CN106468695A - A kind of method simultaneously measuring bioactive peptide and free amino acid - Google Patents
A kind of method simultaneously measuring bioactive peptide and free amino acid Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
- G01N2030/8804—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 automated systems
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
- G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
- G01N2030/8813—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials
- G01N2030/8818—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials involving amino acids
Abstract
The invention belongs to analytical chemistry field, it is related to nutrient substance assay method, disclose the method simultaneously measuring bioactive peptide and free amino acid, by to the buffer composition in amino acid analysises analyzer standard test program, elution program, the adjustment of eluting temperature, several amino acids peak is separated with bioactive peptide peak, achieve and using automatic amino acid analyzer, the one-step method of free amino acid and bioactive peptide is measured, establish the method simultaneously preparing aminoacid and peptides, overcome nontraditional amino acid analyser method to separate bioactive peptide and multiple free amino acid well, and test the shortcoming that time-consuming.
Description
Technical field
The present invention relates to analysis detection field, specially a kind of nutrition and flavor substance method for measuring components, more particularly, to
A kind of method simultaneously measuring bioactive peptide and free aminoacid content.
Background technology
Free amino acid is aquatic food freshness and the important evaluation index of local flavor, is also the important composition of non-protein nitrogen
Part；Different free amino acids has different tastes, assumes the various tastes such as fresh, sweet, sour, bitter, can directly embody water outlet
Product taste of food and delicious degree.As threonine, serine, proline have sweet taste, and aspartic acid, glutamic acid are then
Play important delicate flavour effect.The content of therefore free amino acid can be used as the important freshness of many aquatic products quality evaluations
Potential index.
Bioactive peptide refers to the peptides to the vital movement of organism with physiologically active, generally refer to by 2 to
The specific protein fragments of 20 amino acid residue compositions, because the difference of construction also has different physiological functions.Activity
Peptide also has multiple biological functions and physiological effect in addition to trophic function, for example：Antioxidation, antibacterial, blood pressure lowering, free radical are clear
Except agent etc..
Shellfish is a kind of important Aquatic product kind, bioactive peptide and free amino acid in the shellfish body under different living environments
Composition and content are also not quite similar.The content of free amino acid is to evaluate the important potential index of aquatic products quality, bioactive peptide
There is the trophic functions such as antioxidation, antibacterial, blood pressure lowering, free radical scavenging, the therefore content of bioactive peptide also becomes sign shellfish etc.
The important parameter of aquatic products quality.
Bioactive peptide mostly is the Amino acid score subchain of 2 to 10 aminoacid compositions, similar to free amine group acid molecular structures, changes
Learn property close.Nutrient substance extracting solution is not easy separate the two, then measures its concentration respectively.Therefore, prior art
In many contents simultaneously measuring free amino acid and bioactive peptide in shellfish using one-step method.
The method of related assays bioactive peptide and free amino acid has much both at home and abroad at present, mainly has high performance liquid chromatography
With colorimetry, spectrophotography, high performance capillary electrophoresis.
Although high performance liquid chromatography, colorimetry, spectrophotography, high performance capillary electrophoresis can to bioactive peptide or
Free amino acid is measured, but these methods exist that complex operation, instrument and equipment are relatively costly, be easily subject to impurity interference,
Measurement result is inaccurate and the shortcomings of test approaches are limited, fails fully to be promoted.
Measuring the topmost method of free amino acid at present is automatic amino acid analyzer method, and in prior art, also someone adopts
Measure Glutathione and free amino acid with automatic amino acid analyzer, but the corresponding standard method of amino-acid analyzer can not
Enough well bioactive peptide and free amino acid are separated, and the testing time is long.There is presently no a kind of method, ammonia can be utilized
Base acid automatic analyzer effectively, quickly measures bioactive peptide and multiple free aminoacid content simultaneously.
Therefore, develop one kind and measure multiple free amino acids, Glutathione and flesh using automatic amino acid analyzer simultaneously
The method of peptide content, is the key solving problem in prior art.
Content of the invention
The present invention is intended to provide a kind of method simultaneously measuring bioactive peptide and free amino acid, to solve in prior art not
Can accurate using amino-acid analyzer, the quick problem simultaneously measuring bioactive peptide and free aminoacid content.
The technical scheme is that, using the free amino acid in the elution program separation sample after optimizing and activity
Peptide, draws standard curve, then adopts standard curve method to measure free amino acid and active peptide concentration in testing sample solution.
