CN102323341A - Method for detecting 18 varieties of protein hydrolytic amino acids in milk powder through high-performance liquid chromatographic method - Google Patents
Method for detecting 18 varieties of protein hydrolytic amino acids in milk powder through high-performance liquid chromatographic method Download PDFInfo
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Abstract
The invention belongs to the technical field of amino acid detection, in particular relates to a method for detecting 18 varieties of protein hydrolytic amino acids in milk powder through a high-performance liquid chromatographic method, solving the problems that the traditional amino acid determination method has high cost and poor universality and the qualitative and quantitative analysis of the 18 varieties of standard amino acids after proteolysis cannot be quickly and accurately performed through a high-performance liquid chromatograph at present. The method comprises the steps of: pre-treating the milk powder through three different hydrolysis methods; performing pre-column derivatization of a hydrolyzed milk powder smaple through a CNBF (4-chloro-3,5-dinitrobenzotrifluoride) derivatizing agent; optimizing derivatization conditions and chromatographic fractionation detection conditions by adopting the HPLC (High-performance Liquid Chromatography) and a universal C18 (250mm*4.6mm, 5mu m) liquid chromatography column; and separating and determining all 18 varieties of proteolyzed amino acids in the milk powder. The method has the advantages of stronger universality and no need of the precious amino acid analyzer and/or the precious amino acid determination reagent kit, thereby being suitable for more common laboratories.
Description
Technical field
The invention belongs to amino acid detection technique field, be specially the high-efficiency liquid chromatography method for detecting of 18 kinds of protein hydrolytic amino acids in the milk powder.
Background technology
In recent years, dairy products food security incident causes social extensive concern, because the food origin disease showed increased that dairy products cause makes the quality security problem of dairy products receive extensive attention.The development of food science and technology is when enriching human food's consumption, and also production has brought safety problem to dairy products.In order to strengthen the quality of dairy products supervision; Rules such as " food security method ", " dairy food quality safety supervision management regulations ", " 2010 editions enterprise production dairy products permissive condition examination detailed rules and regulations ", " 2010 editions enterprise production baby formula milk powder permissive condition examination detailed rules and regulations " are put into effect in succession, and the dairy products industry presses for more total solution to satisfy the detection demand that increases day by day.Numerous detection demands and rules demand have been brought also for simultaneously the dairy products assay laboratory and how to be improved a laboratory efficient and a difficult problem that reduces analysis cost.
Detect to amino acid; Existing determined amino acid method, most use triketohydrindene hydrates dye or derive, and adopt spectrophotometer or amino-acid analyzer to carry out amino acid whose analysis; Also there is part to adopt high performance liquid chromatograph, but also need uses special ion-exchange chromatography.Current national standard GB/T 5009.124-2003 " amino acid whose mensuration in the food " is used for measuring food 16 seed amino acids (not comprising tryptophane and (partly) cystine); GB/T18246-2000 " amino acid whose mensuration in the feed " can measure whole 18 kinds of hydrolysis amino acids, but the two all adopts full-automatic amino-acid analyzer; ISO13903:2005 (E) " animal feed-Determination of Amino Acids Content " uses amino-acid analyzer or high performance liquid chromatograph to measure 17 seed amino acids (do not comprise tryptophane, can not distinguish cystine and halfcystine) in the animal feed; AOAC official method 994.12 " amino acid-performic oxidation and sodium metabisulfite-acid-hydrolysis method in the feed " is applicable to amino-acid analyzer measures 16 seed amino acids (comprise methionine and cystine, do not comprise tyrosine and tryptophane).
When adopting high performance liquid chromatograph to detect amino acid; Because most of amino acid do not have uv absorption and fluorescent emission characteristic; For the sensitivity that improves analyzing and testing with separate selectivity characteristic; Usually carry out before the post amino acid or post-column derivation, column front derivation adopts OPA (OPA), phenyl isothiocyanate (PITC), chloro-carbonic acid fluorenes methyl esters (FMOC-Cl), dansyl chloride (DANSYL-Cl), 2,4-dinitrofluorobenzene (DNFB), aminoquinoline base-N-hydroxy-succinamide carbamate (AQC) etc. usually; Post-column derivation is most widely used to be triketohydrindene hydrate, yet above-mentioned derivative reagent respectively has quality.The derivating agent 4-chloro-3 that receives much concern at present; 5-dinitro trifluoromethylbenzene (CNBF); Shi Tianyu etc. (2009) use CNBF derivating agent and column front derivation method that 19 kinds of free amino acids (comprising 18 kinds of standard amino acids that remove glutamine (Gln) and arginine (Arg)) are measured, and are to adopt column front derivation to measure a kind of new trial of amino acid content.
