CN107739748A - A kind of assay method of Pepsin digestibility and its application - Google Patents
A kind of assay method of Pepsin digestibility and its application Download PDFInfo
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- CN107739748A CN107739748A CN201710951110.XA CN201710951110A CN107739748A CN 107739748 A CN107739748 A CN 107739748A CN 201710951110 A CN201710951110 A CN 201710951110A CN 107739748 A CN107739748 A CN 107739748A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
- C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N2333/90—Enzymes; Proenzymes
- G01N2333/914—Hydrolases (3)
- G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
- G01N2333/95—Proteinases, i.e. endopeptidases (3.4.21-3.4.99)
- G01N2333/964—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue
- G01N2333/96425—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals
- G01N2333/96427—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general
- G01N2333/9643—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general with EC number
- G01N2333/96472—Aspartic endopeptidases (3.4.23)
- G01N2333/96475—Aspartic endopeptidases (3.4.23) with definite EC number
- G01N2333/96477—Pepsin (3.4.23.1; 3.4.23.2; 3.4.23.3)
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Abstract
The present invention relates to a kind of assay method of Pepsin digestibility and its application, belong to the detection technique field of chemical composition.This method comprises the following steps：Same sample is taken to be divided into the first sample and the second sample, the first sample determines gross protein value after degreasing, and the second sample obtains residue, determine gross protein value in residue through degreasing, pepsin enzymolysis, centrifugation after washing.Calculate as follows and produce Pepsin digestibility：Digestibility=sample gross protein value × 100% of (gross protein value in first sample gross protein value the second sample residue)/first.Above-mentioned gross protein value is calculated in mass percent.Assay method operation is efficiently convenient, and agents useful for same species is few, source is wide, and the result through assay method measure gained is reproducible, accurate and effective.This method is used to determine Pepsin digestibility, batch processing can be achieved, improves efficiency, it is cost-effective.
Description
Technical field
The present invention relates to the detection technique field of chemical composition, and more particularly to a kind of measure side of Pepsin digestibility
Method and its application.
Background technology
In recent years, the assay method on digestibility received significant attention, main assay method have digestive juice determination method,
Digest enzyme extraction method, intestines isolation method, indicator method and isotope-labelling method etc..Wherein, pepsin determination method is because with quick
The easy, advantage such as stability is good, and be widely used in fish meal and other animal protein feeds.At present, fish meal is evaluated
The index of quality mainly has sense organ, thick protein, crude fat, moisture, salinity, ash content and sanitary index etc., but is only referred to these
Mark is difficult the real quality of thoroughly evaluating fish meal, and can not especially reflect in fish meal can be contained by the protein of livestock and poultry digestibility and utilization
Amount.It is evaluation and the measure of fish meal Pepsin digestibility is that the true reflection for absorbing protein content can be digested in fish meal
The important indicator of quality of fishmeal.Therefore, the measure of fish meal Pepsin digestibility is also increasingly taken seriously.The inventive method energy
Enough rapidly and accurately to determine fish meal Pepsin digestibility, buying and examination to fish meal provide reference data, while to feed
Quality Safety is significant.
Not yet clear and definite to fish meal Pepsin digestibility measure based on country, everybody is to fish meal Pepsin digestibility at present
Assay method slightly difference, therefore be necessary the method for releasing an accurate easy measure fish meal Pepsin digestibility.
The content of the invention
It is an object of the invention to provide a kind of assay method of Pepsin digestibility, assay method operation is efficient just
Victory, agents useful for same species is few, source is wide, and the result through assay method measure gained is reproducible, accurate and effective.
Another object of the present invention is to provide a kind of assay method of above-mentioned Pepsin digestibility in measure stomach cardia
Application in enzymic digestion rate, the especially application in the Pepsin digestibility of measure fish meal, can be real with above-mentioned assay method
The batch processing of existing testing sample, efficiency is improved, it is cost-effective.
The present invention is solved its technical problem and realized using following technical scheme：
The present invention proposes a kind of assay method of Pepsin digestibility, and it comprises the following steps：
Same sample is taken to be divided into the first sample and the second sample, the first sample determines gross protein value after degreasing, the
Two samples obtain residue, determine gross protein value in residue through degreasing, pepsin enzymolysis, centrifugation after washing.
Calculate as follows and produce Pepsin digestibility：Digestibility=(the first sample gross protein value-the second tries
Gross protein value in sample residue)/first sample gross protein value × 100%.
