CN106124429B - A kind of Bionic digestion measuring method of feed digestible carbohydrate total amount - Google Patents
A kind of Bionic digestion measuring method of feed digestible carbohydrate total amount Download PDFInfo
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Abstract
A kind of Bionic digestion measuring method of feed digestible carbohydrate total amount, includes the following steps：1）The preparation of sample；2）Prepare the buffer and simulated digestive juice and other reagents of stomach and small intestine；3）Bionic digestive system for monogastric animals preheating；4）Loading；5）Stomach simulates digestion process；6）Small intestine simulates digestion process；Software is controlled by Bionic digestive system for monogastric animals, small intestine simulation digestion parameter is set, start to carry out small intestine simulation digestion process；7）Slaking residue processing；8）Using DNS colorimetric method for determining control sample and the content of reducing sugar in sample is analyzed, and the digestible carbohydrate total amount being calculated in feed.A kind of Bionic digestion measuring method of feed digestible carbohydrate total amount of the present invention, using During In Vitro Enzymatic Hydrolysis is automatically controlled in Bionic digestive system for monogastric animals, the repeatability and precision of test result are high；The workload for reducing operator, eliminates the systematic error of manual operation introducing, and detection efficiency is high.
Description
Technical field
The present invention relates to a kind of measuring methods of digestible carbohydrate, can digest carbon water more particularly, to a kind of feed
The Bionic digestion measuring method of total amount of compound.
Background technique
Carbohydrate is made of three kinds of carbon, hydrogen and oxygen elements, and the carbohydrate in feed is in animal stomach, small enteral
By physics digestion, digestion enzymic digestion, it can gradually be degraded into the glucose containing free aldehyde or ketone group, fructose, galactolipin etc.
Monosaccharide and on a small quantity containing the disaccharide of free aldehyde（Lactose, maltose）.
Biological value that is objective, accurately assessing all feeds raw material is that Animal nutrition needs quantity research, optimization feed to match
The basis of side is the decision-making foundation for improving livestock and poultry diet utilization rate, reducing daily ration cost and aquaculture energy-saving and emission-reduction.Carbon in feed
The assessment method of hydrate biological value mainly has in vivo method (in vivo) and in vitro method (in vitro).In vivo method is just
It is to carry out zoopery, usually cost, laborious, time-consuming, systematic error and accidental error influence factor are more, under different space-time conditions
Measured value repeatability, comparativity difference is not able to satisfy needed for research and production application.And in vitro method has quick, simplicity, expense low
The advantages that, various countries nutritionist has many researchs to report.In recent years, the external assessment technology of Dietary carbohydrates is in the world
On more and more attention has been paid to push the fast development of correlative study, however these in vitro methods are mostly with triangular flask, examination
Based on the simple experiments equipment such as pipe, dialysis tubing, due to the systematic error that a large amount of manual operation introduces, acquired results are repeatable
Property and precision are poor.
Summary of the invention
The technical problem to be solved by the present invention is to：Overcome the deficiencies of the prior art and provide a kind of easy to operate, test result
The Bionic digestion measuring method of accuracy rate height, reproducible feed digestible carbohydrate total amount.
The technical solution adopted by the present invention to solve the technical problems is：A kind of feed digestible carbohydrate total amount
Bionic digestion measuring method, includes the following steps：
1）The preparation of sample：It is sampled by GB/T 14699.1, then the Feed Sample of sampling quartering is divided extremely
200g or so, with plant pulverizer or mortar by sample comminution to crossing 40 ~ 100 meshes（It is preferred that crossing 60 meshes, aperture 0.30mm）,
Enclosed sample sack sealing storage, it is spare to make sample；
2）Prepare the buffer and simulated digestive juice and other reagents of stomach and small intestine；
3）Bionic digestive system for monogastric animals preheating：Stomach buffer, small intestine leading portion buffering are replaced with 1000mL deionized water
Liquid, small intestine back segment buffer are put into the corresponding position of Bionic digestive system for monogastric animals, and by the pipeline of system and buffer bottle
It connects；In control software, the preheating time that Bionic digestive system for monogastric animals is arranged is 60 ~ 90min；To all digestion ranks
After the parameter input of section, running simulation digestion process；
4）Loading：Simulation digest tube in Bionic digestive system for monogastric animals is cleaned up, is dried, and with turned welt silica gel
It fills in one end plug is tight；Weigh step 1）Prepare spare 0.25g ~ 1.00g（It is accurate to 0.0001g, is marked with W）Feed
Sample, and be added in the simulation digest tube of Bionic digestive system for monogastric animals, while measuring the dry matter content of sample（DM）；
5）Stomach simulates digestion process：First simulation digestion that stomach buffer enters into Bionic digestive system for monogastric animals
Pipe（Control sample）Middle addition 10ml deionized water, other, which are added, is separately added into the simulation of 10ml stomach in the simulation digest tube for analyzing samples
Digestive juice；And in the fixed electric mixer of the other end of simulation digest tube, after being preheated to Bionic digestive system for monogastric animals,
Will simulation digest tube place in Bionic digestive system for monogastric animals, and by more simulation digest tubes by lower end into the liquid above brought out
Stream mode is connected in series, and the simulated digestive juice liquid-feeding tube for simulating digest tube passes through quick coupling and Bionic digestive system for monogastric animals
Connection；Software is controlled by Bionic digestive system for monogastric animals, stomach simulation digestion parameter is set, start to carry out at stomach simulation digestion
Reason；
6）Small intestine simulates digestion process：After stomach phase digestion, by simulated digestive juice liquid-feeding tube to small intestine buffer
The first simulation digest tube entered（Control sample）In add 6.0ml and 1.6ml deionized water in two times, other addition analysis examination
6.0ml small intestine buffer and 1.6ml small intestine simulated digestive juice are successively added in the simulation digest tube of sample respectively；Pass through nonruminant
Bionic digestion system controlling software is arranged small intestine and simulates digestion parameter, starts to carry out small intestine simulation digestion process；
7）Slaking residue processing：After being digested to feed sample through stomach and small intestine simulation, by disappearing in glass digest tube
Change liquid to be transferred to without loss in the clean volumetric flask of 200ml, then use deionized water constant volume, sealed membrane sealing shakes up, calmly
30ml digestive juice is taken in measuring bottle, is drawn with disposable syringe, and by membrane filtration, filtrate is spare；The digestive juice of control group is straight
0.22 μm of membrane filtration is connect, it is spare；
8）Using the content of reducing sugar in DNS colorimetric method for determining control sample and analysis sample, and it is calculated in feed
Digestible carbohydrate total amount.