A kind of method simultaneously measuring bioactive peptide and free amino acid, its step includes, and testing sample solution is added to
In amino-acid analyzer, carry out gradient elution with buffer B 1～B5 by following procedure, developed the color using 1,2,3-indantrione monohydrate nitrite ion：
First stage：With buffer B 1 eluting, persistent period 20～21min；37.5～39 DEG C of initial column temperature, the 2.0th～
It is down to 34.5～35.5 DEG C during 2.1min, at the end of the first stage, column temperature is risen to 55.5～56.5 DEG C；
Second stage：With buffer B 1 and B2 eluting, persistent period 11.5～12.5min；First column temperature is risen to 57.5～
58.5 DEG C, instantaneously it is adjusted to B1 78%～82%, B2 18%～22%；At the uniform velocity become again and turn to B1 68%～72%, B2
28%～32%, at the end of second stage, column temperature is down to 55.5～56.5 DEG C；
Phase III：Instantaneously it is adjusted to buffer B 1 8%～12%, buffer B 2 88%～92%, in this ratio
Eluting 9～10min, and column temperature is down to 41.5～42.5 DEG C in 2.8～3.1min；
Fourth stage：With buffer B 2 eluting 5～6min, and at the end of fourth stage, column temperature is risen to 71.5～72.5
℃；
5th stage：With buffer B 3 and B4 eluting, from buffer B3 68%～72%, buffering in 16.5～17.5min
Liquid B4 28%～32% is at the uniform velocity adjusted to buffer B 3 58%～62%, buffer B 4 38%～42%；Column temperature be 71.5～
72.5℃；
6th stage：With buffer B 3 and B4 eluting, in 2～3min, at the uniform velocity it is adjusted to B3 54%～56%, B4 44%
～46%, column temperature is 71.5～72.5 DEG C；
7th stage：With buffer B 4 eluting 17～18min, column temperature is 71.5～72.5 DEG C；
8th stage：With buffer B 5 eluting 4～5min.
Terminate rear pillar temperature drop in the 8th stage to 69.5～71 DEG C.
Aforementioned proportion is volume ratio.Then the standard work according to bioactive peptide and free amino acid under same elution requirement
Free amino acid and active peptide concentration in curve or regression equation calculation testing sample solution.
Preferably elution program is：
First stage is 0～20.8min, with buffer B 1 eluting, 37.5～39 DEG C of initial column temperature, adjusts during 2.1min
Whole is 34.5～35.5 DEG C, at the end of the first stage, column temperature is risen to 55.5～56.5 DEG C；
Second stage is 20.8～32.8min, with buffer B 1 and B2 eluting, column temperature is risen to 57.5～58.5 DEG C,
The ratio of buffer B 1 and B2 is from B1 78%～82%, B2 18%～22%, at the uniform velocity adjustment B168%～72%, B2 28%
～32%；At the end of second stage, column temperature is adjusted to 55.5～56.5 DEG C；
Phase III is 32.8～42.5min, is instantaneously adjusted to buffer B 1 8%～12%, buffer B 2 78%
～82%, and in this ratio eluting；During 35.8min, column temperature is adjusted to 41.5～42.5 DEG C；
Fourth stage is 42.5～48.0min, and with buffer B 2 eluting, at the end of fourth stage, column temperature is adjusted to 71.5
～72.5 DEG C；
5th stage was 48.0～65.0min, from buffer B3 68%～72%, buffer B 4 28%～32%,
At the uniform velocity it is adjusted to buffer B 3 54%～56%, buffer B 4 44%～46%；
6th stage was 65.0～67.5min, and being at the uniform velocity adjusted to buffer B 3 is 54%～56%, and buffer B 4 is
44%～46%, column temperature is 69.5～71 DEG C；
7th stage was 67.5～84.5min, carried out eluting with buffer B 4, and column temperature is 71.5～72.5 DEG C；
8th stage was 84.5～89.0min, carried out eluting with buffer B 5.
Preferred elution program is：
First stage is 0～20.8min, with buffer B 1 eluting, 38 DEG C of initial column temperature, is adjusted to 35 during 2.1min
DEG C, at the end of the first stage, column temperature is risen to 56 DEG C；
Second stage is 20.8～32.8min, with buffer B 1 and B2 eluting, column temperature is risen to 58 DEG C, buffer B 1
For 80%, B2 be 20%, and be at the uniform velocity adjusted to B1 be 70%, B2 be 30%, at the end of second stage, column temperature is adjusted to 56 DEG C；
Phase III is 32.8～42.5min, and being adjusted to buffer B 1 is 10%, and buffer B 2 is 90%, and presses this
One ratio eluting；During 35.8min, column temperature is adjusted to 42 DEG C；
Fourth stage is 42.5～48.0min, and with buffer B 2 eluting, at the end of fourth stage, column temperature is adjusted to 72
℃；
5th stage was 48.0～65.0min, and being instantaneously adjusted to buffer B 3 is 70%, and buffer B 4 is 30%, and
At the uniform velocity it is adjusted to B3 60%, B4 40%；
6th stage was 65.0～67.5min, and being at the uniform velocity adjusted to buffer B 3 is 55%, and buffer B 4 is 45%；
7th stage was 67.5～84.5min, with buffer B 4 eluting；
8th stage was 84.5～89min, with buffer B 5 eluting.Terminate rear pillar temperature drop in the 8th stage to 70 DEG C.
Described buffer B 1～B5 is citric acid-Lithium Citrate de buffer system, and pH value is respectively 2.75～2.85,3.65
～3.75,3.55～3.65,4.05～4.15；Described buffer B 5 is lithium hydroxide aqueous solution.
PH=2.75～2.85 of buffer B 1, citric acid three lithium concentration be 5.5～6g/L, chlorination lithium concentration be 1～
1.5g/L, citric acid concentration is 19～21g/L, alcohol volume content 2.5%～3.5%, sad volume content 0.005%～
0.015%.Preferably, the pH=2.8 of B1, citric acid three lithium concentration is 5.73g/L, and chlorination lithium concentration is 1.24g/L, citric acid
Concentration is 19.9g/L, alcohol volume content 3.0%, sad volume content 0.01%.