Above-mentioned gross protein value is calculated in mass percent.
The present invention also proposes application of the assay method of above-mentioned Pepsin digestibility in Pepsin digestibility is determined,
Such as it can be used for determining the Pepsin digestibility of fish meal.
The assay method of the Pepsin digestibility of the embodiment of the present invention and its beneficial effect of application are：
Replace in national standard " being placed in 45° angle, allow residue to precipitate more than 15min " that experimental period can be shortened with centrifugation, improve
Conventional efficient.In addition, by way of centrifugally operated can overcome and reach separation by suction filtration in the prior art, time-consuming and nothing
Method realizes the problem of batch processing, greatly improves separating effect, saves the time.
The assay method operation of the Pepsin digestibility of the embodiment of the present invention is efficiently convenient, and agents useful for same species is few, comes
Source is wide, and the result through assay method measure gained is reproducible, accurate and effective.Use it for determining Pepsin digestibility,
In the Pepsin digestibility for especially determining fish meal, the batch processing of testing sample can be achieved, improve efficiency, it is cost-effective.
Embodiment
, below will be in the embodiment of the present invention to make the purpose, technical scheme and advantage of the embodiment of the present invention clearer
Technical scheme be clearly and completely described.Unreceipted actual conditions person, builds according to normal condition or manufacturer in embodiment
The condition of view is carried out.Agents useful for same or the unreceipted production firm person of instrument, it is the conventional production that can be obtained by commercially available purchase
Product.
The assay method to the Pepsin digestibility of the embodiment of the present invention and its application are specifically described below.
The assay method of Pepsin digestibility provided in an embodiment of the present invention, the measure that is particularly suitable for use in fish meal pepsin
Digestibility, specifically, comprise the following steps：
Same sample is taken to be divided into the first sample and the second sample.Wherein, the first sample determines thick protein after degreasing and contained
Amount, the second sample obtain residue, determine gross protein value in residue through degreasing, enzymolysis, centrifugation after washing.
Then calculate as follows and produce Pepsin digestibility：Digestibility=(the first sample gross protein value-the
Gross protein value in two sample residues)/first sample gross protein value × 100%.Above-mentioned gross protein value is with matter
Measure percentage meter.
Optionally, above-mentioned sample before the assay, through pulverization process.Such as can by sample comminution to 0.6-1mm, or
20 mesh sieves are crossed, to improve grease removal and hydrolysis result.
Because crude fat content is in 7-10% in fish meal, if without ungrease treatment, the part crude fat is easily to pepsin
The measurement result of digestibility has an impact.Therefore in embodiments of the present invention, the sample of preferred pair first and the second sample are taken off
Fat processing.
It is preferred that the skimming processes of the first sample and the second sample are drenched with degreasing agent in the embodiment of the present invention
Wash.Optionally, degreasing agent can be petroleum ether or ether.Preferably petroleum ether.
Specifically, the first sample or the second sample can be added and are converted into funnelform filter paper, then carried out with degreasing agent
Elute degreasing.Air-dried after elution, remove degreasing agent.Air-dry can be in carrying out, to avoid solvent etc. from distributing in air in fume hood.
Optionally, the amount of weighing of above-mentioned second sample can be 0.7-1.1g, preferably 0.9-1.0g, the weighing model
The result determined in enclosing is the most stable, and error is minimum.The volume of degreasing agent per the samples of 1g first or used in the second sample is
8-12mL, preferably repeat elution twice, elute altogether three times, to reduce the residual volume of fat.
What deserves to be explained is in the embodiment of the present invention in the case of petroleum ether is used as degreasing agent, when degreasing room temperature is low
When 30 DEG C, it is preferred to use boiling point is 30-60 DEG C of petroleum ether；When degreasing room temperature is higher than 30 DEG C, it is preferred to use boiling point 60-
90 DEG C of petroleum ether, to avoid petroleum ether from being evaporated in skimming processes, influence grease removal effect.
To hold, the embodiment of the present invention is degreasing agent preferably with the petroleum ether for being not belonging to easily make malicious reagent type, and purchase is convenient,
And can be according to the petroleum ether that room temperature height corresponding selection boiling point is 30 DEG C -60 DEG C or 60 DEG C -90 DEG C.State is replaced by eluting mode
Leaching mode in mark, it is efficiently convenient to can reach, and saves time cost and the significant effect of degreasing effect.
