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4(5)-methylimidazole (4-MeI) is a potential carcinogen widely used in food colours. EU regulations specify a maximum allowable concentration of 200 ppm for 4-MeI in caramel colours. This study reports an electrochemical determination technique for 4-MeI in caramel colours for the first time. The effect of pH and interference from air were studied to optimize the detection conditions on a glassy carbon electrode in aqueous alkaline solutions using square wave voltammetry (SWV) technique. The concentration of 4-MeI was quantitatively measured down to 10 µM (∼0.8 ppm). Traditional methods such as HPLC, GC, spectrometry and immunoassays involve either expensive instrumentation and reagents or time consuming preparation and detection processes. This study demonstrates the possibility of rapid and simple electrochemical determination of (4-MeI) in food colours with minimum workup using a portable potentiostat.

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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.

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Nutraceutical activity of food is analysed to promote the healthy characteristics of diet where additives are highly used. Caramel is one of the most worldwide consumed additives and it is produced by heating natural carbohydrates. The aim of this study was to evaluate the food safety and the possible nutraceutical potential of caramel colour class IV (CAR). For this purpose, in vivo toxicity/antitoxicity, genotoxicity/antigenotoxicity and longevity assays were performed using the Drosophila melanogaster model. In addition, cytotoxicity, internucleosomal DNA fragmentation, single cell gel electrophoresis and methylation status assays were conducted in the in vitro HL-60 human leukaemia cell line. Our results reported that CAR was neither toxic nor genotoxic and showed antigenotoxic effects in Drosophila. Furthermore, CAR induced cytotoxicity and hipomethylated sat-α repetitive element using HL-60 cell line. In conclusion, the food safety of CAR was demonstrated, since Lethal Dose 50 (LD50) was not reached in toxicity assay and any of the tested concentrations induced mutation rates higher than that of the concurrent control in D. melanogaster. On the other hand, CAR protected DNA from oxidative stress provided by hydrogen peroxide in Drosophila. Moreover, CAR showed chemopreventive activity and modified the methylation status of HL-60 cell line. Nevertheless, much more information about the mechanisms of gene therapies related to epigenetic modulation by food is necessary.

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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.

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A survey of 2-acetyl-4-tetrahydroxybutylimidazole (THI) and 4-methylimidazole (4-MeI) concentrations in caramel colours, vinegar and beverages from the Chinese market were performed by ultrahigh-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). In total, 117 samples, 78 caramel colour samples, 23 vinegar samples and 16 beverage samples, were investigated. The results indicated that 4-MeI was found in all samples. THI was found in a part of the samples and also the level range was lower compared to 4-MeI. In caramel colour samples, the concentration level range of THI was 1.0-74.3 mg/kg and of 4-MeI was 1.5-1291.8 mg/kg. In vinegar samples, the concentration level range of THI was 13.3-119.2 µg/L and for 4-MeI 111.2-2077.8 µg/L. In beverage samples, THI was only found in two samples and the concentration level range of 4-MeI was 10.8-307.1 µg/L. THI and 4-MeI levels in vinegar and beverages were rather low compared with those in caramel colour samples. These observations can be helpful for evaluating individual exposure to THI and 4-MeI from caramel colours, vinegar and beverages in China.

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Supercritical carbon dioxide fluid extraction was used to extract 5-hydroxymethyl-2-furfural from caramel colour (solid content was about 75%). The procedure was carried out by response surface methodology using a quadratic polynomial model. Extraction pressure, time, temperature and ethanol content were selected as the independent variables. Conditions to obtain the highest extraction ratio of 5-hydroxymethyl-2-furfural were determined to be an extraction pressure of 21.65MPa, time of 46.7min, temperature of 35°C and 70% ethanol content of caramel colour. The predicted 5-hydroxymethyl-2-furfural extraction ratio was 87.42%. Under the conditions stated above, the experimental value of 5-hydroxymethyl-2-furfural extraction ratio was 86.98%, which was similar to the predicted value by the model. This study indicated that supercritical carbon dioxide fluid extraction can effectively reduce 5-hydroxymethyl-2-furfural from caramel colour, which can help food industry to improve the safety of the food material, as well as provide more healthy caramel colour for human beings.

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