Effect of Ascorbic Acid on the Properties of Ammonia Caramel Colorant Additives and Acrylamide Formation C: Food Chemistry

Hongxing Chen and Zhengbiao Gu

Ammonia caramels are among the most widely used colorant additives in the food industry. They are commonly prepared through the Maillard reaction and caramelization of mixtures of reducing sugars with ammonia or ammonium salts. Antioxidants are known to inhibit acrylamide formation during the Maillard reaction, and they may affect the properties of the ammonia caramel products. Thus, the objective of this study was to investigate the effect of the antioxidant ascorbic acid on the properties of ammonia caramel. A mixture of glucose and ammonia was allowed to react at 120 °C for 60 min in the presence of ascorbic acid at final concentrations of 0 to 0.08 M. The ammonia caramels obtained from these reactions were all positively charged. As the concentration of ascorbic acid increased, the color intensity of the ammonia caramel showed a decreasing trend, while the intensity of the fluorescence and total amount of pyrazines in the volatiles showed a tendency to increase. The addition of ascorbic acid did not result in obvious changes in the UV-visible spectra of the ammonia caramels and the types of pyrazines in the volatiles were also unchanged. It is noteworthy that the addition of 0.02 to 0.08 M ascorbic acid did reduce the formation of the by-product acrylamide, a harmful substance in food. When the concentration of ascorbic acid added reached 0.04 M, the content of acrylamide in the ammonia caramel was 20.53 μg/L, which was approximately 44% lower than that without ascorbic acid. As a result, ascorbic acid can be considered to improve the quality and safety of ammonia caramels.

Abstract:

Keywords: acrylamide, ammonia caramel, ascorbic acid, color intensity, pyrazines

Introduction Among all food colorants, caramel is used most widely and in the largest quantities. Caramels are divided into 4 categories based upon the conditions of their preparation and their physicochemical properties. Ordinary caramel (Class I) contains no added compounds and has a slightly negative net ionic charge; caustic sulfite caramel (Class II) contains added sulfite compounds and has a negative net ionic charge; ammonia caramel (Class III) contains added ammonium compounds and has a positive net ionic charge; and acid caramel (Class IV) contains both ammonium and sulfite compounds and has a net negative net ionic charge (Myers and Howell 1992). Ammonia caramel, which constitutes about 3 quarters of all caramel coloring produced in China, is mainly used to color soy sauce and vinegar. Generally, ammonia caramels are produced by adding 2% to 6% (w/w) ammonium salts or ammonia to a reducing sugar, then heating the mixture at a defined pressure and temperature. The ammonia caramel forms through the Maillard reaction and caramelization. The Maillard reaction occurs between the carbonyl group of the reducing sugar and an amino compound (Friedman 1996; Mottram and others 2002; Guan and others 2011). One of the most complex processes in food chemistry (Moreno and others 2002), the Maillard reaction can form Amadori rearrangement products

or other intermediate products, including furfurals, reductones, pyrazine, along with a variety of other cyclic substances (Guan and others 2011). Finally, brown polymers known as melanoidins are formed (Rufi´an-Henares and others 2006; Rufian-Henares and Morales 2007). Acrylamide, which is a toxic compound that can cause damage to DNA, has also been reported in the products of the Maillard reaction (Mottram and others 2002; Stadler 2005). At high doses, acrylamide can affect the nervous and reproductive systems of humans and other animals. Dearfield and others (1995) have reported that acrylamide is also carcinogenic to rodents. In recent years, the mechanisms of acrylamide formation in foods and the ways in which acrylamides can be eliminated from foods have been extensively investigated. The addition of antioxidants was found to reduce the content of acrylamide in products of the Maillard reaction (Ou and others 2010). However, the presence of antioxidants may affect both the production of ammonia caramel and its properties. In the present study, ascorbic acid was added to a mixture of glucose and ammonia and allowed to react at 120 °C for 60 min to produce ammonia caramel. The effects of ascorbic acid on the color intensity, UVvisible absorption spectrum, fluorescence spectrum, charge, flavor, and acrylamide content of the ammonia caramel were examined. This investigation was designed to provide the scientific basis for developing the highest quality ammonia caramel.

