Influence of heating time on the color and acrylamide formation

Shi Zhihong, Zhao Xi, Zhang Hongyi, Zhou Jianke, Zhang Li
(College of Chemistry and Environmental Science, Hebei University; Key Laboratory of Analytical Science and Technology of Hebei Province, Baoding, 071002, China)

Abstract The effect of heating time on the formation of color and acrylamide was studied. Radix Asparagi, a kind of traditional Chinese herb, which contains free amino acid asparagine and reducing sugars in relatively high amounts，was selected as a model. The content of acrylamide formed during the heating process was determined by GC-ECD and color changes were detected by a Model 721 spectrophotometer. Experimental results showed that the formation of acrylamide at 190ºC increased rapidly at the onset of heating, reaching an apparent maximum value at 15 min and decreasing rapidly afterwards. The same trend was observed in color changes.
Keywords Acrylamide, heating time, color, Radix Asparagi, GC-ECD

1. INTRODUCTION
In April 2002, detection of high concentrations of acrylamide in common heated starch-rich foodstuffs by the Swedish National Food Administration attained considerable public concern, since acrylamide was found to be carcinogenic in rodents and was classified as a probable human carcinogen ^[1,2]. Researches have shown that acrylamide formation is closely linked to the Maillard reaction, which is the non-enzymatic browning reaction. The free amino acid asparagine and reducing sugars are considered as the main precursors of acrylamide ^[3,4]. Radix Asparagi, Chinese name Tiandong, is a kind of traditional Chinese herb which contains these compounds in relatively high amounts. So, Tiandong was chosen as a model plant to study on the relationship between heating time and the formation of acrylamide. The content of acrylamide was determined by GC-ECD after extraction and derivatization.
    Colored products are also formed in foods during heating as a result of Maillard reaction. Through our experiment, we found that the aqueous extract of the heating processed herb had different colors at different heating time, so we used spectrophotometer to detect the aqueous extract to demonstrate the difference between samples heated for different period of time.
    This paper presents the changes in colour and acrylamide levels with heating time in traditional Chinese medicine Radix Asparagi during heating process.

2. EXPERIMENTAL
2.1 Chemicals and materials
Acrylamide (>99.9%) was purchased from Amresco (Solon, Ohio, USA). Stock solution of acrylamide (0.1mg/ml) was prepared by dissolving the compound in distilled water. All other reagents used were of analytical grade. Traditional Chinese herb Radix Asparagi was purchased from Yixiaotang drug store (Baoding, China).
2.2 Instrumentation
GC analyses were performed on an SP 3400 GC chromatograph (Varian, USA) which consisted of an electron capture detector (ECD), a 1075 split/splitless injector and an N-2000 dual channel chromatographic data station. Absorbance analyses were performed on a 721 spectrophotometer (Shanghai Optical Instrument Factory, China).
2.3 Heating process for Radix Asparagi 
The Raw material of traditional Chinese herb was cut into slices by stainless steel knife. Then the pieces were put in a culture dish to form a thin layer. Parallel samples were baked in a thermostatic-electric oven at 190ºC for 5, 10, 15, 20, 25 min, respectively.
2.4 Sample extraction and derivatization 
The baked Chinese herb was pulverized into powder and homogenized. 1.0 g of the powder was accurately weighed and put in a centrifuge tube, 10ml of 2 mol/L NaCl solution was added. The centrifuge tube was votexed for 10min and then it was centrifuged at 4000 rpm for 20 min. 5 ml of the supernatant was put in a 10 ml brown volumetric flask, 0.6 ml of 10 % H₂SO₄ was added, then 2 mol/L NaCl was added to the mark. After being kept at 4ºC in a fridge for 15 min, the centrifuge tube was taken out and 1.0 ml of 0.1mol/L KBrO₃ and 1.5 g KBr was added as derivative reagents ^[5]. The derivatization reaction was completed at 4ºC over night. 0.7 ml of 0.1mol/L Na₂S₂O₃ was added to remove excess bromine. 4ml of the reaction solution was extracted twice with 4ml of ethyl acetate. The organic phase was dried with anhydrous Na₂SO₄. 1 m l of the final sample solution was injected for GC-ECD analysis.
2.5 GC analysis of acrylamide
Separation was performed on an FFAP capillary column (50m × 0.22mm × 0.2mm，S.G. E, Australia) using nitrogen as the carrier gas at a flow rate of 1.25 ml/min, applying the following temperature program: 120ºC (hold time 0.5 min), then rises at 10ºC·min^-1 to 180ºC. The sample was injected by using splitless injection mode, valve time was 0.5min, injection volume was 1m L. The injector temperature was held at 220ºC and the ECD detector temperature was held at 300ºC. Peak area was used for quantification.
2.6 Spectrophotometric determination of the color of the sample                     
0.25g of the powder was accurately weighed and transfered to a centrifuge tube, and then it was extracted with 10 ml water for 10 min on a vortex oscillator. The homogenate was centrifuged at 5000 rpm for 10 min, and the supernatant was separated and detected at 660nm by using a 721 spectrophotometer.

