CN106468695B - Method that is a kind of while measuring active peptide and free amino acid - Google Patents
Method that is a kind of while measuring active peptide and free amino acid Download PDFInfo
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- CN106468695B CN106468695B CN201610858349.8A CN201610858349A CN106468695B CN 106468695 B CN106468695 B CN 106468695B CN 201610858349 A CN201610858349 A CN 201610858349A CN 106468695 B CN106468695 B CN 106468695B
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- G—PHYSICS
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- 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
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- G01N30/06—Preparation
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- G—PHYSICS
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- 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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- 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
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Abstract
The invention belongs to analytical chemistry field, it is related to nutriment assay method, disclose while measure active peptide and the method for free amino acid, by being formed to the buffer solution in amino acid analysis analyzer standard test program, elution program, the adjustment of eluting temperature, so that a variety of amino acid peaks are separated with active peptide peak, realize and the one-step method of free amino acid and active peptide is measured using automatic amino acid analyzer, establish while prepare amino acid and the method for peptides, overcoming nontraditional amino acid analyzer method well can not separate active peptide and a variety of free amino acids, and test the shortcomings 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
Method that is a kind of while measuring active peptide and free aminoacid content.
Background technology
Free amino acid is aquatic food freshness and the important evaluation index of flavor, and the important composition of nonprotein nitrogen
Part；Different free amino acids has different tastes, and the various tastes such as fresh, sweet tea, acid, hardship are presented, 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.Therefore the content of free amino acid can be used as the important freshness of many aquatic products quality evaluations
Potential index.
Active peptide refers to the peptides for having physiological activity to the vital movement of organism, 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 a variety of biological functions and physiological effect in addition to trophic function, such as：Anti-oxidant, antibacterial, blood pressure lowering, free radical are clear
Except agent etc..
Shellfish is a kind of important aquatic products kind, active peptide and free amino acid in the body of shellfish 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, active peptide
With trophic functions such as anti-oxidant, antibacterial, blood pressure lowering, radicals scavengings, therefore the content of active peptide also becomes characterization shellfish etc.
The important parameter of aquatic products quality.
Active peptide is mostly the Amino acid score subchain of 2 to 10 amino acid compositions, similar to free amine group acid molecular structures, is changed
Property is learned to approach.It is not easy to separate the two in nutriment extracting solution, then measures its concentration respectively.Therefore, the prior art
In more the content of free amino acid and active peptide in shellfish is measured using one-step method at the same time.
The method of related assays active peptide and free amino acid has much both at home and abroad at present, mainly there is high performance liquid chromatography
With colorimetric method, spectrophotometry, high performance capillary electrophoresis.
Although high performance liquid chromatography, colorimetric method, spectrophotometry, high performance capillary electrophoresis can to active peptide or
Free amino acid is measured, but these methods there are it is complicated, instrument and equipment cost is higher, easily be subject to impurity interference,
The shortcomings of measurement result inaccuracy and limited test approaches, fail fully to be promoted.
The measure most important method of free amino acid is automatic amino acid analyzer method at present, and also someone adopts in the prior art
Glutathione and free amino acid are measured with automatic amino acid analyzer, but the corresponding standard method of amino-acid analyzer cannot
It is enough well to separate active peptide and free amino acid, and testing time length.There is presently no a kind of method, can utilize ammonia
Base acid automatic analyzer effectively, quickly measures active peptide and a variety of free aminoacid contents at the same time.
Therefore, exploitation one kind measures a variety of free amino acids, glutathione and flesh at the same time using automatic amino acid analyzer
The method of peptide content, is the key solved the problems, such as in the prior art.
The content of the invention
The present invention is intended to provide method that is a kind of while measuring active peptide and free amino acid, to solve in the prior art not
Can the problem of active peptide and free aminoacid content measured at the same time accurate using amino-acid analyzer, quick.
The technical scheme is that using free amino acid and activity in the elution program separation sample after optimization
Peptide, draws standard curve, then measures free amino acid and active peptide concentration in testing sample solution using calibration curve method.
