分光光度法测定焦糖色中4-甲基咪唑

本采用分光光度法对焦糖色中4-甲基咪唑进行了定量测定，克服了传统薄层法的精密度、准确度不高的弱点。实验结果表明，4-甲基咪唑经重氮化后在波长440nm处具有最大吸收，据此进行准确定量测定，重现性好。该方法灵敏度高，其线性方程为：C=0．0717A 0．0007，线性相关系数为0．999，检出限为1．4μg，RSD为3．5N。加标回收率为96N～102％。将本法用于实际样品的测定获得满意的结果。     

Development of caramel colour with improved colour stability and reduced 4-methylimidazole

ABSTRACT

This study investigated the effect of chemical substances, such as Mg²⁺ (as magnesium sulphate), Zn²⁺ (as zinc sulphate), Fe²⁺ (as iron sulphate), ascorbic acid, and citric acid, on the formation of 4-methylimidazole (4-MI) and colour stability of caramel colour. The 4-MI concentration in the caramel colour without chemical substances was 963.10 μg/g, and this decreased the most (67.7%) with the addition of 0.1 molar ratio of citric acid. Colour stability was evaluated by measuring the total colour change (ΔE) after storage, and heating in pH 2, 4, and 7 solutions and 50% solution in ethyl alcohol. Among the chemical substances added to reduce 4-MI in the caramel colour, Mg²⁺ (0.1 molar ratio) and ascorbic acid improved the colour stability more than the others.     

顶空进样-毛细管气相色谱法检测饮料用焦糖色素中的4-甲基咪唑

建立了顶空进样-毛细管气相色谱法测定饮料用焦糖色素中4-甲基咪唑含量的方法。采用HP-5毛细管柱、氮磷检测器,溶剂为二氯甲烷,内标为N,N-二甲基苯胺。方法的线性范围为0．025μg／ml-0．295μg／ml,最低检测浓度为0．01μg/ml;回收率在99％以上,相对标准偏差在2％之内。该方法重现性好、简单、快速、准确,能满足分析检测的需要。     

Analysis and risk assessment of 4(5)-methylimidazole in brown colored foods and beverages

In this study, the 4(5)-methylimidazole (4(5)-MI) levels in various 144 brown coloured foods and beverages were determined. The brown coloured foods and beverages were 62 processed sauces, 40 coffee, 9 caramel syrups, 18 red ginseng juice and 15 Japanese apricot fruit juice. The amount of 4(5)-MI in brewed coffee (1821.3 ng/g) was the highest level among the samples. The 4(5)-MI concentration in processed sauce (47.6 ng/g) was the lowest level among the samples. The levels of 4(5)-MI in various samples were found as follows: 47.6–1748.5 ng/g in processed sauces, 64.1–1821.3 ng/g in commercial coffee, 115.5–491.9 ng/g in caramel syrups, 91.0–854.1 ng/g in red ginseng juice and 137.6–587.4 ng/g in Japanese apricot fruit juice. Based on the 4(5)-MI levels, the estimated daily intake (EDI) and chronic daily intake (CDI) were calculated. EDI and CDI of red ginseng juice was the highest among all samples, and they were 1618.6 and 1256.8 ng/kg bw/day, respectively.     

Determination of 4(5)-methylimidazole in carbonated beverages by isotope-dilution liquid chromatography-tandem mass spectrometry

The purpose of this study was to develop a method to quantify 4(5)-methylimidazole (4-MEI), a suspected carcinogen, in carbonated beverages by simple sample dilution and isotope-dilution reverse-phase LC-MS/MS. Isotope dilution using hexa-deuterated methylimidazole (d₆-4-MEI) was used to quantify native 4-MEI and to assess matrix effects quantitatively. The accuracy of the method was assessed by intentionally fortifying a negative control sample at three doses: low, medium and high (replicates of n = 5 each) with a known amount of 4-MEI. The respective absolute error in each case was 18.7 ± 0.7%, 14.6 ± 2.8% and 21.1 ± 9.7%. Within-day (intra-) and day-to-day (inter-) repeatability, determined as the relative standard deviation by fortifying a negative control sample (n = 5), were 9.5% and 15.4%, respectively. Average ion suppression of d₆-4-MEI in beer was 63.9 ± 3.2%, while no suppression or enhancement was seen in non-alcoholic samples. The instrument and method limit of detection were calculated as 0.6 and 5.8 ng ml^–1, respectively. 4(5)-Methylimidazole was quantified in a variety of store-bought consumer beverages and it was found that in many of the samples tested consuming a single can of beer would result in intake levels of 4-MEI that exceed the no significant risk guideline of 29 µg day^–1. Conversely, 4-MEI in the samples was orders of magnitude smaller than the European Food Safety Authority acceptable daily intake threshold value of 100 mg kg^–1 bw day^–1.     