MS 20140395 Submitted 3/10/2014, Accepted 6/12/2014. Authors Gu and Chen are with School of Food Science and Technology, Jiangnan Univ., Wuxi 214122, Materials and Methods China. Authors Chen is also with Yancheng Inst. of Technology, Yancheng, 224051, China. Authors Gu is also with State Key Laboratory of Food Science and Technology, Reagents All chemical reagents used were of analytical grade and purJiangnan Univ., Wuxi 214122. China and Synergetic Innovation Center of Food Safety and Nutrition, Jiangnan Univ., Wuxi 214122, China. Direct inquiries to chased from China Pharmaceutical Group, Shanghai Chemical author Gu (E-mail: [email protected]). Reagent Co. (Shanghai, China).

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R  C 2014 Institute of Food Technologists

doi: 10.1111/1750-3841.12560 Further reproduction without permission is prohibited

Preparation of ammonia caramel Raw materials and the formula used to prepare ammonia caramel were based on current methods for the industrial production of ammonia caramel. Ascorbic acid was added to mixtures containing 100 mL of 2.22 M glucose and 7 mL of 13.33 M ammonia to achieve final ascorbic acid concentrations of 0, 0.02, 0.04, 0.06, and 0.08 M. The mixtures were placed in a GSH-2 high-pressure reactor (Weihai Chemical Machinery Co., Ltd., Weihai, Shandong, China) at 120 °C for 60 min. After the reaction was complete, the reaction products were removed from the reactor and cooled in an ice bath. After cooling, the ammonia caramel was recovered and stored at 4 °C until it was analyzed. Analysis of the general properties of ammonia caramel First, a 1.0-g sample of each ammonia caramel was dissolved in 100 mL distilled water and subjected to centrifugation at 1000 × g for 10 min. Then 10 mL of each supernatant was collected and diluted to 100 mL with distilled water. The UV spectrum (200 to 800 nm) of each diluted solution was determined using a UV752N UV-visible spectrophotometer (Shanghai Yoke Instrument Co., Ltd., Shanghai, China). The absorbance at 610 nm was used to define the color intensity of the ammonia caramel. The fluorescence intensity of the diluted sample, using an excitation wavelength of 375 nm and emission wavelength of 490 nm, was measured with a JASCO FP-6500 fluorescence spectrometer (Jasco Corp., Tokyo, Japan). The gelatin method was used to determine the charge of the ammonia caramel (Greenshields and others 1969).

out, allowing adsorption for 30 min. Then the extraction fiber was inserted into the inlet of a Finnigan Trace MS spectrometer (Finnigan, San Jose, CA, USA ) for analysis. GC-MS conditions were based on those reported by Cao and others (2010), with minor modifications. GC conditions: column: DB-Wax capillary column (30 m × 0.25 mm; 0.25 μm; J&W Scientific, Folsom, Calif., U.S.A.); temperature program: an initial temperature of 40 °C was maintained for 3.5 min, then increased to 120 °C at 6 °C/min, then increased further to 230 °C at 12 °C/min, and finally maintained for 7 min at a vaporizer temperature of 250 °C; injection volume: 1 μL; carrier gas: He at a flow rate of 0.9 mL/min. MS conditions: electron ionization at 70 eV; ion source temperature: 200 °C; interface temperature: 250 °C; scanning range: 33 to 450 amu. The flavor compounds were identified by comparing their mass spectra simultaneously to those in both the Nist05 and Wiley275 standard libraries. The relative content of each component was determined using a peak area normalization method.

Analysis of acrylamide The acrylamide content of the ammonia caramels was determined using the GC-MS method described by Zamora and others (2010).