Method that is a kind of while measuring active peptide and free amino acid, its step include, testing sample solution are added to
In amino-acid analyzer, gradient elution is carried out by following procedure with 1~B5 of buffer B, is developed the color using ninhydrin nitrite ion：
First stage：Eluted with buffer B 1,20~21min of duration；37.5~39 DEG C of initial column temperature, the 2.0th~
34.5~35.5 DEG C are down to during 2.1min, column temperature is risen to 55.5~56.5 DEG C at the end of the first stage；
Second stage：Eluted with buffer B 1 and B2,11.5~12.5min of duration；Column temperature is first 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%, column temperature is down to 55.5~56.5 DEG C at the end of second stage；
Phase III：Buffer B 1 8%~12%, buffer B 2 88%~92% instantaneously are adjusted to, in this ratio
9~10min is eluted, and column temperature is down to 41.5~42.5 DEG C in 2.8~3.1min；
Fourth stage：5~6min is eluted with buffer B 2, and column temperature is risen to 71.5~72.5 at the end of fourth stage
℃；
5th stage：Eluted with buffer B 3 and B4, from buffer solution 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 for 71.5~
72.5℃；
6th stage：Eluted with buffer B 3 and B4, B3 54%~56%, B4 44% are at the uniform velocity adjusted in 2~3min
~46%, column temperature is 71.5~72.5 DEG C；
7th stage：17~18min is eluted with buffer B 4, column temperature is 71.5~72.5 DEG C；
8th stage：4~5min is eluted with buffer B 5.
Column temperature is down to 69.5~71 DEG C after the 8th stage.
Aforementioned proportion is volume ratio.Then worked according to the standard of active 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, is eluted with buffer B 1,37.5~39 DEG C of initial column temperature, tune during 2.1min
Whole is 34.5~35.5 DEG C, and column temperature is risen to 55.5~56.5 DEG C at the end of the first stage；
Second stage is 20.8~32.8min, is eluted with buffer B 1 and B2, column temperature is risen to 57.5~58.5 DEG C,
Buffer B 1 and the ratio of B2 at the uniform velocity adjust B168%~72%, B2 28% from B1 78%~82%, B2 18%~22%
~32%；Column temperature is adjusted to 55.5~56.5 DEG C at the end of second stage；
Phase III is 32.8~42.5min, is instantaneously adjusted to buffer B 1 8%~12%, buffer B 2 88%
~92%, and eluted in this ratio；Column temperature is adjusted to 41.5~42.5 DEG C during 35.8min；
Fourth stage is 42.5~48.0min, is eluted with buffer B 2, column temperature is adjusted to 71.5 at the end of fourth stage
~72.5 DEG C；
5th stage was 48.0~65.0min, from buffer solution 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 it is 54%~56% to be at the uniform velocity adjusted to buffer B 3, and buffer B 4 is
44%~46%, column temperature is 69.5~71 DEG C；
7th stage was 67.5~84.5min, was eluted with buffer B 4, and column temperature is 71.5~72.5 DEG C；
8th stage was 84.5~89.0min, was eluted with buffer B 5.
Preferred elution program is：
First stage is 0~20.8min, is eluted with buffer B 1,38 DEG C of initial column temperature, and when 2.1min is adjusted to 35
DEG C, column temperature is risen to 56 DEG C at the end of the first stage；
Second stage is 20.8~32.8min, is eluted with buffer B 1 and B2, column temperature is risen to 58 DEG C, buffer B 1
For 80%, B2 20%, and be at the uniform velocity adjusted to B1 be 70%, B2 30%, column temperature is adjusted to 56 DEG C at the end of second stage；
Phase III is 32.8~42.5min, and it is 10% to be adjusted to buffer B 1, and buffer B 2 is 90%, and presses this
One ratio elutes；Column temperature is adjusted to 42 DEG C during 35.8min；
Fourth stage is 42.5~48.0min, is eluted with buffer B 2, column temperature is adjusted to 72 at the end of fourth stage
℃；
5th stage was 48.0~65.0min, and it is 70% to be instantaneously adjusted to buffer B 3, 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 it is 55% to be at the uniform velocity adjusted to buffer B 3, and buffer B 4 is 45%；
7th stage was 67.5~84.5min, was eluted with buffer B 4；
8th stage was 84.5~89min, was eluted with buffer B 5.Column temperature is down to 70 DEG C after the 8th stage.
1~the B5 of buffer B is citric acid-lithium citrate buffer system, and pH value is respectively 2.75~2.85,3.65
~3.75,3.55~3.65,4.05~4.15；The buffer B 5 is lithium hydroxide aqueous solution.
PH=2.75~2.85 of buffer B 1, three lithium concentration of citric acid are 5.5~6g/L, chlorination lithium concentration for 1~
1.5g/L, citric acid concentration are 19~21g/L, alcohol volume content 2.5%~3.5%, sad volume content 0.005%~
0.015%.Preferably, the pH=2.8 of B1, three lithium concentration of citric acid are 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 the buffer B 2, three lithium concentration of citric acid are 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, three lithium concentration 9.8g/L of citric acid, chlorination lithium concentration are 6.36g/L,
Citric acid concentration 12.0g/L, alcohol volume content 3%, sad volume content 0.01%.