First sample after degreasing, can use 0.3000g ± 0.0002g according to《GB/T 6432》Middle gross protein value
Assay method determines gross protein value.
Second sample is digested after degreasing with pepsin to it.Enzymolysis can be used under conditions of 42-48 DEG C
Above-mentioned second sample after pepsin solution processing degreasing, enzymolysis time is preferably 14-18h.It is preferred that above-mentioned pepsin
The concentration of pepsin is 18-22IU/mL in solution, and more preferably, concentration of the pepsin in pepsin solution is 20IU/
mL。
In order that enzymolysis is more abundant, enzymolysis process can also be uses stomach egg under conditions of rotating speed is 180-220r/min
The second sample after white enzyme solutions processing degreasing.Optionally, the ratio of the second sample and pepsin solution can be
150mL：0.9-1.0g.
Specifically, such as the second sample connection filter paper funnel after can degreasing be air-dried be put into 250mL tool plug ground
In bottle, pepsin solution that is that 150mL is newly configured and being pre-heated to 42-48 DEG C is then added, bottle cap is covered tightly and is placed in 42-48
DEG C constant-temperature table in, the vibration insulation enzymolysis, digestion 14-18h under conditions of rotating speed is 180-220r/min.It is worth explanation
It is in enzymolysis process, the second sample is soaked completely by pepsin solution, to ensure hydrolysis result.
Wherein, concentration can be prepared through in the following manner for 20IU/mL pepsin solution and obtained：Dilute the dense salt of 6.1mL
For acid into 1000mL water, regulation pH value is 1-2, is then heated to 42-48 DEG C.It is 1 to add 6.67g activity：3000 biochemical level
Pepsin (is pressed before use《Republic of China Veterinary Pharmacopoeia》Middle prescriptive procedure determines pepsin activity).
Further, centrifugally operated is carried out to the second sample after degreasing.Specifically, will be after pepsin solution digests
The second sample take out, be sub-packed in 50mL centrifuge tube, 8-12min then separated under 3800-4200r/min rotating speed, it is excellent
Select and separate 10min under 4000r/min rotating speed, obtain residue.It " is in 45 ° to be replaced in the embodiment of the present invention with centrifugation in national standard
Angle is placed, and allows residue to precipitate more than 15min " experimental period can be shortened, improve conventional efficient.Further, since lead in the prior art
Cross filter reach separation the mode time it is longer, and batch processing can not be realized, therefore by above-mentioned centrifugally operated, can carry significantly
High effect, the time is saved, realizes sample batch processing.
Further, with the content of thick protein in the filtrate of gained after 42-48 DEG C of the above-mentioned residue of water washing to washing
It is stable.Specifically, above-mentioned residue can be filtered on the Buchner funnel for be covered with fast grade filter paper, first with a small amount of warm water (42-48 DEG C)
Residue on bottle cap is washed till on filter paper, then the supernatant after centrifugation is slowly poured out.After supernatant is by filter paper, with equipped with
The plastic squeeze bottle of warm water (42-48 DEG C) cleans in centrifuge tube and port grinding bottle the residue not poured out repeatedly, is then pressed again with glass bar
The residue that firmly filter paper in port grinding bottle will be attached in port grinding bottle bottle wall elutes, and cleans centrifuge tube and port grinding bottle repeatedly, directly
Extremely all residues are transferred in the Buchner funnel for being covered with fast grade filter paper.After whole liquid are by Buchner funnel, warm water is used
(42-48 DEG C) repeatedly residue in washing Buchner funnel on filter paper, and drain moisture as far as possible.
What deserves to be explained is replace the acetone in national standard to wash residue with warm water (42-48 DEG C) in the present embodiment, first,
For warm water is compared with acetone, safety and cost is low；Second, acetone can be avoided, because evaporation rate is very fast, to be reached during suction filtration
Less than the effect of cleaning residue, so as to improve the degree of accuracy of measurement result.
After washing terminates, self-test is carried out to residual washing-out result, i.e., all poured out the filtrate in bottle,suction, and will filtered
Bottle washes clean, then residue is washed with warm water (42-48 DEG C), filtrate is collected, and gross protein value measure is carried out to filtrate.
The step of by newly-increased residual washing-out result self-test, it may be determined that the soluble crude protein in thick protein is through stomach egg
Whether eluted totally after white enzyme solutions enzymolysis, filled up in national standard and do not lacked using acetone washing residue to what whether residue was cleaned
Fall into.