PH=3.65～3.75 of described buffer B 2, citric acid three lithium concentration is 9.5～11g/L, chlorination lithium concentration
For 6～7.5g/L, citric acid concentration is 11.5～13g/L, alcohol volume content 2.5%～4%, sad volume content
0.005%～0.015%.Preferably, the pH=3.7 of B2, citric acid three lithium concentration 9.8g/L, chlorination lithium concentration is 6.36g/L,
Citric acid concentration 12.0g/L, alcohol volume content 3%, sad volume content 0.01%.
PH=3.55～3.65 of described buffer B 3, citric acid three lithium concentration is 8.5～9.6g/L, chlorination lithium concentration
For 25～28g/L, citric acid concentration is 11～12g/L, alcohol volume content 8%～12%, phenethanol volume content 3.5%～
5%, sad volume content 0.005%～0.0.15%.Preferably, the pH=3.6 of buffer B 3, citric acid three lithium concentration is
8.79g/L；Chlorination lithium concentration is 26.62g/L, citric acid concentration is 11.27g/L, and alcohol volume content is 10%, phenethanol body
Long-pending content is 0.4%, and sad volume content is 0.01%.
PH=4.05～4.15 of described buffer B 4, citric acid three lithium concentration is 9.5～11g/L, chlorination lithium concentration
For 37.5～40g/L, citric acid concentration is 3～4g/L, sad volume content 0.005%～0.015%.Preferably, buffer
The pH=4.1 of B4, citric acid three lithium concentration is 9.8g/L, chlorination lithium concentration is 38.15g/L, citric acid concentration is 3.3g/L；Pungent
Sour volume content is 0.01%.
In described buffer B 5, Lithium hydrate concentration is 8～10g/L, preferably 8.4g/L；Alcohol volume content
2.5%～4%, preferably 3%；Sad volume content 0.005%～0.015%, preferably 0.01%.
The product of eluting adopts 1,2,3-indantrione monohydrate derivative reagent to develop the color, and developing wavelength is 440nm and 570nm.
1,2,3-indantrione monohydrate derivative reagent includes ninhydrin reaction liquid, reaction buffer and cleaning mixture, and concrete composition is as follows：
Ninhydrin reaction liquid (R1) is the solution of 1,2,3-indantrione monohydrate and sodium borohydride, and solvent is selected from ethylene glycol single methyl ether or the third two
Alcohol monomethyl ether；Described 1,2,3-indantrione monohydrate concentration is 35～42g/L, preferably 38～40g/L；Described sodium borohydride concentration be 80～
85mg/L, preferably 80～82mg/L.
Reaction buffer (R2) is sodium acetate, the mixed solution of glacial acetic acid, water and organic solvent；Described organic solvent is selected from
Ethylene glycol single methyl ether or propylene glycol monomethyl ether；Described organic solvent is 1 with the volume ratio of glacial acetic acid, water：0.2～0.4：
0.7～1, preferably 1：0.25～0.35：0.8～0.85；Described sodium acetate is 1 with the mass ratio of ultra-pure water：1.5～2.0, excellent
Elect 1 as：1.6～1.7.
Cleaning mixture (R3) is ethanol water；Described ethanol is 1 with the volume ratio of water：18～20, preferably 1：18.5～
19.5.Developed the color with ninhydrin reaction liquid and reaction buffer when separating eluting, cleaned with cleaning mixture at the end of elution program.
In this method, described bioactive peptide is at least one in Glutathione and carnosine；Described free amino acid bag
Include GSH (Glutathione), Car (carnosine), P-Ser (phosphoserine), Tau (taurine), PEA (phosphoethanolamine), Urea
(carbamide), Asp (aspartic acid), Hypro (hydroxyproline), Thr (threonine), Ser (serine), AspNH2(Radix Asparagi acyl
Amine), Glu (glutamic acid), GluNH2 (L-Glutamine), Sar (sarcosine), a-AAA (Amicar), Pro (proline),
Gly (glycine), Ala (alanine), Cit (citrulline), a-ABA (butyrine), Val (L-Valine), Cys (Guang ammonia
Acid), Met (methionine), Cysthi (cystathionie), Ile (isoleucine), Leu (leucine), Tyr (tyrosine), Phe (benzene
Alanine), b-Ala (Beta-alanine), b-AiBA (B-AIB), GABA (γ-aminobutyric acid), Trp (tryptophan),
EOHNH2(ethanolamine), Hylys (oxylysine), Orn (ornithine), Lys (lysine), 1Mehis (1- methyl groups ammonia
Acid), His (histidine), at least one in 3Mehis (3-Methyl histidine), Arg (arginine).
The preparation method of described testing sample solution is：
A. sample (as aquatic products, meat etc.) is mixed with hydrochloric acid solution and antioxidant, supersound process after homogenizing, and solid-liquid divides
From；Antioxidant is 1 with the mass ratio of aquatic products sample to be measured：50～500；Described antioxidant is dithiothreitol, DTT, connects two
Sodium sulfite or vitamin C,
B. the solid of step a is extracted 1～5 time with hydrochloric acid solution again；
C. the clear liquid that a, b step obtain uses hydrochloric acid solution constant volume after merging, and is subsequently adding sulfosalicylic acid, centrifugation, mistake
Filter, takes filtrate.
