《食品中低聚糖的测定 第1部分：母乳低聚糖含量的测定》行业标准（征求意见稿）编制说明

《食品中低聚糖的测定第1部分：母乳低聚糖含量的测定》行业标准编制说明（征求意见稿）2023年5-11月，开展标准制定的前期工作，查阅了大量国内外文2024年11月召开第二次标准工作会议，讨论标准草案，组织实验室间比糖含量的测定方法。其中第一法为液相色谱-（2’-FL）、3-岩藻糖基乳糖（3-FL）、乳糖-N-四糖（LNT）起草组对比分析了国内外食品中母乳低聚糖含量测定方法（具体详见附表液相荧光测定方法和3种母乳低聚糖含量的离子色谱测定方法进行了方法优化HMOs的化学结构中都含有强极性的羟基基团，这些羟基注：IS：甘露三糖内标。图26种HMOs在酰胺色谱柱（a）和HILIC-Amide色谱柱（b）中分离效果1.1.2衍生试剂的选择光信号，与2-AA相比，2-AB的衍生产物响应值显著增强，且衍生产物具有更a.未衍生；b.2-AA；c.2-AB。图32-AA和2-AB衍生效果比较Fig.3Comparisonof2-AAand2-ABderivedeffects图4流动相的选择Fig.4Selectionofmobilephase因此，消除麦芽糊精的干扰对于确保试验结果的准确性至关重要。如图5和6a.麦芽糊精；b.加标样品；c.DFM-1；d.DFM-2；e.DFM-3；f.DFM-4；IS.甘露三糖内标。图5含麦芽糊精样品的处理图6种HMOs是与混合标准溶液峰面积之比；麦芽糊精色谱峰是与样品＋标中总麦芽糊精的峰面积之比。图6酶解前后百分比对比2.2.2FOSa.加标样品；b.加标酶解后样品；IS：甘露三糖内标。图7奶粉酶解前后对比图Fig.7Comparisonofinfantformulabeforeandafterenzymaticdigestiona.混标；b.酶解后混标；IS：甘露三糖内标。图8果聚糖酶酶解前后混标的对照图Fig.8Controldiagramofmixedlabelsbeforeandafterfructokinasedigestion2.2.3GOS干扰的去除、β-半乳糖苷酶酶解率及其适用性判别（1）GOS干扰的去除图9β-半乳糖苷酶酶解LNT和LNnT图Fig.9LNTandLNnTcleavagebyβ-galactosidaseFigure23～33min仍存在GOS干扰，影响甘露三糖、3,-SL、LNT、LNnT和6,-SL图10奶粉的酶解色谱图Fig.10Enzymaticchromatographyofinfantformulaa.样品＋RTM-1、样品＋RTM-2、样品＋RTM-3；b.样品＋RTM-4、样品＋RTM-5、样品＋RTM-6；c.样品＋RTM-7、样品+RTM-8、样品＋RTM-9。图11酶解前后百分比对比Fig.11Percentagecomparisonbeforeandafterenzymaticdigestion图12LNnT酶解产物标准曲线Fig.12StandardcurveofLNnTenzymaticproductsa.SCFF-HMOs-IF-05；b.SCFF-HMOs-IF-06；c.SCFF-HMOs-IF-16；d.SCFF-HMOs-IF-29；e.SCFF-HMOs-IF-32；f.SCFF-HMOs-IF-35。IS：甘露三糖内标。图13奶粉酶解前后对比图由于商品化的β-半乳糖苷酶试剂含有的其他酶可能对乳糖-N-四糖有一定乳糖-N-四糖（酶解前）乳糖-N-四糖（酶解后）移取200µL乳糖-N-四糖标准溶液于2.0mL离心管中加入200µL醋酸钠缓冲液，混匀并密封加入150µL醋酸钠缓冲液和50µLβ-半乳糖苷酶溶液，混匀并密封置于60℃±2℃的水浴锅中，孵育60min，随后沸水浴2min冷却至室温后，加入100µL内标溶液，混匀按照4.6.5和4.6.6衍生和净化后，按照4.6.7进行测定记录内标（AIS）、乳糖-N-四糖（A1）的峰面记录内标（AIS’）、乳糖-N-四糖（A1’）的峰面积，以及酶解产物的保留时间。表26种HMOs线性范围、线性方程、决定系数Table2Linearityofcalibration,linearequations,andcoefficientsofdeterminationfor6HMOs线性方程R22,-FL0.35～20.00y＝4×106x－4344313-FL0.35～20.00y＝3×106x＋773463,-SL0.14～8.00y＝2×106x＋679246,-SL0.14～8.00y＝2×106x＋59569LNT0.23～13.33y＝1×106x－88977LNnT酶解产物0.23～13.33y＝3×106x＋142556图146种HMOs混合标准溶液的色谱图表36种HMOs的分离度2,-FL3-FL3,-SL6,-SLLNTLNnT酶解产物分离度2.122.517.882.019.17选取奶粉样品进行加标回收率试验作为评价标准来考察方法的正确度，表4正确度试验结果浓度水平HMOs检测含量（g/100g）HMOs含量平均值加标回收率/%1232,-FL空白—1.962.032.01—2.18低浓度2.702.702.702.70101.530.12中浓度1.353.323.283.393.3397.311.66高浓度2.034.124.114.10103.290.513-FL空白—0.220.230.22—1.56低浓度0.110.330.330.330.3395.910.52中浓度0.23104.420.16高浓度0.570.57103.621.233,-SL空白—0.120.120.120.12—1.27低浓度0.190.190.190.19中浓度0.15104.450.22高浓度0.230.350.350.35101.741.766,-SL空白——1.72低浓度0.35中浓度1.26高浓度0.110.110.110.11104.81LNT空白—0.100.100.100.10—1.26低浓度0.180.170.170.18103.42中浓度0.150.25105.03高浓度0.230.320.320.330.321.88解产物空白—0.310.310.310.31—0.73低浓度0.1597.201.88中浓度96.550.55高浓度0.731.73注：—.未添加HMOs或未进行加标回收率测定。