CN110045046A - A kind of preprocess method and Contents of Amino Acids method of fresh food materials - Google Patents
A kind of preprocess method and Contents of Amino Acids method of fresh food materials Download PDFInfo
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- CN110045046A CN110045046A CN201910243746.8A CN201910243746A CN110045046A CN 110045046 A CN110045046 A CN 110045046A CN 201910243746 A CN201910243746 A CN 201910243746A CN 110045046 A CN110045046 A CN 110045046A
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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
- G01N30/62—Detectors specially adapted therefor
- G01N30/74—Optical detectors
Abstract
The invention discloses a kind of preprocess method of fresh food materials and Contents of Amino Acids method, the preprocess methods of fresh food materials are as follows: carries out sour water solution to fresh food materials using micro-wave digestion, comprising the following steps: (a) sample extraction；(b) microwave hydrolysis；(c) bigness scale hydrolyzate concentration；(d) measurement liquid preparation.The invention also discloses a kind of fresh food materials Contents of Amino Acids method, this method is derived from the measurement liquid of preprocess method preparation, measures amino acid content using full-automatic amino-acid analyzer, includes the following steps, machine liquid configures in (1) standard；(2) machine liquid measures in standard；(3) in fresh food materials amino acid content measurement.Preprocessing process of the present invention is simple, sample hydrolysis rate is fast, has many advantages, such as safe operation, pollution-free.
Description
Technical field
The present invention relates to fresh food materials preconditioning technique field more particularly to a kind of preprocess methods and ammonia of fresh food materials
Base acid content measuring method.
Background technique
Amino acid is the compound after the hydrogen atom on carboxylic acid carbon atom is replaced by amino, contains amino in amino acid molecular
With carboxyl Liang Zhong functional group.It is similar with carboxylic acid, different location that amino acid can be connected in carbochain according to amino and be divided into α~, β
~, γ~... w~amino acid, but the amino acid obtained after protein hydrolyzes is all α~amino acid, and only twenties
Kind, they are the basic units for constituting protein, are the base substances of protein needed for constituting Animal nutrition.
Amino acid score is essential amino acid and nonessential amino acid.Essential amino acid refers to human body itself (or other vertebras
Animal) it cannot synthesize or aggregate velocity is not able to satisfy human body needs, it is necessary to the amino acid absorbed from food.It is human body
(or other vertebrates) is essential, and what body cannot synthesize, it is necessary to the amino acid supplemented from food, it is referred to as required
Amino acid.Nonessential amino acid can synthesize in animal body, not need the amino acid from external complement as nutrient source.
Existing amino acid sample hydrolysis is in hydrolysis 10~15ml of Guan Zhongjia 6mol/L hydrochloric acid solution, and phenol 3~4 drips,
Water adapter tube is put into refrigerant, 3~5min is freezed, is connected on the exhaust tube of vacuum pump, vacuumizes (close to 0Pa), then fill
Enter nitrogen, after repetition vacuumizes~be filled with nitrogen 3 times, in nitrogen charging gaseity lower sealing or the tight screw cap of lemon, the water that will have been sealed
Solution pipe is placed in 110 DEG C ± 1 DEG C of electric heating air blast insulating box or hydrolysis furnace, after hydrolyzing 22h, is taken out, is cooled to room temperature.Step phase
To cumbersome complexity, the pretreatment process time is longer, causes the minute of amino acid longer, increases testing cost, lowers inspection
Survey efficiency.
Therefore, the preprocess method and Contents of Amino Acids method that inventor is dedicated to designing a kind of fresh food materials are to solve
The certainly above problem.
Summary of the invention
In order to overcome shortcoming and defect existing in the prior art, it is an object of the invention to: a kind of fresh food materials are provided
Preprocess method, fast hydrolyzing sample, prepare sample measurement liquid, shorten pretreatment time, reduce pretreatment cost.
It is another object of the present invention to: a kind of Contents of Amino Acids method of fresh food materials is provided, quickly, accurately
And efficiently measure amino acid content.
The purpose of the invention is achieved by the following technical solution:
A kind of preprocess method of fresh food materials, comprising the following steps:
(a) sample extraction: weighing 0.1~2.0g sample, be placed in micro-wave diminishing pot, and 5~15ml volume fraction is added and is
50% aqueous hydrochloric acid solution adds 30~50mg phenol, is uniformly mixed；
In the step (a), the effect that phenol is added is: phenol is reacted with the dissolved oxygen in solution generates quinone, thus
Protect the amino acid of hydrolysis.
(b) microwave hydrolysis: micro-wave digestion is arranged in carrying out microwave hydrolysis on microwave dissolver in installation micro-wave diminishing pot rapidly
Instrument keeps reaction in 50 DEG C~70 DEG C temperature, prevents solution bumping, then rises to again in 140 DEG C~160 DEG C of hydrolysis temperature
Hydrolysis is kept, after the reaction was completed, stops heating, when temperature display to room temperature, takes out micro-wave diminishing pot, water is made in hydrolysis
Solve liquid；
Microwave dissolver principle: under the action of microwave electric field, it is positive and negative that its is changed constantly with 24.5 hundred million rates per second
Direction, make molecule generate high speed collision and friction and generate high fever, while under the action of microwave electric field, in solution system
Ion directed flow, forms ionic current, and ion occurs high-speed friction with the molecule of surrounding and ion in flow process and touches
It hits, microwave energy is made to switch to thermal energy.
The present invention utilizes the microwave heating advantage and characteristic of microwave dissolver, and salt is added in sample wait hydrolyze into counteracting tank
Acid forms strong acid solution, heats property using microwave body, solution is inside and outside to be heated simultaneously, and heating more rapidly, more evenly, improves
Efficiency, meanwhile, it is carried out in the micro-wave diminishing pot of enclosed high pressure, pressure system can generate superheating phenomenon and (be heated to than under normal pressure
The higher temperature of boiling point), greatly improve hydrolysis rate.
(c) bigness scale hydrolyzate concentration: hydrolyzate is transferred in 50ml colorimetric cylinder, the hydrochloric acid water for being 50% with volume fraction
Solution is settled to scale, and prepare liquid is made, and prepare liquid and titer are carried out colorimetric, measure prepare liquid concentration roughly；
(d) measurement liquid preparation: 1~2ml prepare liquid is accurately pipetted in 15ml test tube, nitrogen is blown under 40~50 DEG C of water-baths
It is dry, residue is formed, 1~3ml deionized water is added, dissolution residual substance, nitrogen dries up under water-bath again, repeats that deionization is added
Water and the drying of water-bath nitrogen, are added 1~2ml pH2.2 sodium citrate buffer, and test tube is placed in ultrasonic wave dissolution instrument, is surpassed
Sound wave dissolution process is filtered with the polyethersulfone membranes that aperture is 0.22 μm, and sample measurement liquid is made.