Preferably, the preparation method of testing sample solution is as follows：
A. take aquatic products sample to be measured, mix with hydrochloric acid solution, add antioxidant dithiothreitol, DTT, after abundant homogenizing,
Supersound process 3～20min, takes supernatant after 0～20 DEG C of centrifugation；Dithiothreitol, DTT is 1 with the mass ratio of aquatic products sample to be measured：
100～400；Described testing sample tissue and hydrochloric acid solution amount ratio are 1g：1～15mL, preferably 1g：5～10mL.
B. according to aquatic products sample to be measured and hydrochloric acid solution 1g：The amount ratio of 8～15mL, take step a centrifugation obtain consolidate
Body and hydrochloric acid solution mix and blend again, centrifuging and taking supernatant；And repeat aforesaid operations 1～5 time, merge supernatant；
C. the supernatant of combining step a and b, with hydrochloric acid solution constant volume and add sulfosalicylic acid, the containing of sulfosalicylic acid
Measure as 2wt%～3wt%；After centrifugation, prepare liquid is obtained using 0.22 μm of membrane filtration.
Concentration of hydrochloric acid used by each step is 0.005～0.1mol/L, preferably 0.01～0.05mol/L above.
This method is passed through to optimize elution program, buffer composition, eluting temperature it is achieved that adopting automatic amino acid analyzer
To food, especially in the nutrient substance of aquatic products etc., the one-step method of aminoacid and active polypeptide measures, and overcomes traditional amino
Bioactive peptide and free amino acid separately and can not be tested the shortcoming that time-consuming by acid analysis instrument well.
The beneficial effects of the present invention is, being capable of fast and effectively the containing of various aminoacid and bioactive peptide in determination sample
Amount；Detection time is less than 90 minutes, and can be kept completely separate Glutathione with free amino acid and be measured.Using the party
Method, between 84.6%～103.60%, in a few days relative standard deviation exists mixed standard solution each component mark-on average recovery rate
Between 0.31%-0.71%, relative standard deviation, between 1.23%～2.38%, meets Good Laboratory and controls rule in the daytime
The related request of model-food Physico-chemical tests；In this assay method, have reasonable linear between the area of chromatographic peak and concentration
Relation, the quantitative limit of each component in mixed standard solution between 0.35～20.12 μm of ol/L, lowest detectable limit 0.12～
Between 6.25 μm of ol/L.Show that this full-automatic amino-acid analyzer method has good sensitivity.
Using the method for the present invention, in 30min, can be well by Glutathione and carnosine isoreactivity peptide and free ammonia
Base acid separate it is adaptable to the mixed determining of peptide containing various active and free amino acid, particularly aquatic products such as shellfish these
Sample containing various active peptide and free amino acid, shortens detection time, and improves sensitivity.
Brief description
Fig. 1 is in embodiment 1, analyzes bioactive peptide and free amino acid mixed standard solution using prior art elution program
Collection of illustrative plates
Fig. 2 is in embodiment 1, elution program analysis bioactive peptide and free amino acid hybrid standard after being optimized using the present invention
Solution collection of illustrative plates
Fig. 3 is the detection separating spectrum of scallop in embodiment 4
Fig. 4 is the detection separating spectrum of Sinonovacula constricta in embodiment 4
Fig. 5 is the detection separating spectrum of Concha Ostreae in embodiment 4
Fig. 6 is the impact to bioactive peptide and amino acid content in flower clam for three kinds of antioxidants
Fig. 7 is the impact of bioactive peptide and amino acid content in three kinds of antioxidant dialogue clams
Specific embodiment
With reference to specific embodiments and the drawings, the present invention is expanded on further.In following examples, institute's accepted standard product
It is active poly saccharide peptide standard product (Glutathione, carnosine) and several amino acids mixing standard liquid；The INSTRUMENT MODEL being adopted is L-8800 ammonia
Base acid fully-automatic analyzer；The instrument parameter setting as：Detached dowel (4.6mm × 60mm) resin is cation exchange resin；Inspection
Survey wavelength：570nm (proline is 440nm)；Sample size 20 μ L；Buffer flow rate is：0.35mL/min；Nitrite ion：1,2,3-indantrione monohydrate
Derivative reagent (flow 0.35mL/min；135 DEG C of cell temperature)
The analysis of embodiment 1 standard solution measures
(1) preparation of elution buffer and 1,2,3-indantrione monohydrate derivative reagent
Prepare buffer B 1～B5 according to table 1；Prepare 1,2,3-indantrione monohydrate derivative reagent (ninhydrin reaction liquid R1, reaction according to table 2
Buffer R2, cleaning mixture R3).
The composition of table 1 B1～B5 buffer
The composition of table 2 1,2,3-indantrione monohydrate derivative reagent
(2) aminoacid and peptides standard solution are prepared
Prepare GSH (Glutathione), Car (carnosine), P-Ser (phosphoserine), Tau (taurine), PEA (phosphoric acid second
Hydramine), Urea (carbamide), Asp (aspartic acid), Hypro (hydroxyproline), Thr (threonine), Ser (serine), AspNH2
(agedoite), Glu (glutamic acid), GluNH2 (L-Glutamine), Sar (sarcosine), a-AAA (Amicar), Pro (dried meat
Propylhomoserin), Gly (glycine), Ala (alanine), Cit (citrulline), a-ABA (butyrine), Val (L-Valine), Cys
(cystine), Met (methionine), Cysthi (cystathionie), Ile (isoleucine), Leu (leucine), Tyr (tyrosine),
Phe (Phenylalanine), b-Ala (Beta-alanine), b-AiBA (B-AIB), GABA (γ-aminobutyric acid), Trp (color ammonia
Acid), EOHNH2(ethanolamine), Hylys (oxylysine), Orn (ornithine), Lys (lysine), 1Mehis (1- methyl groups
Propylhomoserin), His (histidine), 3Mehis (3-Methyl histidine), the standard solution of Arg (arginine), and it is molten to prepare hybrid standard
Liquid.