PH=3.55~3.65 of the buffer B 3, three lithium concentration of citric acid are 8.5~9.6g/L, chlorination lithium concentration
For 25~28g/L, citric acid concentration is 11~12g/L, alcohol volume content 8%~12%, benzyl carbinol volume content 3.5%~
5%, sad 0.005%~0.0.15% of volume content.Preferably, the pH=3.6 of buffer B 3, three lithium concentration of citric acid are
8.79g/L；Chlorination lithium concentration is 26.62g/L, citric acid concentration 11.27g/L, alcohol volume content 10%, benzyl carbinol body
Product content is 0.4%, and sad volume content is 0.01%.
PH=4.05~4.15 of the buffer B 4, three lithium concentration of citric acid are 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 solution
The pH=4.1 of B4, three lithium concentration of citric acid is 9.8g/L, chlorination lithium concentration is 38.15g/L, citric acid concentration 3.3g/L；It is pungent
Sour volume content is 0.01%.
In the buffer B 5, lithium hydroxide concentration is 8~10g/L, is preferably 8.4g/L；Alcohol volume content
2.5%~4%, it is preferably 3%；Sad volume content 0.005%~0.015%, is preferably 0.01%.
The product of elution is developed the color using ninhydrin derivative reagent, and developing wavelength is 440nm and 570nm.
Ninhydrin derivative reagent includes ninhydrin reaction liquid, reaction buffer and cleaning solution, and concrete composition is as follows：
Ninhydrin reaction liquid (R1) is the solution of ninhydrin and sodium borohydride, and solvent is selected from ethylene glycol single methyl ether or the third two
Alcohol monomethyl ether；The ninhydrin concentration is 35~42g/L, is preferably 38~40g/L；The sodium borohydride concentration for 80~
85mg/L, is preferably 80~82mg/L.
Reaction buffer (R2) is the mixed solution of sodium acetate, glacial acetic acid, water and organic solvent；The organic solvent is selected from
Ethylene glycol single methyl ether or propylene glycol monomethyl ether；The volume ratio of the organic solvent and glacial acetic acid, water is 1：0.2~0.4：
0.7~1, it is preferably 1：0.25~0.35：0.8~0.85；The mass ratio of the sodium acetate and ultra-pure water is 1：1.5~2.0, it is excellent
Elect 1 as：1.6~1.7.
Cleaning solution (R3) is ethanol water；The volume ratio of the ethanol and water is 1：18~20, it is preferably 1：18.5~
19.5.Developed the color with ninhydrin reaction liquid and reaction buffer during separation elution, cleaned at the end of elution program with cleaning solution.
In this method, the active peptide is at least one of glutathione and carnosine；The free amino acid bag
Include P-Ser (phosphoserine), Tau (taurine), PEA (phosphoethanolamine), Urea (urea), Asp (aspartic acid),
Hypro (hydroxyproline), Thr (threonine), Ser (serine), AspNH2(asparagine), Glu (glutamic acid), GluNH2
(glutamine), Sar (methyl amimoacetic acid), a-AAA (Amicar), Pro (proline), Gly (glycine), Ala (alanine),
Cit (citrulling), a-ABA (butyrine), Val (valine), Cys (cystine), Met (methionine), Cysthi (Guangs
Thioether), Ile (isoleucine), Leu (leucine), Tyr (tyrosine), Phe (phenylalanine), b-Ala (Beta-alanine), b-
AiBA (B-AIB), GABA (γ-aminobutyric acid), Trp (tryptophan), EOHNH2(hydroxyl relies by (monoethanolamine), Hylys
Propylhomoserin), Orn (ornithine), Lys (lysine), 1Mehis (1-Methyl histidine), His (histidine), 3Mehis (3- methyl
Histidine), at least one of Arg (arginine).
The preparation method of the testing sample solution is：
A. sample (such as aquatic products, meat) is mixed with hydrochloric acid solution and antioxidant, is ultrasonically treated after homogeneous, solid-liquid point
From；The mass ratio of antioxidant and aquatic products sample to be measured is 1：50~500；The 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；
The clear liquid that c.a, b step obtain uses hydrochloric acid solution constant volume after merging, and then adds sulfosalicylic acid, and centrifugation, filter,
Take filtrate.
Preferably, the preparation method of testing sample solution is as follows：
A. aquatic products sample to be measured is taken, is mixed with hydrochloric acid solution, adds antioxidant dithiothreitol (DTT), after abundant homogeneous,
3~20min is ultrasonically treated, supernatant is taken after 0~20 DEG C of centrifugation；The mass ratio of dithiothreitol (DTT) and aquatic products sample to be measured is 1：
100~400；The sample to be tested tissue and hydrochloric acid solution amount ratio are 1g：1~15mL, is preferably 1g：5~10mL.
