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食品安全国家标准 食品中氨基酸的测定
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GB 5009.124-2016
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标准编号: GB 5009.124-2016 (GB5009.124-2016)
中文名称: 食品安全国家标准 食品中氨基酸的测定
英文名称: National Food Safety Standard -- Determination of Amino Acid in Foods
行业: 国家标准
中标分类: X09
字数估计: 8,837
发布日期: 2016-12-23
实施日期: 2017-06-23
旧标准 (被替代): GB/T 5009.124-2003
标准依据: National Health and Family Planning Commission Notice No.17 of 2016
GB 5009.124-2016: 食品安全国家标准 食品中氨基酸的测定
GB 5009.124-2016 英文名称: National Food Safety Standard -- Determination of Amino Acid in Foods
1 范围
本标准规定了用氨基酸分析仪(茚三酮柱后衍生离子交换色谱仪)测定食品中氨基酸的方法。
本标准适用于食品中酸水解氨基酸的测定,包括天冬氨酸、苏氨酸、丝氨酸、谷氨酸、脯氨酸、甘氨
酸、丙氨酸、缬氨酸、蛋氨酸、异亮氨酸、亮氨酸、酪氨酸、苯丙氨酸、组氨酸、赖氨酸和精氨酸共16种氨基酸。
2 原理
食品中的蛋白质经盐酸水解成为游离氨基酸,经离子交换柱分离后,与茚三酮溶液产生颜色反应,
再通过可见光分光光度检测器测定氨基酸含量。
3 试剂和材料
除非另有说明,本方法所用试剂均为分析纯,水为GB/T 6682中规定的一级水。
3.1 试剂
3.1.1 盐酸(HCl):浓度≥36%,优级纯。
3.1.2 苯酚(C6H5OH)。
3.1.3 氮气:纯度99.9%。
3.1.4 柠檬酸钠(Na3C6H5O7·2H2O):优级纯。
3.1.5 氢氧化钠(NaOH):优级纯。
3.2 试剂配制
3.2.1 盐酸溶液(6mol/L):取500mL盐酸加水稀释至1000mL,混匀。
3.2.2 冷冻剂:市售食盐与冰块按质量1∶3混合。
3.2.3 氢氧化钠溶液(500g/L):称取50g氢氧化钠,溶于50mL水中,冷却至室温后,用水稀释至
100mL,混匀。
3.2.4 柠檬酸钠缓冲溶液[c(Na+)=0.2mol/L]:称取19.6g柠檬酸钠加入500mL水溶解,加入
16.5mL盐酸,用水稀释至1000mL,混匀,用6mol/L盐酸溶液或500g/L氢氧化钠溶液调节pH至
2.2。
3.2.5 不同pH和离子强度的洗脱用缓冲溶液:参照仪器说明书配制或购买。
3.2.6 茚三酮溶液:参照仪器说明书配制或购买。
3.3 标准品
3.3.1 混合氨基酸标准溶液:经国家认证并授予标准物质证书的标准溶液。
3.3.2 16种单个氨基酸标准品:固体,纯度≥98%。
3.4 标准溶液配制
3.4.1 混合氨基酸标准储备液(1μmol/mL):分别准确称取单个氨基酸标准品(精确至0.00001g)于
同一50mL烧杯中,用8.3mL6mol/L盐酸溶液溶解,精确转移至250mL容量瓶中,用水稀释定容至
刻度,混匀(各氨基酸标准品称量质量参考值见表1)。
3.4.2 混合氨基酸标准工作液(100nmol/mL):准确吸取混合氨基酸标准储备液1.0mL于10mL容
量瓶中,加pH2.2柠檬酸钠缓冲溶液定容至刻度,混匀,为标准上机液。
4 仪器和设备
4.1 实验室用组织粉碎机或研磨机。
4.2 匀浆机。
4.3 分析天平:感量分别为0.0001g和0.00001g。
4.4 水解管:耐压螺盖玻璃试管或安瓿瓶,体积为20mL~30mL。
4.5 真空泵:排气量≥40L/min。
4.6 酒精喷灯。
4.7 电热鼓风恒温箱或水解炉。
4.8 试管浓缩仪或平行蒸发仪(附带配套15mL~25mL试管)。
