不同处理方式对红肉中N-羟乙酰神经氨酸解离的影响

应用不同方法处理红肉(猪肉与牛肉),研究能够有效解离红肉中N-羟乙酰神经氨酸(N-glycolylneuraminic acid,Neu5Gc)的预处理方式。将红肉通过水煮、微波加热和有机酸腌制等不同方式进行处理,利用酸水解法释放红肉中的两种唾液酸成分Neu5Gc和N-乙酰神经氨酸(N-acetylneuraminic acid,Neu5Ac),经1,2-二氨基-4,5-亚甲基二氧苯(1,2-diamino-4,5-methylenedioxybenzene,DMB)衍生化后用高效液相色谱检测其含量;另外还采用β-半乳糖苷酶水解红肉,再测定水解液中的Neu5Gc和Neu5Ac。结果表明:沸水浴处理红肉后其Neu5Gc和Neu5Ac都有一定程度的解离,水煮时间越长,红肉中Neu5Gc和Neu5Ac解离效果越好,且猪肉中的Neu5Gc相比牛肉的更容易水煮解离。采用微波炉高温处理红肉也能够解离Neu5Gc,但是解离率不超过70.0%,且处理时间影响较小;而Neu5Ac的解离率都低于Neu5Gc。红肉通过不同的弱有机酸腌制处理后Neu5Gc和Neu5Ac解离率差别不大,且醋酸腌制处理后Neu5Gc解离效果较好。另外,β-半乳糖苷酶能有效解离猪肉中Neu5Gc,水解时间越长,解离率越高。对于牛肉,Neu5Gc解离率最高为84.0%,解离效果不如猪肉。     

不同加工处理方式对牛肉中Neu5Gc解离的影响

研究不同红肉及加工肉制品的N-羟乙酰神经氨酸（N-glycolylneuraminic acid,Neu5Gc）含量,不同油炸温度对牛肉中Neu5Gc含量的影响,不同蒸煮时间对牛肉汤中Neu5Gc含量的影响以及不同酶制剂对Neu5Gc的解离效果并从中筛选出能够解离Neu5Gc的酶进行进一步探究。结果表明,牛肉中的Neu5Gc含量最高为（58.45±0.98）μg/g。当油炸温度达到150?℃时,牛肉中Neu5Gc的损失随着温度的升高而增大。随着蒸煮时间的延长,牛肉汤中Neu5Gc的含量逐渐增加。此外,研究结果表明菊粉酶对Neu5Gc有解离作用,且通过正交试验获得菊粉酶作用于Neu5Gc标准品的最适条件为水浴时间30?min、水浴温度50?℃、菊粉酶添加量为质量分数0.8%,对Neu5Gc标准品的解离率可达到（50.52±0.88）%。但是,由于牛肉基质成分复杂,筛选出的菊粉酶最适作用条件作用于牛肉时,解离率仅为（7.29±2.67）%。本实验旨在为人们的日常饮食提供科学指导,并为后续开展红肉中Neu5Gc安全、稳妥的解离方法提供实验依据。     