B. according to aquatic products sample to be measured and hydrochloric acid solution 1g：The amount ratio of 8~15mL, takes what step a centrifuged to consolidate
Body is mixed again with hydrochloric acid solution, 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 adds sulfosalicylic acid, sulfosalicylic acid contains
Measure as 2wt%~3wt%；Prepare liquid is obtained using 0.22 μm of membrane filtration after centrifugation.
Concentration of hydrochloric acid used in each step is 0.005~0.1mol/L above, is preferably 0.01~0.05mol/L.
This method is by optimizing elution program, buffer solution forms, eluting temperature, realizes using automatic amino acid analyzer
The one-step method of amino acid and active peptides in the nutriment of food, especially aquatic products etc. is measured, overcomes traditional amino
Active peptide and free amino acid can not be separated and test the shortcomings that time-consuming by acid analysis instrument well.
The beneficial effects of the present invention are can fast and effectively various amino acid and active peptide contain in determination sample
Amount；Detection time is less than 90 minutes, and glutathione can be kept completely separate with free amino acid and be measured.Using the party
Method, mixed standard solution each component mark-on average recovery rate is between 84.6%~103.60%, and in a few days relative standard deviation exists
Between 0.31%-0.71%, relative standard deviation meets Good Laboratory control rule between 1.23%~2.38% in the daytime
The related request of model-food Physico-chemical tests；In the assay method, have between the area and concentration of chromatographic peak relatively good linear
Relation, the quantitative limit of each component in mixed standard solution between 0.35~20.12 μm of ol/L, minimum detection limit 0.12~
Between 6.25 μm of ol/L.Show that the full-automatic amino-acid analyzer method has good sensitivity.
Method using the present invention, can be well by glutathione and carnosine isoreactivity peptide and free ammonia in 30min
Base acid separates, suitable for peptide containing various active and the mixed determining of free amino acid, particularly aquatic products such as shellfish these
Sample containing various active peptide and free amino acid, shortens detection time, and improve sensitivity.
Brief description of the drawings
Fig. 1 is in embodiment 1, and active peptide and free amino acid mixed standard solution are analyzed using prior art elution program
Collection of illustrative plates
Fig. 2 is elution program analysis active peptide and free amino acid hybrid standard after being optimized using the present invention in embodiment 1
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 razor clam of hanging in embodiment 4
Fig. 5 is the detection separating spectrum of oyster in embodiment 4
Fig. 6 is influence of three kinds of antioxidants to active peptide and amino acid content in flower clam
Fig. 7 is the influence of active peptide and amino acid content in three kinds of antioxidant dialogue clams
Embodiment
With reference to specific embodiments and the drawings, the present invention is further explained.In following embodiments, used standard items
Standard liquid is mixed for active poly saccharide peptide standard product (glutathione, carnosine) and a variety of amino acid；Used instrument model is L-8800 ammonia
Base acid fully-automatic analyzer；The instrument parameter set as：Splitter (4.6mm × 60mm) resin is cation exchange resin；Inspection
Survey wavelength：570nm (proline 440nm)；20 μ L of sample size；Buffer flow rate is：0.35mL/min；Nitrite ion：Ninhydrin
Derivative reagent (flow 0.35mL/min；135 DEG C of cell temperature)
The analysis measure of 1 standard solution of embodiment
(1) preparation of elution buffer and ninhydrin derivative reagent
1~B5 of buffer B is prepared according to table 1；Ninhydrin derivative reagent (ninhydrin reaction liquid R1, reaction are prepared according to table 2
Buffer solution R2, cleaning solution R3).
The composition of table 1B1~B5 buffer solutions
The composition of 2 ninhydrin derivative reagent of table
(2) amino acid and peptides standard solution are prepared
Prepare GSH (glutathione), Car (carnosine), P-Ser (phosphoserine), Tau (taurine), PEA (phosphoric acid second
Hydramine), Urea (urea), Asp (aspartic acid), Hypro (hydroxyproline), Thr (threonine), Ser (serine), AspNH2
(asparagine), Glu (glutamic acid), GluNH2 (glutamine), Sar (methyl amimoacetic acid), a-AAA (Amicar), Pro (dried meat
Propylhomoserin), Gly (glycine), Ala (alanine), Cit (citrulling), a-ABA (butyrine), Val (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(monoethanolamine), 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：GSH 200 μm of ol/L, Tau 50 μm of ol/L, Tau
50 μm of ol/L, AspNH2200 μm of ol/L, GluNH2200 μm of ol/L, Sar 200 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 ratios are 100 μm of ol/L.Above-mentioned solution uses the dilute salt of 0.02mol/L
Acid is prepared.
