1、中华人民共和国出入境检验检疫行业标准SN/T 1868-2007 进出口油菜籽及其饼来自中硫代葡萄糖昔总量的测定方法2007-04-06发布Determination of total glucosinolates in rapeseed and its meal for import and export 2007-10-16实施中华人民共和国发布国家质量监督检验检疫总局目。昌本标准由国家认证认可监督管理委员会提出并归口。本标准起草单位:中华人民共和国上海出入境检验检疫局。本标准主要起草人:倪昕路、禧庆华。本标准系首次发布的出入境检验检疫行业标准。SN/T 1868-2007 SN/T 18
2、68-2007 进出口油菜籽及其饼来自中硫代葡萄糖昔总量的测定方法1 范围本标准规定了进出口油菜籽及其饼柏中硫代葡萄糖昔总量的测定方法。本标准适用于油菜籽及其饼柏中硫代葡萄糖昔总量的测定。2 测定方法2.1 方法提要样品加热使自然态酶灭活，经沸水提取后，用弱阴离子交换树脂柱层析净化，添加黑芥子硫昔酸酶水解硫代葡萄糖背得到葡萄糖并用水洗脱。加入葡萄糖显色试剂，使样品溶液呈色，在505nm波长测定吸光度。根据葡萄糖标准曲线校正，计算样品中硫代葡萄糖昔总量。2.2 试剂和材料除另有规定外，试剂为分析纯，水为蒸馆水。2.2.1 元水磷酸氢二铀。2.2.2 氧化铀。2.2.3 鸽酸铀。2.2.4 苯盼。
3、2.2.5 浓盐酸。2.2.6 弱阴离子交换树脂，DEAESephadex A-25 0 2.2.7 氢氧化铀。2.2.8 冰乙酸。2.2.9 元水乙酸纳。2.2. 10 三起甲基氨基甲:境。2.2.11 黑芥子硫昔酸酶(EC3.2. 1. 147)，活力单位200U/go 2.2.12 4氨基安替毗啡。2.2.13 葡萄糖氧化酶(EC1. 1. 3. 4) ，活力单位20000 U/66. 67 mg 0 2.2.14 过氧化物酶(EC1. 11. 1. 7)，活力单位10000 U/52. 6 mg o 2.2.15 盐酸榕液(2mol/L):量取17.2mL浓盐酸倒入100mL水中。2.
4、2.16 苯酣鸪酸榕液:称取5.0g鸽酸纳、5.0g无水磷酸氢二铀和9.0g氧化铀于500mL容量瓶中，用约350mL水溶解，用2mol/L盐酸溶液调节pH至3.0，加入2.0g苯酌，用水定容至500mLo 2.2.17 氢氧化纳溶液(0.5mol/L) :称取20g氢氧化铀溶解于约800mL水中，以水定容至1L2.2. 18 乙酸铀缓冲液(0.2mol/L) :称取16.5g无水乙酸铀和11.5 mL冰乙酸溶解于约950mL水中，用2mol/L盐酸溶液调节pH至4.9，用水定容至1L2.2.19 Tris-HCl缓冲液:称取1.6g三起甲基氨基甲:民榕解于约900mL水中，用2mol/L盐酸
5、榕液调节pH至7.0，用水定容至1Lo 2.2.20 黑芥子硫昔酸酶榕液:称取500mg黑芥子硫昔酸酶于10mL容量瓶中，加水溶解并定容，实验当日制备。2.2.21 葡萄糖显色、溶液:称取10g元水磷酸氢二铀于盛有约750mL水的1L容量瓶中，加入13 1. 7 mg葡萄糖氧化酶于容量瓶中并摇匀;称取16.7mg过氧化物酶和333mg 4氨基安替毗琳于盛SN/T 1868-2007 有约10mL水的烧杯中并搅拌使其、溶解，移入容量瓶并用水定容;溶液保存在棕色瓶中并于4C冷藏。2.2.22 苯甲酸。2.2.23 无水葡萄糖标准品:纯度二三99%。2.2.24 葡萄糖标准储备液:将无水葡萄糖于40
6、C真空烘箱中干燥4h。冷却后称取1.000 0 g葡萄糖于烧杯内，加入1g苯甲酸防腐，用水溶解并转移至1L容量瓶内，用水稀释定容。葡萄糖储备液中葡萄糖浓度为5.56mol/mL。2.2.25 试管:13 mmX 100 mm，具有螺旋盖。2.2.26 Poly-prep层析柱:称取0.1g(准确至O.001 g) DEAE Sephadex A-25树脂装填入Poly-prep层析柱。柱下装好活塞，使活塞处于关闭。在每支层析柱上沿柱壁加入10mL水，打开活塞，使约2mL 水流过柱的填充料部分。关上活塞，将层析柱加盖，放置过夜，使层析柱内填充物膨润。打开活塞，依次用下列榕液淋洗层析柱使其活化:5