A U.S. population dietary exposure assessment for 4-methylimidazole (4-MEI) from foods containing caramel colour and from formation of 4-MEI through the thermal treatment of food

ABSTRACT

4-Methylimidazole (4-MEI) is formed in caramel colours produced using ammonium compounds (Class III and Class IV caramel colours). 4-MEI can also form in food through Maillard reactions between reducing sugars and amino acids during cooking, roasting or dry-heating. The USFDA has analysed over 700 food and beverage samples collected from 2013 to 2015 for the presence of 4-MEI. These samples include foods containing added caramel colour and foods that are not labelled as containing added caramel colour, but which may contain 4-MEI resulting from thermal treatment. The 4-MEI levels in all food samples were quantified using LC-MS/MS. These data were used to develop a comprehensive dietary exposure assessment for 4-MEI for the U.S. population aged 2 years or more and several sub-populations, using two non-consecutive days of food consumption data from the combined 2009–2012 National Health and Nutrition Examination Survey and 10–14-day food consumption survey data for 2009–2012 from the NPD Group, Inc. National Eating Trends–Nutrient Intake Database. Dietary exposure estimates were prepared for each category of foods labelled as containing added caramel colour and of foods not labelled as containing added caramel colour, but which may contain 4-MEI from thermal treatment. Exposure to 4-MEI from consumption of foods containing added caramel colour was higher than that from foods that contain 4-MEI from thermal treatment for all population groups. Cola-type carbonated beverages were the highest contributors for most populations from foods containing added caramel colour. Coffee was the highest contributor for most populations from foods in which 4-MEI could be formed from thermal treatment. An overall combined exposure to 4-MEI was also estimated that included all foods identified as containing added caramel colour and foods in which 4-MEI could be formed by thermal treatment.     

Dietary intake assessment of caramel colours and their processing by-products 4-methylimidazole and 2-acetyl-4-tetrahydroxy-butylimidazole for the Chinese population

ABSTRACT

The aim of the study was to assess the dietary intake of caramel colours and their by-products 4-methylimidazole (4-MEI) and 2-acetyl-4-tetrahydroxybutylimidazole (THI) for the Chinese population. Based on the typical and maximum reported use levels of caramel colours in 15 food categories, the dietary intakes of combined and single-class caramel colours of Classes I, III and IV were estimated with the food consumption data from the China National Nutrient and Health Survey. Using the mean values of 4-MEI and THI contents in Class III and Class IV Caramel colour samples, the exposures to 4-MEI and THI from dietary caramel colours were derived. The results showed that the combined and individual average dietary caramel colour intakes for the Chinese population of different age groups were estimated to be 232–60.3 mg kg^?1 bw day^?1 for combined caramels, 5.9–29.2 mg kg^?1 bw day^?1 for Class I, 7.7–29.6 mg kg^?1 bw day^?1 for Class III, 21.2–54.3 mg kg^?1 bw day^?1 for Class IV, which were far below the group acceptable daily intake (ADI) and respective ADIs. The combined intake of 4-MEI from Class III and IV caramel colours was estimated to be 3.8–5.2 μg kg^?1 bw day^?1 on average, and 12.9–27.1 μg kg^?1 bw day^?1 at 95th-97.5th percentile for the general population. The anticipated exposure to THI from Class III caramel colours was estimated to be 0.1–0.3 μg kg^?1 bw day^?1 on average and 0.5–1.7 μg kg^?1 bw day^?1 at 95th–97.5th percentile for the general population. The dietary caramel colours intakes and the exposures to 4-MEI and THI from dietary caramel colour for the Chinese population were considered to be of low health concern based on the present toxicological data. Soy sauce, vinegar and compound seasonings were found to be the main contributors to the dietary intake of caramel colours.     

Supercritical fluid extraction (SFE) of 4(5)-methylimidazole (4-MeI) and 2-acetyl-4(5)-(1,2,3,4)-tetrahydroxybutyl-imidazole (THI) from ground-coffee with high-performance liquid chromatographic-electrospray mass spectrometric quantification (HPLC/ESI-MS).