(3) detection of standard solution and mixed standard solution
By mixed standard solution and various amino acid and the standard solution sample introduction of peptides, eluted and root according to the program of table 3
The various composition of mixing mark solution is determined according to the retention time of standard solution.
The amino acid being eluted is reacted with the heating of ninhydrin derivative reagent, and product detects fixed under 570nm and 440nm
Amount.
The gradient elution and heating schedule of amino-acid analyzer after table 3 optimizes
The gradient elution and heating schedule of 4 amino-acid analyzer of table
The spectrogram afforded using elution program in the prior art (table 4) to titer is as shown in Figure 1；After optimization
Elution program (table 3) to carry out the obtained spectrogram of gradient elution to titer as shown in Figure 2.
As it can be seen that can not be by glutathione and free ammonia according to gradient elution program of the prior art according to Fig. 1 and Fig. 2
Base acid separates, can not Accurate Determining solution GSH-PX activity content.Gradient elution program after present invention optimization can be
In 30min, the peak of glutathione and carnosine is preferably separated with free amino acid, realize to glutathione and carnosine concentration
Measure, suitable for the mixed determining of glutathione, carnosine and free amino acid.And the elution program after optimizing can shorten separation
Detection time.
The determination experiment of 2 range of linearity of embodiment and test limit
Standard mixed solution is diluted into certain multiple with the hydrochloric acid solution of 0.02mol/L, the solution of each concentration is using excellent
The elution program of table 3 carries out gradient elution test three times after change.
Qualitatively foundation is used as by the retention time of appearance, is made with the peak area and its corresponding concentration of institute's colour examining spectrum
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 minimum detection limit (LOD) of its hybrid standard component, when chromatographic peak peak height for 10 times of noise when (S/N
=10), determine that its hybrid standard measured portions limits (LOQ), the least concentration value needed for the analyte can be quantified.
Specific data are as shown in table 5：
5 active peptide of table and a variety of amino acid standard mixed solution linearly dependent coefficient, test limit and quantitative limits
By table 5 as it can be seen that two kinds of active peptides and free amino acid are in the corresponding range of linearity, linearly dependent coefficient R2 exists
Between 0.9991~0.9999, show in the full-automatic amino-acid analyzer method after optimizing between the area of chromatographic peak and concentration
There is a relatively good linear relationship, the quantitative limit of each component in mixed standard solution is between 0.35~20.12 μm of ol/L, most
Low test limit is between 0.12~6.25 μm of ol/L.Show that the full-automatic amino-acid analyzer method after the optimization has good spirit
Sensitivity.
The determination experiment of 3 rate of recovery of embodiment and precision
The sample tissue of 2g shellfishes is taken, adds the mixed standard solution of high, normal, basic three kinds of various concentrations in sample respectively, then
Add the dilute hydrochloric acid of 15mL 0.02mol/L and dithiothreitol (DTT) that 1mL concentration is 1wt%, ultrasonic cleaning after abundant homogeneous
5min, then centrifuges 10min with refrigerated centrifuge (5000r, 4 DEG C), collects supernatant.It is by residual residue addition 10mL concentration
Stirred after 0.02mol/L dilute hydrochloric acid, centrifuge (5000r, 4 DEG C) 5min again, merged supernatant, be settled to 50mL.Moved after constant volume
Take 2mL, add the sulfosalicylic acid of 2mL 5wt%, centrifuge (10000r, 4 DEG C) 10min again, then with 0.22 μm of water mutually mistake
Membrane filtration, obtains prepare liquid.
The prepare liquid of each concentration carries out five subgradient elutions using elution program after optimization, its reality is calculated with external standard method
Content and mark-on average recovery rate.Precision test carried out 5 repetition analysis of experiments in 1 day to same prepare liquid and in a few days becomes
Change, to same prepare liquid METHOD FOR CONTINUOUS DETERMINATION 5 days, Daytime varieties were analyzed in measure 1 time daily, were determined respectively in a few days and day to day precision.
Rate of recovery calculation formula is：The rate of recovery=(adding content-sample size after mark product)/adds mark product amount * 100%,
Test result is listed in table 6.
6 active peptide of table and amino acid mixed standard solution recovery of standard addition and accuracy experiment
It can be drawn according to 6 data of table, mixed standard solution each component mark-on average recovery rate is 84.6~103.60%
Between, in a few days relative standard deviation is between 0.31~0.71%, and relative standard deviation is between 1.23~2.38% in the daytime, symbol
Close the related request of Good Laboratory control specification-food Physico-chemical tests.Also illustrate this method rate of recovery and accuracy rate at the same time
It is higher, active peptide and a variety of free amino acids suitable for the aquatic products such as measure shellfish.