In above-mentioned steps (d), the polyethersulfone membranes that aperture is 0.22 μm are water system filter membrane, since the measurement liquid main body is
Water, therefore water system filter membrane is selected, the main function of filter membrane is the solid impurity removed in measurement liquid, prevents impurity from blocking chromatography
Column.
Ultrasonic wave solution principle: mainly caused by " cavitation effect ", cavitation effect makes solution generate localized hyperthermia and height
It presses, after cavitation effect bubbles burst formed in solution, forms powerful shock wave, accelerate solution turbulent flow, meanwhile, ultrasound
Wave causes the mechanical oscillation of solvent, can accelerate hot transmitting, be heated evenly solvent, be heated evenly solute integrally, adds simultaneously
Fast Dispersion of Solute Matter, also accelerates dissolution.
Further, the step (b) is specifically, installation micro-wave diminishing pot is in carrying out Microwave Water rapidly on microwave dissolver
Solution, microwave dissolver are set in 4~6min and gradually rise from room temperature to 50 DEG C~70 DEG C, keep 50 DEG C~70 DEG C continuous heatings
3~10min prevents bumping, adjusts microwave dissolver temperature, its temperature is made to rise to 140 DEG C from 50 DEG C~70 DEG C in 3~5min
~160 DEG C, 140 DEG C~160 DEG C standing waves is kept to heat 15~25min, stops heating, when temperature display to room temperature, take
Micro-wave diminishing pot out.
Further, in the step (b), microwave hydrolysis power is that 800~1200W makes simultaneously to improve hydrolysis efficiency
Hydrolytic process more energy-saving and environmental protection, which chooses according to the sample size of processing, when microwave dissolver is transported at full capacity
When row, microwave hydrolysis power selection 1200W, when sample size is lower than 1/3, microwave hydrolysis power selection 800W.
Further, in the step (c), method that hydrolyzate is transferred to 50ml colorimetric cylinder are as follows: will be in micro-wave diminishing pot
Hydrolyzate pours into 50ml colorimetric cylinder, at least washs microwave in three times with the aqueous hydrochloric acid solution that 15~25ml volume fraction is 50%
Counteracting tank makes the residual sample in micro-wave diminishing pot be hydrolyzed into amino acid rapidly under acidic environment, by washing for micro-wave diminishing pot
It washs liquid to pour into 50ml colorimetric cylinder, be uniformly mixed.
Further, in the step (d), the ultrasonic intensity that ultrasonic wave dissolves instrument is 0.3~1W/cm2, ultrasonic wave dissolution
Temperature is 20~60 DEG C, and sonication treatment time is 3~10min.
Another object of the present invention is achieved through the following technical solutions: a kind of Contents of Amino Acids side of fresh food materials
Method, which, which is derived from the measurement liquid of sample made from above-mentioned fresh food materials preprocess method and measures the amino acid of fresh food materials, contains
Amount, the Contents of Amino Acids method of the fresh food materials include the following steps:
(1) machine liquid configures in standard: taking the kilnitamin standard reserving solution 0.5ml of 2.5 μm of ol/ml in 5mL volumetric flask
In, it is settled to scale with the hydrochloric acid solution of 0.1mol/L, is uniformly mixed, concentration is made as machine liquid in 100nmol/ml standard or divides
Be not made concentration be 5,25,100,250, machine in the standard of 1000nmol/ml (as 0.1,0.5,2,5,20nmol/20 μ L)
Liquid；
(2) machine liquid measures in standard: drawing machine liquid in the standard that concentration is 100nmol/ml and injects full-automatic Amino acid score
Analyzer measures peak area using external standard method single point correction, or the full-automatic ammonia of machine liquid injection in the standard of absorption various concentration respectively
Base acid analysis instrument, using external standard method peak area and makes standard curve；
(3) in fresh food materials amino acid content measurement: take sample made from the preprocess method to measure liquid, injection is complete
Automatic amino acid analyser calculates the content of amino acid in sample using quantified by external standard method.
Further, in the step (2) and (3), the chromatographic condition of full-automatic amino-acid analyzer is that chromatographic column fills material
Material is sulfonic acid cation resin, and column temperature is 45~65 DEG C, 15~25uL of sampling volume, and Detection wavelength is 440nm and 570nm.
Further, in the step (2) and (3), full-automatic amino-acid analyzer uses gradient elution, and derivatization occurs
Reaction, elution flow rate are 0.3~0.5ml/min, and gradient elution program is as follows:
, wherein B1 be 5.8~6.5g sodium citrate, 0.22~0.25g sodium hydroxide, 5.4~5.9g sodium chloride, 19~
21g citric acid, 128~142ml ethyl alcohol and 3.5~4.5ml polyoxyethylene laurel ether are uniformly mixed and are settled to 1000mL with water
The mixed solution being prepared, B2 are 7.0~8.0g sodium citrate, 0.7~0.9g sodium hydroxide, 6.7~7.4g sodium chloride, 21
~23g citric acid, 23~26ml ethyl alcohol and 3.8~4.2ml polyoxyethylene laurel ether are uniformly mixed and are settled to 1000mL with water
The mixed solution being prepared, B3 be 12~14g sodium citrate, 3.5~4.0g sodium chloride, 12~13g citric acid, 8.5~
9.5ml ethyl alcohol and 3.5~4.5ml polyoxyethylene laurel ether are uniformly mixed and to be settled to the mixing that 1000mL is prepared with water molten
Liquid, B4 be 25~28g sodium citrate, 52~57g sodium chloride, 5~6.5g citric acid, 4.5~5.5ml benzyl alcohol and 3.5~
4.5ml polyoxyethylene laurel ether is uniformly mixed and is settled to the mixed solution that 1000mL is prepared with water, and B5 is volume fraction
For 5% ethanol water, B6 is 7.5~8.5g sodium hydroxide, 95~105ml ethyl alcohol and 3.5~4.5ml polyoxyethylene laural
Ether is uniformly mixed and is settled to the mixed solution that 1000mL is prepared with water.
Preferably, B1 is 6.19g sodium citrate, 0.24g sodium hydroxide, 5.66g sodium chloride, 19.8g citric acid, 135ml
Ethyl alcohol and 4ml polyoxyethylene laurel ether are uniformly mixed and are settled to the mixed solution that 1000mL is prepared, B2 7.74g with water
Sodium citrate, 0.8g sodium hydroxide, 7.07g sodium chloride, 22g citric acid, 25ml ethyl alcohol and the mixing of 4ml polyoxyethylene laurel ether are equal
Even and be settled to the mixed solution that 1000mL is prepared with water, B3 is 13.3g sodium citrate, 3.74g sodium chloride, 12.8g lemon
Lemon acid, 9ml ethyl alcohol and 4ml polyoxyethylene laurel ether are uniformly mixed and are settled to the mixed solution that 1000mL is prepared with water,
B4 is that 26.67g sodium citrate, 54.35g sodium chloride, 6.1g citric acid, 5ml benzyl alcohol and the mixing of 4ml polyoxyethylene laurel ether are equal
Even and be settled to the mixed solution that 1000mL is prepared with water, B5 is the ethanol water that volume fraction is 5%, and B6 is 8g hydrogen
Sodium oxide molybdena, 100ml ethyl alcohol and 4ml polyoxyethylene laurel ether are uniformly mixed and to be settled to the mixing that 1000mL is prepared with water molten
Liquid.
