CN106872614A - Composition, kit and its detection method for detecting donkey-hide gelatin content in compound donkey-hide gelatin preparation - Google Patents
Composition, kit and its detection method for detecting donkey-hide gelatin content in compound donkey-hide gelatin preparation Download PDFInfo
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Abstract
The invention discloses a kind of composition for detecting donkey-hide gelatin content in compound donkey-hide gelatin preparation, kit and its detection method.The composition is made up of polypeptide I, polypeptide II, polypeptide III and reference substance detection matrix, the amino acid sequence of the polypeptide I is shown in SEQ ID NO.1, the amino acid sequence of polypeptide II is shown in SEQ ID NO.2, the amino acid sequence of polypeptide III is that shown in SEQ ID NO.3, reference substance detection matrix is fish glue from skin.The detected value of polypeptide II is subtracted by the detected value of polypeptide I, and detection error is corrected using the detected value of polypeptide III, it is achieved thereby that the detection to donkey-hide gelatin content in compound donkey-hide gelatin preparation.The method has the advantages that content detection is accurate, simple to operate, will be with a wide range of applications in the quality monitoring field of donkey-hide gelatin compound preparation.
Description
Technical field
The present invention relates to a kind of composition and kit for detecting donkey-hide gelatin content in compound donkey-hide gelatin preparation, should also including use
The method that composition or kit carry out donkey-hide gelatin content detection in compound donkey-hide gelatin preparation, the invention belongs to medicine and food inspection skill
Art field.
Background technology
Donkey-hide gelatin with enriching yin of enriching blood, is moisturized, is stopped blooding as a kind of traditional rare Chinese medicine, the edible history of existing thousands of years
Effect.Chinese Pharmacopoeia specifies that donkey-hide gelatin dries skin or fresh hide through decocting, concentrating for equine species donkey Equus asinus L.'s
The solid gum being made.Pure donkey-hide gelatin must can ensure effect of product from donkey hide very.From raw material biological classification
Seen on, donkey belongs to Perissodactyla equine donkey category, the kind such as including African wild donkey, Equus kiang, onager, family donkey.It is wherein Chinese
The family donkey of raising include Region in Guanzhong Donkey, Dezhou donkey, Guangling donkey, good rice donkey, Miyang donkey, Huaiyang donkey, Qingyang donkey, North China donkey, Xinjiang Donkey,
Numerous local varieties such as southwestern donkey.Because the herding value of donkey constantly declines, and its economic worth has no raising, the quantity of donkey
Worldwide drastically decline, the supply of donkey hide is also nervous year by year, and price rises steadily.In the market occurs in that many illegal point
Son, when donkey-hide gelatin is produced, partially or completely replaces donkey with various other Animal Skins including horse skin, mule hide, ox-hide, pigskin etc.
Skin is boiled, and what is had is even directly incorporated into industrial gelatine.The presence severe jamming of these adulterants market normal order, infringement
Consumer's interests, influence the prestige of regular product.
Donkey-hide gelatin is formed through the concentration of long-time thermophilic digestion, and main component is very close and production technologies of different manufacturers have
Difference, using the methods such as infrared, near-infrared, X- diffraction, HPLC donkey-hide gelatin is carried out the true and false differentiate be commonly present judge it is inaccurate, shortage
Enough convincingnesses etc..The method mainly DNA molecular authentication method and the inspection that differentiate that authenticity of hide glue is more authoritative reported at present
Survey the LC-MS method of characteristic peptide fragment.The both approaches reported cannot all be compared to donkey hide derived components in donkey-hide gelatin
Accurate quantitative determination, but by detect in donkey-hide gelatin whether the composition containing donkey, and exclude other animal derived components and judge
Authenticity of hide glue；But the adulterated of donkey-hide gelatin may be multifarious, it is therefore desirable to which the animal derived components of exclusion can be varied, utilize
There is major defect differentiating authenticity of hide glue in exclusive method, its accuracy and reliability can have a greatly reduced quality, and such case is very unfavorable
In the supervision to market donkey-hide gelatin product.Judge authenticity of hide glue using donkey hide derived components content, can fundamentally limit donkey-hide gelatin
Imitation behavior.In the document reported, donkey hide derived component is detected, horse skin derived component is not all excluded substantially, though therefore
Donkey hide derived components are claimed as, are actually that donkey and horse have.