In standard solution and mixed standard solution, each component content is：200 μm of ol/L of GSH, Tau50 μm of ol/L, Tau
50 μm of ol/L, AspNH2200 μm of ol/L, GluNH2200 μm of ol/L, Sar200 μm of ol/L, a-AAA 50 μm of ol/L, a-ABA
50 μm of ol/L of 50 μm of ol/L, Cysthi, other constituents ratio are 100 μm of ol/L.Above-mentioned solution is all with the dilute salt of 0.02mol/L
Acid is prepared.
(3) detection of standard solution and mixed standard solution
By the standard solution sample introduction of mixed standard solution and various aminoacid and peptides, according to program eluting and the root of table 3
Determine the various composition of mixing mark solution according to the retention time of standard solution.
The aminoacid being eluted and 1,2,3-indantrione monohydrate derivative reagent reacting by heating, product detects fixed under 570nm and 440nm
Amount.
The gradient elution of amino-acid analyzer and heating schedule after table 3 optimization
The gradient elution of table 4 amino-acid analyzer and heating schedule
Spectrogram titer being afforded using elution program in prior art (table 4) is as shown in Figure 1；After optimizing
Elution program (table 3) that titer is carried out with the spectrogram that gradient elution obtains is as shown in Figure 2.
Visible according to Fig. 1 and Fig. 2, can not be by Glutathione and free ammonia according to gradient elution program of the prior art
Base acid is separated it is impossible to the content of Accurate Determining solution GSH-PX activity.Gradient elution program after the present invention optimizes can be
In 30min, so that Glutathione and the peak of carnosine is preferably separated with free amino acid, realize to Glutathione and carnosine concentration
Measure it is adaptable to the mixed determining of Glutathione, carnosine and free amino acid.And the elution program after optimizing can shorten separation
Detection time.
Embodiment 2 range of linearity and the determination experiment of test limit
With the hydrochloric acid solution of 0.02mol/L, standard mixed solution is diluted certain multiple, the solution of each concentration is using excellent
After change, the elution program of table 3 carries out three subgradient eluting tests.
Qualitatively foundation is used as by the retention time of appearance, is made with the peak area of institute's colour examining spectrum and its corresponding concentration
Go out standard curve, evaluated with linearly dependent coefficient (R2)；According to signal to noise ratio, when the peak height of surveyed material chromatographic peak is 3 times of noise
When (S/N=3), determine the lowest detectable limit (LOD) of its hybrid standard component, (the S/N when the peak height of chromatographic peak is 10 times of noise
=10) its hybrid standard measured portions limit (LOQ), least concentration value that is, can quantitatively needed for this analyte, are determined.
Concrete data is as shown in table 5：
Table 5 bioactive peptide and several amino acids standard mixed solution linearly dependent coefficient, test limit and quantitative limit
From table 5, in the corresponding range of linearity, linearly dependent coefficient R2 exists for two kinds of bioactive peptide and free amino acid
Between 0.9991～0.9999, in the full-automatic amino-acid analyzer method after showing to optimize between the area of chromatographic peak and concentration
Have reasonable linear relationship, the quantitative limit of each component in mixed standard solution between 0.35～20.12 μm of ol/L,
Low test limit is between 0.12～6.25 μm of ol/L.Show that the full-automatic amino-acid analyzer method after this optimization has good spirit
Sensitivity.
Embodiment 3 response rate and the determination experiment of precision
Take the sample tissue of 2g shellfish, in sample, add the mixed standard solution of high, normal, basic three kinds of variable concentrations respectively, then
The dilute hydrochloric acid of 15mL 0.02mol/L and 1mL concentration is added to be the dithiothreitol, DTT of 1wt%, ultrasound wave cleaning after abundant homogenizing
5min, then uses refrigerated centrifuger (5000r, 4 DEG C) to be centrifuged 10min, collects supernatant.Residual residue addition 10mL concentration is
Stir after 0.02mol/L dilute hydrochloric acid, recentrifuge (5000r, 4 DEG C) 5min, merge supernatant, be settled to 50mL.Move after constant volume
Take 2mL, add the sulfosalicylic acid of 2mL 5wt%, recentrifuge (10000r, 4 DEG C) 10min, then with 0.22 μm of aqueous phase mistake
Membrane filtration, obtains prepare liquid.
The prepare liquid of each concentration carries out five subgradient eluting using elution program after optimizing, and calculates it with external standard method actual
Content and mark-on average recovery rate.Precision test carried out 5 repetition analysis of experimentss in 1 day and in a few days becomes to same prepare liquid
Change, to same prepare liquid METHOD FOR CONTINUOUS DETERMINATION 5 days, daily measure 1 time, analyze Daytime varieties, determine in a few days and day to day precision respectively.