Statistical analysis Experimental results are reported as the averages of triplicate measurements. Statistical analyses were carried out using the statistical package from SPSS Inc. (Chicago, IL, USA). Significant differences (P < 0.05) between means were identified by one-way Flavor analysis Headspace solid-phase microextraction (HS-SPME) and gas analysis of variance (ANOVA) and the Student-Newman-Keuls chromatography-mass spectrometry (GC-MS) were used to an- (SNK) procedure. alyze the flavor compounds in the ammonia caramel. A 75-μm CAR-PDMS extraction fiber (Supelco, Bellefonte, Pa., U.S.A.) Results and Discussion was subjected to aging in the GC inlet at 250 °C for 10 min. Meanwhile, an 8-mL sample of diluted caramel solution and 2.0 g Effect of ascorbic acid on the color intensity of ammonia NaCl were added to an extraction flask and equilibrated in a caramel The color intensity of an ammonia caramel, which is an indiwater bath at 60 °C. The extraction fiber was inserted into the headspace of the extraction flask, and then the fiber tip was pushed cator of its overall color, is defined as the absorbance of a 1.0 g/L

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Figure 1–The intensity of the color (absorbance at 610 nm) of ammonia Wavelength (nm) caramels prepared by adding different concentrations of ascorbic acid to the glucose–ammonia system and allowing reaction to proceed at 120 °C for 60 min. Each value represents the mean of three independent measure- Figure 2–UV-visible absorption (200 to 800 nm) of ammonia caramels ments and bars with different letters on the top are significantly different prepared with or without 0.04 M ascorbic acid added to the glucose– (P < 0.05). ammonia system and allowed to react at 120 °C for 60 min. Vol. 79, Nr. 9, 2014 r Journal of Food Science C1679

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Effect of ascorbic acid on caramel properties . . .

Effect of ascorbic acid on caramel properties . . . ascorbic acid concentration increased, the maximum fluorescence intensity of the ammonia caramel increased. An Amadori product, which has no color, no fluorescence, and no UV absorption, is produced during the initial stage of the Maillard reaction. As the reaction proceeds, the Amadori product is dehydrated and cleaved to produce reductone, fluorescent substances, and some colored substances (Morales and van Boekel 1997). Benjakul and others (2005) proposed that the fluorescent compound might be the precursor to the final brown substance. The addition of ascorbic acid may increase the concentration of unsaturated carbonyl compounds in the reaction system, leading to increases in fluorescence and decreases in the concentration of brown substances in the system. This is consistent with the decrease in color intensity of

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Ascorbic acid and the UV-visible absorption spectrum of ammonia caramel The UV-visible absorption spectrum (200 to 800 nm) of the ammonia caramel prepared with ascorbic acid in the reaction mixture was not notably different from that produced without ascorbic acid (Figure 2). The wavelength of maximum absorption was 273 nm and the absorption peak positions in the near-UV did not differ among different preparations. Therefore, adding ascorbic acid did not affect the characteristics of the chromophore or the auxochrome in the ammonia caramel.

Fluorescence intensity (a.u.)

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solution of the ammonia caramel at 610 nm, measured in a 1-cm cuvette using a spectrophotometer (Licht and others 1992). As the concentration of ascorbic acid increased, the color intensity of ammonia caramel showed a decreasing trend (Figure 1). The mixture of glucose and ammonia that produced ammonia caramel in the absence of ascorbic acid was weakly alkaline. This alkalinity favors the browning reaction. When the ascorbic acid concentration increased, the pH of the system gradually decreased, which inhibited the browning reaction. After the ascorbic acid concentration in the system reached 0.04 M, the color intensity showed almost the same level as ascorbic acid concentration continued to increase. This may have been due to the ascorbic acid undergoing a browning reaction with the reducing sugar or amino compounds (Kambo and Upadhyay 2012; Tan and Yu 2012). This additional browning may have counteracted the decrease in color intensity caused by additional decreases in pH.