After crude protein content constant (stabilization) in filtrate, the residue of gained is taken to carry out gross protein value measure.It is above-mentioned
Crude protein content is constant to refer to that the front and rear crude protein content permission relative deviation measured twice is no more than 3%.Specifically, such as can
To remove the filter paper for being loaded with residue from Buchner funnel, nondestructively move into Foss kelvin digest tubes, add about 6.4g mixing and urge
Agent and the 20mL concentrated sulfuric acids (often add a filter paper need to add the about 5mL concentrated sulfuric acids digested), are subsequently placed in Foss digestings
Upper 420 DEG C of digestion is pressed to transparent blue-green (color of copper sulphate)《GB/T 6432》It is thick in assay method measure residue
Protein content.
What deserves to be explained is be additionally provided with blank control group in the embodiment of the present invention, blank control group except not adding an examination of in addition to sample,
Remaining step is consistent with the processing step of the second sample, and measure income value is blank determination value.First sample gross protein value
Actual to subtract blank determination value for the first sample gross protein value measured value, gross protein value is actual in the second sample residue
Blank determination value is subtracted for gross protein value measured value in the second sample residue.
The feature and performance of the present invention are described in further detail with reference to embodiments.
Embodiment 1
Same fish meal sample is taken to be divided into the first sample and the second sample.The first sample and the second sample are drenched with ether
Fat is eluted, elution altogether three times, is pressed per the samples of 1g first or the second sample is eluted with 8mL ether every time.Wherein, second sample
The amount of weighing is 0.7g.
The first sample after 0.2998g degreasings is weighed, according to《GB/T 6432》The assay method of middle gross protein value is surveyed
Determine gross protein value, obtain the first sample gross protein value.
By the second sample after degreasing under conditions of 42 DEG C, 18h is digested with pepsin solution.Second sample and stomach egg
The ratio of white enzyme solutions is 150mL：0.9g, the concentration of pepsin is 18IU/mL in pepsin solution.
The second sample after enzymolysis is separated into 12min under 3800r/min rotating speed, obtains residue.With 42 DEG C of water washing
Above-mentioned residue, and the stable content of self-test thick protein in the filtrate of gained to after washing is carried out to residual washing-out result, then
With《GB/T 6432》In assay method measure residue in gross protein value.
The Pepsin digestibility of fish meal is produced according to below equation：Digestibility=(the first sample gross protein value-the
Gross protein value in two sample residues)/first sample gross protein value × 100%.Above-mentioned gross protein value is with matter
Measure percentage meter.
Embodiment 2
Same fish meal sample comminution is taken then to be divided into the first sample and the second sample to 0.6mm.With ether to the first sample
Elution degreasing is carried out with the second sample, elution altogether three times, is pressed per the samples of 1g first or the second sample 12mL ether drenches every time
Wash.Wherein, the amount of weighing of the second sample is 1.1g.
The first sample after 0.3002g degreasings is weighed, according to《GB/T 6432》The assay method of middle gross protein value is surveyed
Determine gross protein value, obtain the first sample gross protein value.
By the second sample after degreasing in 48 DEG C, the constant-temperature table that rotating speed is 180r/min, with pepsin solution enzyme
Solve 14h.The ratio of second sample and pepsin solution is 150mL：1g, the concentration of pepsin is in pepsin solution
22IU/mL。
The second sample after enzymolysis is separated into 8min under 4200r/min rotating speed, obtains residue.With in 48 DEG C of water washings
Residue is stated, and the stable content of self-test thick protein in the filtrate of gained to after washing, Ran Houyong are carried out to residual washing-out result
《GB/T 6432》In assay method measure residue in gross protein value.
The Pepsin digestibility of fish meal is produced according to below equation：Digestibility=(the first sample gross protein value-the
Gross protein value in two sample residues)/first sample gross protein value × 100%.Above-mentioned gross protein value is with matter
Measure percentage meter.
Embodiment 3
Same fish meal sample comminution is taken then to be divided into the first sample and the second sample to 1mm.With ether to the first sample and
Second sample carries out elution degreasing, and elution altogether three times, is pressed per the samples of 1g first or the second sample 10mL petroleum ethers drench every time
Wash.Wherein, the amount of weighing of the second sample is 0.9g.
The first sample after 0.3000g degreasings is weighed, according to《GB/T 6432》The assay method of middle gross protein value is surveyed
Determine gross protein value, obtain the first sample gross protein value.