Further, in the derivative reaction, derivative temperature is 130~140 DEG C, and derivative flow velocity is 0.25~0.45ml/
Min, derivative flow velocity are less than elution flow rate, derivative program are as follows:
, wherein R1 is 37~41g ninhydrin, 77~85mg sodium borohydride and 930~1028ml ethylene glycol single methyl ether
Mixture, R2 are 194~214g anhydrous sodium acetate, 117~129ml glacial acetic acid and the mixing of 381~421ml ethylene glycol single methyl ether
It mixes and is settled to the mixed solution that 1000ml is prepared with water, R3 is that volume fraction is 5% ethanol water.
Preferably, R1 is the mixture of 39g ninhydrin, 81mg sodium borohydride and 979ml ethylene glycol single methyl ether, and R2 is
204g anhydrous sodium acetate, 123ml glacial acetic acid and the mixing of 401ml ethylene glycol single methyl ether mix and are settled to 1000ml preparation with water
Made of mixed solution, R3 is that volume fraction is 5% ethanol water.
Further, in the step (3), the amino acid peak sequence in sample are as follows: asparatate, threonine, silk ammonia
Acid, glutamic acid, proline, glycine, alanine, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine,
Lysine, histidine, arginine.
The present invention is combined due to using above technical scheme, by microwave hydrolysis with derivative, has studied sample Microwave Water
The derivatization reaction of various amino acid after solution and hydrolysis, has investigated experiment condition to microwave hydrolysis and amino acid derived influence,
Sample pretreatment and analysis of amino acids time are shortened, traditional sample pretreatment time is 22 hours, and not only step is numerous
It is trivial, and take a long time, amino acid is easy to run off in treatment process, and pretreatment time of the invention is 40 minutes, is greatly shortened pre-
Handle the time, using microwave hydrolysis sample, water, sour isopolarity substance under the action of microwave field, molecule generate high velocity impact and
Friction generates high fever, accelerates Specimen eliminating.
The present invention is made sample using microwave hydrolysis preprocess method and measures liquid, the amino acid sample solution after hydrolysis, warp
Sample injector injects full-automatic amino-acid analyzer, and mobile phase brings system by infusion pump, the amino acid composition in sample solution
Because the distribution coefficient in two-phase is different, by the assigning process of Adsorption and desorption repeatedly, thus after separating on a column
Chromatographic column is successively flowed out, into detector, sample concentration is converted into electric signal, is recorded on signal collector, and data are final
It is showed in the form of map.
The preprocess method of the fresh food materials of the present invention, advantageous effects are: using microwave dissolver to sample into
Row hydrolysis pretreatment substitutes conventional hydrolysis with microwave hydrolysis, and realization is easy to operate, and hydrolysis rate is fast, pollutes small pretreatment side
Method, effectively shortens pretreatment time.
A kind of Contents of Amino Acids method of fresh food materials of the present invention, is hydrolyzed sample using microwave dissolver pre-
Processing carries out amino acid quantitative determination to hydrolyzation sample using full-automatic amino-acid analyzer, and advantageous effects are:
(1) conventional hydrolysis is substituted with microwave hydrolysis, realization is easy to operate, and hydrolysis rate is fast, pollutes small preprocess method；
(2) amino acid quantitative analysis is carried out using the small full-automatic amino-acid analyzer of high sensitivity, interference, improves analysis
Speed and measurement accuracy reduce analysis cost；
(3) entire analytic process is simple, quick, accurate, is widely used in the survey of amino acid content in various fresh food materials
It is fixed, the quality good or not of the various fresh food materials of accurate evaluation.
Detailed description of the invention
Fig. 1 a is in single point correction test of the present invention, with the Detection wavelength test concentrations of 570nm for 100nmol/ml standard
Chromatogram when upper machine liquid；
Fig. 1 b is in single point correction test of the present invention, with the Detection wavelength test concentrations of 440nm for 100nmol/ml standard
Chromatogram when upper machine liquid
Fig. 2 a is in amino acid standard curve making of the present invention, is 5nmol/ml with the Detection wavelength test concentrations of 570nm
Chromatogram in standard when machine liquid；
Fig. 2 b is in amino acid standard curve making of the present invention, is 5nmol/ml with the Detection wavelength test concentrations of 440nm
Chromatogram in standard when machine liquid；
Fig. 3 a is in amino acid standard curve making of the present invention, is 25nmol/ml with the Detection wavelength test concentrations of 570nm
Chromatogram in standard when machine liquid；
Fig. 3 b is in amino acid standard curve making of the present invention, is 25nmol/ml with the Detection wavelength test concentrations of 440nm
Chromatogram in standard when machine liquid；
Fig. 4 a is in amino acid standard curve making of the present invention, is 100nmol/ with the Detection wavelength test concentrations of 570nm
Chromatogram in ml standard when machine liquid；
Fig. 4 b is in amino acid standard curve making of the present invention, is 100nmol/ with the Detection wavelength test concentrations of 440nm
Chromatogram in ml standard when machine liquid；
Fig. 5 a is in amino acid standard curve making of the present invention, is 250nmol/ with the Detection wavelength test concentrations of 570nm
Chromatogram in ml standard when machine liquid；
Fig. 5 b is in amino acid standard curve making of the present invention, is 250nmol/ with the Detection wavelength test concentrations of 440nm
Chromatogram in ml standard when machine liquid；
Fig. 6 a is in amino acid standard curve making of the present invention, is 1000nmol/ with the Detection wavelength test concentrations of 570nm
Chromatogram in ml standard when machine liquid；
Fig. 6 b is in amino acid standard curve making of the present invention, is 1000nmol/ with the Detection wavelength test concentrations of 440nm
Chromatogram in ml standard when machine liquid；
Fig. 7 a is in the present invention, and when sample is chilled beef, Detection wavelength is 570nm, sample measures liquid chromatography figure；
Fig. 7 b is in the present invention, and when sample is chilled beef, Detection wavelength is 440nm, sample measures liquid chromatography figure；
Fig. 8 a is in the present invention, and when sample is more precious fishes, Detection wavelength is 570nm, sample measures liquid chromatography figure；
Fig. 8 b is in the present invention, and when sample is more precious fishes, Detection wavelength is 440nm, sample measures liquid chromatography figure；
Fig. 9 a is in the present invention, and when sample is egg, Detection wavelength is 570nm, sample measures liquid chromatography figure；
Fig. 9 b is in the present invention, and when sample is egg, Detection wavelength is 440nm, sample measures liquid chromatography figure.