Step 1）In, the Feed Sample is pannage, chicken feed or duck feed.
Step 2）In, the preparation method of the stomach buffer：Weigh 1 ~ 3g sodium chloride, 0.25 ~ 5g potassium chloride and the mountain 1 ~ 2g
Potassium sorbate is put into 500mL beaker, 400mL deionized water dissolving is added, and with the hydrochloric acid of 2mol/L（HCL）At 39 DEG C ~ 42 DEG C
Above-mentioned solution is transferred to 500mL volumetric flask after cooling by the lower pH to 3.0 for adjusting solution, and with deionized water constant volume.
Step 2）In, the preparation method of the stomach simulated digestive juice：The determination of peptic activity described according to Wirnt R
Method（Referring to Pepsin, measurements with haemoglobin as substrate [A] In:
Bergmeyer H U. Methods of enzymatic analysis[M].Weinheinm:Verlag chemie.
1974）, measure the activity of pepsin in pepsin.Then, according to simulation according in pig simulate the gastric juice pepsin it is dense
Degree is 737.5U/ ml, and the pepsin for weighing 184.38KU is dissolved in the hydrochloric acid solution of 250ml pH 3.0（39 DEG C of subscripts
Determine pH）, it is slowly stirred until dissolution, overheats when not heating simulate the gastric juice on hot plate, or preparing；Prepared before use.
It is 1550U/ml according to the concentration of pepsin in chicken simulate the gastric juice, the pepsin for weighing 387.5KU is dissolved in
In the hydrochloric acid solution of 250ml pH 2.0（PH is demarcated at 41 DEG C）.
It is 1550U/ ml according to the concentration of pepsin in duck simulate the gastric juice, the pepsin for weighing 387.5KU is dissolved in
In the hydrochloric acid solution of 250ml pH 2.0（PH is demarcated at 42 DEG C）.
Step 2）In, the preparation method of the small intestine buffer：0.5 ~ 5g Anhydrous Disodium Phosphate is weighed, 2.0 ~ 3g is anhydrous
Sodium dihydrogen phosphate, PBS ultimate density are 0.05M, 0.2 ~ 2g potassium sorbate, 120,000 U of penicillin.It is put into 100mL beaker, is added
40mL deionized water dissolving, and adjusted at 39 DEG C or 41 °C or 42 DEG C with the sodium hydroxide of the phosphoric acid of 1mol/L or 1mol/L molten
The pH to 6.30 ~ 6.50 of liquid.Above-mentioned solution is transferred to 50mL volumetric flask after cooling, and with deionized water constant volume.
Step 2）In, the preparation method of the small intestine simulated digestive juice：The alphalise starch enzyme activity described according to Dahlqvist A
Property measuring method is (referring to Dahlqvist A;A method for the determination of amylase in
intestinal content [J]. Scandinavian Journal of Clinical and Laboratory
Investigation, 1962, 14:145-151), Wirnt R description determination of tryptic activity method (referring to
Wirnt R, Trypsin； measurement with nα-wtoluenesulfonyl-l-arginine methyl ester
as substrate [A]. Bergmeyer H U. Methods of enzymatic analysis[M]. Weinheinm:
Verlag chemie. 1974), the chymotrypsin activity measuring method of Wirnt R description is (referring to 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), measure SILVER REAGENT amylase, trypsase, in chymotrypsin corresponding digestive ferment activity；Then, according to
The activity of these three digestive ferments, claims respectively in simulated intestinal fluid（Amount）Take amylase 41.41KU ~ 97.12KU, trypsase 9.54KU
~ 26.32KU, chymotrypsin 1.62KU ~ 9.44KU are dissolved in 17 ~ 20ml deionized water, and are slowly stirred until dissolution（Intestines
The digestive enzyme activity that liquid enzyme powder agent provides is 50% or more）；It is overheated when not heating, or preparing on hot plate；Prepared before use.