Response rate computing formula is：The response rate=(adding content-sample size after mark product)/add mark product amount * 100%,
Test result is listed in table 6.
Table 6 bioactive peptide and aminoacid mixed standard solution recovery of standard addition and degree of accuracy experiment
Can be drawn according to table 6 data, mixed standard solution each component mark-on average recovery rate is 84.6～103.60%
Between, in a few days between 0.31～0.71%, relative standard deviation, between 1.23～2.38%, accords with relative standard deviation in the daytime
Close the related request that Good Laboratory controls specification-food Physico-chemical tests.The method response rate and accuracy rate are also described simultaneously
Higher it is adaptable to measure the bioactive peptide in the aquatic products such as shellfish and multiple free amino acid.
In the fresh shellfish of embodiment 4, bioactive peptide and free aminoacid content measure
Choose relatively common four kinds of live-shellfishs on market：Scallop, Sinonovacula constricta, mussel, Concha Ostreae, every kind of aquatic products respectively do 3
Individual parallel sample.Each sample takes 2g, and then in each sample, the dilute hydrochloric acid of addition 15mL0.02mol/L and 1mL concentration are
The dithiothreitol, DTT of 1wt%, ultrasound wave cleaning 5min after abundant homogenizing, then uses refrigerated centrifuger (5000r, 4 DEG C) to be centrifuged
10min, collects supernatant.Residual residue is added 10mL concentration for stirring after 0.02mol/L dilute hydrochloric acid, recentrifuge
(5000r, 4 DEG C) 5min, merges supernatant, is settled to 50mL.Pipette 2mL after constant volume, add the sulfosalisylic of 2mL 5wt%
Acid, recentrifuge (10000r, 4 DEG C) 10min, then filters membrane filtration with 0.22 μm of aqueous phase, obtains prepare liquid.Every part of prepare liquid
Gradient elution is carried out using elution program after optimizing, spss analysis is carried out to the data of eluting detection, calculates standard deviation, and draw
Significance.As shown in table 7 (in same row, identical Superscript letters represent that difference is not notable, i.e. P ＞ 0.05), spectrogram is such as testing result
Shown in Fig. 3～5.
In the common shellfish meat tissue of table 7 bioactive peptide and free amino acid content (N=3, mg/g)
As can be seen from Table 7, the free amino acid in fresh shellfish meat tissue is abundanter, delicious amino acid content
Height, glutamic acid (Glu), glycine (Gly), alanine (Ala) occupy significant proportion in shellfish meat tissue total free amino acid；
In Concha Ostreae, the content of total free amino acid is 5.93mg/g；In scallop and be not detected by carnosine (Car)；All contain in common shellfish
A certain amount of Glutathione (GSH)；This example demonstrates that the gradient elution program after optimizing can fast and effectively measure aquatic products
In multiple free amino acids and bioactive peptide content.
The impact to Glutathione and multiple free aminoacid content for embodiment 5 antioxidant
Accurately weigh 2g flower clam and white clam meat tissue, be separately added into the dilute hydrochloric acid of 15mL 0.02mol/L and 1mL concentration is
The antioxidant solution of 1wt%, antioxidant is dithiothreitol, DTT (DTT), sodium dithionite (Na2S2O4) or vitamin C (Vc).
Ultrasound wave cleaning 5min fully after homogenizing, then uses refrigerated centrifuger (5000r, 4 DEG C) to be centrifuged 10min, collects
Clear liquid.Residual residue is added 10mL concentration for stirring after 0.02mol/L dilute hydrochloric acid, recentrifuge (5000r, 4 DEG C) 5min, closes
And supernatant, it is settled to 50mL with the dilute hydrochloric acid of 0.02mol/L.
Pipette 2mL after constant volume, add the sulfosalicylic acid of 2mL 5wt%, recentrifuge (10000r, 4 DEG C) 10min, so
Filter membrane filtration with 0.22 μm of aqueous phase afterwards, obtain prepare liquid.
Prepare liquid is respectively adopted the content that the gradient elution program after optimization measures free amino acid and bioactive peptide, test
As shown in Figure 6 and Figure 7 (Kb is blank), the interpolation of antioxidant contains result for free amino acid in shellfish meat tissue
Amount does not affect substantially, and dithiothreitol, DTT has more preferable protection and antioxidation for the content of shellfish meat tissue GSH-PX activity
Effect.
It is pointed out that above-described embodiment technology design only to illustrate the invention and feature, its object is to allow ripe
The personage knowing this Project Technical will appreciate that present disclosure and implements according to this, can not limit the protection model of the present invention with this
Enclose.All equivalence changes made according to spirit of the invention or modification, all should be included within the scope of the present invention.