表5重复性试验RSD%1234562,-FL1.311.311.331.321.321.323-FL0.210.220.220.723,-SL0.190.190.190.190.190.190.316,-SL0.140.140.140.140.140.140.41LNT0.160.170.170.170.170.161.76LNnT酶解产物2.3.5检出限和定量限表6检出限和定量限参数2,-FL3-FL3,-SL6,-SLLNTLNnT酶解产物检出限/（g/100g）定量限/（g/100g）0.02140.01200.01440.01750.0128表8奶粉样品检测Table8Infantformulasampletesting样品编号含HMOs样品的标测定的HMOs与标签添加量的相对相差/%SCFF-HMOs-IF-012,-FL0.510.510.51±0.030.530.150.150.150.15±0.000.16SCFF-HMOs2,-FL0.730.75±0.11SCFF-HMOs2,-FLSCFF-HMOs2,-FL0.61SCFF-HMOsSCFF-HMOsSCFF-HMOsSCFF-HMOsSCFF-HMOsSCFF-HMOs-IF-10SCFF-HMOs-IF-112,-FL0.750.77±0.160.350.350.35±0.070.015SCFF-HMOs-IF-122,-FL0.0190.190.190.180.19±0.030.19SCFF-HMOs-IF-132,-FL0.0210.170.180.180.18±0.020.18SCFF-HMOs-IF-142,-FL0.350.350.370.35SCFF-HMOs-IF-152,-FL0.710.710.710.71±0.010.0150.210.210.210.21±0.01SCFF-HMOs-IF-162,-FL0.67±0.110.190.190.190.19±0.010.19SCFF-HMOs-IF-172,-FL1.791.701.761.75±0.301.760.1520.370.37±0.07SCFF-HMOs2,-FL1.581.591.591.59±0.061.650.148样品编号含HMOs样品的标测定的HMOs与标签添加量的相对相差/%-IF-180.350.350.350.35±0.030.41SCFF-HMOs-IF-192,-FL1.531.531.561.54±0.101.650.1660.350.41SCFF-HMOs-IF-202,-FL0.811.730.1660.350.34±0.030.41SCFF-HMOs-IF-212,-FL1.621.611.601.61±0.091.660.330.410.162SCFF-HMOs-IF-222,-FL1.601.621.641.62±0.161.660.350.410.129SCFF-HMOs-IF-232,-FL0.770.770.77±0.02SCFF-HMOs-IF-242,-FL0.100.100.187SCFF-HMOs-IF-252,-FL0.02±0.110.157SCFF-HMOs-IF-262,-FL0.230.230.23±0.020.180.258SCFF-HMOs-IF-272,-FL0.81±0.080.350.148SCFF-HMOs-IF-282,-FL0.170.170.170.17±0.010.1423-FL0.270.27±0.010.160.7133,-SL0.110.110.110.11±0.016,-SLLNT0.20±0.011.375SCFF-HMOs-IF-292,-FL0.87±0.11SCFF-HMOs-IF-302,-FLSCFF-HMOs-IF-312,-FLSCFF-HMOs-IF-322,-FL0.95±0.111.300.2733-FL0.330.330.33±0.010.353,-SL0.150.150.150.15±0.010.7416,-SL0.13LNTSCFF-HMOs-IF-332,-FL0.710.69±0.140.73具体选择哪种色谱柱，还需根据实际样品检测的分离效果来决定。色谱柱89.583617.29210.53568.333151.8759.5838.60745.00021.458通过对比分析，磺基水杨酸、乙腈和15%亚铁氰化钾+30%乙酸新作为沉淀404.3751.875注：a：加标样品；b：DFM-1；c：DFM-2；d：DFM-3；e：DFM-4。注：a：加标样品；b：加标酶解后样品。本试验以添加了GOS的奶粉作为研究对象，首先采用PA20色谱柱进行分析，并在样品注：a：HMOs；b：RTM-1；c：RTM-2；d：RTM-3；e：RTM-4；f：RTM-5；g：RTM-6；h：RTM-7；i：RTM-8；j：RTM-9。注：a：加标样品；b：RTM-1；c：RTM-2；d：RTM-3；e：RTM-4；f：RTM-5；g：RTM-6；h：RTM-7；i：RTM-8；j：RTM-9。HMOs22’-FL1.0～100.0y=0.7214x+2.26550.99163-FL1.0～100.0y=0.4024x+0.72500.99700.5～50.0y=0.6635x+0.54810.9971称取0.50g（精确至0.01g）固体样品于50mL离心管中，加入20.00g水溶解后，盖盖密封，并在70℃水浴下提取20～30min，冷mL，收集后续的洗脱液待测。如表3-7和图3-17所示，3种母乳低聚糖的分离2ı-FL3-FL10.1910.2011.42HMOs(g/100g)浓度(g/100g)(g/100g)1232’-FL30.8531.2230.7930.950.75HMOs(g/100g)浓度(g/100g)(g/100g)12345.6846.7246.1046.17101.453063.4260.9262.2062.18104.092.013573.6174.3774.3074.0995.870.573-FL35.0235.5835.3035.300.8017.554.8854.4655.1154.82111.530.593570.7870.2168.9469.9899.0952.591.6391.4585.8289.6310