肉制品中唾液酸含量和形态分析及在加工中的变化

本文主要运用了高效液相色谱法测量了我国牛肉、猪肉等常见肉类及常见熟食制品如火腿、香肠、卤牛肉、风干羊肉、香辣鸭脖中的两种唾液酸N-羟乙酰神经氨酸（N-Glycolylneuraminic acid,Neu5Gc）、N-乙酰神经氨酸（N-Acetylneuraminic acid,Neu5Ac）的含量、存在形式以及不同加工方式对其含量及存在形式的影响。结果表明红肉中Neu5Gc含量：牛肉46.57±2.53 μg/g>猪肉28.69±1.03 μg/g>羊肉29.09±2.32 μg/g,红肉中同时含有结合态Neu5Gc和游离态Neu5Gc,结合态Neu5Gc含量普遍高于游离态Neu5Gc。红肉中Neu5Ac含量：羊肉200.15±24.96 μg/g>猪肉110.89±5.71 μg/g>牛肉80.97±5.60 μg/g。白肉中不含Neu5Gc,鸭肉的Neu5Ac含量最高,为185.73±23.11 μg/g。熟食肉制品中,牛肉制品的Neu5Gc含量仍高于其他红肉熟食制品,并在白肉熟食制品中检测出了Neu5Gc的存在。对红肉进行蒸煮、油炸及腌制处理后Neu5Gc和Neu5Ac含量都有所下降,其中经过油炸处理的样品Neu5Gc和Neu5Ac含量下降最为明显,其次是腌制、蒸煮。本文还研究了蒸煮时间对猪肉中唾液酸含量的影响,即随着蒸煮时间的增加,Neu5Gc、Neu5Ac含量变化的总体趋势减少,蒸煮45 min后结合态唾液酸明显减少,游离态增多。即红肉中特异性含有Neu5Gc,白肉类熟食制品中也可能含有Neu5Gc,通过加工可改变肉制品中唾液酸的含量和形式,但是每种加工方式对于唾液酸含量及形式的改变不同。     

红肉中N-羟乙酰神经氨酸在人体中的代谢途径及危害

唾液酸(Sialic acid,Sia)是一类含有9个碳骨架的酸性糖类,在胚胎和中枢神经系统发育、细胞识别、黏附、分化、信号传导等多方面具有重要的生理意义。N-羟乙酰神经氨酸(N-glycolylneuraminic acid,Neu5Gc)是一种含有9个碳原子并具备吡喃糖结构的酸性氨基糖。Neu5Gc作为唾液酸中的一种比较特殊的存在,与人类癌症以及动脉粥样硬化、2型糖尿病等疾病的发生发展有着密切联系。该文主要阐述了Neu5Gc的基本结构以及在人体的来源、代谢、吸收和蓄积的过程,并介绍了红肉特异性致病理论及Neu5Gc的危害。     

红肉、N-羟乙酰神经氨酸与癌症关系的研究进展

世界癌症研究基金会报告指出,过多摄入红肉会增加患癌症的风险,红肉可能是导致某些癌症的原因之一。有研究得出,摄入红肉后身体内产生的一种唾液酸--N-羟乙酰神经氨酸（N-glycolylneuraminic acid,Neu5Gc）可能与癌症的发生有关。本文从红肉与癌的关系,引出对Neu5Gc的讨论,从它的结构、来源、生理功能到临床应用价值,最后是与癌症可能的机制讨论,并重申它的研究价值。     

高效液相色谱-荧光检测法测定发酵红肉制品中Neu5Gc

基于高效液相色谱-荧光检测(HPLC-FLD)技术,建立红肉发酵及制品中N-羟乙酰神经氨酸(N-glycolylneuraminic acid,Neu5Gc)的定量分析方法。成功搭建了以酸水解释放试样中的Neu5Gc,4,5-亚甲二氧基-1,2-邻苯二胺盐(4,5-methylenedioxy-1,2-phenylenediamine dihydrochloride,DMB)为衍生化试剂,并采用安捷伦ZORBAX Eclipse XDB-C18色谱柱等系列条件的HPLC-FLD分析检测方法。该方法经验证:Neu5Gc在25～400μmol/L范围内与Neu5Gc峰面积的线性关系良好,R2=0.999 41;样品加标平均回收率在96.4%～99.07%之间;RSD为1.2%,重复性的RSD为1.5%;仪器检出限为0.001μmol/L,定量限为0.003μmol/L。检测发酵红肉样品中Neu5Gc的质量分数在9.59～20.08μg/g之间,不同的发酵红肉制品Neu5Gc的含量之间均存在显著性差异(p0.05)。本方法能有效分离Neu5Gc目标峰(分离度R1.5)、操作简单、重复性好、灵敏度和精密度高,可广泛用于发酵红肉制品中Neu5Gc的定量测定。     

