Synthesis and application of mycophenolic acid-molecularly imprinted polymer based on silica gel surface
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摘要:目的
合成一种对霉酚酸具有特异性识别的分子印迹聚合物吸附材料，用于青贮饲料中霉酚酸的净化、富集和分析检测。
方法以γ−甲基丙烯酰氧基丙基三甲氧基硅烷(γ-MPS)改性的硅胶为载体、霉酚酸酯为虚拟模板，合成硅胶表面接枝分子印迹聚合物，采用扫描电镜对制备材料进行表征，通过平衡吸附试验评价材料吸附特性。建立基于合成的分子印迹聚合物固相萃取−高效液相色谱(High performance liquid chromatography, HPLC)法检测青贮饲料中霉酚酸。
结果改性硅胶表面成功包裹一层印迹聚合物。静态吸附试验表明，印迹材料饱和吸附量为4.5 mg/g；动态吸附试验表明，该材料吸附速率快，60 min内即达到吸附平衡。基于该印迹材料作为固相萃取吸附剂建立了HPLC法，霉酚酸的回收率为76.0%~81.2%，相对标准偏差小于7%，检测限达60 μg/kg。
结论制备的硅胶表面霉酚酸分子印迹聚合物可特异性吸附霉酚酸，建立的分子印迹聚合物固相萃取–HPLC法可用于日常青贮饲料中霉酚酸的分析测定，了解青贮饲料被霉菌毒素污染的状况。研究结果可为青贮饲料的质量安全控制提供指导。
Abstract:ObjectiveA molecularly imprinted polymer adsorbent with specific recognition for mycophenolic acid was synthesized for purification, enrichment and analysis of mycophenolic acid in silage.
MethodUsing silica gel modified by γ-methacryloxy propyl trimethoxy silane (γ-MPS) as the carrier, mycophenolate mofetil as the virtual template, the surface molecularly imprinted polymers were prepared on silica gel. The prepared materials were characterized by scanning electron microscopy, and the adsorption characteristics were evaluated by equilibrium adsorption test. The solid phase extraction-high performance liquid chromatography (HPLC) method based on the synthesized molecularly imprinted polymer was established to detect the mycophenolic acid in silage.
ResultThe modified silica gel surface was successfully wrapped with a layer of molecularly imprinted polymer. The static adsorption test showed that the saturated adsorption capacity of the imprinted material was 4.5 mg/g, and the dynamic adsorption test showed that the adsorption rate of the material was fast, and the adsorption equilibrium was reached within 60 min. The HPLC method was established using the imprinted material as the solid phase extraction adsorbent. The recovery rates of mycopholanolic acid were in the range of 76.0% to 81.2%, the relative standard deviation was less than 7%, and the detection limit was 60 μg/kg.
ConclusionThe prepared mycophenolic acid molecularly imprinted polymer on the surface of silica gel can specifically adsorb mycophenolic acid. The established molecularly imprinted polymer solid phase extraction-HPLC method can be used for daily determination of mycophenolic acid in silage to know the situation of mycotoxin contamination in feed. The results can provide guidance for quality safety control of silage.
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母猪适宜的体况是保证其繁殖性能的关键，妊娠期是恢复体储的过程，泌乳期为保证泌乳量则是损失体储的过程。研究表明泌乳期体况损失过多会对下一胎次产生不利影响，当母猪采食量满足不了泌乳营养需要时, 母猪就会分解体储以满足泌乳需求，导致母猪背膘损失和体重下降，从而降低其下一胎次的繁殖性能[1]。这往往也是产生“二胎综合征”的原因之一。为研究体况损失对二胎母猪繁殖性能的影响，本研究通过测量一胎母猪的临产背膘厚及其下一胎次的开配背膘厚以计算一胎转二胎的背膘损失，来评估背膘损失对繁殖性能的影响，以期为生产应用提供参考。
1. 材料与方法
1.1 试验材料