Step 5）In, the stomach simulation digestion parameter is：39 ~ 42 DEG C of temperature；Wriggling revolution speed 188rpm/min, 4 ~ 6 h of digestion time.
Step 5）With 6）In, quantity >=3 piece of the simulation digest tube of the addition analysis sample, preferably 4 ~ 6.
Step 6）In, the small intestine simulation digestion parameter：39 ~ 42 °C of temperature, wriggling revolution speed 188rpm/min, small intestine disappears
The change time is 8 ~ 16 h.
Step 6）In, the content of reducing sugar in DNS colorimetric method for determining sample includes the following steps：
1. drawing standard curve：Deionized water 4ml is drawn in 25ml scale test tube, DNS reagent 5ml, boiling water bath is added
5min, tap water are cooled to room temperature, and deionized water is settled to 25ml（Directly add 16ml deionized water）, standard blank sample is made；
Respectively draw 10mg/ml glucose solution 1.00ml, 2.00ml, 3.00ml, 4.00ml, 5.00ml, 6.00ml and
7.00ml is settled to 100ml with deionized water respectively, is configured to the Glucose standards that concentration is 0.10mg/ml~0.70mg/ml
Solution；Each 2ml of glucose standard of above-mentioned concentration is drawn respectively（2 parallel）, it is added in 25ml scale test tube, adds
2.0ml deionized water mixes, and 5.0ml DNS reagent is added, and mixes, and boiling water bath 5min, tap water is cooled to room temperature, and adds 16ml
Deionized water shakes up；It is control zeroing with standard blank sample, absorbance OD value is surveyed at 540nm；Using concentration of glucose as Y-axis,
Absorbance OD value is X-axis, draws standard curve y=aX+b；
2. reducing sugar test in control sample and analysis sample：Take step 7）The digestive juice 5ml of preparation is in test tube, then again
5ml deionized water is added, carries out 2 times of dilutions；Digestive juice after taking 2ml to dilute again is added in 25ml scale test tube, is then added again
2ml deionized water, oscillation mix, and 5mlDNS is added, and mix, boiling water bath 5min.Originally water cooling is gone to room temperature, adds 16ml deionization
Water mixes, and OD value is surveyed at spectrophotometer 540nm, obtains the OD of control sample（Control）With the OD of analysis sample（Sample）。
Step 8）In, the calculation formula of the digestible carbohydrate total amount is：
Digestible carbohydrate total amount (mg/g DM)=【(a×OD（Sample）+b)×2×200-(a×OD（Control）+b)×
17.6】/(w×DM)
In formula：A is standard curve regression coefficient；
B is standard curve regression constant；
OD is measured as the absorbance value of analysis Specimen Determination pipe；
OD blank is the absorbance value of control sample pipe；
W is each replication tube feed material example weight；
DM is the dry matter content of Feed Sample.
A kind of beneficial effect of the Bionic digestion measuring method of feed digestible carbohydrate total amount of the present invention：It overcomes
The outer digestion method of conventional bulk cannot achieve the defect that product separates in real time in digestion process, use real-time dynamic digestion product point
Pass through Bionic digestive system for monogastric animals in conjunction with the method for current in vitro method measurement feed digestible carbohydrate from technology
Middle automatic control During In Vitro Enzymatic Hydrolysis is substantially eliminated the systematic error introduced due to manual operation, improves test result
Repeatability and precision；The workload for reducing operator, improves detection efficiency.
Detailed description of the invention
Fig. 1-is a kind of flow diagram of the Bionic digestion measuring method of feed digestible carbohydrate total amount；
Fig. 2-is that the stereochemical structure of Bionic digestive system for monogastric animals of the present invention is illustrated；
Fig. 3-is the rearview of Bionic digestive system for monogastric animals of the present invention；
Fig. 4-is the enlarged diagram in Fig. 1 at A.
Specific embodiment
The invention will be further described with reference to the accompanying drawings and embodiments.
A kind of equipment that the Bionic digestion measuring method of feed digestible carbohydrate total amount uses of the present invention is simple stomach
Animal Bionic digestion system, including casing 2, constant temperature air bath shaking table 6, Bionic digestion device 4, automatic enzyme device, buffer
The upper left side of circulator, cleaning device, control circuit 5, the casing 2 is equipped with digestion reaction room 3, and the constant temperature air bath shakes
6 devices of bed are set in digestion reaction room 3；The automatic enzyme device is fixedly mounted in casing 2, automatic enzyme device with it is bionical
Slaking apparatus 4 is connected by perfusion tube 58；The buffering liquid circulating device and cleaning device are set to the lower section of casing 2, buffer
Circulator, cleaning device pass through perfusion tube 58 respectively and are connected with Bionic digestion device 4；The control circuit 5 is set to casing 2
It is interior, and be connected by conducting wire with 6 device of constant temperature air bath shaking table, automatic enzyme device, buffering liquid circulating device, cleaning device,
The control circuit 5 is connect with the industrial personal computer being mounted in casing, the industrial personal computer and the display for being mounted on 2 upper right side of casing
1 connection；The Bionic digestion device 4 is vertically installed at 6 top of constant temperature air bath shaking table, the i.e. central axis of Bionic digestion device 4
Line is vertical with constant temperature air bath shaking table 6, as depicted in figs. 1 and 2.