7、 mL O. 5 mol/L氢氧化纳溶液、10mL水、5mL乙酸纳缓冲液、10 mL水，并使排干。关上活塞，将柱加盖准备加入样品。2.2.27 Poly-prep柱双向活塞。2.2.28 层析柱盖帽。2.2.29 容量瓶，10mL 2.3 仪器设备2.3. 1 电子天平:感量O.000 1 g和0.001go 2.3.2 真空烘箱。2.3.3 恒温水浴。2.3.4 紫外/可见分光光度计。2.3.5 粉碎机或相当的设备。2.3.6 试管加热器或相当的设备。2.3.7 离心机:转速5000 r/mino 2.4 分析步骤2.4. 1 样品制备油菜籽样品经除去杂质后，用粉碎机粉碎并通过白mm固孔筛，
8、混匀。油菜籽饼柏样品直接用粉碎机粉碎并通过1mm圆孔筛，混匀。2.4.2 灭酶和提取称取0.2g样品(准确到0.001g)于13mmX 100 mm具螺旋盖i式管中。不加盖，将i式管置于加热器中于100C 120C下加热灭酶1ho在试管中加入2mL沸水，加盖泪匀，再置于加热器中于1000C 1200C下提取1ho将试管取出于室温下冷却，加入2mL常温的水，由匀并在3000 r/min下离心10mino将上清液移入10mL容量瓶内，样品再各以2mL水提取2次。将上清液合并至同一容量瓶内，用水稀释并定容至10.0mL，由匀。2.4.3 水解准确移取适量样品提取榕液(1.0 mL5. 0 mL)于已
9、制备好的层析柱(2.2.26)中，使柱排干。用3 mL Tris-HCl缓冲液沿柱壁淋洗层析柱，并使柱排干。向层析柱中加入1.0 mL黑芥子硫昔酸酶溶液，使黑芥子硫昔酸酶榕液进入柱的填充料部分，并确保使填充料的顶部仍处于酶溶液的凹液面之下。关闭活塞，在柱上加盖，置于37C恒温水浴中水解1h。2.4.4 洗脱取出层析柱，用6mL水分6次洗脱层析柱，将洗脱液收集于10.0mL容量瓶中。加入2.0mL苯酣鸽酸溶液，用水定容到10.0mL，混匀。2.4.5 测定将上述洗脱液移入离心管并于3000 r/min下离心10mino准确移取上清液1.0 mL *容液于2 SN/T 1868-2007 13 m
10、mX 100 mm试管中，加入3.0mL葡萄糖显色、溶液，加盖泪匀，置于37C恒温水浴中保温30mi口，取出，冷却至室温，用紫外/可见分光光度计在505nm波长下测定吸光度。2.4.6 标准曲线制作准确移取0，0.10，0.20，0.40，0.60，1.00 ,1. 50 , 2. 00 mL葡萄糖储备液于10mL容量瓶中，在每个容量瓶中加入2mL苯酣鸪酸试剂并用水定容。分别准确移取葡萄糖标准工作、溶液1.0 mL于13mm X 100 mm试管。在每个试管中加入3.0mL葡萄糖显色溶液并混匀，置于37C恒温水浴中保温30 mi口。将试管取出，冷却至室温，用紫外/可见分光光度计在505口m波长
11、下测定吸光度。以omL葡萄糖管作为空白校正。用葡萄糖系列标准工作溶液的校正吸光度数值作为Y轴，对应葡萄糖系列标准工作溶液中葡萄糖的量(mol)为X轴，绘制标准曲线或计算直线回归方程。2.4.7 空白试验称取0.2g样品(准确到0.001g)于13mmX 100 mm具螺旋盖i式管中，加入2mL常温的水，由匀并在3000 r/min下离心10min，将上清液移入10mL容量瓶内。同样再提取三次，将上清液合并并用水稀释定容至10.0mL，混匀。以下除不加黑芥子硫昔酸酶溶液酶解外，按照上述步骤2.4. 32. 4. 5 操作。2.4.8 结果计算与表述样品中硫代葡萄糖昔总量按式(1)计算:式中:w
12、主兰100rnXV W 样品中硫代葡萄糖昔总量，单位为微摩尔每克(mol/g) ; X 由标准曲线查得或由回归方程算得测定榕液中的葡萄糖量，单位为微摩尔(mol); m 样品质量，单位为克(g);V 用于上柱样品提取液的体积，单位为毫升(mL)3 测定低限、添加回收率3.1 测定低限油菜籽及其饼柏中硫代葡萄糖昔总量的测定低限为1mol/go3.2 添加回收率. ( 1 ) 进出口油菜籽及其饼柏中硫代葡萄糖昔总量(黑芥子硫昔酸饵)添加浓度及其回收率实验数据见表1和表20 表1油菜籽添加浓度/Cmol/g)回收率/c%) 10.0 72. 586. 4 25.0 89. 497. 6 50.0 9
13、4. 8107. 8 表2油菜籽饼柏添加浓度/Cf1 mol/ g) 回收率/C%)10.0 72. 098. 4 25.0 63. O86. 8 50.0 78. 593. 5 3 SN/T 1868-2007 Foreword This standard is proposed band is under the charge of the Certification and Accreditation Adminis tration of the Peoples Republic of China. This standard is drafted by Shanghai Entry-E