4.9 氨基酸分析仪:茚三酮柱后衍生离子交换色谱仪。
5 分析步骤
5.1 试样制备
固体或半固体试样使用组织粉碎机或研磨机粉碎,液体试样用匀浆机打成匀浆密封冷冻保存,分析
用时将其解冻后使用。
5.2 试样称量
均匀性好的样品,如奶粉等,准确称取一定量试样(精确至0.0001g),使试样中蛋白质含量在
10mg~20mg范围内。对于蛋白质含量未知的样品,可先测定样品中蛋白质含量。将称量好的样品
置于水解管中。
很难获得高均匀性的试样,如鲜肉等,为减少误差可适当增大称样量,测定前再做稀释。
对于蛋白质含量低的样品,如蔬菜、水果、饮料和淀粉类食品等,固体或半固体试样称样量不大于
2g,液体试样称样量不大于5g。
5.3 试样水解
根据试样的蛋白质含量,在水解管内加10mL~15mL6mol/L盐酸溶液。对于含水量高、蛋白质
含量低的试样,如饮料、水果、蔬菜等,可先加入约相同体积的盐酸混匀后,再用6mol/L盐酸溶液补充
至大约10mL。继续向水解管内加入苯酚3滴~4滴。
将水解管放入冷冻剂中,冷冻3min~5min,接到真空泵的抽气管上,抽真空(接近0Pa),然后充
入氮气,重复抽真空-充入氮气3次后,在充氮气状态下封口或拧紧螺丝盖。
将已封口的水解管放在110℃±1℃的电热鼓风恒温箱或水解炉内,水解22h后,取出,冷却至
室温。
打开水解管,将水解液过滤至50mL容量瓶内,用少量水多次冲洗水解管,水洗液移入同一50mL
容量瓶内,最后用水定容至刻度,振荡混匀。
准确吸取1.0mL滤液移入到15mL或25mL试管内,用试管浓缩仪或平行蒸发仪在40℃~
50℃加热环境下减压干燥,干燥后残留物用1mL~2mL水溶解,再减压干燥,最后蒸干。
用1.0mL~2.0mLpH2.2柠檬酸钠缓冲溶液加入到干燥后试管内溶解,振荡混匀后,吸取溶液通
过0.22μm滤膜后,转移至仪器进样瓶,为样品测定液,供仪器测定用。
5.4 测定
5.4.1 仪器条件
使用混合氨基酸标准工作液注入氨基酸自动分析仪,参照JJG1064-2011氨基酸分析仪检定规程
及仪器说明书,适当调整仪器操作程序及参数和洗脱用缓冲溶液试剂配比,确认仪器操作条件。
5.4.2 色谱参考条件
a) 色谱柱:磺酸型阳离子树脂;
b) 检测波长:570nm和440nm。
5.4.3 试样的测定
混合氨基酸标准工作液和样品测定液分别以相同体积注入氨基酸分析仪,以外标法通过峰面积计
6 分析结果的表述
6.1 混合氨基酸标准储备液中各氨基酸浓度的计算
各氨基酸标准品称量质量参考值见表1。
7 精密度
在重复性条件下获得的两次独立测定结果的绝对差值不得超过算术平均值的12%。
8 其他
当试样为固体或半固体时,最大试样量为2g,干燥后溶解体积为1mL,各氨基酸的检出限和定量
限见表3。
GB 5009.124-2016
GB
NATIONAL STANDARD OF THE
PEOPLE’S REPUBLIC OF CHINA
National Food Safety Standard
– Determination of Amino Acid in Foods
ISSUED ON. DECEMBER 23, 2016
IMPLEMENTED ON. JUNE 23, 2017
Issued by. National Health and Family Planning Commission of PRC;
China Food and Drug Administration.
Table of Contents
Foreword ... 3
1 Scope ... 4
2 Principle ... 4
3 Reagents and Materials ... 4
4 Instrument and Equipment ... 5
5 Analytical Procedures ... 6
6 Expression of Analytical Results ... 8
7 Precision ... 10
8 Others ... 10
Appendix A Chromatogram ... 12
Foreword
This Standard replaces GB/T 5009.124-2003 Determination of Amino Acid in Foods.
