparation method of duck simulated intestinal fluid |
| CN102715380A (en) * | 2012-07-09 | 2012-10-10 | 山东农业大学 | Method for determining poultry metabolizable energy of corn quickly by utilizing in vitro digestion method |
| CN104605169A (en) * | 2015-02-03 | 2015-05-13 | 广东溢多利生物科技股份有限公司 | Simulated endogenous digestive juice of grass carps |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102313794A (en) * | 2011-05-27 | 2012-01-11 | 中国农业科学院北京畜牧兽医研究所 | Preparation method of duck simulated intestinal fluid |
| CN102715380A (en) * | 2012-07-09 | 2012-10-10 | 山东农业大学 | Method for determining poultry metabolizable energy of corn quickly by utilizing in vitro digestion method |
| CN104605169A (en) * | 2015-02-03 | 2015-05-13 | 广东溢多利生物科技股份有限公司 | Simulated endogenous digestive juice of grass carps |
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