Two polar analytes, 4(5)-methylimidazole (4-MeI) and 2-acetyl-4(5)-(1,2,3,4)-tetrahydroxybutyl-imidazole (THI), were extracted with supercritical carbon dioxide (CO2) modified with aqueous methanol. The method was applied to a roasted coffee powder with good recovery rates. Method efficiency was compared with that of solid-phase extraction using SCX Disc cartridges and validated for spiked solid matrix. The analytes were determined using isocratic liquid chromatography-mass spectrometry (LC/MS) on an Atlantis HILIC Silica column (150 x 2.1 mm, 3 microm) with 80% methanol and 20% 0.01 mol l-1 ammonium formate as the mobile phase. The limit of quantification was around 1.5 pg for 4-MeI and 2.0 pg for THI. The linearity of the calibration curves was satisfactory as indicated by correlation coefficients of >0.999. The coefficient of variation for the intra-day and inter-day precisions was <4% (n = 6). Accuracy was in the range 98-101%; recovery rates were > or = 98 and > or = 99% for THI and 4-MeI, respectively. Several samples of Arabica coffee from various locations and commercially available 'off-the-shelf' coffee products (Arabica/Robusta mixtures) were analysed to test the method.     

高效液相色谱法测定焦糖色素中的4-甲基咪唑

建立了高效液相色谱测焦糖色素中4-甲基咪唑的方法。样品经碱性条件下三氯甲烷-无水乙醇萃取和硫酸水溶液反萃取后,用反向色谱柱HypersilBDSC18(4.6mmi.d.×250mm,5um)分离,以体积比为5∶95的甲醇-0.05mol/L磷酸二氢钾缓冲液(含0.005mol/L庚烷磺酸钠,pH3.5)为流动相,在UV215nm波长条件下检测了自制及几种市售焦糖色素中的4-甲基咪唑的含量。并试验了不同流动相pH值和甲醇浓度对4-甲基咪唑分离的影响。本法平均加标回收率为94.7%,检出限为0.7ng。     

高品质焦糖色素(Ⅳ)的制备工艺优化

为优化制备高色率焦糖色素的工艺,以葡萄糖为反应物,亚硫酸铵为助剂,制备焦糖色素(Ⅳ)。通过控制葡萄糖浓度、铵含量、反应温度、反应时间等因素,以色素色率为主要检测指标,红色指数和黄色指数为辅助检测指标对结果进行优化。结果表明,高品质焦糖色素(Ⅳ)的最佳工艺条件为:葡萄糖浓度1000g/L,亚硫酸铵添加质量分数(以NH4+的量计)为6%,反应温度为150℃,反应时间为60min。制成焦糖色素的色率达7.47×104EBC。     

固相萃取-高效液相色谱法测定酱油专用焦糖中5-羟甲基糠醛和糠醛

建立了固相萃取-高效液相色谱(SPE-HPLC)法测定酱油专用焦糖中5-羟甲基糠醛和糠醛的方法。样品经水稀释,亲水亲油平衡(HLB)固相萃取柱净化,洗脱液静置沉淀后,用水稀释并过0.45μm滤膜。流动相为水和乙腈梯度洗脱,经Symmetry Shield RP18色谱柱分离,在紫外检测器280nm波长条件下进行测定。该方法在0.5~100μg/mL范围内线性关系良好,R2均大于0.999,相对标准偏差(n=6)在2.5%~4.6%之间,加标回收率在87.1%~99.2%之间。5-羟甲基糠醛和糠醛的方法检出限分别为0.039mg/kg和0.041mg/kg。     

柱前衍生法测定4-甲基咪唑条件的研究

用丹磺酰氯(Dns-Cl)柱前衍生的高效液相色谱法来测定4-甲基咪唑.研究了衍生试剂的用量、缓冲体系的PH值、衍生时间等条件的变化对衍生反应的影响,得到了用丹磺酰氯对4-甲基咪唑的最佳衍生条件为最佳衍生条件为:加入100倍的丹磺酰氯在pH10.0的溶液中反应40 min.     

2, 4, 5-TRIHYDROXYBUTYROPHENONE (2, 4, 5-THBP)a AS A SUBSTRATE FOR MUSHROOM TYROSINASEb

2, 4, 5-THBP (2, 4, 5-trihydroxybutyrophenone) is a substrate for mushroom tyrosinase with a K_m value of 1.00 mM. The final product(s) formed is red (λ._max 480–490 nm) and it is rather stable with time. An intermediate red product(s), having a maximum at λ 505–515 nm, was detected and the time course of its conversion to the final red product(s) was studied. The data suggest that the intermediate red product(s) is 2, 4, 5-THBP-quinone which is converted to colorless polymerized 2, 4, 5-THBP-OH, which is then oxidized to a red polymerized 2, 4, 5-THBP-quinone(s), which is the final red product(s).     