Referring to Fig. 4, the Bionic digestion device 4 includes fixed bracket, vertical simulation digester, glass pipe clip 7, stirs
Mix stick 11, motor 15 and transmission device；The vertical simulation digester is by several concatenated dialysis simulation digest tubes and/or bilayer
Glass molds paragastric canals composition, such as 2 groups, every group 5 concatenated dialysis simulation digest tube compositions；The vertical simulation digester is perpendicular
Straight clamping is mounted in the glass pipe clip 7, and the stirring rod 11 is mounted in vertical simulation digest tube, top and biography
Dynamic device is detachably connected；The transmission device is electrically connected with the motor 15 for being mounted on fixed cantilever tip；The glass tube is solid
Clamp 7 is fixedly mounted on fixed mid-stent.
Referring to Fig. 4, the fixed bracket is made of bottom plate 20, lower frame plate 19, backboard 18, side plate 17 and support plate 16, institute
Bottom plate 20 is stated to be fixedly connected with lower frame plate 19；The lower frame plate 19 is formed by one piece of sheet metal bent, including a horizontal fixed plate
The vertical plate erect with two pieces, two vertical plates are located at below horizontal fixed plate, and the left and right ends integrally connected with horizontal fixed plate；It is vertical
The lower end of plate is folded upward at into fixed block, and fixation hole is equipped with location hole；It is offered on horizontal fixed plate for fixed vertical
The first through hole of 8 fixing clamp 7 of glass tube, the backboard 18, side plate 17 are fixed on the back upper place of lower frame plate 19,18 He of backboard
The top of side plate 17 is fixedly connected with support plate 16, and motor fixing threaded hole and motor shaft perforation are additionally provided in the support plate 16.
Embodiment 1
Referring to Fig.1, the Bionic digestion measuring method of a boar food digestible carbohydrate total amount of the present embodiment, packet
Include following steps：
1）The preparation of sample：It is sampled by GB/T 14699.1, then the pannage sample of sampling quartering is divided extremely
200g or so, with plant pulverizer or mortar by sample comminution to crossing 60 meshes（Aperture 0.30mm）, enclosed sample sack, which seals, to be deposited
It puts, it is spare to make sample；
2）Prepare the buffer and simulated digestive juice and other reagents of stomach and small intestine：
Stomach buffer（pH 3.0）：2.59g sodium chloride, 0.25g potassium chloride and 1g potassium sorbate are weighed, 500mL burning is put into
In cup, 400mL deionized water dissolving is added, and with the hydrochloric acid of 2mol/L（HCl）The pH to 3.0 of solution is adjusted under 39 °C.It is cold
But above-mentioned solution is transferred to 500mL volumetric flask afterwards, and with deionized water constant volume.
Stomach simulated digestive juice：
It is 737.5U/ ml according to simulating according to the concentration of pepsin in pig simulate the gastric juice, weighs the stomach egg of 184.38KU
White enzyme is dissolved in the hydrochloric acid solution of 250ml pH 3.0（PH is demarcated at 39 DEG C）, it is slowly stirred until dissolving, not in heating plate
Upper heating simulate the gastric juice, or overheated when preparation；Prepared before use.
Small intestine buffer：Weigh 0.52g Anhydrous Disodium Phosphate, 2.57g anhydrous sodium dihydrogen phosphate, 0.2530g sorbic acid
Potassium, 120,000 U of penicillin.It is put into 100mL beaker, 40mL deionized water dissolving is added, and with the phosphoric acid or 1mol/L of 1mol/L
Sodium hydroxide the pH to 6.30 of solution is adjusted under 39 °C.Above-mentioned solution is transferred to 50mL volumetric flask after cooling, and spend from
Sub- water constant volume.
Small intestine simulated digestive juice：According to the alpha-amylase activity measuring method that Dahlqvist A is described, Wirnt R description
Determination of tryptic activity method, Wirnt R description chymotrypsin activity measuring method, measure SILVER REAGENT amylase, pancreas
The activity of corresponding digestive ferment in protease, chymotrypsin.Then, according to the activity of these three digestive ferments in simulated intestinal fluid, respectively
Claim（Amount）Amylase 41.41KU, trypsase 12.82KU are taken, chymotrypsin 1.62KU is dissolved in 17ml deionized water, and
It is slowly stirred until dissolution（The digestive enzyme activity that erepsin pulvis provides is 50% or more）.It does not heat, or prepares on hot plate
When overheat.Prepared before use.
