Covalent interactions between proteins and oxidation products of caffeoylquinic acid (chlorogenic acid)

BACKGROUND: The interactions between phenolic compounds and proteins can modify protein properties important in the food industry. To understand the effects of these interactions, the covalent interactions between caffeoylquinic acid (chlorogenic acid, CQA) oxidised by polyphenol oxidase (PPO) at acidic pH 6 (pH 6) and α‐lactalbumin, lysozyme and bovine serum albumin (BSA) were compared with non‐enzymatically induced covalent interactions at alkaline pH (pH 9). The effects of these modifications on protein properties were examined. RESULTS: Both ways of modification seemed to result in protein modification mainly via dimeric rather than monomeric CQA quinones. These modifications led to a decrease in the number of free primary amino groups of the proteins. Modification with CQA alone induced a low degree of protein dimerisation, which also occurred through the action of PPO alone. Modification drastically reduced the solubility of lysozyme over a broad pH range, whereas that of α‐lactalbumin was strongly reduced only at pH values close to its pI. The solubility of BSA was much less affected than that of the other proteins and only at acidic pH. CONCLUSION: These results indicate some similarities between modifications at pH 6 and 9 and that both modifications clearly change the functional properties of globular proteins. Copyright © 2007 Society of Chemical Industry     

绿原酸与蛋白质氨基酸残基共价互作效率研究

目的 探究绿原酸(Chlorogenic acid,CGA)氧化形成的CGA醌与氨基酸侧链基团在不同pH条件下的反应效率,为CGA与蛋白质共价互作反应位点的调控提供理论依据。方法 利用循环伏安法研究了酸性(pH 5.0)、中性(pH 7.0)和碱性(pH 8.0)条件下CGA与氨基酸侧链基团的反应效率,采用UPLC-QTOF-MS/MS对反应产物进行结构鉴定。结果 在pH 7.0、扫描速率10 mV/s,0.20 mmol/L CGA与10.00 mmol/L封闭α-氨基的氨基酸的反应效率顺序依次为Nα-乙酰-L-半胱氨酸(~100.00%)≈Nα-乙酰-L-色氨酸(~100.00%)> Nα-乙酰-L-酪氨酸(39.20% ± 2.19%)>Nα-乙酰-L-赖氨酸(10.25% ± 0.83%)>Nα-Boc-L-组氨酸(~0.00%)≈Nα-乙酰-L-精氨酸(~0.00%);缩小扫描电压范围及其他反应条件相同情况下,CGA与Nα-乙酰-L-半胱氨酸、Nα-乙酰-L-色氨酸及Nα-乙酰-L-酪氨酸的共价互作效率分别为~100%、12.83% ± 1.16%及~0%;pH 7.0或8.0时GCA与氨基酸残基的共价互作效率高于pH 5.0;CGA醌与Nα-乙酰-L-赖氨酸、Nα-乙酰-L-精氨酸的反应产物以氧化态的醌-氨基酸形式存在,而CGA醌与Nα-乙酰-L-半胱氨酸、Nα-Boc-L-组氨酸、Nα-乙酰-L-色氨酸的反应产物以还原态的酚-氨基酸形式存在。结论 CGA氧化形成的CGA醌可与蛋白质中半胱氨酸、色氨酸、赖氨酸、组氨酸及精氨酸残基发生反应,其中半胱氨酸残基是CGA醌与蛋白质共价互作的主要位点,可通过调整食品体系的pH值和绿原酸醌浓度实现绿原酸-蛋白质共价互作位点的调控。     

绿原酸抑制猪肉肌原纤维蛋白氧化及NDEA生成的作用研究

在羟自由基氧化体系中,研究不同添加量的绿原酸对猪肉肌原纤维蛋白(MP)理化性质和亚硝基二乙胺(NDEA)生成的影响。结果表明,氧化导致羰基含量升高,疏水性增加,蛋白结构展开。添加绿原酸后,0.1~0.5 mmol/L能有效抑制羰基的生成(p<0.05),而0.8~1.0 mmol/L未能抑制羰基的生成。0.1~2.0 mmol/L的绿原酸使肌原纤维蛋白疏水性增加,内源色氨酸荧光强度降低。绿原酸对NDEA的生成具有浓度依赖性,0.1~0.8 mmol/L的绿原酸促进NDEA生成,而0.8~2.0 mmol/L的绿原酸抑制NDEA生成。结论:适量的绿原酸抑制了肌原纤维蛋白的氧化和NDEA的生成。     