高效液相色谱-荧光检测法测定红肉及其加工肉中唾液酸含量

采用高效液相色谱-荧光检测法对红肉及其加工肉中两种唾液酸N-乙酰神经氨酸（N-acetylneuraminic acid,Neu5Ac）和N-羟乙酰神经氨酸（N-glycolylneuraminic acid,Neu5Gc）进行定性和定量分析。利用单因素试验对衍生化与样品酸解条件进行优化,并借助超声缩短酸解时间。结果表明,以盐酸作为酸解试剂,超声辅助酸解30 min,4,5-亚甲二氧基-1,2-邻苯二胺盐作为衍生化试剂,衍生化试剂浓度为13 mmol/L,样品与衍生化试剂比值为1∶1,衍生化时间120 min时,检测效果最佳。Neu5Ac和Neu5Gc在0.1～10 μg/mL范围内线性良好,回收率为91.2%～119.7%,检出限分别为0.003 mg/kg和0.01 mg/kg,重复性的相对标准偏差（relative standard deviation,RSD）为0.7%～1.8%,精密度RSD分别为1.4%和1.2%。本方法具有灵敏度高、分析时间短、重复性及准确性好等特点。     

9种鱼卵中的游离态及结合态唾液酸含量的分析比较研究

从不同品种鱼卵中提取出游离态唾液酸和总唾液酸，采用DMB试剂对这2种唾液酸分别进行衍生化标记，再利用高效液相色谱(HPLC)系统性对比分析了9种鱼卵中N-乙酰神经氨(Neu5Ac)和N-羟乙酰神经氨酸(Neu5Gc)的存在形式及含量的差异。结果表明，游离态和结合态的Neu5Ac在鲫鱼鱼卵中含量最高，每克鱼卵干重中分别含1.61μg和4.07μg;Neu5Gc仅存在于黄花鱼、鳜鱼和鲂鱼的鱼卵中，其中鲂鱼鱼卵中游离态Neu5Gc含量最高，每克鱼卵干重中含0.57μg；而鳜鱼鱼卵中结合态Neu5Gc含量最高，每克鱼卵干重中含0.24μg。以上结果为鱼卵的深度开发利用提供理论依据。     

传统中式火腿加工过程中N-羟乙酰神经氨酸的动态变化

为探究传统中式火腿加工过程中不同形态N-羟乙酰神经氨酸（N-glycolylneuraminic acid，Neu5Gc）之间的关系，采用高效液相色谱-荧光检测（high performance liquid chromatography-fluorescence detector，HPLC-FLD）法测定样品中总Neu5Gc、游离态Neu5Gc、结合态Neu5Gc的含量。结果表明：上述3 种形态的Neu5Gc含量随着加工时间的延长均呈前期上升后期降低的趋势；浅层肌肉半膜肌和深层肌肉股二头肌中总Neu5Gc和结合态Neu5Gc的含量变化规律相似，因水分含量降低程度的不同而使股二头肌的变化规律滞后。发酵后期水分损失幅度变缓，股二头肌中Neu5Gc解离速率高于半膜肌；新鲜猪后腿原料中游离态Neu5Gc含量极少，甚至低于检测限，结合态Neu5Gc含量与总Neu5Gc含量（15.00～30.62 μg/g）接近。发酵半年、1 年和2 年的火腿中总Neu5Gc含量分别为（15.09±0.39）、（14.52±0.38）、（28.30±0.43）μg/g，均与猪后腿原料中的Neu5Gc含量相近。为进一步探究Neu5Gc的变化，对样品进行冷冻干燥处理以避免水分含量变化对Neu5Gc含量的影响，结果表明，随着加工时间的延长，总Neu5Gc、结合态Neu5Gc的含量逐渐降低，且发酵期降低明显，具有显著差异性（P＜0.05）；游离态Neu5G变化无明显规律，但与空白样品相比，其含量呈增加趋势。     