Active peptide and free aminoacid content measure in 4 fresh shellfish of embodiment
Choose four kinds of relatively common live-shellfishs of in the market：Scallop, razor clam of hanging, freshwater mussel, oyster, every kind of aquatic products respectively do 3
A parallel sample.Each sample takes 2g, then adds the dilute hydrochloric acid of 15mL0.02mol/L into each sample and 1mL concentration is
The dithiothreitol (DTT) of 1wt%, ultrasonic cleaning 5min after abundant homogeneous, is then centrifuged with refrigerated centrifuge (5000r, 4 DEG C)
10min, collects supernatant.Residual residue is added into 10mL concentration to be stirred after 0.02mol/L dilute hydrochloric acid, is centrifuged again
(5000r, 4 DEG C) 5min, merges supernatant, is settled to 50mL.2mL is pipetted after constant volume, adds the sulfosalisylic of 2mL 5wt%
Acid, centrifuges (10000r, 4 DEG C) 10min, then mutually filters membrane filtration with 0.22 μm of water, obtain prepare liquid again.Every part of prepare liquid
Gradient elution is carried out using elution program after optimization, spss analyses are carried out to the data of elution detection, calculate standard deviation, and draw
Conspicuousness.As shown in table 7 (identical Superscript letters represent that difference is not notable in same row, i.e. P ＞ 0.05), spectrogram is such as testing result
Shown in Fig. 3~5.
The content of active peptide and free amino acid in the common shellfish meat tissue of table 7 (N=3, mg/g)
As can be seen from Table 7, the free amino acid in fresh shellfish meat tissue is relatively abundanter, delicious amino acid content
Height, glutamic acid (Glu), glycine (Gly), alanine (Ala) occupy significant proportion in shellfish meat tissue total free amino acid；
The content of total free amino acid is 5.93mg/g in oyster；In scallop and it is not detected by carnosine (Car)；Contain in common shellfish
A certain amount of glutathione (GSH)；This example demonstrates that the gradient elution program after optimization can fast and effectively measure aquatic products
In a variety of free amino acids and active peptide content.
Influence of 5 antioxidant of embodiment to glutathione and a variety of free aminoacid contents
Accurately weigh 2g flower clams and white clam meat tissue, the dilute hydrochloric acid and 1mL concentration for being separately added into 15mL 0.02mol/L are
The antioxidant solution of 1wt%, antioxidant is dithiothreitol (DTT) (DTT), sodium dithionite (Na2S2O4) or vitamin C (Vc).
Ultrasonic cleaning 5min after abundant homogeneous, then centrifuges 10min with refrigerated centrifuge (5000r, 4 DEG C), collects
Clear liquid.Residual residue is added into 10mL concentration to be stirred after 0.02mol/L dilute hydrochloric acid, (5000r, 4 DEG C) 5min is centrifuged again, closes
And supernatant, it is settled to 50mL with the dilute hydrochloric acid of 0.02mol/L.
2mL is pipetted after constant volume, adds the sulfosalicylic acid of 2mL 5wt%, centrifuges (10000r, 4 DEG C) 10min again, so
Membrane filtration is mutually filtered with 0.22 μm of water afterwards, obtains prepare liquid.
Prepare liquid is respectively adopted to the content of the gradient elution program measure free amino acid after optimization and active peptide, test
As a result as shown in Figure 6 and Figure 7 (Kb is blank control), the addition of antioxidant contains for free amino acid in shellfish meat tissue
Amount do not influence substantially, dithiothreitol (DTT) for the content of shellfish meat tissue GSH-PX activity have preferably protection and it is anti-oxidant
Effect.
It is pointed out that the technical concepts and features of above-described embodiment only to illustrate the invention, ripe its object is to allow
Present disclosure can be understood and implement according to this by knowing the personage of this Project Technical, and the protection model of the present invention can not be limited with this
Enclose.Any equivalent change or modification in accordance with the spirit of the invention, should be covered by the protection scope of the present invention.