绿原酸影响氨基酸消减丙烯酰胺的研究

绿原酸是马铃薯中的主要酚酸。试验将丙烯酰胺分别与7种氨基酸进行高温加工处理,研究添加绿原酸对体系中丙烯酰胺消减的影响。结果表明,绿原酸促进丙烯酰胺的消减。加入绿原酸在160℃反应30 min后,丙烯酰胺消减率显著提高,为9.58%(天冬酰胺)～33.46%(半胱氨酸)。采用甘氨酸/丙烯酰胺体系,进一步研究绿原酸添加量、加热温度和时间对丙烯酰胺消减的影响,发现丙烯酰胺消减率随绿原酸添加量、加热温度的增加和时间的延长而提高。绿原酸与丙烯酰胺物质的量比25∶1,180℃反应60 min后,丙烯酰胺消减率达到66.9%。     

Degradation of pelargonidin 3‐glucoside in the presence of chlorogenic acid and blueberry polyphenol oxidase

Oxidation of both chlorogenic acid (CG) and pelargonidin 3‐glucoside (Pg 3‐glc) as well as their mixture, in the presence of blueberry polyphenol oxidase (PPO) was studied in model solutions. The reactions were monitored by spectrophotometric, polarographic and HPLC methods. In the absence of CG, Pg 3‐glc is not oxidised by blueberry PPO, but pigment degradation is induced by the presence of chlorogenic acid. Kinetic studies showed that Pg 3‐glc was degraded by a mechanism involving a reaction between chlorogenoquinone and/or secondary products of oxidation formed from the quinone and the pigment. Therefore, no coupled oxidation mechanisms occurred with Pg 3‐glc, as expected of its structure. The oxidation of CG by PPO in the presence of Pg 3‐glc involved the consumption of 0.6 μmol of oxygen per μmol of CG oxidised, which is close to the stoichiometry calculated from the oxidation of CG alone (0.63 μmol of oxygen per μmol). The initial phase of the pigment degradation showed a delay which did not correspond to cresolase activity. This phase could be explained by a quinone/anthocyanin reaction which is dependent on the chlorogenoquinone concentration and/or a reaction involving secondary products of oxidation (formed from the chlorogenoquinone) and Pg 3‐glc. © 1999 Society of Chemical Industry     

Detection of melamine and cyanuric acid in vegetable protein products used in food production

The multitude of food recalls in 2007 clearly demonstrated that total nitrogen-content (ΣN) determination by means of near-infrared spectroscopy (NIRS) and Kjeldahl-based measurements can be deceived, and should no longer be regarded as a complete quality assurance program for nutritive-protein evaluations. Furthermore, contemporary Canadian-employed analytical tools are precariously limited in their ability to effectively assure a product where there is no a priori knowledge of the environmental toxin(s) involved. In light of these challenges, this study explored a number of analytical techniques used to assess and furthermore assure the quality of vegetable protein products (VPPs). Using liquid chromatography with tandem mass spectrometry (LC/MS/MS) technologies, a combination of VPP-based samples was analyzed for the presence of nitrogen-bearing environmental toxicants. Of the 52 samples tested, involving an assortment of matrices, melamine and cyanuric acid were positively identified (>1 ng/mL) in 22 and 17 samples, respectively. Subsequent high pressure liquid chromatography with ultraviolet/visible (HPLC-UV) amino acid profiling further confirmed the adulteration of those materials contaminated with melamine and melamine-related compounds. Based on the evidence presented herein, LC/MS/MS in combination with HPLC-UV provides for a reliable food safety detection system as applied to VPPs. Moreover, HPLC-UV is indispensable as a stand-alone 1st level of screening to assess the integrity of a VPP or any nutritive protein-based sample.     