Specific embodiment
Below with reference to examples and drawings 1~9, embodiments of the present invention are specifically illustrated, attached drawing is only for reference and explanation
It uses, does not constitute the limitation to the invention patent protection scope.
Embodiment 1
The sample of the present embodiment is derived from the chilled beef in fresh meat food materials.
A kind of preprocess method of fresh food materials, comprising the following steps:
(a) sample extraction: the chilled beef sample after weighing 0.1g grinding is placed in micro-wave diminishing pot, 5ml volume point is added
Number is 50% aqueous hydrochloric acid solution, adds 30mg phenol protective agent, is uniformly mixed；
(b) microwave hydrolysis: installation micro-wave diminishing pot sets microwave hydrolysis in carrying out microwave hydrolysis on microwave dissolver rapidly
Power is 800W, sets microwave dissolver microwave heating program, gradually rises from room temperature to 50 DEG C in 4min, is kept for 50 DEG C continue
10min is heated, microwave dissolver temperature is adjusted, its temperature is made to rise to 140 DEG C from 50 DEG C in 3min, 140 DEG C of holding lasting micro-
Wave hydrolysis heating 25min, stops heating, when temperature display to room temperature, takes out micro-wave diminishing pot, hydrolyzate is made in hydrolysis；
(c) bigness scale hydrolyzate concentration: the hydrolyzate in micro-wave diminishing pot is poured into 50ml colorimetric cylinder, with 15ml volume point
Number at least washs micro-wave diminishing pot for 50% aqueous hydrochloric acid solution in three times, and all cleaning solutions of micro-wave diminishing pot are poured into 50ml
It in colorimetric cylinder, is transferred to all hydrolyzates in 50ml colorimetric cylinder, is uniformly mixed, it is fixed for 50% aqueous hydrochloric acid solution with volume fraction
Hold to scale, prepare liquid is made, prepare liquid and titer are subjected to colorimetric, measure prepare liquid concentration roughly；
(d) measurement liquid preparation: 1ml prepare liquid is accurately pipetted in 15ml test tube, nitrogen dries up under 40 DEG C of water-baths, is formed residual
Object is stayed, 1ml deionized water is added, dissolution residual substance, nitrogen dries up under water-bath again, repeats that deionized water is added and water-bath nitrogen is blown
It is dry, 1ml pH2.2 sodium citrate buffer is added, test tube is placed in ultrasonic wave dissolution instrument, setting ultrasonic wave dissolves instrument
Ultrasonic intensity be 0.3W/cm2, solution temperature is 20 DEG C, is 0.22 μm with aperture after ultrasonic wave dissolution process 10min
Sample measurement liquid is made in polyethersulfone membranes filtering.
A kind of Contents of Amino Acids method of fresh food materials, takes the preprocess method of above-mentioned fresh food materials in the present embodiment
Sample measurement liquid obtained measures the amino acid content of fresh food materials, comprising the following steps:
(1) machine liquid configures in standard:
Take the kilnitamin standard reserving solution 0.5ml of 2.5 μm of ol/ml in 5mL volumetric flask, with the hydrochloric acid of 0.1mol/L
Solution is settled to scale, is uniformly mixed, and it is machine liquid in 100nmol/ml standard that concentration, which is made,；
(2) machine liquid measures in standard: drawing machine liquid in the standard that concentration is 100nmol/ml and injects full-automatic Amino acid score
Analyzer, using external standard method single point correction peak area, chromatogram such as Fig. 1 a and 1b, corresponding peak area result is as shown in table 1 below:
Table 1
(3) in fresh food materials amino acid content measurement: take chilled beef sample made from above-mentioned preprocess method measure liquid,
Inject full-automatic amino-acid analyzer, various amino acid peak sequences in chilled beef are as follows: asparatate, threonine, serine,
Glutamic acid, glycine, alanine, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine, relies proline
Propylhomoserin, histidine, arginine, chilled beef sample measure liquid chromatography figure as illustrated in figs. 7 a and 7b, and chilled beef sample measures liquid peak face
Product is as shown in table 2 below,
Table 2
The concentration C i (nmol/20 μ L) that sample measures amino acid in liquid is calculated by following formula,
Ci=(Cs/AS) Ai,
Wherein,
Ci: sample measures the content of liquid amino acid i, and unit is every 20 microlitres of nanomole (nmol/20 μ L)；
Ai: the peak area of sample measurement liquid amino acid i；
As: the peak area of amino acid standard working solution amino acid s；
Cs: the content of amino acid standard working solution amino acid s, unit are every 20 microlitres of nanomole (nmol/20 μ L)；
The content Xi (g/100g) of each amino acid in sample is calculated by following formula,
(/ (m × 10 (Ci × F × V × M) Xi=9)) × 5000,
In formula:
Xi: the content of amino acid i in sample, unit are gram every hectogram (g/100g)
Ci: the content of amino acid i in Specimen Determination liquid, unit are every 20 milliliters of nanomole (nmol/20 μ L)
F: extension rate；
V: sample hydrolyzate shifts the volume of constant volume, and unit is milliliter (mL)；
M: the molal weight of amino acid i, unit are gram every mole (g/mol), and the title and molal weight of each amino acid are shown in
Table 2；
M: sample weighting amount, unit are gram (g)；
109: sample content is converted to the coefficient of gram (g) by nanogram (ng)；
5000: conversion coefficient.
In the step (2) and (3), the chromatographic condition of full-automatic amino-acid analyzer are as follows: chromatography column material is sulphur
Acid type resin cation, column temperature are 45 DEG C, sampling volume 15uL；
In the step (2) and (3), full-automatic amino acid analysis use gradient elution, elution flow rate 0.3ml/min,
And derivative reaction occurs, derivative temperature is 130 DEG C, and derivative flow velocity is 0.25ml/min, and derivative flow velocity is less than elution flow rate；
The gradient elution program of the present embodiment is as shown in table 3 below:
Table 3
, wherein B1 be 5.8g sodium citrate, 0.22g sodium hydroxide, 5.4g sodium chloride, 19g citric acid, 128ml ethyl alcohol and
3.5ml polyoxyethylene laurel ether is uniformly mixed and is settled to the mixed solution that 1000mL is prepared with water, and B2 is 7.0g lemon
Sour sodium, 0.7g sodium hydroxide, 6.7g sodium chloride, 21g citric acid, 23ml ethyl alcohol and 3.8ml polyoxyethylene laurel ether are uniformly mixed
And be settled to the mixed solution that 1000mL is prepared with water, B3 be 12g sodium citrate, 3.5g sodium chloride, 12g citric acid,
8.5ml ethyl alcohol and 3.5ml polyoxyethylene laurel ether are uniformly mixed and are settled to the mixed solution that 1000mL is prepared, B4 with water
It is uniformly mixed and is used in combination for 25g sodium citrate, 52g sodium chloride, 5g citric acid, 4.5ml benzyl alcohol and 3.5ml polyoxyethylene laurel ether
Water is settled to the mixed solution that 1000mL is prepared, and B5 is the ethanol water that volume fraction is 5%, and B6 is 7.5g hydroxide
Sodium, 95ml ethyl alcohol and 3.5ml polyoxyethylene laurel ether are uniformly mixed and are settled to the mixed solution that 1000mL is prepared with water.