Adopt high performance liquid chromatography-evaporative light-scattering (HPLC-ELSD) detection method to measure amino acid without deriving.The ELSD detecting device is a kind of universal type detecting device, do not derive and promptly can detect materials such as amino acid and carbohydrates, and the wide ranges of detection, it is comparatively convenient to use.But it is relatively stricter that this detecting device requires sample pre-treatments, is applicable to the detection of the sample that composition is more single or cleaner, and the sample of complicated component then can cause bigger interference to the result; In the eluent atomization process, need to consume a large amount of nitrogen, increased cost of determination; Can not use salt solusion as moving phase, can cause interference detection.
Therefore; Most employing spectrophotometer or the automatic amino acid analyzers of needing of existing amino acid detection method; Minority uses high performance liquid chromatograph also to need special ion-exchange chromatography or ELSD detecting device or determined amino acid pack; Do not have universal applicability, cost can not quick and precisely carry out qualitative and quantitative analysis to 18 kinds of standard amino acids behind the proteolysis based on the determined amino acid method of high performance liquid chromatograph at present also than higher.
Summary of the invention
The present invention exists for solving existing determined amino acid method that cost is high, universality difference and can not satisfy the problem of rapidly and accurately 18 kinds of proteolysis standard amino acids being carried out qualitative and quantitative analysis based on the determined amino acid method of high performance liquid chromatograph, and the high-efficiency liquid chromatography method for detecting of 18 kinds of protein hydrolytic amino acids in the milk powder is provided.
For realizing above-mentioned purpose, the present invention adopts following technical scheme: the high-efficiency liquid chromatography method for detecting of 18 kinds of protein hydrolytic amino acids in the milk powder may further comprise the steps:
(1) preparation milk powder hydrolyzation sample:
Adopting three kinds of different pretreatment methods is that routine hydrolysis method, oxidize water solution, alkali hydrolysis method are hydrolyzed to milk powder respectively, makes three kinds of milk powder hydrolyzation samples respectively;
(separate and measure 18 kinds of protein hydrolytic amino acids through different pretreatment; Wherein conventional (acid) Hydrolyze method is applicable to separation and measures except that sulfur-containing amino acid (methionine and halfcystine) and the hydrolysis amino acid the tryptophane, comprises asparatate, glutamic acid, histidine, arginine, serine, threonine, proline, glycocoll, alanine, valine, leucine, isoleucine, phenylalanine, tyrosine, lysine 15 seed amino acids; Oxidation (acid) Hydrolyze method is applicable to separates and mensuration methionine and two kinds of sulfur-containing amino acid of halfcystine; Alkali hydrolysis method is applicable to and separates and the mensuration tryptophane.For those of ordinary skills, above-mentioned three kinds of different method for hydrolysis are to realize easily.）
Deriving of (2) three kinds of milk powder hydrolyzation samples:
Derivating agent is a 4-chloro-3; 5-dinitro trifluoromethylbenzene (CNBF), its concentration is greater than 3 times of every kind of milk powder hydrolyzation sample total amino acid concentration, and the time of deriving is 40min; The temperature of deriving is 65 ℃, with above-mentioned derivatization conditions 18 kinds of kilnitamin standard solution is derived simultaneously;