14、xit Inspection and Ouarantine Bureau of the People s Re public of China. The main drafters of this standard are Ni Xinlu and Chu Oinghua. This standard is a professional standard promulgated for the first time. 4 SN/T 1868-2007 Determination of total glucosinolates in rapeseed and its meal for impor
15、t and export 1 Scope This standard specifies the determination of total glucosinolates in rapeseed and its meal for import and export. This standard is suitable for determination of total glucosinolates in rapeseed and its meal. 2 Determination 2. 1 Principle Heat is applied to rapeseed or their mea
16、ls sample to denature natural myrosinase. Glucosinolates are extracted with boiling water and cleaned by column packed with weak anionic exchange resin. My cosinase (thioglucosidase) is applied to the column ,hydrolyzing glucose from all glucosinolates pres ent. The resulting glucose is eluted from
17、the column with water. The glucose color reagent makes the sample solution take on a pink color which absorbs light in the visible range (= 505 nm) and its col or is proportional to the amount of glucose present in the sample being tested. According to the glu cose calibration curve,calculate total
18、glucosinolates content in the sample. 2. 2 Reagents and materials Unless otherwise specified ,all the reagents used should be analytical grade;water is distilled wa ter. 2.2.1 Disodium hydrogen phosphate,anhydrous. 2.2.2 Sodium chloride. 2.2.3 Sodium tungstate. 2. 2. 4 Pheno l. 2.2.5 Hydrochloric ac
19、id. 2. 2. 6 DEAE Sephadex A25. 5 SN/T 1868-2007 2.2.7 Sodium hydroxide. 2.2.8 Glacial acetic acid. 2.2.9 Sodium acetate ,anhydrous. 2.2.10 Trizma base. 2.2.11 Myrosinase (EC 3. 2.1.147) ,200 U/g. 2.2.12 4-Aminopyrine. 2.2.13 Glucose oxidase (EC 1.1.3.4) ,20000 U/66.67 mg. 2.2.14 Peroxidase (EC 1.11.