DNS is prepared：NaOH 20.0g is weighed, deionized water dissolving is settled to 100ml, and the NaOH for being configured to 200g/L is molten
Liquid weighs 3,5- dinitrosalicylic acid 3.15g (chemistry is pure), adds water 500ml, stir 5s, then water-bath gradually adds to 45 DEG C
Enter 100mlNaOH solution, be stirred continuously simultaneously, until solution clear（Pay attention to：It is added during NaOH, solution temperature is not
It to be more than 48 DEG C）.It is gradually added Rochelle salt 91.0g, phenol 2.50g and anhydrous sodium sulfite 2.50g again, continues 45
DEG C heating water bath, while water supplement 300ml, are stirred continuously, and until the substance of addition is completely dissolved, stops heating, are cooled to room
Wen Hou is settled to 1000ml. with water and is stored in brown bottle, is kept in dark place, and can be used after placing 7d at room temperature, validity period
6 months.
3）Bionic digestive system for monogastric animals preheating：Stomach buffer, small intestine leading portion buffering are replaced with 1000mL deionized water
Liquid, small intestine back segment buffer are put into the corresponding position of Bionic digestive system for monogastric animals, and by the pipeline of system and buffer bottle
It connects；In control software, the preheating time that Bionic digestive system for monogastric animals is arranged is 60min；To all digestion phases
Parameter input after, running simulation digestion process；
4）Loading：Simulation digest tube in Bionic digestive system for monogastric animals is cleaned up, is dried, and with turned welt silica gel
It fills in one end plug is tight；Weigh step 1）Prepare spare 0.25g（It is accurate to 0.0001g, is marked with W）4 parts of feed sample,
And it is added in the simulation digest tube of Bionic digestive system for monogastric animals, while measuring the dry matter content of sample（DM）；
5）Stomach simulates digestion process：First simulation digestion that stomach buffer enters into Bionic digestive system for monogastric animals
Pipe（Control sample）Middle addition 10ml deionized water, 4 simulation digest tubes of other addition analysis samples（Analyze sample）Middle difference
10ml stomach simulated digestive juice is added；And in the fixed electric mixer of the other end of simulation digest tube, digested to monogastric animal bionic
After system warm-up, it will simulate in digest tube placement and Bionic digestive system for monogastric animals, and 5 simulation digest tubes are pressed
It holds into the flow regime above brought out and is connected in series, the simulated digestive juice liquid-feeding tube for simulating digest tube is dynamic by quick coupling and simple stomach
The connection of object Bionic digestion system；Software is controlled by Bionic digestive system for monogastric animals, and stomach simulation digestion parameter is set：39 ° of temperature
C, wriggling revolution speed 188rpm/min, 4 h of digestion time start to carry out stomach simulation digestion process；
6）Small intestine simulates digestion process：After stomach phase digestion, by simulated digestive juice liquid-feeding tube to small intestine buffer
The first simulation digest tube entered（Control sample）In add 6.0ml and 1.6ml deionized water in two times, other addition analysis examination
4 simulation digest tubes of sample（Analyze sample）Middle difference successively adds 6.0ml small intestine buffer and 1.6ml small intestine simulated digestive juice
To simulation digest tube；Software is controlled by Bionic digestive system for monogastric animals, and small intestine simulation digestion parameter is set：39 °C of temperature,
Wriggling revolution speed 188rpm/min, small intestine digestion time are 16 h, start to carry out small intestine simulation digestion process；
7）Slaking residue processing：After being digested to feed sample through stomach and small intestine simulation, by disappearing in glass digest tube
Change liquid to be transferred to without loss in the clean volumetric flask of 200ml, then use deionized water constant volume, sealed membrane sealing shakes up, calmly
30ml digestive juice is taken in measuring bottle, is drawn with disposable syringe, and by membrane filtration, filtrate is spare；The digestive juice of control group is straight
0.22 μm of membrane filtration is connect, it is spare；
8）Using the content of reducing sugar in DNS colorimetric method for determining control sample and analysis sample, and it is calculated in feed
Digestible carbohydrate total amount：
1. drawing standard curve：Deionized water 4ml is drawn in 25ml scale test tube, DNS reagent 5ml, boiling water bath is added
5min, tap water are cooled to room temperature, and deionized water is settled to 25ml（Directly add 16ml deionized water）, standard blank sample is made；
Respectively draw 10mg/ml glucose solution 1.00ml, 2.00ml, 3.00ml, 4.00ml, 5.00ml, 6.00ml and
7.00ml is settled to 100ml with deionized water respectively, is configured to the Glucose standards that concentration is 0.10mg/ml~0.70mg/ml
Solution；Each 2ml of glucose standard of above-mentioned concentration is drawn respectively（2 parallel）, it is added in 25ml scale test tube, adds
2.0ml deionized water mixes, and 5.0ml DNS reagent is added, and mixes, and boiling water bath 5min, tap water is cooled to room temperature, and adds 16ml
Deionized water shakes up；It is control zeroing with standard blank sample, absorbance OD value is surveyed at 540nm；Using concentration of glucose as Y-axis,
Absorbance OD value is X-axis, draws standard curve y=aX+b；
2. reducing sugar test in control sample and analysis sample：Take step 7）The digestive juice 5ml of preparation is in test tube, then again
5ml deionized water is added, carries out 2 times of dilutions；Digestive juice after taking 2ml to dilute again is added in 25ml scale test tube, is then added again
2ml deionized water, oscillation mix, and 5mlDNS is added, and mix, boiling water bath 5min.Originally water cooling is gone to room temperature, adds 16ml deionization
Water mixes, and OD value is surveyed at spectrophotometer 540nm, obtains the OD of control sample（Control）With the OD of analysis sample（Sample）。
Embodiment 2
Referring to Fig.1, the Bionic digestion measuring method of a kind of feed digestible carbohydrate total amount of the present embodiment, with reality
Applying example 1, there are following differences：
Step 1）In, the Feed Sample is chicken feed.