乳酸菌发酵对梨汁的护色作用

梨汁在加工过程中极易褐变,乳酸菌发酵能有效保护梨汁的色泽。本研究测定梨汁发酵及储藏过程中色泽、多酚氧化酶活性、酚类物质、抗坏血酸含量等指标的变化,并与未发酵梨汁和添加柠檬酸的调酸梨汁对比,探索乳酸菌发酵对果蔬汁的护色机理。30天储藏期内,未发酵梨汁褐变指数从0.073变化至0.310,显著高于发酵梨汁(0.134);乳酸菌发酵作用有效抑制了多酚氧化酶的活性,使得发酵梨汁中的酚类物质的保存率(43%)显著高于未发酵梨汁(16%);发酵梨汁中抗坏血酸稳定性更高,保存率(85%)较未发酵梨汁高(50%),减少了非酶褐变。乳酸菌通过在梨汁中发酵产酸,抑制多酚氧化酶的活性,保存酚类、抗坏血酸等物质,从而保护梨汁色泽。     

大麦酚类化合物及相关酶类的研究进展

大麦作为重要的粮食作物和啤酒酿造原料,其中的酚类化合物及其相关酶类（如酚类合成酶——苯丙氨酸解氨酶,酚类氧化酶——过氧化物酶和多酚氧化酶）对大麦的生长发育起重要的调节和保护作用,而在大麦的加工过程中会影响产品质量,如过氧化物酶和多酚氧化酶的氧化产物（苯醌和聚合产物）直接影响食品的质量。本文综述了大麦籽粒中酚类化合物及相关酶的种类和功能,并分析了对食品工业和啤酒酿造的影响。     

磷脂酶A1(Lecitase Ultra)的纯化及氨基酸序列分析

本论文研究了超滤法和RP-HPLC对磷脂酶A1(Lecitase Ultra)的纯化,利用MALDI SYNAPT Q-TOF MS和MS/MS对其氨基酸序列进行分析及推测。纯化去除了Lecitase Ultra中的山梨醇和山梨醇盐,纯度97.7%,酶蛋白和亚基的分子量为30838.0和16536.0。通过MALDI SYNAPT Q-TOF MS和MS/MS对酶蛋白进行分析,确定了Lecitase Ultra的氨基酸序列组成;进行数据库(http: //www.matrixscience.com)比对,发现其氨基酸序列信息与棉状嗜热丝孢菌(T. lanuginosus)脂肪酶和尖孢镰刀菌(F. oxysporum)脂肪酶高度吻合,序列中1-284是T. lanuginosus脂肪酶的氨基酸序列,序列中285-339是脂肪酶F. oxysporum脂肪酶的氨基酸序列,其中113、118和121可能是被基因突变成113 G-A、118 D-W和121 E-K。研究结果为进一步探讨Lecitase Ultra的三维空间结构及其活性中心催化机理提供了数据支撑。     

Decrease of the chlorogenic acid content in commercial sunflower meal using a polyphenol oxidase preparation secreted by the white‐rot fungus Trametes versicolor ATCC 42530

BACKGROUND: Sunflower meal (SFM) is a by‐product from the oil extraction process from sunflower seeds. The meal is used as a protein supplement in the livestock diet. However, relatively high levels of polyphenols, among which chlorogenic (CGA) and caffeic acids are in larger amounts, in the meal compromises its use for animal feed and human consumption. The aim of this work was to investigate an enzymatic process for upgrading the quality of SFM by decreasing its CGA content using an enzyme preparation from the white‐rot fungus Trametes versicolor. RESULTS: The effects of pH, temperature, enzyme and meal concentrations, and mass transfer on the decrease of the CGA content in SFM were investigated. It was found that: (1) the optimum pH and temperature were 3.4 and 45 °C, respectively. (2) The system was saturated with the enzyme when its concentration was 5 nkat/mL of liquid phase; (3) the agitation speed of the system influenced the extraction of CGA from the meal; and (4) the conversion of CGA in the SFM system increased in the presence of larger volumes of liquid phase. CONCLUSIONS: The enzyme preparation used in the experiments is able to decrease successfully the CGA content in SFM. Copyright © 2007 Society of Chemical Industry     