(described derivating agent 4-chloro-3,5-dinitro trifluoromethylbenzene is CNBF, can generate with the amino acid reaction of one-level or secondary not only to have strong uv absorption, but also have the stable product of fluorescent absorption.This derivating agent reaction conditions is gentle; Generate stable N-for-2 with the amino acid reaction, 6-dinitro-4-trifluoromethyl-aniline, derivative products are at room temperature stable to be preserved more than 3 days; Can the crest of 18 seed amino acids fully be separated at 240nm, and help detecting accurately and effectively.The concentration of CNBF has multiple possibility theoretically, yet also will consider cost issues greater than 3 times of every kind of milk powder hydrolyzation sample total amino acid concentration, accomplishes economical and efficient as far as possible.Excessive CNBF derivating agent and water reaction generate the not stable accessory substance 4-ammonia-3 of interference measurement, 5-dinitrofluorobenzene and 3, and the 5-dinitro is to trifluoromethyl phenol.The characteristics of this derivating agent and advantage help determined amino acid accurately and effectively, and as a kind of fine chemical material, low price is buied easily, helps applying of method.）
(3) chromatographic resolution testing conditions:
Detecting instrument: Waters Alliance 2690/2695 highly effective liquid phase chromatographic system; 120 hole automatic sampling apparatus; Waters 2487 UV-detectors, Waters PDAD (PDA) (being used for) to the UV scanning of the absorbing wavelength of CNBF amino acid derivativges and amino acid whose qualitative;
Chromatographic column: general C18 (250 mm * 4.6 mm, 5 μ m) liquid-phase chromatographic column;
Moving phase: mobile phase A is an acetonitrile, and Mobile phase B is the triethylamine of 50mmol/L sodium acetate buffer and 0.24%, and flow velocity is 0.32ml/min, and pH is 5.8;
Detect wavelength: the setting absorbing wavelength is 240nm;
(4) measure:
18 kinds of kilnitamin standard solution after deriving are carried out uv scan and chromatogram detection, obtain 18 kinds of standard amino acid CNBF derivant spectral scan figure and chromatogram; Milk powder hydrolyzation sample to three kinds of different pre-treatments after deriving carries out efficient liquid phase chromatographic analysis, obtains 18 seed amino acid HPLC mensuration curve spectrum in the milk powder.
This method is carried out pre-treatment through three kinds of different method for hydrolysis to milk powder; Adopt HPLC and general C18 (250 mm * 4.6 mm; 5 μ m) liquid-phase chromatographic column can separate and measures proteoclastic whole 18 seed amino acids in the milk powder, and this method universality is stronger; And do not need valuable amino-acid analyzer and/or valuable determined amino acid pack, be applicable to more Routine Test Lab.
Description of drawings
Fig. 1 is the spectral scan figure of 18 kinds of standard amino acid CNBF derivants;
Fig. 2 is 18 kinds of protein hydrolytic amino acid CNBF derivant chromatograms of 2 mmol/ L for concentration;
Fig. 3 measures curve spectrum A for the amino acid HPLC that obtains after adopting the conventional acid Hydrolyze method to the pre-treatment of infant growth milk powder;
Fig. 4 measures curve spectrum B for the amino acid HPLC that obtains after adopting the conventional acid Hydrolyze method to the pre-treatment of infant growth milk powder;
Fig. 5 measures curve spectrum A for the amino acid HPLC that obtains after adopting the oxidizing acid Hydrolyze method to the pre-treatment of infant growth milk powder;
Fig. 6 measures curve spectrum B for the amino acid HPLC that obtains after adopting the oxidizing acid Hydrolyze method to the pre-treatment of infant growth milk powder;
Fig. 7 measures curve spectrum A for the amino acid HPLC that obtains after adopting alkali hydrolysis method to the pre-treatment of infant growth milk powder;
Fig. 8 measures curve spectrum B for the amino acid HPLC that obtains after adopting alkali hydrolysis method to the pre-treatment of infant growth milk powder.
Horizontal ordinate is an absorbing wavelength among Fig. 1, and unit is nm, and ordinate is a peak height, and unit is AU; Horizontal ordinate is a retention time among Fig. 2-8, and unit is min, and ordinate is a peak height, and unit is AU.
Embodiment
Below in conjunction with accompanying drawing and embodiment the present invention is done explanation further.