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Ascorbic acid and the fluorescence spectrum of ammonia 400 450 500 550 caramel Wavelength (nm) The maximum fluorescence intensity of the ammonia caramel prepared at different ascorbic acid concentrations, measured with Figure 3–Fluorescence of ammonia caramels prepared by adding differexcitation at 375 nm, was observed at 457 nm (Figure 3). As the ent concentrations of ascorbic acid to the glucose–ammonia system and allowing them to react at 120 °C for 60 min.

Figure 4–Total ion chromatograms of volatiles produced by adding different concentrations of ascorbic acid to the glucose–ammonia system and allowing them to react at 120 °C for 60 min. Final concentration of ascorbic acid added: (A) 0; (B) 0.02 M; (C) 0.04 M.

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Effect of ascorbic acid on caramel properties . . . Table 1–Peak area percentages of pyrazines in ammonia caramels prepared by adding different concentrations of ascorbic acid to the glucose–ammonia system and allowing reaction at 120 °C for 60 min.

Number

1 Methylpyrazine 2 2,5-Dimethylpyrazine 3 2,3-Dimethylpyrazine 4 Pyrazine 5 Trimethylpyrazine 6 Tetramethylpyrazine 7 2-Ethyl-6-methylpyrazine 8 2-Ethyl-5-methylpyrazine 9 Ethylpyrazine 10 2-Acetyl-3-methylpyrazine 11 Acetylpyrazine 12 Isopropenylpyrazine 13 Pyrrolo(2,3-b)pyrazine 14 2-Ethenyl-6-methylpyrazine Total pyrazines (mean values), % a

0b

0.02 Mb

0.04 Mb

30.45 ± 0.22a 17.80 ± 0.18a 3.67 ± 0.06a 3.63 ± 0.08a 3.04 ± 0.04a 0.09 ± 0.02a 1.11 ± 0.03a 0.52 ± 0.02c NDc 0.29 ± 0.01a 0.20 ± 0.01a 0.07 ± 0.01a 0.11 ± 0.02a 0.01 ± 0.01a 60.99 ± 0.71a

30.94 ± 0.34a 18.46 ± 0.20b 4.01 ± 0.08b 3.63 ± 0.04a 3.45 ± 0.04b 0.21 ± 0.03b 1.20 ± 0.04b 0.33 ± 0.03b NDc 0.40 ± 0.02b 0.26 ± 0.01b 0.13 ± 0.02b 0.15 ± 0.01b 0.04 ± 0.01b 63.21 ± 0.87b

31.14 ± 0.36a 18.71 ± 0.17b 4.28 ± 0.08c 3.65 ± 0.05a 3.79 ± 0.06c 0.23 ± 0.03b 1.23 ± 0.04b 0.28 ± 0.02a 0.14 ± 0.01 0.40 ± 0.02b 0.30 ± 0.02c 0.15 ± 0.01b 0.18 ± 0.02b 0.04 ± 0.01b 64.52 ± 0.90b

Compounds

Values are the means ± SD (n = 3). Means with different superscript lower-case letters within the same line are significantly different (P < 0.05). concentrations of ascorbic acid added. ND, not detected.

b The c

ammonia caramel with the increase of ascorbic acid concentration, pounds to form a dihydropyrazine that spontaneously oxidizes to the corresponding pyrazine (Yu and others 2012). shown in Figure 3.