In the present embodiment derivative reaction, derivative program is as shown in table 4 below:
Table 4
| Time (min) | R1 volume fraction (%) | R2 volume fraction (%) | R3 volume fraction (%) |
| 0.0~32.0 | 50 | 50 | 0 |
| 32.1~37.0 | 0 | 0 | 100 |
| 37.1~53.0 | 50 | 50 | 0 |
, wherein R1 is the mixture of 37g ninhydrin, 77mg sodium borohydride and 930ml ethylene glycol single methyl ether, and R2 is
194g anhydrous sodium acetate, 117ml glacial acetic acid and the mixing of 381ml ethylene glycol single methyl ether mix and are settled to 1000ml preparation with water
Made of mixed solution, R3 is that volume fraction is 5% ethanol water.
Embodiment 2
The sample of the present embodiment is derived from more precious fishes in fresh aquatic product food materials.
A kind of preprocess method of fresh food materials, comprising the following steps:
(a) sample extraction: more precious fish samples after weighing 1g grinding are placed in micro-wave diminishing pot, 10ml volume point are added
Number is 50% aqueous hydrochloric acid solution, adds 40mg phenol protective agent, is uniformly mixed；
(b) microwave hydrolysis: installation micro-wave diminishing pot sets microwave hydrolysis in carrying out microwave hydrolysis on microwave dissolver rapidly
Power is 1000W, sets microwave dissolver microwave heating program, gradually rises from room temperature to 60 DEG C in 5min, is kept for 60 DEG C and held
Continuous heating 6min, adjusts microwave dissolver temperature, its temperature is made to rise to 150 DEG C from 60 DEG C in 4min, and 150 DEG C of holding lasting micro-
Wave hydrolysis heating 20min, stops heating, when temperature display to room temperature, takes out micro-wave diminishing pot, hydrolyzate is made in hydrolysis；
(c) bigness scale hydrolyzate concentration: the hydrolyzate in micro-wave diminishing pot is poured into 50ml colorimetric cylinder, with 20ml volume point
Number at least washs micro-wave diminishing pot for 50% aqueous hydrochloric acid solution in three times, and all cleaning solutions of micro-wave diminishing pot are poured into 50ml
It in colorimetric cylinder, is transferred to all hydrolyzates in 50ml colorimetric cylinder, is uniformly mixed, it is fixed for 50% aqueous hydrochloric acid solution with volume fraction
Hold to scale, prepare liquid is made, prepare liquid and titer are subjected to colorimetric, measure prepare liquid concentration roughly；
(d) measurement liquid preparation: 1.5ml prepare liquid is accurately pipetted in 15ml test tube, nitrogen dries up under 45 DEG C of water-baths, is formed
1ml deionized water is added in residue, and dissolution residual substance, nitrogen dries up under water-bath again, repeats that deionized water and water-bath nitrogen is added
1ml pH2.2 sodium citrate buffer is added in drying, test tube is placed in ultrasonic wave dissolution instrument, setting ultrasonic wave dissolves
The ultrasonic intensity of instrument is 0.6W/cm2, solution temperature is 40 DEG C, is 0.22 μm with aperture after ultrasonic wave dissolution process 6min
Sample measurement liquid is made in polyethersulfone membranes filtering.
A kind of Contents of Amino Acids method of fresh food materials, takes the preprocess method of above-mentioned fresh food materials in the present embodiment
Sample measurement liquid obtained measures the amino acid content of fresh food materials, comprising the following steps:
(1) machine liquid configures in standard:
Take the kilnitamin standard reserving solution 0.5ml of 2.5 μm of ol/ml in 5mL volumetric flask, with the hydrochloric acid of 0.1mol/L
Solution is settled to scale, is uniformly mixed, be respectively prepared concentration be 5,25,100,250, machine liquid in the standard of 1000nmol/ml；
(2) machine liquid measures in standard: drawing machine liquid in the standard of above-mentioned various concentration respectively and injects full-automatic Amino acid score
Analyzer, using external standard method, using the peak area of each amino acid as ordinate (y), using the concentration of each amino acid as abscissa (x,
Nmol/20 μ L)), make standard curve；
When machine liquid is 5nmol/ml (i.e. the every 20 μ L of 0.1nmol) in standard, machine liquid chromatography figure is as schemed in corresponding standard
Shown in 2a and 2b, corresponding peak area result is as shown in table 5 below:
Table 5
When machine liquid is 25nmol/ml (i.e. the every 20 μ L of 0.5nmol) in standard, machine liquid chromatography figure is as schemed in corresponding standard
Shown in 3a and 3b, corresponding peak area result is as shown in table 6 below:
Table 6
When machine liquid is 100nmol/ml (i.e. the every 20 μ L of 2nmol) in standard, machine liquid chromatography figure is as schemed in corresponding standard
Shown in 4a and 4b, corresponding peak area result is as shown in table 7 below:
Table 7
When machine liquid is 250nmol/ml (i.e. the every 20 μ L of 5nmol) in standard, machine liquid chromatography figure is as schemed in corresponding standard
Shown in 5a and 5b, corresponding peak area result is as shown in table 8 below:
Table 8
Machine liquid chromatography figure is such as when machine liquid is 1000nmol/ml (i.e. the every 20 μ L of 20nmol) in standard, in corresponding standard
Shown in Fig. 6 a and 6b, corresponding peak area result is as shown in table 9 below:
Table 9
(3) in fresh food materials amino acid content measurement: take sample made from above-mentioned preprocess method measure liquid, dilution 10
Times, so that final sample is measured liquid in standard curve range, injects full-automatic amino-acid analyzer, various amino in more treasured fishes
Sour peak sequence are as follows: asparatate, threonine, serine, glutamic acid, proline, glycine, alanine, valine, egg ammonia
Acid, isoleucine, leucine, tyrosine, phenylalanine, lysine, histidine, arginine, more treasured fish samples measure liquid chromatography
As shown in figs. 8 a and 8b, more treasured fish sample measurement liquid peak areas are as shown in the following table 10 for figure:
Table 10
External standard method measures the concentration C i of amino acid in liquid by calculated by peak area sample,
The content Xi (g/100g) of each amino acid in more precious fish samples is calculated by following formula,
(/ (m × 10 (Ci × F × V × M) Xi=9)) × 5000,
In formula:
Xi: the content of amino acid i in sample, unit are gram every hectogram (g/100g)
Ci: the content of amino acid i in Specimen Determination liquid, unit are every 20 milliliters of nanomole (nmol/20 μ L)
F: extension rate；
V: sample hydrolyzate shifts the volume of constant volume, and unit is milliliter (mL)；
M: the molal weight of amino acid i, unit are gram every mole (g/mol), and the title and molal weight of each amino acid are shown in
Table 2；
M: sample weighting amount, unit are gram (g)；
109: sample content is converted to the coefficient of gram (g) by nanogram (ng)；
5000: conversion coefficient.