The high-efficiency liquid chromatography method for detecting of 18 kinds of protein hydrolytic amino acids in the milk powder may further comprise the steps:
(1) preparation milk powder hydrolyzation sample:
Adopting three kinds of different pretreatment methods is that routine hydrolysis method, oxidize water solution, alkali hydrolysis method are hydrolyzed to milk powder respectively, makes the milk powder hydrolyzation sample respectively; Milk powder is selected infant growth milk powder for use, can adopt following proposal during practical implementation:
1. routine hydrolysis method: take by weighing 0.0500g (accurately to 0.0001g) milk powder and be transferred in ampere bottle or the hydrolysis pipe; Add 6mol/L hydrochloric acid 10ml; Drip the fresh phenol after 2-3 drips dissolving; Charge into high pure nitrogen 15min, under the inflated with nitrogen state, to the ampere bottle closure or tighten hydrolysis pipe nut rapidly, ampere bottle or hydrolysis pipe are placed thermostatic drying chamber hydrolysis 20 ± 1h of 110 ± 1 ℃; Take out an ampere bottle or a hydrolysis pipe, cooling, mixing, open pipe is transferred to the volumetric flask of 50.0ml with hydrolyzate with the pH7.0 PBS, and constant volume is used filter paper filtering behind the abundant mixing, and supernatant is stored in the refrigerator subsequent use;
2. oxidize water solution: take by weighing 0.0500g (accurately to 0.0001g) milk powder and be transferred in ampere bottle or the hydrolysis pipe; In ice bath, cool off 30min, add the performic acid solutions 2.0ml of precooling, during liquid feeding that sample is all wetting; But do not shake; Build bottle stopper, place 4 ℃ of refrigerators, reaction 16h together with ice bath; Add hydrobromic acid 0.3ml, ice bath is put back in jolting, in vent cabinet, leaves standstill 30min, under not being higher than 60 ℃ temperature, is concentrated into dried (pressure is lower than 3.3 * 103 Pa during rotary evaporation) with Nitrogen evaporator or rotary evaporation; Get 6.0mol/L hydrochloric acid 10ml residue quantitatively is transferred in ampere bottle or the hydrolysis pipe, seal, put thermostatic drying chamber hydrolysis 20 ± 1h of 110 ± 1 ℃; Take out ampere bottle or hydrolysis pipe, cooling quantitatively is transferred to hydrolyzate in the 50.0 ml volumetric flasks with deionized water, and constant volume is fully used filter paper filtering behind the mixing, and supernatant is stored in the refrigerator subsequent use;
3. alkali hydrolysis method: take by weighing 0.0500g (accurately to 0.0001g) milk powder; Be transferred in the teflon bushing pipe, add 10 ml alkaline hydrolysis agent, freezing in liquid nitrogen or dry ice ethanol; The teflon bushing pipe is inserted ampere bottle or hydrolysis pipe; Charge into high pure nitrogen 15 min, under the inflated with nitrogen state, to the ampere bottle closure or tighten hydrolysis pipe nut rapidly, ampere bottle or hydrolysis pipe are placed 110 ± 1 ℃ of thermostatic drying chamber hydrolysis 20 ± 1h; Take out ampere bottle or hydrolysis pipe, be chilled to room temperature, open pipe is transferred to hydrolyzate in the 50.0 ml volumetric flasks with pH 7.0 PBSs, and constant volume is fully used filter paper filtering behind the mixing, and supernatant is stored in the refrigerator subsequent use;
Above-mentioned three kinds of method for hydrolysis are the pre-treating methods to the HPLC method, and original method has been carried out the improvement of part, it is advantageous that to obtain testing result accurately with less sample size, have practiced thrift operation cost and time;
Deriving of (2) three kinds of milk powder hydrolyzation samples:
Derivating agent is a 4-chloro-3; 5-dinitro trifluoromethylbenzene (CNBF), its concentration is greater than 3 times of every kind of milk powder hydrolyzation sample total amino acid concentration, and the time of deriving is 40min; The temperature of deriving is 65 ℃, with above-mentioned derivatization conditions 18 kinds of kilnitamin standard solution is derived simultaneously; Can adopt following proposal during practical implementation:
In 10 ml grinding port plug test tubes, add 1.0 ml milk powder hydrolyzation samples, add 5.0 ml pH, 9.0 borate buffer solutions, with the vortex oscillator 30 s mixings that vibrate; Add 2.0 ml CNBF solution (concentration of CNBF is 50-100 mmol/L, confirms according to amino acid whose concentration in every kind of milk powder hydrolyzation sample, and the total concentration that guarantees CNBF is greater than more than three times of amino acid total concentration in every kind of milk powder hydrolyzation sample); The vortex oscillator fully vibrates behind the mixing, seal the film sealing after, put into 65 ℃ of water-baths, 40 min that derive; Be placed to room temperature after deriving; Be transferred in the 25ml scale test tube, add the pH7.0 PBS, with the vortex oscillator 30 s mixings that vibrate to 25mL; Quiet to 10 min, get an amount of solution with 0.22 μ m membrane filtration; This concrete derivatives has been done partly to improve to the HPLC method, can access the best effect of deriving;
(3) chromatographic resolution testing conditions:
Detecting instrument: Waters Alliance 2690/2695 highly effective liquid phase chromatographic system, 120 hole automatic sampling apparatus, Waters 2487 UV-detectors, Waters PDAD (PDA);
Chromatographic column: general C18 (250 mm * 4.6 mm, 5 μ m) liquid-phase chromatographic column, can select Japanese Shiseido ODS C18 chromatographic column (250 * 4.6mm ID, 5 μ m) for use, Sweden Kromasil ODS C18 chromatographic column (250 * 4.6mm ID, 5 μ m);
Moving phase: mobile phase A is an acetonitrile, and Mobile phase B is the triethylamine of 50mmol/L sodium acetate buffer and 0.24%, and flow velocity is 0.32ml/min, and pH is 5.8;
Detect wavelength: the setting absorbing wavelength is 240nm;
(4) measure:
18 kinds of kilnitamin standard solution after deriving are carried out uv scan and chromatogram detection, obtain 18 kinds of standard amino acid CNBF derivant spectral scan figure (as shown in Figure 1) and chromatogram (as shown in Figure 2); Milk powder hydrolyzation sample to three kinds of different pre-treatments after deriving carries out efficient liquid phase chromatographic analysis, obtains 18 seed amino acid HPLC mensuration curve spectrum (shown in Fig. 3-8) in the infant growth milk powder.
18 kinds of standard amino acid CNBF derivant spectral scan collection of illustrative plates shown in Figure 1, maximum absorption spectrum is respectively: (1) CysA, 239.0nm; (2) Asp, 240.3nm; (3) Glu, 240.3 nm; (4) His, 240.3nm; (5) Arg, 239.7nm; (6) Ser, 239.7nm; (7) Thr, 240.3nm; (8) Pro, 215.9nm-270.3nm; (9) Met (O) 2,239.0; (10) Gly, 237.8nm; (11) Ala, 239.7nm; (12) Val, 240.3nm; (13) Trp, 219.5nm; (14) Leu, 240.3nm; (15) Ile, 240.3nm; (16) Phe, 241.5nm; (17) Tyr, 221.9nm; (18) Lys, 237.8nm.Be mainly used in qualitative to every seed amino acid, confirm that promptly amino acid and the amino acid in the standard items in the sample are with a kind of material, play the effect of affirmation.
18 kinds of standard amino acid CNBF derivant chromatograms shown in Figure 2, peak sequence is followed successively by: (1) CysA, cysteic acid (halfcystine); (2) Asp, asparatate; (3) Glu, glutamic acid; (4) His, histidine; (5) Arg, arginine; (6) Ser, serine; (7) Thr, threonine; (8) Pro, proline; (9) Met (O) 2, methionine sulfone (methionine); (10) Gly, glycocoll; (11) Ala, alanine; (12) Val, valine; (13) Trp, tryptophane; (14) Leu, leucine; (15) Ile.Isoleucine; (16) Phe, phenylalanine; (17) Tyr, tyrosine; (18) Lys, lysine.
The amino acid HPLC that Fig. 3 and Fig. 4 obtain after being respectively and adopting conventional (acid) Hydrolyze method to the pre-treatment of infant growth milk powder measures curve spectrum and amplification thereof; This method is used to measure this method and is used for measuring other the 15 kinds of protein hydrolytic amino acids except that sulfur-containing amino acid (methionine and (partly) cystine) and tryptophane; Comprise asparatate, glutamic acid, histidine,, arginine, serine, threonine, proline, glycocoll, alanine, valine, leucine, isoleucine, phenylalanine, tyrosine, lysine.