Therefore, at present in the urgent need to proposing a kind of side that can quickly, accurately detect donkey-hide gelatin content in compound donkey-hide gelatin preparation
Method, and then realize differentiating the true and false of compound donkey-hide gelatin preparation.
The content of the invention
It is an advantage of the invention to provide the composition of donkey-hide gelatin content in quick, accurate detection compound donkey-hide gelatin preparation.
It is still another object of the present invention to provide for donkey-hide gelatin content in quick, accurate detection compound donkey-hide gelatin preparation
Kit.
It is a further object of the invention to provide the detection of donkey-hide gelatin content in quick, accurate detection compound donkey-hide gelatin preparation
Method.
To achieve these goals, present invention employs following technical scheme：
A kind of composition for detecting donkey-hide gelatin content in compound donkey-hide gelatin preparation of the invention, by polypeptide I, polypeptide II, many
Peptide III and reference substance detection matrix composition, wherein, the amino acid sequence of the polypeptide I is Ser-Gly-Gln-Pro-Gly-
The amino acid sequence of Thr-Val-Gly-Pro-Ala-Gly-Val-Arg (shown in SEQ ID NO.1), polypeptide II is His-Gly-
The amino acid sequence of His-Arg (shown in SEQ ID NO.2), polypeptide III is Gly-Val-Val-Gly-Pro-Gln-Gly-
Ala-Arg (shown in SEQ ID NO.3), reference substance detection matrix is fish glue from skin.
Detection kit containing the composition is also within protection scope of the present invention.
Further, the invention allows for a kind of using in described composition or kit detection compound donkey-hide gelatin preparation
The method of donkey-hide gelatin content, it is characterised in that comprise the following steps：
1) preparation of reference substance solution：
After reference substance detection matrix is crushed, weigh a certain amount of reference substance and detect that matrix in measuring bottle, adds NH4HCO3It is molten
Liquid, ultrasound is completely dissolved sample, obtains matrix solution, and the matrix solution obtained with preparation dissolves load weighted polypeptide I, polypeptide
II and polypeptide III, shakes up, and obtains reference substance mother liquor, using the method for stepwise dilution, with matrix solution as solvent, by reference substance
Mother liquor dilutes 500-105Times, filtering with microporous membrane adds trypsin solution, enzymolysis in subsequent filtrate；
2) preparation of compound donkey-hide gelatin preparation sample solution：
Compound donkey-hide gelatin preparation sample to be detected is taken in measuring bottle, weight is designated as M1, after being dissolved in water, add trichloroacetic acid molten
Liquid, ultrasound, removes supernatant after centrifugation, precipitation wash with acetone, weigh after drying, and weight is designated as M2；Weigh a certain amount of precipitation
Dried object adds NH in measuring bottle4HCO3Solution is diluted to scale, shakes up, and filtering with microporous membrane adds tryptose in subsequent filtrate
Enzyme solutions, enzymolysis；
3) reference substance solution, compound donkey-hide gelatin preparation sample solution to be detected are respectively put into LC-MS instrument, select m/
Z592.2 → 756.4,655.4,499.3, m/z253.8 → 312.2,369.2, m/z421.0 → 684.4,528.3 are used as detection
Ion pair carries out MRM scannings, wherein selection m/z592.2 → 756.4, m/z253.8 → 312.2, m/z421.0 → 684.4 difference
Used as the quota ion pair of polypeptide I, polypeptide II and polypeptide III, the content of polypeptide I is designated as N in donkey-hide gelatin sample to be detected1, polypeptide
The content of II is designated as N2, polypeptide III content be designated as N3；
4) it is calculated as follows out the content of donkey-hide gelatin in sample：
Content (%)=(N of donkey-hide gelatin in compound donkey-hide gelatin preparation1/1182.3–N2/505.5)×840/N3×M2/M1×
100%.
In the present invention, it is preferred to, described NH4HCO3Solution is the NH of 1% (w/w) pH8.04HCO3Solution.