Quantitative UPLC‐MS/MS analysis of chlorogenic acid derivatives in antioxidant fractionates from dandelion (Taraxacum officinale) root

While qualitative studies have identified chlorogenic acids in antioxidant extracts, particularly ethyl acetate‐derived extracts, of Taraxacum officinale, quantitative analysis of these phenolic compounds remains largely unreported for this species. In this study, bioactivity‐guided fractionation of an antioxidant crude ethyl acetate extract (DPPH = 295.481 ± 0.955 mg TE g^?1 extract) from T. officinale root resulted in a number of reverse‐phase fractions that demonstrated high antioxidant activity (DPPH = 1058.733–1312.136 mg TE g^?1 extract), stronger than that of the synthetic antioxidant Trolox^®. UPLC‐MS/MS screening of these fractions for the presence of selected mono‐ and di‐caffeoylquinic acids revealed large quantities of 1,5‐dicaffeoylquinic acid present in several fractions (853.052–907.324 μg mg^?1), respectively. Due to the antioxidant potency and high levels of 1,5‐dicaffeoylquinic acid observed in these fractions, it was concluded that specifically this chlorogenic acid derivative is a major contributor to the antioxidant efficacy of dandelion root.     

Photo-induced changes in the total chlorogenic acid content of potato (Solanum tuberosum) Tubers

The exposure of potato tubers to light resulted in a significant cultivar-dependent increase in chlorogenic acid content. The magnitude of the observed increase resulting from 48 h light exposure was significantly correlated with the initial chlorogenic acid content of the tubers and also with the previously reported photo-induced increases in total glycoalkaloids. Time-course studies indicated that the greatest rate of increase occurred between 8 and 16 h continual exposure. Prolonged storage under light revealed that chlorogenic acid values in some cultivars reached a maximum value after 48 h, whilst in others these continued to increase almost linearly until the end of the experiment 5 days later.     

Reactions of chlorogenic acid and quercetin with a soy protein isolate--influence on the in vivo food protein quality in rats

Plant phenolic compounds are known to interact with proteins producing changes in the food (e. g., biological value (BV), color, taste). Therefore, the in vivo relevance, especially, of covalent phenol-protein reactions on protein quality was studied in a rat bioassay. The rats were fed protein derivatives at a 10% protein level. Soy proteins were derivatized with chlorogenic acid and quercetin (derivatization levels: 0.056 and 0.28 mmol phenolic compound/gram protein). Analysis of nitrogen in diets, urine, and fecal samples as well as the distribution of amino acids were determined. Depending on the degree of derivatization, the rats fed with soy-protein derivatives showed an increased excretion of fecal and urinary nitrogen. As a result, true nitrogen digestibility, BV, and net protein utilization were adversely affected. Protein digestibility corrected amino acid score was decreased for lysine, tryptophan, and sulfur containing amino acids.     

PPO酶促诱发因素对苹果多酚非酶褐变的影响

为探究苹果多酚氧化酶(PPO)酶促诱发因素对苹果多酚参与的非酶褐变的影响,以绿原酸和根皮苷为底物,以磷酸缓冲液为苹果汁模拟体系,通过控制PPO酶促诱发条件,进行酶促诱发反应,然后钝化酶,模拟体系于60℃进行非酶聚合反应,每隔24 h用色差计测定L*值、a*值、b*值和ΔE*值。结果表明,酶促诱发因素对绿原酸和根皮苷参与的非酶褐变的影响存在较大差异。p H在3.5~6.0,含有绿原酸和根皮苷的模拟体系,其褐变程度随酶促诱发p H升高而增大;酶促诱发温度20~60℃,诱发体系温度对绿原酸参与的非酶褐变影响不显著,而根皮苷在40℃时褐变程度较大;酶促诱发时间分别为40 min和30 min时绿原酸和根皮苷参与的非酶褐变程度较大;酶活14.25~171 U,酶活的增大加速两种多酚参与的非酶褐变。结果同时表明,根皮苷非酶褐变与果汁后浑浊有关,且较高的p H、温度、PPO酶活以及较长的酶促诱发时间加速果汁后浑浊形成。     