The amino acid HPLC that Fig. 5 and Fig. 6 obtain after being respectively and adopting the oxidizing acid Hydrolyze method to the pre-treatment of infant growth milk powder measures curve spectrum and amplifies collection of illustrative plates, is mainly used in to measure two kinds of sulfur-containing amino acid of methionine (among the figure by its oxidation product methionine sulfone--Met (O) 2 replaces) and (partly) cystine (among the figure by its oxidation product cysteic acid-CysA replacement).
The amino acid HPLC that Fig. 7 and Fig. 8 obtain after being respectively and adopting alkali hydrolysis method to the pre-treatment of infant growth milk powder measures curve spectrum and amplifies collection of illustrative plates, is mainly used in the mensuration tryptophane.
Above-mentioned amino acid HPLC measures the typical curve that curve spectrum is certain concentration (this method adopts 2mmol/L) from high performance liquid chromatograph output; Explain that this method is to amino acid whose separation degree (diverse ways obtains different curves); Be the most directly sign of method quality, but not as concrete use.When detecting, to do typical curve (selecting five concentration usually), and obtain the amino acid content (concentration) of sample through typical curve.
Claims (6)
1. the high-efficiency liquid chromatography method for detecting of 18 kinds of protein hydrolytic amino acids in the milk powder is characterized in that: may further comprise the steps:
(1) preparation milk powder hydrolyzation sample:
Adopting three kinds of different pretreatment methods is that routine hydrolysis method, oxidize water solution, alkali hydrolysis method are hydrolyzed to milk powder respectively, makes the milk powder hydrolyzation sample respectively;
Deriving of (2) three kinds of milk powder hydrolyzation samples:
Derivating agent is CNBF, and its concentration is greater than 3 times of every kind of milk powder hydrolyzation sample total amino acid concentration, and the time of deriving is 40min, and the temperature of deriving is 65 ℃, with above-mentioned derivatization conditions 18 kinds of kilnitamin standard solution is derived simultaneously;
(3) chromatographic resolution testing conditions:
Detecting instrument: Waters Alliance 2690/2695 highly effective liquid phase chromatographic system, 120 hole automatic sampling apparatus, Waters 2487 UV-detectors, Waters PDAD;
Chromatographic column: general C18 liquid-phase chromatographic column;
Moving phase: mobile phase A is an acetonitrile, and Mobile phase B is the triethylamine of 50mmol/L sodium acetate buffer and 0.24%, and flow velocity is 0.32ml/min, and pH is 5.8;
Detect wavelength: the setting absorbing wavelength is 240nm;
(4) measure:
18 kinds of kilnitamin standard solution after deriving are carried out uv scan and chromatogram detection, obtain 18 kinds of standard amino acid derivant spectral scan figure and chromatogram; Milk powder hydrolyzation sample to three kinds of different pre-treatments after deriving carries out efficient liquid phase chromatographic analysis, obtains 18 seed amino acid HPLC mensuration curve spectrum in the milk powder.
2. the high-efficiency liquid chromatography method for detecting of 18 kinds of protein hydrolytic amino acids in the milk powder according to claim 1 is characterized in that: three kinds of concrete schemes of different pretreatment method do
1. routine hydrolysis method: take by weighing 0.0500g milk powder and be transferred in ampere bottle or the hydrolysis pipe; Add 6mol/L hydrochloric acid 10ml; Drip the fresh phenol after 2-3 drips dissolving; Charge into high pure nitrogen 15min, under the inflated with nitrogen state, to the ampere bottle closure or tighten hydrolysis pipe nut rapidly, ampere bottle or hydrolysis pipe are placed thermostatic drying chamber hydrolysis 20 ± 1h of 110 ± 1 ℃; Take out an ampere bottle or a hydrolysis pipe, cooling, mixing, open pipe is transferred to the volumetric flask of 50.0ml with hydrolyzate with the pH7.0 PBS, and constant volume is used filter paper filtering behind the abundant mixing, and supernatant is stored in the refrigerator subsequent use;