Claims (9)
1. method that is a kind of while measuring active peptide and free amino acid, it is characterised in that step includes：By testing sample solution
It is added in amino-acid analyzer, carries out gradient elution with 1~B5 of buffer B, elution program includes：
First stage：Eluted with buffer B 1,20~21min of duration；37.5~39 DEG C of initial column temperature, the 2.0th~
34.5~35.5 DEG C are down to during 2.1min, column temperature is risen to 55.5~56.5 DEG C at the end of the first stage；
Second stage：Eluted with buffer B 1 and B2,11.5~12.5min of duration；Column temperature is first risen to 57.5~58.5
DEG C, instantaneously it is adjusted to B1 78%~82%, B2 18%~22%；Again at the uniform velocity become turn to B1 68%~72%, B2 28%~
32%, column temperature is down to 55.5~56.5 DEG C at the end of second stage；
Phase III：Buffer B 1 8%~12%, buffer B 2 88%~92% instantaneously are adjusted to, 9 are eluted in this ratio
~10min, and column temperature is down to 41.5~42.5 DEG C in 2.8~3.1min；
Fourth stage：5~6min is eluted with buffer B 2, and column temperature is risen to 71.5~72.5 DEG C at the end of fourth stage；
5th stage：Eluted with buffer B 3 and B4, from buffer solution 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 4 38%~42%；
6th stage：Eluted with buffer B 3 and B4, be at the uniform velocity adjusted in 2~3min B3 54%~56%, B4 44%~
46%；
7th stage：17~18min is eluted with buffer B 4；
8th stage：4~8min is eluted with buffer B 5；
Aforementioned proportion is volume ratio；
The buffer B 1 is citric acid-lithium citrate buffer system of pH=2.75~2.85；
The buffer B 2 is citric acid-lithium citrate buffer system of pH=3.65~3.75；
The buffer B 3 is citric acid-lithium citrate buffer system of pH=3.55~3.65；
The buffer B 4 is citric acid-lithium citrate buffer system of pH=4.05~4.15；
The buffer B 5 is lithium hydroxide aqueous solution；
The active peptide is at least one of glutathione and carnosine.
2. method that is according to claim 1 while measuring active peptide and free amino acid, it is characterised in that the elution
Program includes：
First stage is 0~20.8min, is eluted with buffer B 1,37.5~39 DEG C of initial column temperature, and when 2.1min is adjusted to
34.5~35.5 DEG C, column temperature is risen to 55.5~56.5 DEG C at the end of the first stage；
Second stage is 20.8~32.8min, is eluted with buffer B 1 and B2, column temperature is risen to 57.5~58.5 DEG C, from B1
78%~82%, B2,18%~22%, are 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 88%~
92%, and eluted in this ratio；Column temperature is down to 41.5~42.5 DEG C during 35.8min；
Fourth stage is 42.5~48.0min, is eluted with buffer B 2, column temperature rises to 71.5~72.5 at the end of fourth stage
℃；
5th stage was 48.0~65.0min, from buffer solution 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, was eluted with buffer B 4；
8th stage was 84.5~89.0min, was eluted with buffer B 5.
3. method that is according to claim 1 while measuring active peptide and free amino acid, it is characterised in that the elution
Program includes：
First stage is 0~20.8min, is eluted with buffer B 1,38 DEG C of initial column temperature, and when 2.1min is adjusted to 35 DEG C,
Column temperature is risen to 56 DEG C at the end of first stage；
Second stage is 20.8~32.8min, is eluted with buffer B 1 and B2, column temperature is risen to 58 DEG C, from buffer solution B1
80%th, B2 20% is at the uniform velocity adjusted to B1 70%, B2 30%, and column temperature is adjusted to 56 DEG C at the end of second stage；
Phase III is 32.8~42.5min, and it is 10% to be adjusted to buffer B 1, and buffer B 2 is 90%, and presses this ratio
Example elution；Column temperature is adjusted to 42 DEG C during 35.8min；
Fourth stage is 42.5~48.0min, is eluted with buffer B 2, and column temperature is adjusted to 72 DEG C during 42.0min；
5th stage was 48.0~65.0min, was instantaneously adjusted to buffer B 3 70%, buffer B 4 30%, and at the uniform velocity adjust
It is whole be B3 be 60%, B4 40%；
6th stage was 65.0~67.5min, and it is 55% to be at the uniform velocity adjusted to buffer B 3, and buffer B 4 is 45%；
7th stage eluted for 67.5~84.5min buffer Bs 4；
8th stage was 84.5~89min, was eluted with buffer B 5.
