Kinetic behaviour of ascorbic acid - fructose browning reaction in alkaline medium

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1 Indian Journal of Chemical Technology Vol. 19, March 01, pp inetic behaviour of ascorbic acid - fructose browning reaction in alaline medium Neelu ambo 1 & Santosh Upadhyay * 1 Department of Chemistry, Uttar Pradesh Textile Technology Institute, anpur 08 00, India Department of Chemistry, H B Technological Institute, anpur 08 00, India Received 4 January 011; accepted 0 January 01 Ascorbic acid and fructose react in an aqueous alaline solution at temperature below 50 C in a Maillard-type reaction to from brown coloured end products. The inetics of browning of alaline solution of fructose in presence of ascorbic acid has been studied by measuring the absorbance of browning development in the reaction. The development of brown colour due to heating of either reducing sugar alone or ascorbic acid alone in the presence of alali is found to be very slow even at high temperature (~70 C). However, in the presence of ascorbic acid the development of brown colour due to heating of alaline fructose is sufficiently fast even at low temperature ~50 C. The rate of development of colour (browning reaction) has been found to be first order dependence with respect to [alali and [fructose and fractional order dependence with respect to [ascorbic acid at low concentrations of ascorbic acid. A mechanism consistent with the observed inetic data has also been proposed. eywords: Ascorbic acid, Browning coloration, Browning reaction, Fructose, inetics Non-enzymatic browning reactions, which involve Maillard reactions 1-6 (reaction of amino acids/ proteins with reducing sugars), caramelisation 7 (heating of sugars and reducing sugars in presence or absence of alali at higher temperatures) and degradation of ascorbic acid 8,9 (specially in juices), lead to formation of a wide variety of products which contribute unpleasant changes and deleterious to the nutritional value of food concern. The reactions indicated a very complex nature of system and involve a number of intermediate complexes 10. The reactions between nitrogen free carboxylic acids (oxalic, malic, fumaric, phosphoric acids) and reducing sugars have also been found to follow a Maillard type reaction 11 accompanied by browning and CO production. Chemical inhibitors viz. glutamic and aspartic acids, sodium bisulphate and sulphur dioxide have been used 1,13 to advantage in limiting browning reactions during the production and storage of variety of foods. Many researchers 9,14 have demonstrated that nonenzymatic browning in citrus juices is due to the reactions of sugars, amino acids and ascorbic acid. It leads to the formation of a wide variety of * Corresponding author. upadhyay_s_@rediffmail.com end products. The most common method for characterizing browning is the measurement of colour development as a function of time and its expression in terms of inetics of reactions. Various studies have hypothesized the appearance of brown pigments following either zero order or first order inetics The measurement of the inetics of a reaction provides the most general method of determining its mechanism and can help a food scientist in maing decisions on how to optimize a process. At a fixed temperature, the reaction rate is a function of the concentration of some or all of the components of the system, etc. In preliminary studies it has been observed that the browning development due to heating of reducing sugar alone in presence of alali proceeds at high temperature i.e. above 70 C. The rate of development of colour by heating of reducing sugars in presence of alali may be represented as Rate of browning α [Reducing sugar [OH Further, it has been observed that development of colour due to heating of ascorbic acid alone in presence of alali was negligible up to temperature 80 C. The development of brown colour due to either fructose or ascorbic acid alone is very slow even at 80 C, while in presence of both (fructose and ascorbic