In the present invention, it is preferred to, the preparation of described reference substance solution is comprised the following steps：
1) preparation of matrix solution：Reference substance detection matrix crush after, weigh 0.50g reference substance detection matrix in
In 250ml measuring bottles, plus a small amount of 1% (w/w) pH8.0 NH4HCO3Solution, ultrasound is completely dissolved sample, with 1% (w/w)
The NH of pH8.04HCO3Solution is diluted to scale, shakes up；
2) the polypeptide III of polypeptide II and 16.4mg of polypeptide I, 9.9mg of 23.1mg is weighed in 25ml measuring bottles, uses step
1) matrix solution prepared dissolves and is diluted to scale, shakes up；
3) preparation of reference substance solution：It is respectively that reference substance is female with matrix solution as solvent using the method for stepwise dilution
Liquid dilutes 500-105Times, filtering with microporous membrane, precision measures the trypsin solution 10 μ l of subsequent filtrate 100 μ l plus 2mg/ml, 37
DEG C constant temperature enzymolysis 12h.
In the present invention, it is preferred to, the preparation of described compound donkey-hide gelatin preparation sample solution to be detected is comprised the following steps：
The compound donkey-hide gelatin preparation sample to be detected of 1~5g is weighed, weight is designated as M1, 5ml is complemented to water, add the three of 30% (w/v)
Monoxone 5ml, concussion mixing makes final concentration of 15% (w/v), the ultrasonic 15min of trichloroacetic acid, 4 DEG C of standing 30min, centrifugation
After remove clear liquid, precipitation washed with acetone, dry after weigh, weight is designated as M2, the precipitation dried object of 0.05g is weighed in 25ml amounts
In bottle, the NH of a small amount of 1% (w/w) pH8.0 is added4HCO3After solution dissolving, with the NH of 1% (w/w) pH8.04HCO3Solution dilutes
To scale, shake up, filtering with microporous membrane, precision measures the μ l of trypsin solution 10 of subsequent filtrate 100 μ l plus 2mg/ml, 37 DEG C of perseverances
Temperature enzymolysis 12h.
In the present invention, it is preferred to, step 3) in LC-MS instrument detection liquid-phase condition be：C18Reverse-phase chromatographic column, stream
Dynamic phase A is that, containing 0.1% (v/v) first aqueous acid, Mobile phase B is the acetonitrile solution containing 0.1% (v/v) formic acid, flow velocity
0.3ml/min；Gradient elution：0 → 20min, mobile phase A 100% → 25%, Mobile phase B 0% → 75%.Mass Spectrometry Conditions：Electricity
Spraying positive ion mode (ESI+) carry out multiple-reaction monitoring.
Further, the invention allows for described composition and its kit detect compound donkey-hide gelatin preparation in Ah
Application in glue content.
The composition of donkey-hide gelatin compound preparation is increasingly complex compared with donkey-hide gelatin, therefore carries out content inspection to donkey-hide gelatin in donkey-hide gelatin compound preparation
Survey, its difficulty in simple donkey-hide gelatin than detecting bigger.This not only to find out can indicate donkey hide derived components and different process Ah
The metastable material of content in glue, the adulterant composition of removal and the nearer horse of donkey affiliation, mule etc.；It is also contemplated that detection letter
Number stability, meet in detection with signal response higher, relatively low noise and the interference of other materials etc..These are all needed
Want the work of novelty.In this regard, the present invention is by choosing donkey hide source property characteristic polypeptide, and detected value using polypeptide I is subtracted
The detected value of polypeptide II, and detection error is corrected using the detected value of polypeptide III, so as to realize to donkey in donkey-hide gelatin compound preparation
The content detection of skin derived components.It is demonstrated experimentally that using method of the invention, it is possible to detect donkey hide in donkey-hide gelatin compound preparation exactly
The content of derived components, and it is simple to operate, will be with a wide range of applications in donkey-hide gelatin compound preparation quality monitoring field.
Brief description of the drawings
In order that the object, technical solutions and advantages of the present invention are clearer, below in conjunction with accompanying drawing the present invention is made into
The detailed description of one step, wherein：
Fig. 1 be select under reference substance solution MRM scan patterns ion pair m/z592.2 → 756.4, m/z253.8 →
312.2nd, the mass spectrogram of m/z421.0 → 684.4 monitoring.