菜籽蛋白氨基酸组成分析及与功能特性的相关性研究

选用长江上游、中游和下游的30个菜籽品种的饼提取蛋白质,对蛋白质进行氨基酸组成和功能特性的研究.结果表明:菜籽蛋白中总氨基酸含量为68.67％～84.90％,其中中双11号(湖南)氨基酸总量最高,而丰油730号最低;30个菜籽蛋白中的氨基酸种类齐全,必需氨基酸占氨基酸总量的37％～38％,绝大部分样品的第一限制氨基酸是蛋氨酸,氨基酸比值系数分(SRC)为77.66～91.53.在营养分析的基础上,建立氨基酸组成与功能特性的相关性分析,分析结果为:亲水性氨基酸含量与乳化稳定性呈显著负相关,乳化性与乳化稳定性呈极显著负相关,可溶性蛋白含量与吸油性、吸水性、起泡性呈正相关.菜籽蛋白是营养价值很高的植物蛋白,可广泛应用于食品工业.     

蛋白质降解及其产物氨基酸检测的研究进展

综述了蛋白质的降解方法,包括酸水解、碱水解、酶解以及超声辅助水解,并分析比较了每种方法的优缺点。其次从检测方法的角度综述了蛋白降解产物-氨基酸的分析检测方法,包括化学分析法、电化学分析法和分光光度法。      

Effects of varying extracellular amino acid concentrations on bidirectional amino acid transport and intracellular fluxes in mammary epithelial cells

Understanding the regulation of cellular AA uptake as protein supply changes is critical for predicting milk component yields because intracellular supplies partly regulate protein synthesis. Our objective was to evaluate cellular uptake and kinetic behavior of individual AA when cells are presented with varying extracellular AA supplies. Bovine primary mammary epithelial cells were grown to confluency and transferred to medium with an AA profile and concentration similar to that of plasma from dairy cows for 24 h. Treatments were 4 AA concentrations, 0.36, 2.30, 4.28, and 6.24 mM, which represented 16, 100, 186, and 271% of typical plasma AA concentrations, respectively, in lactating dairy cows. Twenty-four plates of cells (89.4 × 19.2 mm) were assigned to each treatment. Cells were first subjected to treatment medium enriched with ¹⁵N-labeled AA for 24 h and then incubated with treatment medium enriched with ¹³C-labeled AA for 0, 15, 60, 300, 900, 1,800, and 3,600 s. Intracellular free AA, intracellular protein-bound AA, and extracellular medium free AA were analyzed for concentrations and isotopic enrichment using gas chromatography–mass spectrometry. A dynamic, 12-pool model was fitted to the data for 14 AA to derive unidirectional uptake and efflux, protein turnover, transamination, oxidation, and synthesis. The derived concentration for half the maximal uptake (k_m) indicated no saturation of AA uptake at typical in vivo concentrations for 11 of the 14 AA. Arginine, Pro, and Val appeared to exhibit saturation kinetics. Net uptake of all essential AA except Phe was positive across treatments. Most nonessential AA exhibited negative net uptake values. Efflux of AA was quite high, with several AA exhibiting greater than 100% efflux of the respective influx. Intracellular pool turnover was rapid for most AA (e.g., 2 min for Arg), demonstrating plasticity in matching needs for protein translation to supplies. Intracellular AA concentrations increased linearly in response to treatment for most AA, whereas 9 AA exhibited quadratic responses. Amino acid uptake is responsive to varying extracellular supplies to maintain homeostasis. No saturation of uptake was evident for most AA, indicating that transporter capacity is likely not a limitation for most AA except possibly Arg, Val, and Pro in mammary epithelial cells.