2. oxidize water solution: take by weighing 0.0500g milk powder and be transferred in ampere bottle or the hydrolysis pipe, in ice bath, cool off 30min, add the performic acid solutions 2.0ml of precooling; During liquid feeding that sample is all wetting, but do not shake, build bottle stopper; Place 4 ℃ of refrigerators together with ice bath, reaction 16h; Add hydrobromic acid 0.3ml, ice bath is put back in jolting, in vent cabinet, leaves standstill 30min, with Nitrogen evaporator or rotary evaporation be not higher than be concentrated under 60 ℃ the temperature dried; Get 6.0mol/L hydrochloric acid 10ml residue quantitatively is transferred in ampere bottle or the hydrolysis pipe, seal, put thermostatic drying chamber hydrolysis 20 ± 1h of 110 ± 1 ℃; Take out ampere bottle or hydrolysis pipe, cooling quantitatively is transferred to hydrolyzate in the 50.0 ml volumetric flasks with deionized water, and constant volume is fully used filter paper filtering behind the mixing, and supernatant is stored in the refrigerator subsequent use;
3. alkali hydrolysis method: take by weighing 0.0500g milk powder; Be transferred in the teflon bushing pipe, add 10 ml alkaline hydrolysis agent, freezing in liquid nitrogen or dry ice ethanol; The teflon bushing pipe is inserted ampere bottle or hydrolysis pipe; Charge into high pure nitrogen 15 min, under the inflated with nitrogen state, to the ampere bottle closure or tighten hydrolysis pipe nut rapidly, ampere bottle or hydrolysis pipe are placed 110 ± 1 ℃ of thermostatic drying chamber hydrolysis 20 ± 1h; Take out ampere bottle or hydrolysis pipe, be chilled to room temperature, open pipe is transferred to hydrolyzate in the 50.0 ml volumetric flasks with pH 7.0 PBSs, and constant volume is fully used filter paper filtering behind the mixing, and supernatant is stored in the refrigerator subsequent use.
3. the high-efficiency liquid chromatography method for detecting of 18 kinds of protein hydrolytic amino acids in the milk powder according to claim 1 and 2 is characterized in that: the concrete scheme of deriving of three kinds of milk powder hydrolyzation samples is: in 10 ml grinding port plug test tubes, add 1.0 ml milk powder hydrolyzation samples, add 5.0 ml pH, 9.0 borate buffer solutions; With the vortex oscillator 30 s mixings that vibrate, add 2.0 ml CNBF solution, the concentration of CNBF is 50-100 mmol/L; Confirm that according to amino acid whose concentration in every kind of milk powder hydrolyzation sample the total concentration that guarantees CNBF is greater than more than three times of amino acid total concentration in every kind of milk powder hydrolyzation sample, the vortex oscillator fully vibrates behind the mixing; After sealing the film sealing; Put into 65 ℃ of water-baths, 40 min that derive, be placed to room temperature after deriving, be transferred in the 25ml scale test tube; Add the pH7.0 PBS to 25mL; With the vortex oscillator 30 s mixings that vibrate, quiet to 10 min, get an amount of solution with 0.22 μ m membrane filtration.
4. the high-efficiency liquid chromatography method for detecting of 18 kinds of protein hydrolytic amino acids in the milk powder according to claim 1 and 2; It is characterized in that: general C18 liquid-phase chromatographic column, can select Japanese Shiseido ODS C18 chromatographic column, Sweden Kromasil ODS C18 chromatographic column for use.
5. the high-efficiency liquid chromatography method for detecting of 18 kinds of protein hydrolytic amino acids in the milk powder according to claim 3; It is characterized in that: general C18 liquid-phase chromatographic column, can select Japanese Shiseido ODS C18 chromatographic column, Sweden Kromasil ODS C18 chromatographic column for use.
6. the high-efficiency liquid chromatography method for detecting of 18 kinds of protein hydrolytic amino acids in the milk powder according to claim 1 is characterized in that: 18 kinds of standard amino acid derivant chromatograms that obtain, and peak sequence is followed successively by: (1) CysA, cysteic acid; (2) Asp, asparatate; (3) Glu, glutamic acid; (4) His, histidine; (5) Arg, arginine; (6) Ser, serine; (7) Thr, threonine; (8) Pro, proline; (9) Met (O) 2, the methionine sulfone; (10) Gly, glycocoll; (11) Ala, alanine; (12) Val, valine; (13) Trp, tryptophane; (14) Leu, leucine; (15) Ile, isoleucine; (16) Phe, phenylalanine; (17) Tyr, tyrosine; (18) Lys, lysine.
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