Fig. 2 be compound donkey-hide gelatin preparation sample and its adulterant selected under MRM scan patterns ion pair m/z592.2 →
756.4th, m/z253.8 → 312.2, the mass spectrogram of m/z421.0 → 684.4 monitoring.
Specific embodiment
Hereinafter with reference to accompanying drawing, preferred embodiment of the invention is described in detail.It is specific without indicating in preferred embodiment
The experimental technique of condition, is generally carried out according to the condition proposed by normal condition or manufacturer.
Embodiment 1
1 material and reagent
1) material
Various sterling glue, including donkey-hide gelatin, horse skin glue, oxhide gelatin, pig skin gelatin, sheepskin glue, (reference substance detects base to fish glue from skin
Matter), decocted with donkey hide, horse skin, ox-hide, pigskin, sheepskin and fish-skin respectively, concentration, dry after obtain.
Polypeptide I (shown in SEQ ID NO.1), polypeptide II (shown in SEQ ID NO.2), polypeptide III (SEQ ID NO.3 institutes
Show), transfer to biotech firm to synthesize.
Compound donkey-hide gelatin preparation sample：Concentrate solution is extracted with the vegetable drug in complex prescription glue mucilage production process, is added respectively
Enter above-mentioned sterling glue, extracting concentrate solution according to every 9g vegetable drugs adds 1.0g sterling glue samples to be made the compound containing donkey-hide gelatin
Corii asini pulp (preparation 1), the complex prescription glue mucilage adulterant (preparation 2) containing horse skin the glue, (system of the complex prescription glue mucilage adulterant containing oxhide gelatin
Agent 3), the complex prescription glue mucilage adulterant (preparation 4) containing pig skin gelatin, the complex prescription glue mucilage adulterant (preparation 5) containing sheepskin glue.
2) reagent
Ammonium hydrogen carbonate (analysis is pure), trypsase (sequence is pure)
2 detection methods
1) preparation of matrix solution：Weigh 0.50g reference substances and detect matrix in 250ml measuring bottles, plus a small amount of 1%
NH4HCO3Solution (pH8.0), ultrasound is completely dissolved sample, uses 1%NH4HCO3Solution (pH8.0) is diluted to scale, shakes up.
2) preparation of reference substance mother liquor：Weigh polypeptide I, 9.9mg of 23.1mg polypeptide II and 16.4mg polypeptide III in
In 25ml measuring bottles, dissolved with the matrix solution of above-mentioned preparation and be diluted to scale, shaken up.
3) preparation of reference substance solution：It is with matrix solution as solvent, reference substance mother liquor is dilute using the method for stepwise dilution
1000 times are released, filtering with microporous membrane, precision measures the μ l of trypsin solution 10 of subsequent filtrate 100 μ l plus 2mg/ml, 37 DEG C of constant temperature
Enzymolysis 12h.
4) preparation of compound donkey-hide gelatin preparation sample solution：(weight is designated as M to weigh 5g1) compound donkey-hide gelatin preparation sample to be detected
Product, 5ml is complemented to water, adds the trichloroacetic acid 5ml of 30% (w/v), and concussion mixing makes final concentration of the 15% of trichloroacetic acid
(w/v), ultrasonic 15min, 4 DEG C of standing 30min, removes clear liquid after centrifugation, precipitation wash with acetone, dry after weigh (weight note
It is M2), the precipitation dried object of 0.05g is weighed in 25ml measuring bottles, add a small amount of 1% NH4HCO3After solution (pH8.0) dissolving,
With 1% NH4HCO3Solution (pH8.0) is diluted to scale, shakes up, filtering with microporous membrane, and precision measures the μ l of subsequent filtrate 100 and adds
The μ l of trypsin solution 10 of 2mg/ml, 37 DEG C of constant temperature digest 12h；
5) detect：Reference substance solution, each 5 μ l of compound donkey-hide gelatin preparation sample solution to be detected are taken respectively is put into LC-MS instrument
Detected.Liquid-phase condition：C18Reverse-phase chromatographic column (2.1mm × 100mm, 1.8 μm), mobile phase A is the water-soluble of 0.1% formic acid
Liquid, Mobile phase B is the acetonitrile solution of 0.1% formic acid, flow velocity 0.3ml/min；Gradient elution：0 → 20min, mobile phase A 100%
→ 25%, Mobile phase B 0% → 75%.Mass Spectrometry Conditions：Electron spray positive ion mode (ESI+) multiple-reaction monitoring is carried out, select m/
Z592.2 → 756.4,655.4,499.3, m/z253.8 → 312.2,369.2, m/z421.0 → 684.4,528.3 are used as detection
Ion pair carries out MRM scannings, wherein selection m/z592.2 → 756.4, m/z253.8 → 312.2, m/z421.0 → 684.4 difference
As the quota ion pair of polypeptide I, polypeptide II and polypeptide III.