In the step (2) and (3), the chromatographic condition of full-automatic amino-acid analyzer are as follows: chromatography column material is sulphur
Acid type resin cation, column temperature are 57 DEG C, sampling volume 20uL.
In the step (2) and (3), full-automatic amino-acid analyzer uses gradient elution, elution flow rate 0.4ml/
Min, and derivative reaction occurs, derivative temperature is 135 DEG C, and derivative flow velocity is 0.35ml/min, and derivative flow velocity is less than elution stream
Speed.
In the present embodiment, elution program is as shown in the table 3 in embodiment 1, wherein B1 is 6.19g sodium citrate, 0.24g
Sodium hydroxide, 5.66g sodium chloride, 19.8g citric acid, 135ml ethyl alcohol and 4ml polyoxyethylene laurel ether are uniformly mixed and are determined with water
Hold the mixed solution being prepared to 1000mL, B2 is 7.74g sodium citrate, 0.8g sodium hydroxide, 7.07g sodium chloride, 22g lemon
Lemon acid, 25ml ethyl alcohol and 4ml polyoxyethylene laurel ether are uniformly mixed and are settled to the mixed solution that 1000mL is prepared with water,
B3 is that 13.3g sodium citrate, 3.74g sodium chloride, 12.8g citric acid, 9ml ethyl alcohol and 4ml polyoxyethylene laurel ether are uniformly mixed
And it is settled to the mixed solution that 1000mL is prepared with water, B4 is 26.67g sodium citrate, 54.35g sodium chloride, 6.1g lemon
Acid, 5ml benzyl alcohol and 4ml polyoxyethylene laurel ether are uniformly mixed and are settled to the mixed solution that 1000mL is prepared with water,
B5 is the ethanol water that volume fraction is 5%, and B6 is 8g sodium hydroxide, 100ml ethyl alcohol and the mixing of 4ml polyoxyethylene laurel ether
Uniformly and the mixed solution that 1000mL is prepared is settled to water.
In the present embodiment, derivative program is as shown in the table 4 in embodiment 1, wherein R1 is 39g ninhydrin, 81mg hydroboration
The mixture of sodium and 979ml ethylene glycol single methyl ether, R2 are 204g anhydrous sodium acetate, 123ml glacial acetic acid and 401ml ethylene glycol list
Methyl ether mixing mixes and is settled to the mixed solution that 1000ml is prepared with water, and R3 is that volume fraction is that 5% ethyl alcohol is water-soluble
Liquid.
Embodiment 3
The sample of the present embodiment is derived from the egg in fresh eggs food materials.
A kind of preprocess method of fresh food materials, comprising the following steps:
(a) sample extraction: the egg sample after weighing 2g stirring is placed in micro-wave diminishing pot, 15ml volume fraction is added
For 50% aqueous hydrochloric acid solution, 50mg phenol protective agent is added, is uniformly mixed；
(b) microwave hydrolysis: installation micro-wave diminishing pot sets microwave hydrolysis in carrying out microwave hydrolysis on microwave dissolver rapidly
Power is 1200W, sets microwave dissolver microwave heating program, gradually rises from room temperature to 70 DEG C in 6min, is kept for 70 DEG C and held
Continuous heating 3min, adjusts microwave dissolver temperature, its temperature is made to rise to 160 DEG C from 70 DEG C in 5min, and 160 DEG C of holding lasting micro-
Wave hydrolysis heating 15min, stops heating, when temperature display to room temperature, takes out micro-wave diminishing pot, hydrolyzate is made in hydrolysis；
(c) bigness scale hydrolyzate concentration: the hydrolyzate in micro-wave diminishing pot is poured into 50ml colorimetric cylinder, with 25ml volume point
Number at least washs micro-wave diminishing pot for 50% aqueous hydrochloric acid solution in three times, and all cleaning solutions of micro-wave diminishing pot are poured into 50ml
It in colorimetric cylinder, is transferred to all hydrolyzates in 50ml colorimetric cylinder, is uniformly mixed, the aqueous hydrochloric acid solution for being 50% with volume fraction
It is settled to scale, prepare liquid is made, prepare liquid and titer are subjected to colorimetric, measure prepare liquid concentration roughly；
(d) measurement liquid preparation: 2ml prepare liquid is accurately pipetted in 15ml test tube, nitrogen dries up under 50 DEG C of water-baths, is formed residual
Object is stayed, 3ml deionized water is added, dissolution residual substance, nitrogen dries up under water-bath again, repeats that deionized water is added and water-bath nitrogen is blown
It is dry, 2ml pH2.2 sodium citrate buffer is added, test tube is placed in ultrasonic wave dissolution instrument, setting ultrasonic wave dissolves instrument
Ultrasonic intensity be 1W/cm2, solution temperature is 60 DEG C, after ultrasonic wave dissolution process 3min, the polyethers for being 0.22 μm with aperture
Sample measurement liquid is made in sulfone membrane filtration.
A kind of Contents of Amino Acids method of fresh food materials, takes the preprocess method of above-mentioned fresh food materials in the present embodiment
Sample measurement liquid obtained measures the amino acid content of fresh food materials, comprising the following steps:
(1) machine liquid configures in standard:
Take the kilnitamin standard reserving solution 0.5ml of 2.5 μm of ol/ml in 5mL volumetric flask, with the hydrochloric acid of 0.1mol/L
Solution is settled to scale, is uniformly mixed, be respectively prepared concentration be 5,25,100,250, machine liquid in the standard of 1000nmol/ml；
(2) machine liquid measures in standard: drawing machine liquid in the standard of above-mentioned various concentration respectively and injects full-automatic Amino acid score
Analyzer, using external standard method, using the peak area of each amino acid as ordinate (y), using the concentration of each amino acid as abscissa (x,
Nmol/20 μ L)), make standard curve；
Machine liquid chromatography figure (Fig. 2 a to Fig. 6 b) and corresponding peak area result (table 5 to table 9) and reality in the present embodiment standard
Apply that example 2 is identical, and standard curve is also same as Example 2.