Claims (10)
1. a kind of method of mensure bioactive peptide and free amino acid simultaneously is it is characterised in that step includes：By testing sample solution
It is added in amino-acid analyzer, carries out gradient elution with buffer B 1～B5, elution program includes：
First stage：With buffer B 1 eluting, persistent period 20～21min；37.5～39 DEG C of initial column temperature, the 2.0th～
It is down to 34.5～35.5 DEG C during 2.1min, at the end of the first stage, column temperature is risen to 55.5～56.5 DEG C；
Second stage：With buffer B 1 and B2 eluting, persistent period 11.5～12.5min；First column temperature is risen to 57.5～58.5
DEG C, instantaneously it is adjusted to B1 78%～82%, B2 18%～22%；At the uniform velocity become again turn to B1 68%～72%, B2 28%～
32%, at the end of second stage, column temperature is down to 55.5～56.5 DEG C；
Phase III：Instantaneously it is adjusted to buffer B 1 8%～12%, buffer B 2 88%～92%, in this ratio eluting 9
～10min, and column temperature is down to 41.5～42.5 DEG C in 2.8～3.1min；
Fourth stage：With buffer B 2 eluting 5～6min, and at the end of fourth stage, column temperature is risen to 71.5～72.5 DEG C；
5th stage：With buffer B 3 and B4 eluting, from buffer B3 68%～72%, buffer B 4 in 16.5～17.5min
28%～32% is at the uniform velocity adjusted to buffer B 3 58%～62%, buffer B 438%～42%；
6th stage：With buffer B 3 and B4 eluting, be at the uniform velocity adjusted in 2～3min B3 54%～56%, B4 44%～
46%；
7th stage：With buffer B 4 eluting 17～18min；
8th stage：With buffer B 5 eluting 4～8min；
Aforementioned proportion is volume ratio；
Described buffer B 1 is the citric acid-Lithium Citrate de buffer system of pH=2.75～2.85；
Described buffer B 2 is the citric acid-Lithium Citrate de buffer system of pH=3.65～3.75；
Described buffer B 3 is the citric acid-Lithium Citrate de buffer system of pH=3.55～3.65；
Described buffer B 4 is the citric acid-Lithium Citrate de buffer system of pH=4.05～4.15；
Described buffer B 5 is lithium hydroxide aqueous solution.
2. the method simultaneously measuring bioactive peptide and free amino acid according to claim 1 is it is characterised in that described eluting
Program includes：
First stage is 0～20.8min, with buffer B 1 eluting, 37.5～39 DEG C of initial column temperature, is adjusted to during 2.1min
34.5～35.5 DEG C, at the end of the first stage, column temperature is risen to 55.5～56.5 DEG C；
Second stage is 20.8～32.8min, with buffer B 1 and B2 eluting, column temperature is risen to 57.5～58.5 DEG C, from B1
78%～82%, B2,18%～22%, be at the uniform velocity adjusted to B1 68%～72%, B2 28%～32%；At the end of second stage
Column temperature is adjusted to 55.5～56.5 DEG C；
Phase III is 32.8～42.5min, is instantaneously adjusted to buffer B 1 8%～12%, buffer B 2 78%～
82%, and in this ratio eluting；During 35.8min, column temperature is down to 41.5～42.5 DEG C；
Fourth stage is 42.5～48.0min, and with buffer B 2 eluting, at the end of fourth stage, column temperature rises to 71.5～72.5
℃；
5th stage was 48.0～65.0min, from buffer B3 68%～72%, buffer B 4 28%～32%, at the uniform velocity
It is adjusted to buffer B 3 54%～56%, buffer B 4 44%～46%；
6th stage was 65.0～67.5min, at the uniform velocity becomes and turns to buffer B 3 54%～56%, buffer B 4 44%～
46%；
7th stage was 67.5～84.5min, carried out eluting with buffer B 4；
8th stage was 84.5～89.0min, carried out eluting with buffer B 5.
3. the method simultaneously measuring bioactive peptide and free amino acid according to claim 1 is it is characterised in that described eluting
Program includes：
First stage is 0～20.8min, with buffer B 1 eluting, 38 DEG C of initial column temperature, is adjusted to 35 DEG C during 2.1min,
At the end of first stage, column temperature is risen to 56 DEG C；
Second stage is 20.8～32.8min, with buffer B 1 and B2 eluting, column temperature is risen to 58 DEG C, from buffer B1
80%th, B2 20% is at the uniform velocity adjusted to B1 70%, B2 30%, and at the end of second stage, column temperature is adjusted to 56 DEG C；
Phase III is 32.8～42.5min, and being adjusted to buffer B 1 is 10%, and buffer B 2 is 90%, and presses this ratio
Example eluting；During 35.8min, column temperature is adjusted to 42 DEG C；
Fourth stage is 42.5～48.0min, and with buffer B 2 eluting, during 42.0min, column temperature is adjusted to 72 DEG C；
5th stage was 48.0～65.0min, is instantaneously adjusted to buffer B 3 70%, buffer B 430%, and at the uniform velocity adjusts
For B3 be 60%, B4 be 40%；
6th stage was 65.0～67.5min, and being at the uniform velocity adjusted to buffer B 3 is 55%, and buffer B 4 is 45%；
7th stage was 67.5～84.5min buffer B 4 eluting；
8th stage was 84.5～89min, with buffer B 5 eluting.