6) content of donkey-hide gelatin in sample is calculated：
Fig. 1 be select under reference substance solution MRM scan patterns ion pair m/z592.2 → 756.4, m/z253.8 →
312.2nd, the mass spectrogram of m/z421.0 → 684.4 monitoring.Fig. 2 is that compound donkey-hide gelatin preparation and its adulterant are selected under MRM scan patterns
Select ion pair m/z592.2 → 756.4, m/z253.8 → 312.2, the mass spectrogram of m/z421.0 → 684.4 monitoring.
By by the mass spectrogram peak area ratio pair of reference substance and compound donkey-hide gelatin preparation sample and its adulterant, calculate compound Ah
The content of polypeptide I, polypeptide II and polypeptide III in glue formulation samples and its adulterant, the content note of polypeptide I in donkey-hide gelatin sample to be detected
It is N1, polypeptide II content be designated as N2, polypeptide III content be designated as N3；Then, it is calculated as follows out containing for donkey-hide gelatin in each sample
Amount：
Content (%)=(N of donkey-hide gelatin in sample1/1182.3–N2/505.5)×840/N3×M2/M1× 100%.
Content (%)=(N of donkey-hide gelatin in preparation 11/1182.3–N2/505.5)×840/N3×M2/M1× 100%=
(0.3245/1182.3-0/505.5) × 840/0.2319 × 0.5209/5.0810 × 100%=10.19%
Content (%)=(N of donkey-hide gelatin in preparation 21/1182.3–N2/505.5)×840/N3×M2/M1× 100%=
(0.3212/1182.3-0.1388/505.5) × 840/0.2331 × 0.5493/5.0415 × 100%=-0.11% ≈ 0%
Content (%)=(N of donkey-hide gelatin in preparation 31/1182.3–N2/505.5)×840/N3×M2/M1× 100%=(0/
1182.3-0/505.5) × 840/0.2322 × 0.5581/5.0370 × 100%=0%
Content (%)=(N of donkey-hide gelatin in preparation 41/1182.3–N2/505.5)×840/N3×M2/M1× 100%=(0/
1182.3-0/505.5) × 840/0.2306 × 0.5260/5.0561 × 100%=0%
Content (%)=(N of donkey-hide gelatin in preparation 51/1182.3–N2/505.5)×840/N3×M2/M1× 100%=(0/
1182.3-0/505.5) × 840/0.2352 × 0.0.5144/5.0505 × 100%=0%
Above-mentioned result of calculation shows：The donkey-hide gelatin content and addition phase detected in compound ass-hide gelatin slurry samples containing donkey-hide gelatin
Symbol, and it is 0% to be free of the donkey-hide gelatin content detected in the complex prescription glue mucilage adulterant of donkey-hide gelatin, this is consistent with actual conditions, shows the party
Method can detect the donkey-hide gelatin content in donkey-hide gelatin compound preparation.
Embodiment 2
1 material and reagent
1) material
Polypeptide I (shown in SEQ ID NO.1), polypeptide II (shown in SEQ ID NO.2), polypeptide III (SEQ ID NO.3 institutes
Show), transfer to biotech firm to synthesize.
Epoxy glue sample is made up of the donkey-hide gelatin that the sterling glue sample in embodiment 1 is separately added into different quality, including contains
The oxhide gelatin of 15% donkey-hide gelatin, the oxhide gelatin containing 30% donkey-hide gelatin, the oxhide gelatin containing 60% donkey-hide gelatin.