(3) in fresh food materials amino acid content measurement: take sample made from above-mentioned preprocess method measure liquid, dilution 20
Times, so that final sample is measured liquid in standard curve range, injects full-automatic amino-acid analyzer, various amino acid in egg
Peak sequence are as follows: asparatate, threonine, serine, glutamic acid, proline, glycine, alanine, valine, egg ammonia
Acid, isoleucine, leucine, tyrosine, phenylalanine, lysine, histidine, arginine, egg sample measure liquid chromatography figure
As illustrated in figures 9 a and 9b, egg sample measurement liquid test tests peak area as shown in table 11:
Table 11
External standard method measures the concentration C i of amino acid in liquid by calculated by peak area sample, and calculates egg sample by formula
In each amino acid content Xi (g/100g), calculation method is same as Example 2.
In the step (2) and (3), the chromatographic condition of full-automatic amino-acid analyzer are as follows: chromatography column material is sulphur
Acid type resin cation, column temperature are 65 DEG C, sampling volume 25uL.
In the step (2) and (3), full-automatic amino-acid analyzer uses gradient elution, elution flow rate 0.5ml/
Min, and derivative reaction occurs, derivative temperature is 140 DEG C, and derivative flow velocity is 0.45ml/min, and derivative flow velocity is less than elution stream
Speed.
In the present embodiment, elution program is as shown in the table 3 in embodiment 1, wherein B1 is 6.5g sodium citrate, 0.25g hydrogen
Sodium oxide molybdena, 5.9g sodium chloride, 21g citric acid, 142ml ethyl alcohol and 4.5ml polyoxyethylene laurel ether are uniformly mixed and use water constant volume
The mixed solution being prepared to 1000mL, B2 are 8.0g sodium citrate, 0.9g sodium hydroxide, 7.4g sodium chloride, 23g lemon
Acid, 26ml ethyl alcohol and 4.2ml polyoxyethylene laurel ether are uniformly mixed and are settled to the mixed solution that 1000mL is prepared with water,
B3 is that 14g sodium citrate, 4.0g sodium chloride, 13g citric acid, 9.5ml ethyl alcohol and 4.5ml polyoxyethylene laurel ether are uniformly mixed simultaneously
It is settled to the mixed solution that 1000mL is prepared with water, B4 is 28g sodium citrate, 57g sodium chloride, 6.5g citric acid, 5.5ml
Benzyl alcohol and 4.5ml polyoxyethylene laurel ether are uniformly mixed and are settled to the mixed solution that 1000mL is prepared with water, and B5 is
The ethanol water that volume fraction is 5%, B6 are 8.5g sodium hydroxide, 105ml ethyl alcohol and the mixing of 4.5ml polyoxyethylene laurel ether
Uniformly and the mixed solution that 1000mL is prepared is settled to water.
In the present embodiment, derivative program is as shown in the table 4 in embodiment 1, wherein R1 is 41g ninhydrin, 85mg hydroboration
The mixture of sodium and 1028ml ethylene glycol single methyl ether, R2 are 214g anhydrous sodium acetate, 129ml glacial acetic acid and 421ml ethylene glycol
Monomethyl ether mixing mixes and is settled to the mixed solution that 1000ml is prepared with water, and R3 is that volume fraction is 5% ethanol water
Solution.
In the present invention, when Detection wavelength is 570nm, asparatate, Soviet Union is can be detected in full-automatic amino-acid analyzer
Propylhomoserin, serine, glutamic acid, glycine, alanine, valine, methionine, isoleucine, leucine, tyrosine, phenylpropyl alcohol ammonia
15 kinds of acid, lysine, histidine and arginine amino acid, and can not detect proline (Pro), when Detection wavelength is 440nm
When, full-automatic amino-acid analyzer can be detected proline (Pro), therefore the full-automatic amino-acid analyzer Detection wavelength of the present invention is
440nm and 570nm.
The preparation method of kilnitamin standard reserving solution in the present invention are as follows:
According to the form below 12 accurately weighs single amino acids standard items in the volumetric flask of same 100ml respectively；
Table 12
It is dissolved with the hydrochloric acid solution of 0.1mol/L, and is settled to scale, be uniformly mixed, obtain 2.5 μm of ol/ml mixed aminos
Sour standard reserving solution.
Chilled beef of the present invention, the various amino acid contents of three kinds of samples of more precious fishes and egg are as shown in table 13 below:
Table 13
The amino acid content of the above table 13 is as the result is shown: chilled beef Glutamic Acid content highest, methionine content is minimum,
Total amino acid content is 16.933g in 100g chilled beef, and more treasured fishes and egg Glutamic Acid content highest, histidine content is minimum,
Total amino acid content is 13.931g in the more precious fishes of 100g, and total amino acid content is 10.490g in 100g egg, in three kinds of fresh food materials,
The amino acid content highest of chilled beef, the amino acid content of egg is minimum, and chilled beef nutritive value is higher.
Above disclosed is only presently preferred embodiments of the present invention, cannot limit rights protection model of the invention with this
It encloses, therefore according to equivalent variations made by scope of the present invention patent, is still within the scope of the present invention.
Claims (10)
1. a kind of preprocess method of fresh food materials, which comprises the following steps:
(a) sample extraction: weighing 0.1~2.0g sample, be placed in micro-wave diminishing pot, and it is 50% that 5~15ml volume fraction, which is added,
Aqueous hydrochloric acid solution adds 30~50mg phenol, is uniformly mixed；
(b) microwave hydrolysis: installation micro-wave diminishing pot is arranged microwave dissolver and exists in carrying out microwave hydrolysis on microwave dissolver rapidly
Reaction is kept in 50 DEG C~70 DEG C temperature, then rises to again and keeps reaction in 140 DEG C~160 DEG C of hydrolysis temperature, and reaction is completed
Afterwards, stop heating, when temperature display to room temperature, take out micro-wave diminishing pot, hydrolyzate is made in hydrolysis；
(c) bigness scale hydrolyzate concentration: hydrolyzate is transferred in 50ml colorimetric cylinder, fixed for 50% aqueous hydrochloric acid solution with volume fraction
Hold to scale, prepare liquid is made, prepare liquid and titer are subjected to colorimetric, measure prepare liquid concentration roughly；
(d) measurement liquid preparation: 1~2ml prepare liquid is accurately pipetted in 15ml test tube, nitrogen dries up under 40~50 DEG C of water-baths, shape
At residue, 1~3ml deionized water is added, dissolution residual substance, nitrogen dries up under water-bath again, repeats that deionized water is added simultaneously
The drying of water-bath nitrogen, is added 1~2ml pH2.2 sodium citrate buffer, test tube is placed in ultrasonic wave dissolution instrument, ultrasonic wave
Dissolution process is filtered with the polyethersulfone membranes that aperture is 0.22 μm, and sample measurement liquid is made.