Step 2）In, the preparation method of the stomach buffer：Weigh 0.5425g sodium chloride, 0.3925g potassium chloride and 1.5g
Potassium sorbate is put into 500mL beaker, 400mL deionized water dissolving is added, and with the hydrochloric acid of 2mol/L（HCL）Under 41 °C
It adjusts the pH to 3.0 of solution, above-mentioned solution is transferred to 500mL volumetric flask after cooling, and with deionized water constant volume.
Step 2）In, the preparation method of the stomach simulated digestive juice：The determination of peptic activity described according to Wirnt R
Method measures the activity of pepsin in pepsin.It then, is 1550U/ according to the concentration of pepsin in chicken simulate the gastric juice
Ml, the pepsin for weighing 387.5KU are dissolved in the hydrochloric acid solution of 250ml pH 2.0（PH is demarcated at 41 DEG C）.It is slowly stirred
Until dissolution, overheats when not heating simulate the gastric juice on hot plate, or preparing；Prepared before use.
Step 2）In, the preparation method of the small intestine buffer：Weigh 0.5837g Anhydrous Disodium Phosphate, 2.5061g without
Water sodium dihydrogen phosphate, 0.25g potassium sorbate, 120,000 U of penicillin.It is put into 100mL beaker, 40mL deionized water dissolving is added,
And the pH to 6.50 of solution is adjusted under 41 °C with the sodium hydroxide of the phosphoric acid of 1mol/L or 1mol/L.By above-mentioned solution after cooling
It is transferred to 50mL volumetric flask, and with deionized water constant volume.
Step 2）In, the preparation method of the small intestine simulated digestive juice：The alphalise starch enzyme activity described according to Dahlqvist A
Property measuring method, Wirnt R description determination of tryptic activity method, Wirnt R description chymotrypsin activity measurement side
Method, measurement SILVER REAGENT amylase, trypsase, in chymotrypsin corresponding digestive ferment activity；Then, according to this in simulated intestinal fluid
The activity of three kinds of digestive ferments, claims respectively（Amount）Take amylase 8 8.32KU, trypsase 10.84KU, chymotrypsin 2.48KU dissolution
In 20ml deionized water, and it is slowly stirred until dissolution（The digestive enzyme activity that erepsin pulvis provides is 50% or more）.Not
It is overheated when heating, or preparing on hot plate.Prepared before use.
Step 5）In, the stomach simulation digestion parameter is：41 DEG C of temperature；Wriggling revolution speed 188rpm/min, digestion time 4
~6 h。
Step 5）With 6）In, the quantity of the simulation digest tube of the addition analysis sample is 5.
Step 6）In, the small intestine simulation digestion parameter：41 °C of temperature, wriggling revolution speed 188rpm/min, small intestinal digestion
Time is 8 ~ 12h.
Embodiment 3
Referring to Fig.1, the Bionic digestion measuring method of a kind of feed digestible carbohydrate total amount of the present embodiment, with reality
Applying example 2, there are following differences：
Step 1）In, the Feed Sample is duck feed.
Step 2）In, the preparation method of the stomach simulated digestive juice：The determination of peptic activity described according to Wirnt R
Method measures the activity of pepsin in pepsin.It then, is 1550U/ according to the concentration of pepsin in duck simulate the gastric juice
Ml, the pepsin for weighing 387.5KU are dissolved in the hydrochloric acid solution of 250ml pH 2.0（PH is demarcated at 42 DEG C）.It is slowly stirred
Until dissolution, overheats when not heating simulate the gastric juice on hot plate, or preparing；Prepared before use.
The preparation method of the small intestine buffer：Weigh 0.6067g Anhydrous Disodium Phosphate, 2.4867g anhydrous phosphoric acid two
Hydrogen sodium.
Step 2）In, the preparation method of the small intestine simulated digestive juice：The alphalise starch enzyme activity described according to Dahlqvist A
Property measuring method, Wirnt R description determination of tryptic activity method, Wirnt R description chymotrypsin activity measurement side
Method, measurement SILVER REAGENT amylase, trypsase, in chymotrypsin corresponding digestive ferment activity；Then, according to this in simulated intestinal fluid
The activity of three kinds of digestive ferments, claims respectively（Amount）Take amylase 8 8.34KU, trypsase 23.92KU, chymotrypsin 8.58KU dissolution
In 20ml deionized water, and it is slowly stirred until dissolution（The digestive enzyme activity that erepsin pulvis provides is 50% or more）.Not
It is overheated when heating, or preparing on hot plate.Prepared before use.
