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National Standard of the People's Republic of China
Determination of available lysine in feedstuffs
Determination of available lysine in feedstuffs This standard adopts the international standard ISO5510--1984 "Determination of available lysine in feedstuffs". Subject content and scope of application
This standard specifies the determination method of available lysine in feedstuffs. This standard is applicable to compound (mixed) feeds and single feeds containing animal or vegetable proteins. 2 Reference standards
GB6432 Determination of crude protein in feed
3 Terminology
GB/T 15398-94
Available lysine refers to the difference between total lysine and non-effective lysine measured under specified determination conditions. 4 Principle
The e-amino group of available lysine in protein can react with 2.4-dinitrofluorobenzene to generate dinitrophenyllysine after acid hydrolysis, while the other non-effective parts generate lysine. Therefore, the samples without dinitrofluorobenzene treatment and the samples with dinitrofluorobenzene treatment were hydrolyzed respectively, and the lysine content of each was determined by ion exchange chromatography, and the difference between them could be used to obtain the effective lysine content in the sample. 5 Reagents and solutions
All reagents used in this standard are analytically pure, and the water is deionized water. 5.1 Ether (HG3--1002).
5.2 Sodium bicarbonate (GB640) solution: 80g/L. 5.3 2.4-Dinitrofluorobenzene (DNFB) ethanol solution: Dissolve a certain amount of DNFB in 95% ethanol at a volume ratio of 0.15:12. The solution is prepared before use. 5.4 Hydrochloric acid (GB622) solution: 6.0mol/L. 5.5 Hydrochloric acid (GB622) solution: 6.5mol/L. 5.6 Sodium citrate buffer for dilution: pH about 2.2. Dissolve the following reagents in appropriate amount of water:
20g hydrated citric acid (HG3-1108)
8g sodium hydroxide (GB629)
16mL concentrated hydrochloric acid (GB622)bzxz.net
0.1ml. Caprylic acid
20ml thiodiglycol
Dilute with water to 1000ml.
Approved by the State Administration of Technical Supervision on December 30, 1994 and implemented on July 1, 1995
5.7 Sodium citrate buffer for chromatography:
GB/T 15398—94
The pH value and preparation method vary according to different models of amino acid analyzers. Please refer to the instrument manual for preparation. Section 5.8 Triketone Reagent
Prepare according to the instrument manual.
5.9 Lysine standard solution: 50nmol/mL or other concentrations. First prepare 2.5μmol/ml lysine standard stock solution: weigh 45.6mg lysine monohydrochloride and dissolve it in 0.1mol/L hydrochloric acid, dilute to 100ml, and mix well.
Use this standard stock solution or commercial 2.5μmol/mL standard amino acid mixture to prepare 50. ()nmol/ml standard solution with pH2.2 sodium citrate buffer as solvent. It can also be prepared into other optimal concentrations according to the instrument manual. 6 Instruments and equipment
6.1 Laboratory sample crusher: It can crush samples quickly and consistently and avoid overheating of samples and minimize contact with the outside world. 6.2 Sample sieve: aperture 0.25mm (60 mesh). 6.3 Analytical balance: sensitivity 0.0001g.
6.4 Oil bath: The temperature can be stabilized between 120~~130℃. 6.5 Reflux hydrolysis device: 150ml., 500mL short-necked flasks are connected to the reflux condenser (about 35~40cm long) with a glass ground joint. 6.6 Centrifuge: 4000r/min.
6.7 Rotary evaporator.
6.8 Amino acid automatic analyzer.
6.9 Reflux hydrolysis device: 50mL hydrolysis tube or digestion tube, connected to the reflux condenser with a ground joint. 6.10 Metal block digestion furnace: The temperature is required to be maintained between 120 and 130C. If necessary, a relay and contact thermometer can be added for control. The heating depth of the digestion tube in the metal block should be higher than or equal to the liquid level of 25mL digestion liquid. 6.11 Water bath
Sample preparation
Collect representative compound (mixed) feed or single feed, and prepare laboratory samples by quartering. Take 10~~20g and crush it so that it can all pass through the sample sieve (6.2). Mix thoroughly and put it into a sealed container. 8 Analysis steps
8.1.1 Total lysine
8.1.1.1 Acid hydrolysis
Weigh two samples containing about 50mg crude protein, place them in a 500mL flask (6.5), add 250mL hydrochloric acid (5.4), install a reflux condenser, place it in a 120~130℃ oil bath (6.4), let it boil slowly, and reflux hydrolysis for 24h. Remove the flask and cool it. If necessary, re-adjust the volume of the acid hydrolyzate (together with the insoluble matter after hydrolysis) to 250mL. Filter, take 5 ml of the filtrate, evaporate to dryness on a rotary evaporator (6.7) at about 60°C. Add a little water and repeat evaporation 1-2 times. Dissolve the residue in 5 ml of sodium citrate buffer for dilution (5.6), centrifuge for 10 min (4000 r/min), take the supernatant and put it on the analyzer or store it in a stoppered tube and refrigerate for later use.
8.1.1.2 Determination
Turn on the amino acid automatic analyzer (6.8), make the column temperature, reaction bath temperature, buffer and triketone flow rate and other operating parameters meet the predetermined requirements, and take a quantitative lysine standard solution (5.9) for calibration according to the instrument manual. Then take the sample solution obtained in 8.1.1.1 under the same conditions for determination.