4. measuring the method for active peptide and free amino acid at the same time according to claims 1 to 3 any one of them, its feature exists
In：
The buffer B 1 contains 5.5~6g/L citric acids, three lithium, 1~1.5g/L lithium chlorides, 19~21g/L citric acids, body
The ethanol of product 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 acids, three lithium, 6~7.5g/L lithium chlorides, 11.5~13g/L citric acids, volume
Measure 2.5%~4% ethanol and the octanoic acid of volume content 0.005%~0.015%；
Buffer B 3 contains containing 8.5~9.6g/L citric acids, three lithium, 25~28g/L lithium chlorides, 11~12g/L citric acids, volume
Measure 8%~12% ethanol, volume content 3.5%~5% 0.005%~0.0.15% of benzyl carbinol and volume content octanoic acid；
Buffer B 4 contains containing 9.5~11g/L citric acids, three lithium, 37.5~40g/L lithium chlorides, 3~4g/L citric acids and volume
The octanoic acid of amount 0.005%~0.015%；
Buffer B 5 containing 8~10g/L lithium hydroxides, 2.5%~4% ethanol of volume content and volume content 0.005%~
0.015% octanoic acid.
5. method that is according to claim 4 while measuring active peptide and free amino acid, it is characterised in that
The pH=2.8 of the buffer B 1, contains three lithium of 5.73g/L citric acids, 1.24g/L lithium chlorides, 19.9g/L lemons
Acid, 3.0% ethanol of volume content and the octanoic acid of volume content 0.01%；
The pH=3.7 of buffer B 2, contains containing three lithium of 9.8g/L citric acids, 6.36g/L lithium chlorides, 12.0g/L citric acids, volume
Measure 3% ethanol and the octanoic acid of volume content 0.01%；
The pH=3.6 of buffer B 3, contains three lithium of 8.79g/L citric acids, 26.62g/L lithium chlorides, 11.27g/L citric acids, body
Ethanol, the benzyl carbinol of volume content 0.4% and the octanoic acid of volume content 0.01% of product content 10%；
The pH=4.1 of buffer B 4, contains three lithium of 9.8g/L citric acids, 38.15g/L lithium chlorides, 3.3g/L citric acids and volume
The octanoic acid of content 0.01%；
Buffer B 5 contains 8.4g/L lithium hydroxides, 3% ethanol of volume content and the octanoic acid of volume content 0.01%.
6. the method for measuring active peptide and free amino acid at the same time such as claims 1 to 3 any one of them, it is characterised in that
Developed the color using ninhydrin derivative reagent；Developing wavelength is respectively 440nm and 570nm.
7. the method as claimed in claim 6 for measuring active peptide and free amino acid at the same time, it is characterised in that the ninhydrin
Derivative reagent includes ninhydrin reaction liquid, reaction buffer and cleaning solution；
Ninhydrin reaction liquid is ninhydrin and the solution of sodium borohydride, and solvent is selected from ethylene glycol single methyl ether or propylene glycol monomethyl
Ether, ninhydrin concentration are 38~40g/L, and sodium borohydride concentration is 80~82mg/L；
Reaction buffer is the mixed solution of sodium acetate, glacial acetic acid, water and organic solvent；The organic solvent is selected from ethylene glycol list
Methyl ether or propylene glycol monomethyl ether；The volume ratio of the organic solvent and glacial acetic acid, water is 1：0.25~0.35：0.8~
0.85；The mass ratio of the sodium acetate and ultra-pure water is 1：1.6~1.7；
Cleaning solution is ethanol water；The volume ratio of the ethanol and water is 1：18.5~19.5.
8. method that is according to claim 1 while measuring active peptide and free amino acid, it is characterised in that described to be measured
The preparation method of sample solution comprises the following steps：
A. sample is mixed with hydrochloric acid solution, antioxidant, is ultrasonically treated after homogeneous, separation of solid and liquid；Antioxidant and aquatic products to be measured
The mass ratio of product sample is 1：50~500；The 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. hydrochloric acid solution constant volume is used after the supernatant of combining step a, b, then adds sulfosalicylic acid, centrifugation, filtering, take filter
Liquid；
Concentration of hydrochloric acid solution used in above steps is 0.005~0.1mol/L.
9. method that is according to claim 1 while measuring active peptide and free amino acid, it is characterised in that described to be measured
The preparation method of sample solution comprises the following steps：
A. by sample to be tested and mixed in hydrochloric acid, dithiothreitol (DTT) is added, 3~20min is ultrasonically treated after homogeneous, 0~20 DEG C centrifuges,
Take supernatant；The mass ratio of dithiothreitol (DTT) and aquatic products sample to be measured is 1：100~400；The dosage of sample to be tested and hydrochloric acid
Than for 1g：5~10mL；
B. the solid for taking step a to centrifuge, with stirring, centrifuging and taking supernatant after mixed in hydrochloric acid, repeats 1~5 time；Sample to be tested
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 adds 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 in above steps is 0.01~0.05mol/L.
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| CN107632094A (en) * | 2017-09-18 | 2018-01-26 | 成都中医药大学 | A kind of method for determining free amino acid in the coptis |
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