羟自由基与Neu5Gc抽氢反应的理论动力学研究

红肉风险物质N-羟乙酰神经氨酸（Neu5Gc）可诱发炎症，但与食品消费代谢和加工贮藏过程相关的羟自由基（OH·）互作机制未知。体外OH·模拟试验表明过氧化氢（H2O2）添加量2%，254 nm紫外灯15 处理60 min时对Neu5Gc标准品含量降低效果最佳为76.23%±2.17%。为探究该过程机制，首先在气相、水和苯相下以M062X/6-31+G（d, p）理论水平对OH·作用Neu5Gc分子中羟基氢和非羟基氢抽提反应的各驻点进行优化和焓值校正，M062X/def2TZVP水平计算能垒。其次，298 K~453 K内以过渡态理论和三参数阿伦尼乌斯方程分别获得反应速率和动力学参数。结果表明：气相和苯相下Neu5Gc中H24位抽氢能垒最低，分别为13.20 kJ/mol和19.54 kJ/mol；水相下H 29位能垒最低6.25 kJ/mol；气相下活化能最低为H 39位8.09 kJ/mol，苯相下最低为H 38位1.72 kJ/mol，水相最低为H 24位14.3 kJ/mol；273到453 K间，Neu5Gc分子中非羟基氢处抽氢速率最大可高于羟基氢108倍。综上，OH·主要通过对Neu5Gc非羟基氢原子的氢抽提反应进行相互作用。     

Determination of sialic acids in infant formula by chromatographic methods: a comparison of high-performance anion-exchange chromatography with pulsed amperometric detection and ultra-high-performance liquid chromatography methods

Sialic acid determination in an infant formula presents many challenges, including efficient sialic acid release from glycoconjugates, effective sample preparation, and rugged chromatography. This work compares 2 chromatographic assays developed for determination of sialic acids in infant formula. Prior to chromatography, both assays release sialic acids by acid hydrolysis and treat the hydrolysate with a subsequent anion-exchange sample preparation. Both high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) and fluorescence ultra-high-performance liquid chromatography (UHPLC) sample analysis methods were evaluated to compare assay performance and convenience. Calibration ranges were chosen to encompass the expected amounts of 2 sialic acids in infant formula: N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc). Response was linear by either method with coefficients of determination of 1.00 by HPAEC-PAD between 5.0 and 100pmol of Neu5Ac and between 0.34 and 6.8 pmol of Neu5Gc and >0.99 by UHPLC between 5.0 and 260 pmol of Neu5Ac and between 0.20 and 9.8 pmol of Neu5Gc. Both methods had sufficient sensitivity to determine these sialic acids in infant formula. Three infant formulas were analyzed to evaluate accuracy and precision of the assays. The HPAEC-PAD assay was found to be faster overall and the UHPLC assay was more sensitive. Reaction efficiency, and therefore sensitivity, was dependent on the sample matrix. This work illustrates sample-specific complexity that must be considered in choosing an assay.     

Comparison of spectrophotometric and HPLC methods for determining sialic acid in infant formulas

Two methods for determining sialic acid in infant formulas – spectrophotometry and HPLC with fluorescence detection – have been optimised and validated, the first one allows to determine total sialic acid while the second allows to differentiate the two main forms of sialic acid (N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc)). A common sample preparation procedure (hydrolysis and purification) for both methods has been proposed. The linearity (from 6 to 150 μg of total sialic acid in the assay for spectrophotometry, and from 12.5 to 250 ng and 1 to 5 ng of Neu5Ac and Neu5Gc, respectively, for HPLC) is adequate. The detection and quantification limits (0.29 and 0.97 mg of total sialic acid/L of reconstituted sample, respectively, for spectrophotometry, and 0.03 and 0.08 mg Neu5Ac/L; 0.003 and 0.009 mg Neu5Gc/L of reconstituted sample, respectively, for HPLC) are low enough for the determination of sialic acid in infant formulas. The precision of both methods, expressed as relative standard deviation, is less than 6%, and the accuracy evaluated by recovery assays show 104% recovery for spectrophotometry; 95% for Neu5Ac and 109% for Neu5Gc for HPLC. Samples analysed show no significant differences (α < 0.05) attributable to the method used; consequently, both of them could be applied after common sample preparation, the choice of technique depending on the facilities available in the laboratory.     