2 AMBO & UPADHYAY: INETIC BEHAVIOUR OF ASCORBIC ACID - FRUCTOSE BROWNING REACTION 19 acid) the development of brown colour proceeds with a measurable speed even at low temperature (~50 C). It is, therefore, thought worthwhile to study the inetics of fructose-ascorbic acid browning reaction in detail to understand the mechanism of the reaction. The findings are reported in the present paper. Experimental Procedure All the solutions were prepared in double distilled water. Aqueous solutions of D (-) fructose, ascorbic acid (Thomas Baer, Mumbai, India) were prepared just before the experiments. All the chemicals used were of AR grade. The required quantities of solution of fructose and ascorbic acid were placed in 100 dm 3 Jena glass vessel. The requisite amount of double distilled water was added to eep the volume of reaction mixture 50 dm 3 after adding the NaOH. After equilibration at the desired temperature ± 0.1 C, the reaction was initiated by adding the requisite amount of NaOH, placed separately in the same water bath. The most common method for the characterization of browning is the measurement of the developed colour in the reaction mixture as a function of time. Therefore, the inetics of the reaction have been followed spectrophotometrically by monitoring the absorbance of the developed brown colour in the reaction mixture at 40 nm using the spectrophotometer (Toshniwal TVSP 5, India). Results and Discussion It has been observed that the statistical difference between a zero order fit of inetic data and a first order fit is very slight due to formation of a variety of browning pigments with varying stabilities and, therefore, it has been hypothesized that either model could be used without introducing significant error. In the present investigation, it is observed that initially the absorbance (i.e. development of colour) increases linearly with time and then tends to become constant (Fig.1). This is expected because on completion of the reaction there will be no further development in the colour. The pseudo zero-order rate constants ( obs ) have been evaluated from the slopes of linear portion of the plots of absorbance versus time at different initial concentrations of the reactants. The rate constants obs are found to be reproducible within 5% in replicate runs. The rate constants have been obtained at various initial concentrations of the reactants. The observed rate constant ( obs ) increases with an increase in [fructose and a plot of rate constant versus [fructose is found to be linear (r >0.997, s = 0.01) passing through the origin (Fig. ), suggesting a first-order dependence of rate with respect to fructose. The effect of ascorbic acid is found to be interesting. On increasing the ascorbic acid Fig.1 Plots of absorbance versus time at 50 C [[fructose = mol dm -3, [ascorbic acid = mol dm -3 and [OH - = 4.0, 6.0, 8.0, 10.0, and mol dm -3 for a, b, c, d and e respectively Fig. Plot of obs versus [fructose at 50 C [[ascorbic acid = mol dm -3 and [OH - = mol dm -3

3 130 INDIAN J. CHEM. TECHNOL, MARCH 01 concentration, an increase in obs is observed at lower concentrations of ascorbic acid. However, at higher [ascorbic acid the effect of ascorbic acid on obs becomes negligible. A plot of obs versus [ascorbic acid is linear (r >0.998, s = 0.01), with an intercept at lower [ascorbic acid while at higher [ascorbic acid there is no change in obs (Fig. 3). The value of intercept of this plot matches with obs obtained in absence of ascorbic acid, i.e. when [ascorbic acid is equal to zero. The effect of alali was studied at a fixed ionic strength (μ = 0.4 mol dm -3 ) maintained by NaClO 4. The plot of obs versus [OH - (Fig. 4) is linear (r >0.997, s = 0.01) passing through the origin, suggesting a first-order dependence of the rate with respect to OH. The effect of alali was also studied at higher concentration of ascorbic acid (> mol dm -3 ), where the plot of obs versus [OH is found to deviate from the linearity at higher [OH. Therefore, it appears that at higher [ascorbic acid, the order of reaction in alali also decreases from unity. The reaction has been studied at different temperatures such as 50, 60 and 70 C and energy of activation (ΔE # ) is evaluated as ± 1.0 Jmol -1. Although the mechanism of the browning involves the various steps, the evaluation of other activation parameters is not of much importance, even then the activation parameters have been evaluated and are found to be ΔH # = 79.5 ± 0.5 J mol -1, ΔS # = ± 1.0 J -1 mol -1 and ΔG # = 58.5 ± 0.5 J mol -1. A high negative value of ΔS # indicates the compactness of the transition state. The effect of glutamic acid, aspartic acid, sodium bisulphate and sulphur dioxide on the rate of development of brown colour in the reaction has also been studied at 50 C. It is observed that the rate of development of colour is inhibited in presence of the above substances. In alaline medium, the formation of an enediol anion 19-1 of monosaccharides taes place as follows: In the absence of other reactants, these anion undergo epimerization and isomerization to form a mixture of aldoses and etoses (Labryde-Bruyn- Albenda Van Eenstein transformation). Aldoses and etoses generally yield mixtures of Z and E enediols, the proposition of which differs from sugar to sugar and experimental conditions such as strength, Fig.3 Plot of obs versus [ascorbic acid at 50 C [[fructose = mol dm -3 and [OH - = mol dm -3 Fig.4 Plot of obs versus [OH - at 50 C [(µ = 0.40 mol dm -3 ) other conditions same as in Fig.1