4. the method measuring bioactive peptide and free amino acid while according to any one of claims 1 to 3, its feature exists
In：
Described buffer B 1 contains 5.5～6g/L citric acid three lithium, 1～1.5g/L lithium chloride, 19～21g/L citric acid, body
The ethanol of long-pending content 2.5%～3.5% and the octanoic acid of volume content 0.005%～0.015%；
Buffer B 2 contains containing 9.5～11g/L citric acid three lithium, 6～7.5g/L lithium chloride, 11.5～13g/L citric acid, volume
Amount 2.5%～4% ethanol and volume content 0.005%～0.015% octanoic acid；
Buffer B 3 contains containing 8.5～9.6g/L citric acid three lithium, 25～28g/L lithium chloride, 11～12g/L citric acid, volume
The octanoic acid of amount 8%～12% ethanol, the phenethanol of volume content 3.5%～5% and volume content 0.005%～0.0.15%；
Buffer B 4 contains 9.5～11g/L citric acid three lithium, 37.5～40g/L lithium chloride, 3～4g/L citric acid and volume and contains
Amount 0.005%～0.015% octanoic acid；
Buffer B 5 contain 8～10g/L Lithium hydrate, volume content 2.5%～4% ethanol and volume content 0.005%～
0.015% octanoic acid.
5. the method simultaneously measuring bioactive peptide and free aminoacid content according to claim 4 it is characterised in that
The pH=2.8 of described buffer B 1, containing 5.73g/L citric acid three lithium, 1.24g/L lithium chloride, 19.9g/L Fructus Citri Limoniae
Acid, volume content 3.0% ethanol and volume content 0.01% octanoic acid；
The pH=3.7 of buffer B 2, contains containing 9.8g/L citric acid three lithium, 6.36g/L lithium chloride, 12.0g/L citric acid, volume
Measure 3% ethanol and volume content 0.01% octanoic acid；
The pH=3.6 of buffer B 3, containing 8.79g/L citric acid three lithium, 26.62g/L lithium chloride, 11.27g/L citric acid, body
The octanoic acid of the ethanol, the phenethanol of volume content 0.4% and volume content 0.01% of long-pending content 10%；
The pH=4.1 of buffer B 4, containing 9.8g/L citric acid three lithium, 38.15g/L lithium chloride, 3.3g/L citric acid and volume
Content 0.01% octanoic acid；
Buffer B 5 contains 8.4g/L Lithium hydrate, volume content 3% ethanol and volume content 0.01% octanoic acid.
6. while as described in any one of claims 1 to 3, measure the method for bioactive peptide and free amino acid it is characterised in that
Using the colour developing of 1,2,3-indantrione monohydrate derivative reagent；Developing wavelength is respectively 440nm and 570nm.
7. the as claimed in claim 6 method simultaneously measuring bioactive peptide and free amino acid is it is characterised in that described 1,2,3-indantrione monohydrate
Derivative reagent includes ninhydrin reaction liquid, reaction buffer and cleaning mixture；
Ninhydrin reaction liquid is the solution of 1,2,3-indantrione monohydrate and sodium borohydride, and solvent is selected from ethylene glycol single methyl ether or propylene glycol monomethyl
Ether, 1,2,3-indantrione monohydrate concentration is 38～40g/L, and sodium borohydride concentration is 80～82mg/L；
Reaction buffer is sodium acetate, the mixed solution of glacial acetic acid, water and organic solvent；Described organic solvent is selected from ethylene glycol list
Methyl ether or propylene glycol monomethyl ether；Described organic solvent is 1 with the volume ratio of glacial acetic acid, water：0.25～0.35：0.8～
0.85；Described sodium acetate is 1 with the mass ratio of ultra-pure water：1.6～1.7；
Cleaning mixture is ethanol water；Described ethanol is 1 with the volume ratio of water：18.5～19.5.
8. the method measuring bioactive peptide and free amino acid while according to any one of claims 1 to 3, its feature exists
In described bioactive peptide includes at least one in Glutathione and carnosine.
9. the method simultaneously measuring bioactive peptide and free amino acid according to claim 1 is it is characterised in that described to be measured
The preparation method of liquid comprises the following steps：
A. sample is mixed with hydrochloric acid solution, antioxidant, supersound process after homogenizing, solid-liquid separation；Antioxidant and Aquatic product to be measured
The mass ratio of product sample is 1：50～500；Described antioxidant is dithiothreitol, DTT, sodium dithionite or vitamin C,
B. the solid of step a is extracted 1～5 time with hydrochloric acid solution again；
C. use hydrochloric acid solution constant volume after the supernatant of combining step a, b, be subsequently adding sulfosalicylic acid, centrifugation, filtration, take filter
Liquid；
Concentration of hydrochloric acid solution used by above steps is 0.005～0.1mol/L.
10. the method for mensure bioactive peptide and free amino acid simultaneously according to claim 1 is it is characterised in that described treat
The preparation method surveying liquid comprises the following steps：
A. by testing sample and mixed in hydrochloric acid, dithiothreitol, DTT, supersound process 3～20min after homogenizing are added, 0～20 DEG C is centrifuged,
Take supernatant；Dithiothreitol, DTT is 1 with the mass ratio of aquatic products sample to be measured：100～400；Testing sample and the consumption of hydrochloric acid
Than for 1g：5～10mL；
B. take the step a solid that obtains of centrifugation, with mixed in hydrochloric acid after stirring, centrifuging and taking supernatant, repeat 1～5 time；Testing sample
Amount ratio with hydrochloric acid is 1g：3～8mL；
C. the supernatant of combining step a and step b, with hydrochloric acid solution constant volume and add sulfosalicylic acid, makes sulfosalicylic acid
Content is 2wt%～3wt%；Filtrate is taken to obtain prepare liquid with 0.2～0.5 μm of membrane filtration after centrifugation；
Concentration of hydrochloric acid solution used by above steps is 0.01～0.05mol/L.
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