20、1.7) ,10000 U/52. 6 mg. 2.2.15 Hydrochloric acid (2 mol/L) :Add 17.2 mL 36% hydrochloric acid into 100 mL water. 2.2. 16 Phenol tungstate reagent: Dissolve 5.0 9 sodium tungstate ,5.0 9 disodium hydrogen phos phate anhdrous and 9.0 9 sodium chloride with approximately 350 mL water in a 500 mL volume
21、tric flask. Adjust pH to 3.0 with 2 mol/L hydrochloric acid. Add 2.0 9 phenol and top up to 500 mL with water. 2.2. 17 Sodium hydroxide (0. 5 mol/U: Dissolve 20 9 sodium hydroxide with approximately 800 mL water in a 1 L volumetric flask and top up to 1 L with water. 2.2. 18 Sodium acetate buffer (0
22、. 2 mol/U: Dissolve 16. 5 9 sodium acetate anhydrous and 11.5 mL glacial acetic acid with 950 mL water in a 1 L volumetric flask. Adjust pH to 4. 9 with 2 mol/L hydrochloric acid and top up to 1 L with water. 2.2. 19 Tris-HCI buffer: Dissolve 1.6 9 trizma base with 900 mL water in a 1 L volumetric f
23、lask. Ad just to pH 7.0 with 2 mol/L hdrochloric acid and top up to 1 L with water. 2.2.20 Mrosinase solution: Dissolve 500 mg mrosinase with little water in a 10 mL volumetric flask and top up to 10 mL with water. Prepare it on the day when it is required for experiment. 2.2.21 Glucose color reagen
24、t: Dissolve 10 9 disodium hydrogen phosphate anhydrous with approxi mately 750 mL water in a 1 L volumetric flask. Add 131. 7 mg glucose oxidase to the volumetric flask and shake to mix. Dissolve 16.7 mg peroxidase and 333 mg 4-aminopyrine in a beaker with approxi mately 10 mL water. Transfer the so
25、lution to the volumetric flask and top up to 1 L with water. Store the solution in a brown bottle and refrigerate it at 40C. 6 SN/T 1868-2007 2. 2. 22 Benzoic acid. 2.2.23 Dextrose anhdrous (0 - ( + ) - glucose) standa时，purit;?:99%. 2.2.24 Glucose stock solution: Dry 0 - ( +) glucose in vacuum oven
26、at 400C for 4 hours. Take out and cool it. Weigh 1.000 0 9 dry 0一(+)glucose into a beaker,and add 1 9 benzoic acid into it in or der to preserve. then dissolve and transfer them into a 1 L volumetric flask with water and top up to 1 L with water. The concentration of glucose in the stock solution is
27、 5.56mol/L. 2.2.25 Test tubes , 13 mm x 100 mm, with screw cap. 2.2.26 Poly-prep chromatography column: Place O. 1 9 (exactly weigh to 0.001 g) DEAE Sephadex A-25 into column. Attach stopcock to the column. Fill each column with approximatel10 mL water , and run approximately 2 mL through the open s
28、topcock. Close the stopcock and cap the column , then let column packing swell overnight. Run the 5 mL O. 5 mol/L Sodium hdroxide， 10 mL wat町，5mL 0.2 mol/L Sodium Acetate Buffer and 10 mL water through the column one by one and drain the col umn every time. Dont open the stopcock and cap of the colu
29、mn until sample is applied. 2.2.27 Poly-prep column 2-way stopcocks. 2. 2. 28 Column stack caps. 2. 2. 29 Volumetric flasks , 10 mL. 2.3 Instruments and apparatus 2.3. 1 Electric balances , with the minimum weighing value of 0.000 1 9 and 0.001 9 respectivel. 2.3. 2 Vacuum oven. 2.3.3 Water bath. 2.
30、3.4 Ultraviolet/visible spectrometer. 2.3.5 Grinder or the other equivalent equipment. 2.3.6 Heat Block or the other equivalent equipment. 2.3.7 Centrifuger, rotate speed 5 000 r/min. 2.4 Procedure 2.4. 1 Sample Preparation 7 SN/T 1868-2007 Rapeseed (free from admixture) and its meal should be crash
31、ed by grinder and mixed thoroughly and pass through 1 mm round hole sieve. 2.4. 2 Enzyme denaturation and extraction Weigh approximately O. 2 9 (exact to 0.001 g) ground sample into a 13 mm x 100 mm test tube with screw cap. Place the test tube, uncapped , in a heat block at 1 OOoC .120oC for 1 h. A
32、dd 2 mL boiling water to the test tube , cap ,vortex and let the test tube stand in heat block at 1000C .120oC for an other 1 h. Remove the test tube from the heat block and cool it at room temperature. Add 2 mL room temperature water to the test tube, vortex and centrifuge it at 3 000 r/min for 10