By the second sample after degreasing in 45 DEG C, the constant-temperature table that rotating speed is 220r/min, with pepsin solution enzyme
Solve 16h.The ratio of second sample and pepsin solution is 150mL：0.95g, the concentration of pepsin in pepsin solution
For 20IU/mL.
The second sample after enzymolysis is separated into 10min under 4000r/min rotating speed, obtains residue.With 45 DEG C of water washing
Above-mentioned residue, and the stable content of self-test thick protein in the filtrate of gained to after washing is carried out to residual washing-out result, then
With《GB/T 6432》In assay method measure residue in gross protein value.
The Pepsin digestibility of fish meal is produced according to below equation：Digestibility=(the first sample gross protein value-the
Gross protein value in two sample residues)/first sample gross protein value × 100%.Above-mentioned gross protein value is with matter
Measure percentage meter.
Embodiment 4
Same fish meal sample comminution is taken then to be divided into the first sample and the second sample to 0.8mm.With ether to the first sample
Elution degreasing is carried out with the second sample, elution altogether three times, is pressed per the samples of 1g first or the second sample 10mL petroleum ethers every time
Elution.Wherein, the amount of weighing of the second sample is 1.0g.
The first sample after 0.3000g degreasings is weighed, according to《GB/T 6432》The assay method of middle gross protein value is surveyed
Determine gross protein value, obtain the first sample gross protein value.
By the second sample after degreasing in 45 DEG C, the constant-temperature table that rotating speed is 200r/min, with pepsin solution enzyme
Solve 16h.The ratio of second sample and pepsin solution is 150mL：0.9g, the concentration of pepsin is in pepsin solution
20IU/mL。
The second sample after enzymolysis is separated into 10min under 4000r/min rotating speed, obtains residue.With 45 DEG C of water washing
Above-mentioned residue, and the stable content of self-test thick protein in the filtrate of gained to after washing is carried out to residual washing-out result, then
With《GB/T 6432》In assay method measure residue in gross protein value.
The Pepsin digestibility of fish meal is produced according to below equation：Digestibility=(the first sample gross protein value-the
Gross protein value in two sample residues)/first sample gross protein value × 100%.Above-mentioned gross protein value is with matter
Measure percentage meter.
Embodiment 5
Take same fish meal sample comminution to cross 20 mesh sieves, be then divided into the first sample and the second sample.Tried with ether first
Sample and the second sample carry out elution degreasing, and elution altogether three times, is pressed per the samples of 1g first or the second sample 10mL oil every time
Ether elutes.Wherein, the amount of weighing of the second sample is 0.95g.
The first sample after 0.3000g degreasings is weighed, according to《GB/T 6432》The assay method of middle gross protein value is surveyed
Determine gross protein value, obtain the first sample gross protein value.
By the second sample after degreasing in 45 DEG C, the constant-temperature table that rotating speed is 200r/min, with pepsin solution enzyme
Solve 16h.The ratio of second sample and pepsin solution is 150mL：0.9g, the concentration of pepsin is in pepsin solution
20IU/mL。
The second sample after enzymolysis is separated into 10min under 4000r/min rotating speed, obtains residue.With 45 DEG C of water washing
Above-mentioned residue, and the stable content of self-test thick protein in the filtrate of gained to after washing is carried out to residual washing-out result, then
With《GB/T 6432》In assay method measure residue in gross protein value.
The Pepsin digestibility of fish meal is produced according to below equation：Digestibility=(the first sample gross protein value-the
Gross protein value in two sample residues)/first sample gross protein value × 100%.Above-mentioned gross protein value is with matter
Measure percentage meter.
Test example 1
Different amounts of same fish meal sample is taken to be divided into 1-5 groups, every group is divided into the first sample and the second sample, in every group
The amount of first sample is equal, and the amount of the second sample then distinguishes the sequence number 1-5 in corresponding table 1, and every group of setting 2 is parallel.Namely fish
The sample weighting amount of the sample of powder second is respectively 0.7g, 0.8g, 0.9g, 1.0g and 1.1g, and when weighing the second sample weight error
No more than 0.01g.The pepsin digestion of above-mentioned 1-5 groups fish meal sample is determined according to the determination method method of the embodiment of the present invention 4
Rate, its result are as shown in table 1.