本试验在温氏种猪公司下属的核心育种场开展，试验母猪为大白原种猪，试验从2017年7月开始，到2018年5月结束，全程测量一胎母猪的临产背膘厚及其下一胎次的开配背膘厚。
1.2 试验方法
使用超声波背膘仪(美国Renco)测量一胎母猪妊娠第110天及其下一胎次开配的P2点(最后肋骨上距背中线约6.5 cm处)背膘厚，计算母猪一胎转二胎的背膘损失，将背膘损失分为6个组，分别为掉膘≥5 mm，掉膘3~4 mm，掉膘2 mm，掉膘1 mm，不变和增膘。
1.3 数据处理
母猪分娩后收集相应的繁殖数据，包括总产仔和健仔数(初生重≥0.8 kg)，并计算无效仔率，记录初生窝重并计算初生均重。
无效仔率=(总仔数-健仔数)/总仔数×100%。
使用Excel 2013进行数据的初步整理，采用SAS 9.2统计软件对试验数据进行单因素方差分析，并用Duncan's法进行多重比较，由于母猪损耗等原因，共有666头母猪的完整数据进入后续的分析。
2. 结果与分析
2.1 母猪一胎转二胎背膘损失情况
一胎母猪临产背膘厚越高，其在产房损失的背膘就会越多，当一胎猪临产背膘厚超过16 mm时，其在产房就会出现掉膘现象，临产背膘厚达到19 mm时，背膘损失高达5 mm以上；反之，一胎母猪临产背膘越薄，其在产房往往会增膘，详情见表 1。
表 1 母猪一胎转二胎背膘损失情况mm 背膘损失/mm 数量 1胎临产
背膘厚2胎开配
背膘厚背膘厚变化 ≥5 32 19.03 13.78 -5.25 3~4 171 17.54 14.16 -3.38 2 141 16.55 14.55 -2.00 1 126 16.11 15.11 -1.00 0 105 15.97 15.97 0.00 ＜0 91 16.00 17.58 1.58 合计 666 16.67 15.16 -1.52 2.2 一胎转二胎背膘损失对母猪繁殖性能的影响
一胎转二胎背膘损失对二胎总产仔数有显著的影响，掉膘≥5和掉膘3~4 mm组总产仔数显著少于掉膘1 mm、不变和增膘组，掉膘2 mm组与其他各组差异均不显著。掉膘≥5 mm组健仔数显著少于掉膘1 mm、不变和增膘组，掉膘3~4 mm和掉膘2 mm组健仔与其他各组差异均不显著。掉膘≥5 mm组的无效仔率显著高于其他组，除掉膘≥5 mm组外，其他各组无效仔率间无显著差异。掉膘1 mm和不变组窝重显著高于掉膘3~4 mm和增膘组的，掉膘≥5 mm和掉膘2 mm组和其他各组窝重无显著差异。掉膘≥5 mm组仔猪均重显著高于掉膘3~4 mm、掉膘1 mm、不变和增膘组，掉膘2 mm组仔猪均重显著高于不变组，具体数据见表 2。
表 2 一胎转二胎背膘损失对二胎繁殖性能的影响1)背膘损失/mm 母猪/头 总仔/头 健仔/头 无效仔率/% 初生窝重/kg 初生均重/kg ≥5 32 14.59b 11.56b 21.04a 20.62ab 1.53a 3~4 171 14.89b 12.38ab 15.87b 19.11b 1.43bc 2 141 15.21ab 12.37ab 18.07ab 19.97ab 1.47ab 1 126 16.05a 12.94a 17.91ab 20.20a 1.43bc 0 105 15.70a 12.57a 18.59ab 20.95a 1.39c ＜0 91 15.97a 12.86a 18.72ab 18.92b 1.41bc 平均标准误 0.39 0.30 0.01 0.46 0.03 P 0.03 0.11 0.15 0.01 0.07 1)同列数据后凡具有一个相同小写字母者表示差异不显著(P>0.05，Duncan's法) 2.3 一胎猪临产背膘厚和背膘损失的线性回归分析
本试验数据显示，大白高产母猪在泌乳期摄入的营养物质满足不了泌乳需求，会通过损失体况来满足泌乳需要，平均背膘损失高达1.52 mm。对临产背膘(x)和背膘损失(y)进行一元线性回归分析，回归方程为y=-0.468 7x+6.279 6(图 1)。回归方程拟合度较好(R2=0.944 9)，该方程显示一胎母猪临产背膘厚与背膘损失呈高度负相关，当背膘损失为2 mm时，临产背膘厚为17.67 mm，为了避免因产房背膘损失过大而带来的不良后果，一胎母猪的临产背膘厚不宜超过18 mm。
3. 结论与讨论
经过多年的选育，大白母猪的繁殖性能已得到了极大的提高，窝产总仔和健仔数显著增多，但在母猪育种过程中，母猪泌乳期采食量并没有得到相应提高，为了满足仔猪生长发育的需要，母猪需要更多的营养物质来维持泌乳，泌乳期往往会造成体况的损失。体况包含体蛋白和体脂肪，由于体蛋白不易测量，所以通常用背膘(体脂肪)来代表体况，背膘反映的是母猪在不同生理阶段的体况和能量储备情况，母猪背膘变化与繁殖性能密切相关。研究表明母猪泌乳期体重损失很小时，不会影响母猪下一胎次的繁殖性能，但当母猪体重损失超过其体重的10%时，将会延长母猪下一胎次断奶到发情的时间间隔、阻碍卵母细胞发育和降低排卵率[2-3]。
本研究表明，一胎转二胎背膘损失对二胎母猪总产仔数有显著的影响，掉膘≥5 mm和掉膘3~4 mm组的总产仔数少于15头且显著低于掉膘1 mm、不变和增膘组，掉膘2 mm组总产仔数虽与其他各组差异不显著，但掉膘2 mm组与掉膘1 mm相比总产仔数有减少的趋势。一胎母猪在产房有一定的背膘损失是正常而且可以接受的，当背膘损失超过2 mm时，意味着在二胎开配前的母猪体况储备不足，会导致母猪排卵数减少，从而降低二胎的总产仔数。从健仔数来看，虽然掉膘3~4 mm组的总产仔数较少，但其无效仔率是最少的，说明其健仔数保持在较高的水平，可能是因为其弱仔死胎数比较少，具体原因还有待进一步的研究。初生窝重和均重的差异主要是由于总产仔数的差异导致的，掉膘1 mm、不变和增膘组仔猪重均无显著差异。
线性回归分析结果表明，为了避免因产房背膘损失过大而带来的不良后果，一胎母猪的临产背膘厚不宜超过18 mm。这提示在生产上，减少泌乳母猪体重和背膘损失是饲养母猪的重要目标，一胎母猪在妊娠期背膘增长不能过多，临产背膘厚不宜超过18 mm，同时在泌乳期要提高母猪的采食量以减少背膘损失，确保背膘损失不超过2 mm，以保证下一胎次的繁殖性能。
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图 1 不同单体种类(A)和不同模板单体摩尔比(B)对印迹因子的影响
各小图折线图上的不同小写字母表示处理间差异显著(P<0.05，Duncan’s法)
Figure 1. The influence of different monomer types (A) and different mole ratios of template to monomer (B) on impringting factor