Compound donkey-hide gelatin preparation sample：Concentrate solution is extracted with the vegetable drug in complex prescription glue mucilage production process, is added respectively
Enter above-mentioned epoxy glue sample, according to every 9g vegetable drugs extract concentrate solution add 1.0g epoxy glue samples be made 3 compound Ah
Rubber cement adulterant, is respectively designated as preparation 1, preparation 2, preparation 3, and donkey-hide gelatin content is respectively 1.5%, 3.0%, 6.0%.
2) reagent
Ammonium hydrogen carbonate (analysis is pure), trypsase (sequence is pure)
2 detection methods
1) preparation of matrix solution：Weigh 0.50g reference substances and detect matrix in 250ml measuring bottles, plus a small amount of 1%
NH4HCO3Solution (pH8.0), ultrasound is completely dissolved sample, uses 1%NH4HCO3Solution (pH8.0) is diluted to scale, shakes up.
2) preparation of reference substance mother liquor：Weigh polypeptide I, 9.9mg of 23.1mg polypeptide II and 16.4mg polypeptide III in
In 25ml measuring bottles, dissolved with the matrix solution of above-mentioned preparation and be diluted to scale, shaken up.
3) preparation of reference substance solution：Using the method for stepwise dilution, with matrix solution as solvent, by reference substance mother liquor point
Not Xi Shi 500 times, 1000 times, 2000 times, 4000 times, 8000 times, 10000 times, filtering with microporous membrane, precision measures subsequent filtrate
100 μ l add the μ l of trypsin solution 10 of 2mg/ml, 37 DEG C of constant temperature to digest 12h.
4) preparation of compound donkey-hide gelatin preparation sample solution：(weight is designated as M to weigh 5g1) compound donkey-hide gelatin preparation sample to be detected
Product, 5ml is complemented to water, adds the trichloroacetic acid 5ml of 30% (w/v), and concussion mixing makes final concentration of the 15% of trichloroacetic acid
(w/v), ultrasonic 15min, 4 DEG C of standing 30min, removes clear liquid after centrifugation, precipitation wash with acetone, dry after weigh (weight note
It is M2), the precipitation dried object of 0.05g is weighed in 25ml measuring bottles, add a small amount of 1% NH4HCO3After solution (pH8.0) dissolving,
With 1% NH4HCO3Solution (pH8.0) is diluted to scale, shakes up, filtering with microporous membrane, and precision measures the μ l of subsequent filtrate 100 and adds
The μ l of trypsin solution 10 of 2mg/ml, 37 DEG C of constant temperature digest 12h；
5) detect：Each 5 μ l of reference substance solution, detected sample solution are taken respectively be put into LC-MS instrument detected.Liquid
Phase condition：C18Reverse-phase chromatographic column (2.1mm × 100mm, 1.8 μm), mobile phase A is 0.1% first aqueous acid, and Mobile phase B is
The acetonitrile solution of 0.1% formic acid, flow velocity 0.3ml/min；Gradient elution：0 → 20min, mobile phase A 100% → 25%, flowing
Phase B 0% → 75%.Mass Spectrometry Conditions：Electron spray positive ion mode (ESI+) carry out multiple-reaction monitoring, selection m/z592.2 →
756.4th, 655.4,499.3, m/z253.8 → 312.2,369.2, m/z421.0 → 684.4,528.3 are entered as detection ion pair
Row MRM is scanned, wherein selection m/z592.2 → 756.4, m/z253.8 → 312.2, m/z421.0 → 684.4 are respectively as polypeptide
The quota ion pair of I, polypeptide II and polypeptide III.
6) in sample donkey hide derived components content calculating
With the content of each polypeptide of reference substance solution as ordinate, peak area the drafting of standard curve is carried out for abscissa,
Linearly dependent coefficient R >=0.998 of each polypeptide, thus calculates polypeptide I, polypeptide II and polypeptide in compound donkey-hide gelatin preparation sample
The content of III, the content of polypeptide I is designated as N in compound donkey-hide gelatin preparation sample to be detected1, polypeptide II content be designated as N2, polypeptide III
Content be designated as N3, then, it is calculated as follows out the content of donkey-hide gelatin in each sample：
Content (%)=(N of donkey-hide gelatin in sample1/1182.3–N2/505.5)×840/N3×M2/M1× 100%.