8.1.2 Ineffective lysine
8.1.2.1. Dinitrobenzenization reaction
GB/T 15398—94
Weigh two samples containing about 50 mg crude protein, add 4 ml sodium bicarbonate solution (5.2) into a 150 ml flask (6.5), and place for 10 minutes, shaking from time to time. Then add 6 mL DNFB ethanol solution (5.3), cover, shake, and be careful not to let the sample particles adhere to the wall of the flask. Place in a dark place at room temperature overnight. 8.1.2.2 Purification
Evaporate the above reactants on a rotary evaporator at a temperature not higher than 40°C, add 35 ml ether (5.1), shake or stir, wait for the solid to settle fully, discard most of the ether, and be careful not to bring out the solid sample particles. Repeat the above operation twice, adding 25 ml of ether each time (the ether layer in this step may be turbid sometimes. As long as there are no particles at the sample outlet when pouring, centrifugation is not necessary). Finally, evaporate to remove the remaining ether.
8.1.2.3 Acid hydrolysis
Add 75 ml of hydrochloric acid (5.4) while rinsing the bottle mouth and bottle wall, so that the sample is completely immersed in acid, install the reflux condenser, place it in a 120~130℃ oil bath, let it boil slowly, and reflux hydrolysis for 24 hours. Remove the flask and cool it. Transfer the hydrolyzate quantitatively to a 100 mL volumetric flask, make up to volume with water, and filter. Take 5 ml of the filtrate and evaporate it to dryness on a rotary evaporator at about 60°C, add a little water, and repeat evaporation to dryness 1~2 times. Dissolve the residue in 3 ml of diluent sodium citrate buffer (5.6), centrifuge it in a centrifuge (6.6) for 10 minutes, take the supernatant and put it on the centrifuge or refrigerate it in a stoppered tube for later use.
8.1.2.4 Determination
Week 8. 1. 1. 2.
The peak of ineffective lysine is low and easily interfered by adjacent peaks or miscellaneous peaks. For example, some amino acid automatic analyzers cannot separate this peak well. It is necessary to lengthen the resin bed (or analytical column) or change the parameters and procedures. 8.2B Method - Simplified Method
8.2.1 Total Lysine
Accurately weigh two samples containing about 50 mg of crude protein, place them in a 50 mL hydrolysis tube, add 25 mL of hydrochloric acid (5.4), install a reflux condenser and place it on a metal block digestion furnace (6.10) preheated to 120-130°C, slowly boil and hydrolyze for 24 hours. Cool, make the hydrolyzate volume to 50 mL, mix well, and filter. Take 2 mL of the filtrate, concentrate to dryness at 60°C on a rotary evaporator, add a little water, and repeat evaporation to dryness 1-2 times.
Add an appropriate amount of sodium citrate buffer (5.6) to the residue (add 2 mL for samples with a crude protein content of 10% to 20%, and 5 mL for samples with a crude protein content of 30% to 50%), centrifuge after full dissolution, and take the supernatant for analysis. 8.2.2 Ineffective lysine
8.2.2.1 Dinitrobenzenization reaction
Weigh two samples containing about 20 to 25 mg of protein, place them in a 50 mL hydrolysis tube, add 2 mL of sodium bicarbonate solution (5.2). Let stand for 10 minutes (shake occasionally), add DNFB ethanol solution (5.3) 3mL, shake to mix, and place in dark place at room temperature overnight. Place the above hydrolysis tube in a 85-90°C water bath to evaporate the ethanol until no foam is produced when shaking. At this time, the hydrolysis tube should lose 2.5-3 g in weight.
8.2.2.2° Purification
Add 20mL of ether (5.1) to the hydrolysis tube after alcohol removal, add a stopper, shake vigorously to separate, discard the ether layer, and extract twice with ether, 10mL each time. After discarding the ether layer, place the hydrolysis tube in a 60-70°C water bath to remove the remaining ether. 8.2.2.3 Acid hydrolysis
Add 23ml of hydrochloric acid (5.5) while rinsing the tube mouth and tube wall. Install the condenser and place it on a 120-130°C metal block digestion furnace (6.10) with the total lysine sample, and reflux hydrolysis for 24h. Cool. Make the volume of hydrolyzate to 50mL, mix well, and filter. Take 5mL of filtrate and evaporate to dryness on a 60C rotary evaporator, add a little water, and repeat the evaporation 1 to 2 times.
Add appropriate sodium citrate buffer (5.6) to the residue (add 3ml for protein content 10% to 20%, and 211
5ml for 30% to 50%). Centrifuge after full dissolution. Take the supernatant and put it on the machine. 9 Expression of analysis results
9.1 Total lysine
GB/T 15398--94
The total lysine content W in the sample is expressed as its mass percentage, that is: Total lysine W (%) =
Where: A-lysine content in each liter of liquid on the machine, ng; -sample mass, mg;
dilution multiple.
9.2 Ineffective lysine
× 10-6 × 100 ×D
The ineffective lysine content W in the sample is expressed as its mass percentage, that is: Ineffective lysine W (%) =
× 10-6 × 100 × D
Where: 4—lysine content in each milliliter of upper liquid, ng; m2
Sample mass in the ineffective lysine determination, mg; D dilution multiple.
9.3 Effective lysine
The effective lysine content in the sample is W:, expressed as a mass percentage, that is: effective lysine W, = W-W,
If the effective lysine content is expressed as its mass percentage in the total lysine, then Wl-W2×100
Ws(%) =
10Repeatability
The relative difference between the values of two parallel determinations of the same sample should not exceed 10% of their average value. Note: Compared with the bioassay method, the determination results of this method are higher, and attention should be paid when analyzing and evaluating the experimental results. Additional notes:
This standard was proposed by the Ministry of Agriculture of the People's Republic of China. This standard is under the jurisdiction of the National Agricultural Standardization Technical Committee. This standard was drafted by the Analysis and Testing Center of the Chinese Academy of Agricultural Sciences. The drafters of this standard are Chang Biying, Yan Huiwen, and Zuo Jiangwan. 21
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