The Pepsin digestibility of the fish meal sample of table 1
As can be seen from Table 1, when the sample weighting amount of the second sample is 0.7-1.1g scopes, the stomach cardia of same fish meal sample
The measurement result of enzymic digestion rate is as a result more consistent 96% or so.But by contrast, the sample weighting amount of the second sample is 0.9-
When in the range of 1.0g, the measurement result of the pepsin effect rate of fish meal sample is the most stable, namely between two groups of parallel samples
Measurement result difference is minimum.Therefore, the sample weighting amount Optimal Control of the second sample of the fish meal sample in the embodiment of the present invention exists
0.9-1.0g。
Test example 2
This test example is provided with 3 groups of test groups, and the quality and weight of the fish meal sample of every group of test group are consistent, every group of experiment
Group sets 5 parallel repeating groups.Test group 1 carries out ungrease treatment by the way of ether extraction to fish meal sample, and test group 2 is adopted
The mode eluted with ether carries out ungrease treatment to fish meal sample, and test group 3 is by the way of petroleum ether elution to fish meal sample
Carry out ungrease treatment.The crude fat content (%) of the sample after the processing of test group 1 is determined respectively, after the processing of test group 2
Sample residue crude fat content (%) and the sample residue crude fat content (%) after the processing of test group 3.Its result such as table 2
It is shown.
The crude fat content of table 2
As can be seen from Table 2, compared with test group 1, can effectively be removed using the elution mode of test group 2 and test group 3
The crude fat gone in fish meal sample, its remaining crude fat content can reach measure Pepsin digestibility to sample after processing
The requirement of crude fat content, and the cumbersome of leaching mode can be saved, while time cost is saved, improve test efficiency.It is in addition, right
Than test group 2 and test group 3 as can be seen that same fish meal sample sample residue fat content and warp after petroleum ether elutes degreasing
Sample residue fat content is consistent substantially after ether elution degreasing, illustrates petroleum ether is substituted into national standard in the embodiment of the present invention
In ether it is effective and feasible.And petroleum ether is safer compared with ether use, and different boilings can be selected according to different degreasing conditions
The oil ether product of point.
Test example 3
This test example is provided with 2 groups of test groups, and the quality and weight of the fish meal sample of every group of test group are consistent.Wherein, try
The assay method that group 1 is the fish meal Pepsin digestibility in embodiment 4 is tested, unique difference of test group 2 and embodiment 4 is
" being placed in 45° angle, allow residue to precipitate more than 15min " in national standard is used to replace the centrifugation step in embodiment 4 in test group 2
(separating 10min under 4000r/min rotating speed).Every group of test group sets 5 parallel repeating groups.Two groups of test group fishes are determined respectively
Powder Pepsin digestibility (%).Its result is as shown in table 3.
The fish meal Pepsin digestibility of table 3
As can be seen from Table 3, test group 1 and the fish meal Pepsin digestibility result of the gained of test group 2 keep one substantially
It is fixed, illustrate " being placed in 45° angle, allow residue to precipitate more than 15min " in centrifugally operated replacement national standard in the embodiment of the present invention
It is effective and feasible.Also, for test group 2, the test period can also be shortened using the centrifugally operated in test group 1, improve experiment
Efficiency.
Test example 4
This test example is provided with 2 groups of test groups, and test group 1 is consistent with the quality and weight of the fish meal sample of test group 2.Its
In, test group 1 is unique area of the assay method of the fish meal Pepsin digestibility in embodiment 4, test group 2 and embodiment 4
The warm water (45 DEG C) in embodiment 4 is not being replaced to wash residue with " acetone " in national standard in test group 2.Every group of examination
Group 5 parallel repeating groups of setting are tested, determine two groups of test group fish meal Pepsin digestibilities (%), its result such as institute of table 4 respectively
Show.
The Pepsin digestibility of table 4 and gross protein value
As can be seen from Table 4, gross protein value is higher in filtrate after washing residue with acetone in test group 2, therefore stomach
Protease digestion rate is relatively low, and its reason may be very fast due to the evaporation rate of acetone so that residue fails to be washed totally, causes
Residue gross protein value is higher, causes final result relatively low.Thick protein in filtrate after washing residue with warm water in test group 1
Content is compared with lower in test group 2, namely Pepsin digestibility is higher, illustrates to replace national standard with warm water in the embodiment of the present invention
In " acetone " washing residue and the newly-increased operation that self-test is carried out to residue can improve the accuracy of measurement result.