2. the preprocess method of fresh food materials according to claim 1, which is characterized in that the step (b) is specifically, fast
Speed installation micro-wave diminishing pot in carrying out microwave hydrolysis on microwave dissolver, microwave dissolver be set in 4~6min from room temperature by
It edges up up to 50 DEG C~70 DEG C, keeps 50 DEG C~70 DEG C 3~10min of continuous heating, adjust microwave dissolver temperature, make its temperature
Rise to 140 DEG C~160 DEG C from 50 DEG C~70 DEG C in 3~5min, keep 140 DEG C~160 DEG C standing waves heating 15~
25min stops heating, when temperature display to room temperature, takes out micro-wave diminishing pot.
3. the preprocess method of fresh food materials according to claim 1, which is characterized in that in the step (b), Microwave Water
Solution power is 800~1200W.
4. the preprocess method of fresh food materials according to claim 1, which is characterized in that in the step (c), hydrolyzate
The method for being transferred to 50ml colorimetric cylinder are as follows: pour into the hydrolyzate in micro-wave diminishing pot in 50ml colorimetric cylinder, with 15~25ml body
The aqueous hydrochloric acid solution that fraction is 50% at least washs micro-wave diminishing pot in three times, and the cleaning solution of micro-wave diminishing pot is poured into 50ml
In colorimetric cylinder, it is uniformly mixed.
5. the preprocess method of fresh food materials according to claim 1, which is characterized in that in the step (d), ultrasonic wave
The ultrasonic intensity for dissolving instrument is 0.3~1W/cm2, ultrasonic wave solution temperature be 20~60 DEG C, sonication treatment time be 3~
10min。
6. a kind of Contents of Amino Acids method of fresh food materials, which is characterized in that take such as any one of Claims 1 to 5 institute
The amino acid content that the measurement liquid of sample made from the preprocess method of fresh food materials measures fresh food materials is stated, the fresh food materials
Contents of Amino Acids method includes the following steps:
(1) machine liquid configures in standard: taking the kilnitamin standard reserving solution 0.5ml of 2.5 μm of ol/ml in 5mL volumetric flask, uses
The hydrochloric acid solution of 0.1mol/L is settled to scale, is uniformly mixed, and concentration is made as machine liquid in 100nmol/ml standard or makes respectively
It is 5 at concentration, 25,100,250, machine liquid in the standard of 1000nmol/ml；
(2) machine liquid measures in standard: it draws machine liquid in the standard that concentration is 100nmol/ml and injects full-automatic amino-acid analyzer,
Peak area is measured using external standard method single point correction, or the full-automatic Amino acid score of machine liquid injection in the standard of absorption various concentration respectively
Analyzer using external standard method peak area and makes standard curve；
(3) in fresh food materials amino acid content measurement: take sample made from the preprocess method to measure liquid, injection is full-automatic
Amino-acid analyzer calculates the content of amino acid in sample using quantified by external standard method.
7. the Contents of Amino Acids method of fresh food materials according to claim 6, which is characterized in that the step (2) and
(3) in, the chromatographic condition of full-automatic amino-acid analyzer is that chromatography column material is sulfonic acid cation resin, and column temperature is
45~65 DEG C, 15~25uL of sampling volume, Detection wavelength is 440nm and 570nm.
8. the Contents of Amino Acids method of fresh food materials according to claim 6, which is characterized in that the step (2) and
(3) in, full-automatic amino-acid analyzer uses gradient elution, and derivative reaction occurs, and elution flow rate is 0.3~0.5ml/
The gradient elution program of min, full-automatic amino-acid analyzer are as follows:
Wherein, B1 is 5.8~6.5g sodium citrate, 0.22~0.25g sodium hydroxide, 5.4~5.9g sodium chloride, 19~21g lemon
Lemon acid, 128~142ml ethyl alcohol and 3.5~4.5ml polyoxyethylene laurel ether be uniformly mixed and be settled to 1000mL preparation with water and
At mixed solution, B2 be 7.0~8.0g sodium citrate, 0.7~0.9g sodium hydroxide, 6.7~7.4g sodium chloride, 21~23g
Citric acid, 23~26ml ethyl alcohol and 3.8~4.2ml polyoxyethylene laurel ether be uniformly mixed and be settled to 1000mL preparation with water and
At mixed solution, B3 be 12~14g sodium citrate, 3.5~4.0g sodium chloride, 12~13g citric acid, 8.5~9.5ml ethyl alcohol
It is uniformly mixed with 3.5~4.5ml polyoxyethylene laurel ether and is settled to the mixed solution that 1000mL is prepared, B4 25 with water
~28g sodium citrate, 52~57g sodium chloride, 5~6.5g citric acid, 4.5~5.5ml benzyl alcohol and 3.5~4.5ml polyoxy second
Alkene laurel ether is uniformly mixed and is settled to the mixed solution that 1000mL is prepared with water, and B5 is the ethyl alcohol that volume fraction is 5%
Aqueous solution, B6 are that 7.5~8.5g sodium hydroxide, 95~105ml ethyl alcohol and 3.5~4.5ml polyoxyethylene laurel ether are uniformly mixed
And the mixed solution that 1000mL is prepared is settled to water.
9. the Contents of Amino Acids method of fresh food materials according to claim 8, which is characterized in that the derivative reaction
In, derivative temperature is 130~140 DEG C, and derivative flow velocity is 0.25~0.45ml/min, and derivative flow velocity is less than elution flow rate, derivative
Program are as follows:
Wherein, R1 is the mixing of 37~41g ninhydrin, 77~85mg sodium borohydride and 930~1028ml ethylene glycol single methyl ether
Object, R2 are that 194~214g anhydrous sodium acetate, 117~129ml glacial acetic acid and the mixing of 381~421ml ethylene glycol single methyl ether mix
And it is settled to the mixed solution that 1000ml is prepared with water, R3 is that volume fraction is 5% ethanol water.
10. the Contents of Amino Acids method of fresh food materials according to claim 6, which is characterized in that the step (3)
In, amino acid peak sequence in sample are as follows: asparatate, threonine, serine, glutamic acid, proline, glycine, the third ammonia
Acid, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine, lysine, histidine, arginine.
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| CN112985970A (en) * | 2021-03-01 | 2021-06-18 | 上海屹尧仪器科技发展有限公司 | Method for rapidly hydrolyzing protein in dairy product by using inflatable microwave digestion device |
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Address after: No.7, Gongye North 4th Road, Songshanhu high tech Industrial Development Zone, Dongguan City, Guangdong Province, 523000 Applicant after: Guangdong Zhongding Testing Technology Co.,Ltd. Address before: No.7, North 4th Road, Songshanhu high tech Industrial Development Zone, Dongguan City, Guangdong Province, 523000 Applicant before: DONGGUAN CONSUMER TESTING TECHNOLOGY Co.,Ltd. |
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Application publication date: 20190723 |