33、min. Pour the su pernatant into a clean 10 mL volumetric flask. Extract the sample residue with 2 x 2 mL water again. Transfer the supernatant to the same 10 mL volumetric flask and top up to 10 mL with water, mix throughly. 2.4.3 Hydrolysis Accurately apply suitable aliquot of supernatant (1.0 mL.5
34、. 0 mL) to a prepared column (2.2.26) and drain the column. Rinse the column with 3 mL Tris-HCI buffer (rinsing sides of column) and drain it. Carefully add 1.0 mL myrosinase solution to the column and run it into the column packing , leaving a slight meniscus above the packing. Close the column sto
35、pcock and cap the column. Incubate the col umn in water bath at 3rC for 1 h. 2.4.4 Elution Take out and rinse the column with 6 x 1 mL water and catch the eluent in a clean 10.0 mL volumetric flask. Add 2.0 mL phenol tungstate reagent and top up to 10. 0 mL with water, mix throughl. 2.4. 5 Determina
36、tion Transfer the contents of the 10 mL volumetric flask to a centifugal tube, centrifuge at 3 000 r/min for 10 min. Accurately transfer 1.0 mL aliquot of the supernatant into a clean 13 mm x 100 mm test tube with screw cap ,add 3. 0 mL glucose color reagent into it, then cap and vortex. Incubate in
37、 water bath at 3rC for 30 min. Take out and cool it to room temperature, read absorbance of it at 505 nm with Ultraviolet/visible spectrometer. 2.4. 6 Calibration curve Accurately measure 0 ,0.10 ,0.20,0.40 ,0.60, 1.00, 1.50 and 2.00 mL of glucose stock solution into 10 mL volumetric flasks. Add 2 m
38、L phenol tungstate reagent to each 10 mL volumetric flask and top up to 10 mL with water. Accurateltransfer 1.0 mL aliquots of each glucose standard working solu tion into 13 mm x 100 mm test tubes with screw cap. Add 3.0 mL glucose color reagent into each test tube, then cap and vortex. Incubate th
39、e test tubes in water bath at 370C for 30 min. Take out and 8 SN/T 1868-2007 cool them to room tempreture. Read absorbance of them at 505 nm with Ultraviolet/visible spec trometer. Correct the absorbance by reagent blank Cthe tube with 0 mL of glucose stock solution). Plot corrected absorbance of te
40、st aliquot at 505 nm CY axis) vs. glucose (mol) (X axis) and get a linear relationship. Calculate the slope and y-intercept of the best fit line relating glucose Cmol) with absorbance. 2.4. 7 Reagent blank Weigh approximately O. 2 9 (exact to 0.001 g) ground sample into a 13 mm X 100 mm test tube wi
41、th screw cap. Add 2 mL room temperature water into the test tube, then vortex and centrifuge at 3 000 r/min for 10 min. Pour the supernatant into a clean 10 mL volumetric flask. Extract the sample residue with 3 X 2 mL water again. Transfer the supernatant to the same 10 mL volumetric flask and top
42、up to 10 mL with water. Process as 2.4.3.2.4.5 except not adding mrosinase solution to hdrolsis. 2.4.8 Calculation and expression of result The content of total glucosinolates in the sample is calculated as formula (1). 叭/二xX 100 mXV 、，F两EI/h . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
43、 where W-Value of totle glucosinolates content of original sample，mol/ X -Value of glucose content of test solution got from calibration curve or linear equation，mol; m-Value of original sample mass， V-Value of suitable aliquot of supernatant to a prepared column , m L. 3 Limit of determination and
44、recovery 3. 1 limit of determination The limit of determination of totle glucosinolates in rapeseed and its meal is 1mol/g. 3. 2 Recovery According to the experiment data, the fortifying concentrations of total glucosinolates and their re coveries in rapeseed and its meal see table 1 and table 2. 9
45、SN/T 1868-2007 Table 1-Rapeseed Fortifying concentrations/ (f1mol/g) Recovery / ( % ) 10.0 72. 586. 4 25.0 89. 497. 6 50.0 94. 8107. 8 Table 2-Rapeseed meals Fortifying concentrations/ (mol/g) Recovery / ( % ) 10.0 72. 098. 4 25.0 63. 086. 8 50.0 78. 593. 5 10 khCON-问Z中华人民共和国出入境检验检疫行业标准进出口油菜籽及其饼靠自中硫代葡萄糖昔总量的测定方法SN/T 1868-2007 -兴中国标准出版社出版北京复兴门外三里河北街16号邮政编码:100045网址电话:6852394668517548 中国标准出版社秦皇岛印刷厂印刷当咛开本880X12301/16 印张1字数19千字2007年6月第一版2007年6月第一次印刷印数1-2000 定价10.00元-兴书号:155066 2-17797 SN/T 1868-2007