3. RESULTS AND DISCUSSION
3.1 The GC analysis of acrylamide
Typical chromatograms for the derived products of acrylamide standard and the sample are shown in Fig. 1. From Fig. 1, it can be seen that the retention time of the derivative of acrylamide is 16.965 min. The peak of the derivative of acrylamide was not interfered by the co-existed components in the sample.
    To prepare the calibration curve, 0.015, 0.030, 0.075, 0.150 and 0.750 mg/ml of acrylamide standard solution was derivatized for GC-ECD analysis according to the procedures described in 2.5 and 2.6. The regression equation was Y= 134947.4 X + 29.4799, correlation coefficient R = 0.99993, LOD was calculated to be 0.5 mg/kg by signal to noise ratio of 3.

                   (A)

                  (B)
Fig.1 Chromatograms for derivatization results of acrylamide standard and the sample. (A) Chromatogram of the derivative of acrylamide standard, (B) Chromatogram of the derivative of the sample, the retention time of the derivative of acrylamide is 16.965 min.

3.2 The effect of heating time on the formation of acrylamide 
Fig. 2 shows the relationship between acrylamide formation and the heating time for the herb material baked at 190ºC. From Fig 2, it can be seen that when the material was heated for 5 min, the acrylamide formed was only 853.4 m g/kg, but with the increase of the heating time, the acrylamide content increased significantly. When the heating time was prolonged to 15 min, the content of acrylamide in the sample reached 10709.2 m g/kg, which increased for 12.5 times. While when the heating time was prolonged to 20 min, the acrylamide content decreased rapidly. After 20 min, the acrylamide content decreased gradually.

Fig.2 The effect of heating time on the formation of acrylamide

3.3 The effect of heating time on the color formation                                      
Acrylamide formation was found to occur during the browning process by Maillard reaction of reducing sugars with asparagine at temperatures above 120ºC. Colored products are also formed in the sample during heating process as a result of Maillard reaction. The heating time also affects the color formation as it does on the formation of acrylamide. From our experimental results, we found that samples baked at 190ºC for different period of time presented different depth of colors. To quantitatively demonstrate the difference of the color of the samples, we extracted the samples with water and determined the absorbance of the extraction solution at 660 nm by using a 721 spectrophotometer. The results are shown in Fig.3. While the absorbance of the solution increased rapidly at the onset of heating, reaching an apparent maximum value at 15 min and decreasing rapidly afterwards.

Fig. 3 The effect of heating time on the color formation

4. CONCLUSIONS
The effect of heating time on the formation of color and acrylamide was investigated in this paper. It was interesting to see that changes in acrylamide levels and the absorbance of the aqueous extracts of the sample followed almost the same trend during heating at 190ºC for different period of time, and an initial increase to a maximum followed by a subsequent decrease was observed in both cases.

ACKNOWLEDGMENTS　Financial support from the National Natural Science Foundation of China (20575016) and the Natural Science Foundation of Hebei Province China (B2006000953) are gratefully acknowledged.

REFERENCES
[1] Friedman M. J. Agric. Food Chem., 2003, 51: 4504-4526.
[2] Senyuva H. Z., GokmenV. Food Addit. Contam., 2005, 22(3): 214-220.
[3] Mottram D. S., Wedzicha B. L., Dodson A. T. Nature, 2002, 419:448-449.
[4] Stadler R. H., Blank I., Varga N. et al. Nature, 2002, 419: 449.
[5] Zhang Y., Dong Y., Ren Y. P. et al. J. Chromatogr.A, 2006, 1116:209-216.

加热时间对丙烯酰胺形成和颜色变化的影响研究
石志红， 赵喜， 张红医， 周建科， 张立
（河北大学化学与环境科学学院, 河北省分析科学技术重点实验室 河北 保定 071002）
摘要  本文以富含天门冬酰胺和还原性糖的药用植物天冬作为研究模型，考察了加热时间对丙烯酰胺的形成和颜色变化的影响。样品中形成的丙烯酰胺经提取、衍生后采用GC-ECD法测定，颜色变化通过测定水提液的吸光度值来表征。研究结果表明：在190ºC，随着加热时间的延长，丙烯酰胺的含量逐渐升高，15 min达最大值，随后丙烯酰胺的含量急遽降低，颜色变化呈现相似趋势。
关键词  丙烯酰胺，加热时间，颜色，天冬，GC-ECD

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