Step 5）In, the stomach simulation digestion parameter is：42 DEG C of temperature；Wriggling revolution speed 188rpm/min, digestion time 4
h。
Step 5）With 6）In, the quantity of the simulation digest tube of the addition analysis sample is 6.
Step 6）In, the small intestine simulation digestion parameter：42 °C of temperature, wriggling revolution speed 188rpm/min, small intestinal digestion
Time is 16h.
A kind of reagent that the Bionic digestion measuring method of feed digestible carbohydrate total amount uses of the present invention is not spy
Not Zhu Ming be analytical reagents, experimental water meets the specification of tertiary effluent in GB/T6682-2008.
Claims (10)
1. a kind of Bionic digestion measuring method of feed digestible carbohydrate total amount, which is characterized in that include the following steps：
1）The preparation of sample：Sampling, then by the Feed Sample of sampling plant pulverizer or mortar by sample comminution to crossing 60 mesh
Sieve encloses sample sack sealing storage, it is spare to make sample；
2）Prepare the buffer and simulated digestive juice and other reagents of stomach and small intestine；
3）Bionic digestive system for monogastric animals preheating：Preheating time is 60 ~ 90min；
4）Loading：Simulation digest tube in Bionic digestive system for monogastric animals is cleaned up, is dried, and will with turned welt silica gel plug
One end plug is tight；Weigh step 2）Spare 0.25g ~ 1.00g feed sample is prepared, and monogastric animal bionic Digestive is added
In the simulation digest tube of system, while measuring the dry matter content of feed sample；
5）Stomach simulates digestion process：In first simulation digest tube that stomach buffer enters into Bionic digestive system for monogastric animals
10ml deionized water is added, is separately added into 10ml stomach simulated digestive juice in the simulation digest tube of other addition analysis samples；Pass through
Bionic digestive system for monogastric animals controls software setting stomach and simulates digestion parameter, starts to carry out stomach simulation digestion process；
6）Small intestine simulates digestion process：After stomach phase digestion, entered by simulated digestive juice liquid-feeding tube to small intestine buffer
First simulation digest tube in add 6.0ml and 1.6ml deionized water in two times, other addition analysis samples simulations digestion
6.0ml small intestine buffer and 1.6ml small intestine simulated digestive juice are successively added in pipe respectively；Pass through Bionic digestive system for monogastric animals
It controls software setting small intestine and simulates digestion parameter, start to carry out small intestine simulation digestion process；
7）Slaking residue processing：To feed sample through stomach and small intestine simulation digestion after, by the digestive juice in glass digest tube,
After dilution is handled, by membrane filtration, filtrate is spare；The direct membrane filtration of the digestive juice of control group, it is spare；
8）Using the content of reducing sugar in DNS colorimetric method for determining control sample and analysis sample, and disappearing in feed is calculated
Change carbohydrate total amount.
2. the Bionic digestion measuring method of feed digestible carbohydrate total amount as described in claim 1, which is characterized in that step
Rapid 1）In, the Feed Sample is pannage, chicken feed or duck feed.
3. the Bionic digestion measuring method of feed digestible carbohydrate total amount as claimed in claim 1 or 2, feature exist
In step 2）In, the preparation method of the stomach buffer：Weigh 1 ~ 3g sodium chloride, 0.25 ~ 5g potassium chloride and 1 ~ 2g sorbic acid
Potassium is put into 500mL beaker, and 400mL deionized water dissolving is added, and is adjusted at 39 DEG C ~ 42 DEG C with the hydrochloric acid of 2mol/L molten
Above-mentioned solution is transferred to 500mL volumetric flask after cooling by the pH to 3.0 of liquid, and with deionized water constant volume；
The preparation method of the stomach simulated digestive juice：Measure the activity of pepsin in pepsin；Then, according to simulate the gastric juice
The concentration of middle pepsin be 737.5 ~ 1550U/ml, weigh 184.38KU ~ 387.5KU pepsin be dissolved in 250ml,
In the stomach buffer of pH2.0 ~ 3.0, it is slowly stirred until dissolution, mistake when not heating simulate the gastric juice on hot plate, or preparing
Heat；Prepared before use.
4. the Bionic digestion measuring method of feed digestible carbohydrate total amount as claimed in claim 1 or 2, feature exist
In step 2）In, the preparation method of the small intestine buffer：Weigh 0.5 ~ 5g Anhydrous Disodium Phosphate, 2.0 ~ 3g anhydrous phosphoric acid
Sodium dihydrogen, PBS ultimate density are 0.05M, 0.2 ~ 2g potassium sorbate, 120,000 U of penicillin；It is put into 100mL beaker, 40mL is added
Deionized water dissolving, and adjusted at 39 DEG C ~ 42 DEG C with the sodium hydroxide of the phosphoric acid of 1mol/L or 1mol/L the pH of solution to
6.30~6.50；Above-mentioned solution is transferred to 50mL volumetric flask after cooling, and with deionized water constant volume；
The preparation method of the small intestine simulated digestive juice：Measurement SILVER REAGENT amylase, trypsase accordingly digest in chymotrypsin
The activity of enzyme；Then, according to the activity of these three digestive ferments in simulated intestinal fluid, amylase 41.41KU ~ 97.12KU is weighed respectively,
Trypsase 9.54KU ~ 26.32KU, chymotrypsin 1.62KU ~ 9.44KU are dissolved in 17 ~ 20ml deionized water, and slowly stir
It mixes until dissolution；It is overheated when not heating, or preparing on hot plate；Prepared before use.