Ascorbic acid and the flavor of ammonia caramel The color intensities of the ammonia caramels prepared when the ascorbic acid concentration of the reaction system was greater than 0.04 M did not differ much from each other, indicating the almost complete Maillard browning reaction. Therefore, volatiles in the ammonia caramels prepared at ascorbic acid concentrations of 0, 0.02, and 0.04 M were analyzed in this study. The peaks in the total ion chromatograms of the volatile products from these 3 samples did not differ notably, indicating that the types of volatiles in the 3 samples were not significantly different (Figure 4). The volatile products were mainly pyrazine compounds, which possess strong flavor (Seifert and others 1972). The types and content of pyrazines are critical for the flavor of ammonia caramel. Table 1 lists the pyrazine compounds found in the volatile products and their relative peak areas in the ion chromatograms. The results show that when ascorbic acid was added to the mixture of glucose and ammonia, the total amount of pyrazine in the volatile components of the ammonia caramel increased, and the flavor was enhanced. The total amount of pyrazine may increase because the ascorbic acid reacted with NH3 in the reaction system and produced pyrazine. The most accepted mechanism for pyrazine formation involves the condensation of 2 α-amino carbonyl com-

Elimination of acrylamide by addition of ascorbic acid The amounts of acrylamide formed when different levels of ascorbic acid were added to the glucose–ammonia system are shown in Figure 5. The acrylamide content decreased as the ascorbic acid concentration increased to 0.04 M, and then increased again as the ascorbic acid concentration increased to 0.08 M. The addition of 0.04 M of ascorbic acid produced the greatest reduction in the level of acrylamide—approximately 44%. Antioxidants are believed to reduce free electrons in the free radical intermediates formed during the Maillard reaction (Friedman 1996), thereby inhibiting the formation of acrylamide. Ascorbic acid was also found to inhibit acrylamide formation during potato processing

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Acrylamide content (μg/l)

Ascorbic acid and the charge of ammonia caramel Caramel charge is a very important indicator of quality. Typically, the pH of the ammonia caramel used in sauce or vinegar is 3.8 to 5.0, and the ammonia caramel carries a positive charge. This can avoid turbidity in the soy sauce or vinegar caused by an attraction between dissimilar charges. In the present study, the gelatin method was used to determine the charge of ammonia caramel. The pH of gelatin is 4.7. When the pH of a gelatin solution is lower than 4.7, it is positively charged. When ammonia caramels prepared with different concentrations of ascorbic acid were mixed with gelatin at pH lower than 4.7, no turbidity was observed. This indicated that all the ammonia caramels had positive charges.

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Figure 5–Acrylamide contents of ammonia caramels prepared by adding different concentrations of ascorbic acid to the glucose–ammonia system and allowing the reaction to proceed at 120 °C for 60 min. Each value represents the mean of 3 independent measurements and bars with different letters on the top are significantly different (P < 0.05).

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Peak area percentage (%)a

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(Biedermann and others 2002). Levine and Smith (2005) also drew similar conclusions when studying cracker production. It is worthy of note that, the acrylamide content of ammonia caramels showed the opposite concentration-dependent relationships at different levels of ascorbic acid (Figure 5). The addition of a higher concentration of ascorbic acid resulted in higher acrylamide content than that using 0.04 M of ascorbic acid. Li and others (2012) also observed the same phenomenon while studying the effect of antioxidants from bamboo leaves on the formation of acrylamide during cookie processing. These are the examples of the so-called “antioxidant paradox” (Halliwell 2000).

Conclusion As the concentration of ascorbic acid added to the mixture of glucose and ammonia increased, the color intensity of the resulting ammonia caramel showed a decreasing trend, but the intensity of fluorescence and total amount of pyrazine (main flavor substance) in the volatile products showed an increasing trend. Within the tested range of ascorbic acid concentration, the resulting ammonia caramels were all positively charged. Neither the UV-visible absorption spectra nor the types of pyrazine compounds in the volatiles were significantly different. Furthermore, the addition of 0.02 to 0.08 M ascorbic acid did reduce formation of the byproduct acrylamide, a harmful substance in food. As a result, the addition of ascorbic acid can improve the quality and safety of ammonia caramels.

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Effect of ascorbic acid on the properties of ammonia caramel colorant additives and acrylamide formation.

Ammonia caramels are among the most widely used colorant additives in the food industry. They are commonly prepared through the Maillard reaction and ...
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