Compared with GB/T 5009.124-2003, this Standard has the major changes as follows.
--- Modify the standard name into “National Food Safety Standard – Determination
of Amino Acid in Foods”;
--- Expand the applicable scope;
--- Add the limit of detection and quantitative limit of the method;
--- Modify the result calculation formula.
National Food Safety Standard
– Determination of Amino Acid in Foods
1 Scope
This Standard specifies using the amino acid analyzer (ninhydrin post-column
derivatization ion exchange chromatography) to determine the amino acid in foods.
This Standard is applicable to determine the amino acid hydrolyzed by acid in foods,
there are totally 16 kinds of amino acids such as aspartic acid, threonine, serine,
glutamic acid, proline, glycine, alanine, valine, methionine, isoleucine, leucine, tyrosine,
phenylalanine, histidine, lysine, and arginine.
2 Principle
The protein in food is hydrolyzed by hydrochloric acid into free amino acid; after being
separated by ion exchange column, it occurs color reaction with ninhydrin solution;
then determine the amino acid content through the visible light spectrophotometer.
3 Reagents and Materials
Unless otherwise specified, the reagents used in this method are analytically pure;
while water is Class-I water stipulated in GB/T 6682.
3.1 Reagents
3.1.1 Hydrochloric acid (HCl). concentration≥36%, guarantee reagent.
3.1.2 Phenol (C6H5OH).
3.1.3 Nitrogen. with purity of 99.9%.
3.1.4 Sodium citrate (Na3C6H5O7•2H2O). guarantee reagent.
3.1.5 Sodium hydroxide (NaOH). guarantee reagent.
3.2 Reagents preparation
3.2.1 Hydrochloric acid solution (6mol/L). take 500mL of hydrochloric acid, dilute with
water to 1000mL, mix evenly.
3.2.2 Refrigerant. mix the commercial salt and ice by the mass ratio of 1.3.
Place the hydrolysis tube into the refrigerant, frozen for 3min~5min; connect to the
suction tube of vacuum pump; pump vacuum (close to 0Pa); then inject nitrogen;
repeatedly pump vacuum, inject nitrogen, after 3 times, seal the cap or tighten the
screw cap under nitrogen injecting state.
Place the sealed hydrolysis tube into 110°C±1°C electric blower thermostat or
hydrolysis furnace; hydrolyze for 22h, then take out; cooling off to the room
temperature.
Open the hydrolysis tube, filter the hydrolysate into 50mL volumetric flask; use small
amount of water to wash the hydrolysis tube for several times; transfer the washing
liquid into the same 50mL volumetric flask; finally use water to make constant volume
to the scale; shake and mix evenly.
Accurately pipette 1.0mL of filtrate into 15mL or 25mL test tube; use the test tube
concentrator or parallel evaporator to reduce pressure to dry at the 40°C~50°C heating
temperature environment; after drying, the residuals shall use 1mL~2mL of water to
dissolve; then reduce pressure to dry; finally evaporate to dryness.
drying to dissolve; after shaking and mixing evenly; absorb the solution to get through
the 0.22µm filter film; transfer into the instrument sample-injecting bottle; it is the
sample assay solution for the measurement of the instrument.
5.4 Determination
5.4.1 Instrument conditions
Inject the mixed amino acid standard working solution into the automatic amino acid
analyzer; refer to the test protocol and instrument manual of the amino acid analyzer
stipulated in JJG1064-2011; appropriately adjust the instrument operation procedures,
parameters, and reagent ratio of buffer solution; confirm the instrument operating
5.4.2 Chromatographic reference conditions
a) chromatographic column. sulfonic acid cation resin;
b) detection wavelength. 570nm and 440nm.
5.4.3 Determination of specimen
Inject the same volume of amino acid standard working solution and sample assay
solution into the amino acid analyzer; calculate the concentration of amino acid in the
sample assay solution through the peak area by external standard method.
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