Formation of protein bound lysine-derived galactosyl and glucosyl pyrroles in heated model systems

Residues of lysine-substituted AGEs (advanced glycosylation end-products) arising from the Maillard reaction and containing the carbon backbone of lactose or maltose, thus deriving from 1-desoxy-ketose degradation, were produced when casein was heated in the presence of the corresponding U14C labelled disaccharides. The enzymatic hydrolysates of the washed casein were purified by SPE and submitted to HPLC on a C18 column flushed with diluted acetic acid. A specific chromatographic peak (lambda max, 288.1 nm; MW, 416.2 Da) with a different retention time was obtained for each disaccharide reacted. On the basis of the value of the specific radioactivity, the two compounds appeared to contain the whole carbon backbone of the parent sugar. Analyses by MS/MS and NMR performed on the same two compounds extracted at preparative scale from lysine-lactose and lysine-maltose model systems allowed the structure assignment of 6-[2-acetyl-3-(beta-D-galactopyranosyloxy)-1-pyrrolyl] 2-amino hexanoic acid and 6-[2-acetyl-3-(alpha-D-glucopyranosyloxy)-1-pyrrolyl] 2-amino hexanoic acid, respectively. Both compounds submitted to enzymatic deglycosylation by specific alpha- or beta-glucosidases produced the lysine-derived acetyl pyrrole 6-(2-acetyl-3-hydroxy-1-pyrrolyl) 2-amino hexanoic acid (lambda max, 288.1 nm; MW, 254.1 Da). Galactosyl- and glucosyl-isomaltoles, extracted from the lysine-containing systems, identified with the reference molecules and heated in the presence of lysine under slightly alkaline conditions, gave the expected lysine-derived glycosyl pyrroles as identified above. The HPLC conditions were optimized by adjusting the composition of the eluting solvent and temperature of the column to achieve the best separation and identification of the AGEs in mixtures such as foods with possible interfering molecules like Trp and lysyl pyrrole aldehyde. Because of the reported presence of the two precursors isomaltol glycosydes in some foods, the corresponding lysine-derived glycosyl pyrroles can occur as both protein bound and in free form.     

Effect of Storage on 5-Hydroxymethylfurfural (HMF) Formation and Color Change in Jams

In this study, the effects of storage temperature (10, 20, and 37°C) and time (0, 2, 4, and 6 months) on the HMF and color values in strawberry, cherry and apricot jams were studied. Results showed that the HMF and total color difference (ΔE) values of all jams were increased linearly with storage time and higher values were found at higher storage temperature. Kinetic model was applied to changes in HMF and ΔE. Changes in HMF and ΔE were explained using zero-order reaction kinetics and the dependence of the rate constant on temperature was described by the Arrhenius equation     

A new enzymatic method for determination of sulphite in food

A novel enzymatic method for determining sulphite is described. Using the enzyme sulphite oxidase (EC 1.8.3.1) isolated from chicken liver, sulphite is oxidised to sulphate and hydrogen peroxide is formed. This is allowed to react with reduced nicotinamide-adenine dinucleotide (NADH) in the presence of NADH-peroxidase from microorganisms (EC 1.11.1.1). The decrease of NADH, which is proportional to the concentration of sulphite, is measured photometrically.In spite of doubts about determining sulphite quantitatively according to this principle, the reaction conditions could be optimised: sulphite is oxidised in triethanolamine buffer (TEA) at pH8·0 with 40 mU/ml sulphite oxidase and 47 mU/ml NADH-peroxidase quantitatively within a period of 20 min. The precision of measurement is very high in the range 13–205 μmol sulphite/litre test solution. The coefficient of variation is found to be CV = 0·67% in a solution of 67 μmol SO₂/litre.The accuracy of the enzymatic measurements in pure aqueous sulphite solutions is confirmed by comparison with chemical determination. The correlation with the iodimetric method is R = 0.995.High specificity for sulphite is found when applying the enzymatic test described here. Carbonyl/sulphite addition compounds (e.g. those occurring in wine) are converted to sulphate and the free carbonyl compound. The majority of sulphur-containing compounds do not react in the test. Only glycosides of isothiocyanates (e.g. in mustard) are oxidised like sulphite, though more slowly.The majority of substances occurring in food do not interfere significantly with the test. Ascorbic acid influences the determination if it is present in high concentrations. Therefore, it should be removed from the sample before measuring sulphite.The method has been proven in use for a large number of foodstuffs. By using the method described here, sulphite can be determined in food rapidly and with high reliability.