Content (%)=(N of donkey-hide gelatin in preparation 11/1182.3–N2/505.5)×840/N3×M2/M1× 100%=
(0.0496/1182.3-0/505.5) × 840/0.2341 × 0.5065/5.0521 × 100%=1.51%
Content (%)=(N of donkey-hide gelatin in preparation 21/1182.3–N2/505.5)×840/N3×M2/M1× 100%=
(0.0992/1182.3-0/505.5) × 840/0.2413 × 0.5256/5.0535 × 100%=3.04%
Content (%)=(N of donkey-hide gelatin in preparation 31/1182.3–N2/505.5)×840/N3×M2/M1× 100%=
(0.1968/1182.3-0/505.5) × 840/0.2398 × 0.5116/5.0445 × 100%=5.91%
Above-mentioned result of calculation shows：Using three polypeptides disclosed by the invention and reference substance detection matrix, using liquid matter
Combined instrument, can accurately detect the content of donkey-hide gelatin in donkey-hide gelatin compound preparation.
It should be noted that above example is only used to illustrate the technical scheme of invention and unrestricted, although by referring to
Invention has been described for the preferred embodiments of the present invention, but the various changes that those skilled in the art make to the present invention
Or modification, without departing from spirit and scope of the invention, these equivalent form of values are also fallen within the scope of the present invention.
Sequence table
<110>Donga donkey-hide gelatin limited company
<120>Composition, kit and its detection method for detecting donkey-hide gelatin content in compound donkey-hide gelatin preparation
<130> KLPI161261
<160> 3
<170> PatentIn 3.5
<210> 1
<211> 13
<212> PRT
<213>Polypeptide I
<400> 1
Ser Gly Gln Pro Gly Thr Val Gly Pro Ala Gly Val Arg
<210> 2
<211> 4
<212> PRT
<213>Polypeptide II
<400> 2
His Gly His Arg
<210> 3
<211> 9
<212> PRT
<213>Polypeptide III
<400> 3
Gly Val Val Gly Pro Gln Gly Ala Arg
Claims (9)
1. the composition of donkey-hide gelatin content in compound donkey-hide gelatin preparation is detected, it is characterised in that：The composition by polypeptide I, polypeptide II,
Polypeptide III and reference substance detection matrix composition, wherein, the amino acid sequence of the polypeptide I is Ser-Gly-Gln-Pro-
The amino acid sequence of Gly-Thr-Val-Gly-Pro-Ala-Gly-Val-Arg, polypeptide II is His-Gly-His-Arg, polypeptide
The amino acid sequence of III is Gly-Val-Val-Gly-Pro-Gln-Gly-Ala-Arg, and reference substance detection matrix is fish glue from skin.
2. the detection kit of composition described in claim 1 is contained.
3. a kind of method that usage right requires donkey-hide gelatin content in the combination analyte detection compound donkey-hide gelatin preparation described in 1, its feature exists
In comprising the following steps：
1) preparation of reference substance solution：
After reference substance detection matrix is crushed, weigh a certain amount of reference substance and detect that matrix in measuring bottle, adds NH4HCO3Solution, surpasses
Sound is completely dissolved sample, obtains matrix solution, with prepare the matrix solution that obtains dissolve load weighted polypeptide I, polypeptide II and
Polypeptide III, shakes up, and obtains reference substance mother liquor, using the method for stepwise dilution, with matrix solution as solvent, by reference substance mother liquor
Dilution 500-105Times, filtering with microporous membrane adds trypsin solution, enzymolysis in subsequent filtrate；
2) preparation of compound donkey-hide gelatin preparation sample solution：
Compound donkey-hide gelatin preparation sample to be detected is taken in measuring bottle, weight is designated as M1, after being dissolved in water, solution of trichloroacetic acid is added, surpass
Sound, removes supernatant after centrifugation, precipitation washed with acetone, dry after weigh, weight is designated as M2；Weigh a certain amount of precipitation dried object
In measuring bottle, NH is added4HCO3Solution is diluted to scale, shakes up, and filtering with microporous membrane adds trypsin solution in subsequent filtrate,
Enzymolysis；
3) reference substance solution, compound donkey-hide gelatin preparation sample solution to be detected are respectively put into LC-MS instrument, select m/z592.2
→ 756.4,655.4,499.3, m/z253.8 → 312.2,369.2, m/z421.0 → 684.4,528.3 as detection ion pairs
MRM scannings are carried out, wherein selection m/z592.2 → 756.4, m/z253.8 → 312.2, m/z421.0 → 684.4 are respectively as more
The quota ion pair of peptide I, polypeptide II and polypeptide III, the content of polypeptide I is designated as N in donkey-hide gelatin sample to be detected1, polypeptide II contains
Amount is designated as N2, polypeptide III content be designated as N3；
4) it is calculated as follows out the content of donkey-hide gelatin in sample：
Content (%)=(N of donkey-hide gelatin in compound donkey-hide gelatin preparation1/1182.3–N2/505.5)×840/N3×M2/M1× 100%.