5. the Bionic digestion measuring method of feed digestible carbohydrate total amount as claimed in claim 1 or 2, feature exist
In step 5）With 6）In, quantity >=3 piece of the simulation digest tube of the addition analysis sample.
6. the Bionic digestion measuring method of feed digestible carbohydrate total amount as claimed in claim 5, which is characterized in that step
Rapid 5）With 6）In, the quantity of the simulation digest tube of the addition analysis sample is 4 ~ 6.
7. the Bionic digestion measuring method of feed digestible carbohydrate total amount as claimed in claim 1 or 2, feature exist
In step 5）In, the stomach simulation digestion parameter is：39 ~ 42 DEG C of temperature；Wriggling revolution speed 188rpm/min, digestion time 4 ~ 6
h。
8. the Bionic digestion measuring method of feed digestible carbohydrate total amount as claimed in claim 1 or 2, feature exist
In step 6）In, the small intestine simulation digestion parameter：39 ~ 42 °C of temperature, wriggling revolution speed 188rpm/min, when small intestinal digestion
Between be 8 ~ 16 h.
9. the Bionic digestion measuring method of feed digestible carbohydrate total amount as claimed in claim 1 or 2, feature exist
In step 6）In, the content of reducing sugar in DNS colorimetric method for determining sample includes the following steps：1. drawing standard curve：It draws
DNS reagent 5ml, boiling water bath 5min is added in 25ml scale test tube in deionized water 4ml, and tap water is cooled to room temperature, deionization
Water is settled to 25ml, and standard blank sample is made；Draw respectively glucose solution 1.00ml, 2.00ml of 10mg/ml, 3.00ml,
4.00ml, 5.00ml, 6.00ml and 7.00ml are settled to 100ml with deionized water respectively, and being configured to concentration is 0.10mg/ml
The glucose standards solution of~0.70mg/ml；Each 2ml of glucose standard for drawing above-mentioned concentration respectively, is added to 25ml scale
In test tube, 2.0ml deionized water is added, is mixed, 5.0ml DNS reagent is added, is mixed, boiling water bath 5min, tap water is cooling
To room temperature, adds 16ml deionized water, shake up；It is control zeroing with standard blank sample, absorbance OD value is surveyed at 540nm；With Portugal
Grape sugar concentration is Y-axis, and absorbance OD value is X-axis, draws standard curve y=aX+b；
2. reducing sugar test in control sample and analysis sample：Take step 7）Then the digestive juice 5ml of preparation is added in test tube
5ml deionized water carries out 2 times of dilutions；Digestive juice after taking 2ml to dilute again is added in 25ml scale test tube, then adds 2ml to go again
Ionized water, oscillation mix, and 5ml DNS is added, and mix, boiling water bath 5min；Tap water is cooled to room temperature, and adds 16ml deionized water,
It mixes, OD value is surveyed at spectrophotometer 540nm, obtains the OD of control sample（Control）With the OD of analysis sample（Sample）。
10. the Bionic digestion measuring method of feed digestible carbohydrate total amount as claimed in claim 1 or 2, feature exist
In step 8）In, the calculation formula of the digestible carbohydrate total amount is：
Digestible carbohydrate total amount (mg/g DM)=【(a×OD（Sample）+b)×2×200-(a×OD（Control）+b)×
17.6】/(w×DM)
In formula：A is standard curve regression coefficient；
B is standard curve regression constant；
OD is measured as the absorbance value of analysis Specimen Determination pipe；
OD blank is the absorbance value of control sample pipe；
W is each replication tube feed material example weight；
DM is the dry matter content of Feed Sample.
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| CN105891428B (en) * | 2016-06-16 | 2018-05-25 | 湖南中本智能科技发展有限公司 | A kind of full-automatic Bionic digestive system for monogastric animals |
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| 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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| CN111175166A (en) * | 2020-01-06 | 2020-05-19 | 华南农业大学 | In-vitro bionic digestion method for determining nutrient utilization rate of duck feed |
| CN113341059B (en) * | 2021-05-31 | 2023-09-22 | 中国农业科学院北京畜牧兽医研究所 | Bionic digestion method for growing pig stomach-small intestine-large intestine and application of bionic digestion method to estimation of effective value of feed |
| CN114657230B (en) * | 2022-02-23 | 2023-11-24 | 中国农业科学院北京畜牧兽医研究所 | Method for evaluating fermentation characteristics of fiber raw materials by combining bionic digestion and in-vitro fermentation |
| CN117491351B (en) * | 2023-12-27 | 2024-04-05 | 中国农业科学院北京畜牧兽医研究所 | Bionic digestion determination kit and determination method for effective phosphorus of broiler feed |
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