Test example 5
This test example is provided with 2 groups of test groups, and test group 1 is consistent with the quality and weight of the fish meal sample of test group 2.Its
In, test group 1 is the assay method of the fish meal Pepsin digestibility (%) in embodiment 4, and test group 2 and embodiment 4 are only
One difference is using national standard in test group 2《GB/T 17811-2008》Carry out the measure of Pepsin digestibility (%).Every group of examination
Test group 5 parallel repeating groups of setting.Its result is as shown in table 5.
The Pepsin digestibility of table 5
| Sequence number | Test group 1 | Test group 2 |
| 1 | 94.52 | 94.05 |
| 2 | 94.08 | 93.86 |
| 3 | 96.68 | 96.42 |
| 4 | 96.03 | 95.57 |
| 5 | 95.14 | 94.66 |
As can be seen from Table 5, assay method and national standard of the embodiment of the present invention to fish meal Pepsin digestibility《GB/T
17811-2008》Assay method result difference (there was no significant difference) in allowed band, illustrates provided in an embodiment of the present invention
The assay method of Pepsin digestibility is effective and feasible.
In summary, efficient convenient, the agents useful for same of assay method operation of the Pepsin digestibility of the embodiment of the present invention
Species is few, source is wide, and the result through assay method measure gained is reproducible, accurate and effective.Use it for determining stomach cardia
In enzymic digestion rate, the especially Pepsin digestibility of measure fish meal, the batch processing of testing sample can be achieved, improve efficiency,
It is cost-effective.
Embodiments described above is part of the embodiment of the present invention, rather than whole embodiments.The reality of the present invention
The detailed description for applying example is not intended to limit the scope of claimed invention, but is merely representative of the selected implementation of the present invention
Example.Based on the embodiment in the present invention, what those of ordinary skill in the art were obtained under the premise of creative work is not made
Every other embodiment, belongs to the scope of protection of the invention.
Claims (10)
1. a kind of assay method of Pepsin digestibility, it is characterised in that comprise the following steps：
Same sample is taken to be divided into the first sample and the second sample, first sample determines gross protein value after degreasing, institute
The second sample is stated through degreasing, pepsin enzymolysis, centrifugation, residue is obtained after washing, determines gross protein value in the residue；
Calculate as follows and produce Pepsin digestibility：Digestibility=(first the-the second sample of sample gross protein value is residual
Gross protein value in slag)/first sample gross protein value × 100%；The gross protein value is with mass percent
Meter.
2. the assay method of Pepsin digestibility according to claim 1, it is characterised in that first sample and institute
The degreasing for stating the second sample is eluted with degreasing agent, and the degreasing agent is selected from petroleum ether or ether.
3. the assay method of Pepsin digestibility according to claim 1, it is characterised in that enzymolysis is at 42-48 DEG C
Under conditions of with pepsin solution handle degreasing after the second sample 14-18h, stomach described in the pepsin solution
The concentration of protease is 18-22IU/mL.
4. the assay method of Pepsin digestibility according to claim 3, it is characterised in that enzymolysis is to be in rotating speed
Under conditions of 180-220r/min second sample after degreasing is handled with the pepsin solution.
5. the assay method of Pepsin digestibility according to claim 3, it is characterised in that the pepsin solution
Ratio with second sample is 0.9-1.0g：150mL.
6. the assay method of Pepsin digestibility according to claim 1, it is characterised in that centrifugation is in 3800-
8-12min is separated under 4200r/min rotating speed.
7. the assay method of Pepsin digestibility according to claim 1, it is characterised in that also include using 42-48 DEG C
Water washing described in residue to wash after gained filtrate in thick protein content it is constant.
8. the assay method of Pepsin digestibility according to claim 1, it is characterised in that the matter of second sample
Measure as 0.9-1.0g.
9. the assay method of Pepsin digestibility according to claim 1, it is characterised in that before measure, described in crushing
Sample is to 0.6-1mm.
10. the assay method of the Pepsin digestibility as described in claim any one of 1-9 is in measure Pepsin digestibility
In application.
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| CN113702568A (en) * | 2021-08-06 | 2021-11-26 | 福建傲农生物科技集团股份有限公司 | Method for measuring pepsin digestibility of animal protein feed |
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| CN113702568A (en) * | 2021-08-06 | 2021-11-26 | 福建傲农生物科技集团股份有限公司 | Method for measuring pepsin digestibility of animal protein feed |
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