Exposure to heterocyclic aromatic amines from the consumption of cooked red meat and its effect on human cancer risk: A review

This review covers the bibliographic data from the last 10 years on the possible carcinogenicity of heterocyclic aromatic amines (HAAs) in humans. Aspects such as red meat intake, cooking methods applied to red meat, and doneness of cooking are discussed from an epidemiological point of view. The role in the carcinogenicity of the HAAs has been assigned to two main factors: first, the very high frequency of consumption of red meat; and, second, very darkly browned meats from cooking. However, there are some uncertainties associated with epidemiological results such as the presence of other carcinogens, co-carcinogens and anti-carcinogens in the diet, analytical results on the content of HAAs in foods, food frequency questionnaires, and mainly genetic susceptibility to HAAs. It is concluded that there is not sufficient scientific evidence to support the hypothesis that human cancer risk is due specifically to the intake of HAAs in the diet.     

Exposure to heterocyclic aromatic amines from the consumption of cooked red meat and its effect on human cancer risk: A review

This review covers the bibliographic data from the last 10 years on the possible carcinogenicity of heterocyclic aromatic amines (HAAs) in humans. Aspects such as red meat intake, cooking methods applied to red meat, and doneness of cooking are discussed from an epidemiological point of view. The role in the carcinogenicity of the HAAs has been assigned to two main factors: first, the very high frequency of consumption of red meat; and, second, very darkly browned meats from cooking. However, there are some uncertainties associated with epidemiological results such as the presence of other carcinogens, co-carcinogens and anti-carcinogens in the diet, analytical results on the content of HAAs in foods, food frequency questionnaires, and mainly genetic susceptibility to HAAs. It is concluded that there is not sufficient scientific evidence to support the hypothesis that human cancer risk is due specifically to the intake of HAAs in the diet.     

牛免疫球蛋白G的体外人源唾液酸化及Fc片段的制备

建立将牛免疫球蛋白G (bovine immunoglobulin G,bIgG)糖链末端N-羟乙酰神经氨酸酶切并连接人源N-乙酰神经氨酸(N-acetylneuraminic acid,Neu5Ac)的方法,在实现bIgG转化为人源IgG (human IgG,hIgG)的基础上,研究hIgG可结晶(Fc)片段的制备...     

高效液相色谱法测定母乳中唾液酸含量

建立荧光高效液相色谱(fluorescence detector-high performance liquid chromatography,HPLC-FLD)测定母乳中唾液酸N-乙酰神经氨酸(N-acetylneuraminic acid,Neu5Ac)和N-羟乙酰神经氨酸(N-glycolyl neuraminic acid,Neu5Gc)含量的分析方法。利用酸水解法释放出母乳中的唾液酸,以4,5-亚甲二氧基-1,2-邻苯二胺盐(4,5-methylenedioxy-1,2-phenylenediamine dihydrochloride,DMB)为衍生化试剂,50℃避光衍生150min,采用荧光高效液相色谱仪检测。色谱条件：LiChrosorb RP-18柱(250mm×4mm,5μm),流动相为甲醇-乙腈-超纯水(7:8:85),流速0.9mL/min,进样体积10μL,柱温30℃,荧光检测器激发波长373nm,发射波长448nm。结果表明：唾液酸在50～400μmol/L范围内与唾液酸峰面积的线性关系良好,平均回收率为94.0%,精密度的相对标准偏差(relative standard deviation,RSD)为0.4%,稳定性RSD为1.0%,重复性RSD为0.8%,Neu5Ac的最低检出限为0.02μmol/L,Neu5Gc的最低检出限位0.03μmol/L。该方法简单、重复性好、灵敏度高,可广泛用于奶粉、牛奶及母乳中唾液酸含量测定。