4. method as claimed in claim 3, it is characterised in that described NH4HCO3Solution is 1% (w/w) pH8.0's
NH4HCO3Solution.
5. method as claimed in claim 3, it is characterised in that the preparation of described reference substance solution is comprised the following steps：
1) preparation of matrix solution：After reference substance detection matrix is crushed, the reference substance detection matrix for weighing 0.50g is measured in 250ml
Bottle in, plus a small amount of 1% (w/w) pH8.0 NH4HCO3Solution, ultrasound is completely dissolved sample, with 1% (w/w) pH8.0's
NH4HCO3Solution is diluted to scale, shakes up；
2) the polypeptide III of polypeptide II and 16.4mg of polypeptide I, 9.9mg of 23.1mg is weighed in 25ml measuring bottles, with step 1) match somebody with somebody
The matrix solution of system dissolves and is diluted to scale, shakes up；
3) preparation of reference substance solution：It is respectively that reference substance mother liquor is dilute with matrix solution as solvent using the method for stepwise dilution
Release 500-105Times, filtering with microporous membrane, precision measures the μ l of trypsin solution 10 of subsequent filtrate 100 μ l plus 2mg/ml, 37 DEG C of perseverances
Temperature enzymolysis 12h.
6. method as claimed in claim 3, it is characterised in that step 2) described in compound donkey-hide gelatin preparation sample to be detected it is molten
The preparation of liquid is comprised the following steps：1~5g compound donkey-hide gelatin preparation samples to be detected are weighed, weight is designated as M1, complemented to water
5ml, adds the trichloroacetic acid 5ml of 30% (w/v), and concussion mixing makes final concentration of 15% (w/v) of trichloroacetic acid, ultrasound
15min, 4 DEG C of standing 30min, removes supernatant after centrifugation, precipitation wash with acetone, weigh after drying, and weight is designated as M2, weigh
The precipitation dried object of 0.05g adds the NH of a small amount of 1% (w/w) pH8.0 in 25ml measuring bottles4HCO3After solution dissolving, with 1%
(w/w) NH of pH8.04HCO3Solution is diluted to scale, shakes up, filtering with microporous membrane, and precision measures subsequent filtrate 100 μ l plus 2mg/
The μ l of trypsin solution 10 of ml, 37 DEG C of constant temperature digest 12h.
7. method as claimed in claim 3, it is characterised in that step 3) in the liquid-phase condition of LC-MS instrument detection be：C18
Reverse-phase chromatographic column, mobile phase A is that, containing 0.1% (v/v) first aqueous acid, Mobile phase B is to contain 0.1% (v/v) formic acid
Acetonitrile solution, flow velocity 0.3ml/min；Gradient elution：0 → 20min, mobile phase A 100% → 25%, Mobile phase B 0% →
75%.Mass Spectrometry Conditions：Electron spray positive ion mode (ESI+) carry out multiple-reaction monitoring.
8. application of the composition described in claim 1 in donkey-hide gelatin content in detecting compound donkey-hide gelatin preparation.
9. application of the kit described in claim 2 in donkey-hide gelatin content in detecting compound donkey-hide gelatin preparation.
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| CN109142598A (en) * | 2018-09-10 | 2019-01-04 | 上海市食品药品检验所 | A kind of method of one group of donkey source property feature peptide and identification donkey-hide gelatin or donkey-hide gelatin product |
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| CN109142598B (en) * | 2018-09-10 | 2021-07-30 | 上海市食品药品检验研究院 | Donkey-derived characteristic peptides and method for identifying donkey-hide gelatin or donkey-hide gelatin product |
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