Different lowercase letters on line chart in each figure indicate significant differences among treatments (P<0.05, Duncan’s method)
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图 2 活化硅胶(A)、SiO2-MPS (B)、SiO2-MPS@MIP(C)和SiO2-MPS@NIP(D)的扫描电镜图
Figure 2. Scanning electron microscope images of activated silica gel (A), SiO2-MPS (B), SiO2-MPS@MIP (C) and SiO2-MPS@NIP (D)
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图 3 SiO2-MPS@MIP和SiO2-MPS@NIP的静态(A)和动态(B)吸附曲线
Figure 3. Static (A) and dynamic (B) adsorption curves of SiO2-MPS@MIP and SiO2-MPS@NIP
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图 4 不同上样溶液(A)及不同pH水(B)对霉酚酸回收率的影响
Figure 4. The influences of different loading solutions (A) and different pH water (B) on mycophenolic acid recovery rate
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图 5 不同淋洗溶液(A)和洗脱溶液(B)对霉酚酸回收率的影响
a：1% ($\varphi $)甲醇溶液；b：5% ($\varphi $)甲醇溶液；c：10% ($\varphi $)甲醇溶液；d：20% ($\varphi $)甲醇溶液；e：10% ($\varphi $)乙腈溶液；f：20% ($\varphi $)乙腈溶液；g：1% ($\varphi $)乙酸甲醇溶液；h：2% ($\varphi $)乙酸甲醇溶液；i：5% ($\varphi $)乙酸甲醇溶液；j：8% ($\varphi $)乙酸甲醇溶液
Figure 5. The influences of different washing solutions (A) and elution solutions (B) on mycophenolic acid recovery rate
a: 1% ($\varphi $) methanol in water; b: 5% ($\varphi $) methanol in water; c: 10% ($\varphi $) methanol in water; d: 20% ($\varphi $) methanol in water; e: 10% ($\varphi $) acetonitrile in water; f: 20% ($\varphi $) acetonitrile in water; g: 1% ($\varphi $) acetic acid in methanol; h: 2% ($\varphi $) acetic acid in methanol; i: 5% ($\varphi $) acetic acid in methanol; j: 8% ($\varphi $) acetic acid in methanol
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图 6 过分子印迹固相萃取柱前(A)和经分子印迹固相萃取柱净化后(B)的加标青贮饲料(2 000 μg/kg)以及相应的标准溶液(C)HPLC-UVD色谱图
Figure 6. HPLC-UVD chromatograms of the spiked silage (2 000 μg/kg) before (A) and after (B) purification by the molecularly imprinted polymer solid phase extraction column and the corresponding standard solution (C)
表 1 乙腈溶液中不同三氯甲烷添加比例对SiO2-MPS@MIP和SiO2-MPS@NIP吸附量的影响
Table 1 The influence of different addition proportions of chloroform in acetonitrile on the adsorption capacity of SiO2-MPS@MIP and SiO2-MPS@NIP
φ(三氯甲烷)/%
Chloroform
content吸附量/(mg·g−1)
Adsorption capacity印迹因子
Impringting factorSiO2−MPS
@MIPSiO2−MPS
@NIP10 2.9 1.8 1.6 20 5.9 4.2 1.4 30 4.7 4.4 1.1 40 4.6 4.7 1.0 50 4.7 4.7 1.0 
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表 2 空白样品中霉酚酸的加标回收率及相对标准偏差(RSD)
Table 2 Recovery rates of spiked mycophenolic acid and the relative standard deviation (RSD) in the blank sample
w/(μg·kg−1)
Spiked日内(n=5) Intraday 日间(n=15) Interday 回收率/%
Recovery rateRSD/% 回收率/%
Recovery rateRSD/% 200 81.2 3.7 79.2 4.8 2 000 79.6 3.3 76.0 4.3 8 000 78.8 6.6 77.3 6.4
下载: 导出CSV
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