4 AMBO & UPADHYAY: INETIC BEHAVIOUR OF ASCORBIC ACID - FRUCTOSE BROWNING REACTION 131 nature of alali and temperature. In the presence of an oxidant or a catalyst the enediol anion has been considered as the reactive species of the reducing sugar. First order dependence of rate with respect to each fructose and OH, clearly indicates that the formation of enediol anion is the rate-determining step during the colour development. The enediol anion leading to the formation of an intermediate and then coloured product in the fast steps. However, in the presence of ascorbic acid, the enediol anion may react with ascorbic acid to give an intermediate which is finally converted to a coloured product. This is also supported by a fractional order dependence of rate with respect to ascorbic acid at lower [ascorbic acid. On the basis of experimental results and above facts, the mechanism for the browning development may be suggested as follows: In the absence of ascorbic acid in absence of ascorbic acid. The experimental results in the absence of ascorbic acid are in agreement with rate law [Eq (3). In presence of ascorbic acid The enhancement of rate of development of brown colour due to fructose even at ~50 C in presence of ascorbic acid indicates the interactions between fructose and ascorbic acid. Ascorbic acid is very closely related to the monosaccharide and behaves as an unsaturated compound, a strong reducing agent 3. The salt forming property of ascorbic acid has been explained due to the presence of an enol group. It has been shown that in alaline solution, the hydrogen atom of the C -enol group ionizes and is replaced by the metal. Therefore, in alaline solution the formation of enediol anion of ascorbic acid is considered as As + OH 3 (Ascorbic acid) As + H O (a) (Enediol anion of ascorbic acid) The presence of ascorbic acid increases the rate of development of brown colour, and therefore, it is possible that enediol anions of reducing sugars and ascorbic acid interact to give an intermediate which is finally converted to a coloured product, as shown below: On the basis of mechanism (Scheme 1) the rate of browning in the absence of ascorbic acid may be given as d [ colour = [F (1) By applying the steady state conditions with respect to [F, the rate law may be written as d [ colour 1[F[OH = [H O + 1 () Taing a suitable approximation i.e. >> -1 [H O, because step [Eq (ii) is fast, the rate law [Eq () reduces to d [ colour = 1 [F[OH (3) Rate law [Eq (3) suggests a first order dependence of rate with respect to each reducing sugar and alali On the basis of Scheme, the rate of colour development in presence of ascorbic acid may be given as

5 13 INDIAN J. CHEM. TECHNOL, MARCH 01 d[ colour / = [X I + 5 [X or = [F [X + 5 (4) The formation of F is governed by slow rate determining step (i), and therefore [F may be obtained by applying steady state conditions with respect to steps (i) and (ii). The [F is given as 1[F[OH [F = [H O + _1 Since step (ii) is fast, >> 1 [H O may be taen as suitable approximation, thus [F may be obtained as [F [F[OH 1 = (5) Further, considering the equilibrium step (iii) we can get [As = 3 [As [OH where 3 also includes water molecule. Similarly from equilibrium steps (iii) and (iv), we get [X = 4 [As [F = 4 3 [As[OH [F (6) On substituting the value of [F from Eq (5) into (6), we obtain [ X [As[F[OH = (7) The total concentration of fructose at any time i.e. [F T is given as [F T = [F + [F + [X (8) On substituting the value of [F and [X from Eqs (5) and (7) respectively in Eq (8), after solving the values of [F and [X in terms of [F T may be obtained as [F 1 T [F = + 1[OH [As[OH and [OH [AS[F T [X = + 1[OH [As[OH [OH (9) (10) On substituting the values of [F and [X in Eq (4), the rate law is obtained as d[colour 1[F T[OH { [As[OH } = + 1[OH {1+ 3 4[As[OH } (11) The rate law [Eq (11) explains the experimental results. In absence of ascorbic acid, where, the steps (iii)-(v) do not exist, the rate law reduces to d[colour = 1 [F T [OH, (1) which suggests first order dependence of rate of development of colour with respect to each alali and fructose in absence of ascorbic acid. At low concentrations of ascorbic acid, the rate law [Eq (11) reduces to d[colour = [F T { [As[OH } (13) where 1 [OH >> and 1 >> 3 4 [As[OH have been taen as suitable approximations. The rate law [Eq (13) predicts first order dependence of rate with respect to each fructose, alali and ascorbic acid. The plot of ( obs ) versus [ascorbic acid should also give an intercept which has been observed experimentally. At very high concentrations of ascorbic acid and alali, where rate of browning in absence of ascorbic acid becomes negligible, the rate law [Eq (11) reduces to d[colour 5 3 4[F T[As[OH = 1+ [As[OH 3 4 (14) Under these conditions, taing suitable approximation as 3 4 [As[OH >>1, the rate law [Eq (14) becomes d[colour = 5 [F T (15) This suggests that the rate of development of colour does not depend on ascorbic acid and alali at higher concentrations of ascorbic acid and alali. The experimental results are in agreement with the proposed rate laws. On the basis of above studies it may be concluded that the rate of browning, which is negligible or very

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