Evaluating Stability of Vitamin C in Fortified Formula Using Water Activity and Glass Transition

Abstract

Stability of vitamin C in fortified formula food at constant temperature, as affected by the water activity (a_w ) and glass transition (T_g ), was investigated in an attempt to differentiate the effects of a_w and T_g on the degradation reaction. Fortified formula samples, equilibrated at different moisture contents/water activity levels, were stored for more than 10 weeks at the room temperature, 23°C. The water activity and glass transition temperatures as a function of moisture content were determined for fortified formula to compare the two distinct criteria of food stability. The adsorption experiments, which were carried out at controlled water activities, at 23°C using an isopiestic method, were modeled using the GAB equation. The glass transition temperatures, which were measured by modulated differential scanning calorimeter, were modeled using the Gordon-Taylor equation. The loss of vitamin C followed a zero-order kinetic model. The vitamin C retention after storage of 73 days at water activities of 0.11 and 0.87 was 40 and 7%, respectively. The rate constants ranged from 5.8 to 10 mgd⁻¹, depending on the water activities. The constants were correlated with water activities and glass transition temperatures. Results indicated that reactions rates correlated with both water activity and the glass transition temperatures. In the storage and processing of fortified food products, both the water activity and glass transition should be recognized as important criteria for gaining a better understanding of vitamin stability.

Keywords:

• Differential scanning calorimeter
• Neutraceuticals
• Reaction rate kinetics
• Water sorption isotherms

INTRODUCTION

In recent years, vitamins have been incorporated into more and more varieties of food formulations as the market for neutraceuticals and supplemented food keeps on growing. Vitamin C is one of the most frequently added vitamins in commercially available fortified formulas. L-Ascorbic acid is the main biologically active form of vitamin C. As an antioxidant, vitamin C plays several roles in human health. It is now well established that it helps in neutralizing free radicals, which play an important role in cell degradation. It readily scavenges reactive oxygen and nitrogen species, and thereby, prevents oxidative damage to important biological macromolecules such as DNA, lipids, and protein. It also reduces redox active transition metal ions in the active sites of specific biosynthesis enzymes and allows for lowering of blood pressure.[1]

Vitamin C is unstable and its degradation depends upon many factors such as oxygen, heat, light, storage temperature, and storage time.[2] The stability of vitamin C has been evaluated in fresh fruits during processing and storage.[2–7] The stability of vitamin C in fortified formula has not been evaluated using water activity and glass transition concepts.

Water activity has traditionally been used to describe microbial and chemical stability of food products.[8–10] During the last two decades, attempts have also been made to apply glass transition concept to explain the physical, chemical, and microbial stability of food, biological, and pharmaceutical product.[11–20] In the glassy state, reactions that depend on molecular diffusion, such as chemical and enzymatic changes, are reduced significantly. Despite the research effort, the significance and the mechanism of the glass transition as an indicator of chemical and biochemical stability is still not clear.[18] Due to the interrelation among factors (i.e. temperature, water activity, and pH) influencing chemical reactions, it is difficult to interpret the results.[21] Food materials are heterogeneous systems; these may contain local regions of different molecular mobility. The use of a single glass transition temperature measured using existing methods of mechanical and thermal measurements may not be taken as an absolute reference for stability.[22]

Few studies have compared both criteria (i.e., water activity and glass transition) from the viewpoint of product stability. Roos[23] evaluated these concepts in relation to the molecular weight of materials of a homologous family. He defined the critical water content that depressed the glass transition temperature to 25°C. It was indicated that increasing molecular weight of the material required a rise in critical water activity from 0.07 to 0.71 in order to depress the T_g to ambient temperature. Schaller-Povolny et al.[24] compared water activity and glass transition data of four different molecular weights of inulin. Similarly, their study argued that increasing molecular weight increased the critical water activity from 0.44 to 0.61 required at a given T_g . These studies compared the a _w/T_g requirement at ambient temperature (i.e., between 23 and 25°C). Sablani et al.[25] examined the two concepts with several food materials. They indicated that based on the sorption isotherm criterion, for most food materials (expect sugar rich fruits), the predictions of the glass transition model overestimate the stable temperature range at each level of solids.

Most of the studies reported have used model food systems to evaluate chemical changes while applying glass transition and water activity concepts.[15,16,18,33] Information on stability of bioactive compounds in food materials such as fruits, vegetables, and dairy products; and, neutraceuticals formulations as influenced by water activity and glass transition is scarce.

The objective of the present article was to evaluate the storage stability of vitamin C in fortified formula using water activity and glass transition concepts. This would enhance the knowledge of the necessary requirements for proper storage conditions and chemically stable formulations.

MATERIALS AND METHODS

Material and Vitamin C Analysis

A commercial fortified formula manufactured by a multinational company was selected for the evaluation of the stability of vitamin C during storage at room temperature. The shelf life of fortified formula as specified on the label was 2 years. At the time of purchase, the formula powder was four months old and its initial moisture content was 5.4% (w.b.). The ingredients of the fortified formula listed on the bottle label were milk solids, wheat flour, malted barley, malt extract, sugar, 16 different minerals, and vitamins. Immediately after purchase, the formula samples were equilibrated at different moisture contents using water activity jars. These were then stored at room temperature for 73 days. The moisture content of equilibrated samples was calculated gravimetrically by drying samples in the air convection drier at 105°C for at least 24 hours.[26] The vitamin C content in fortified powder was determined using a titration method with 2–6 dichloro-phenol-indophenol reagents.[26] The loss of vitamin C in fortified formula was modeled using the standard equation of zero- and first-order reaction given below:

(1)

where C, the concentration at time t; k, the reaction rate constant; t, the storage time (day); and n is 0 or 1.

Sorption Isotherms

The water sorption isotherm of fortified formula powder was obtained using an isopiestic method.[28,29] Five replicates of powder were placed in open weighing bottles and stored in air-sealed glass jars while maintaining equilibrium relative humidity with saturated salt solutions. The salts were: LiCl, CH₃COOK, MgCl₂, K₂CO₃, Mg(NO₃)₂, NaBr, SrCl₂, and KCl (BDH, Laboratory Supplies, Poole, England). Relative humidity values for these solutions were obtained from Greenspan,[29] and Spiess and Wolf.[30] The jars were kept at room temperature of 23°C.

The most commonly used BET and GAB equations were employed for modeling of the sorption isotherm.[28] The BET equation is given by:

(2)

where M_b is the BET monolayer moisture content (dry basis) and B is a constant related to the net heat of sorption. The BET isotherm holds well between water activities of 0.05 and 0.45, an adequate range for the calculation of parameters M_b and B. The GAB equation is given by:

(3)

where M_g is the GAB monolayer moisture content (dry basis). The GAB isotherm equation is an extension of the BET model taking into account the modified properties of the solute in the multilayer region and the bulk liquid water properties through the introduction of a third constant K. In theory, both methods should provide the same monolayer value. However, estimation of the three parameters in GAB using two variables (i.e., water content and water activity) leads to a non-linear optimization may cause different localized optimization.[32] Evaluation of predictions from BET and GAB models with experimental data was carried out using the mean relative error (MRE).

Thermal Analysis

A modulated differential scanning calorimeter (MDSC, Q1000, TA Instruments, New Castle, DE) equipped with a mechanical cooling system was used to perform experiments. Temperature modulation (sinusoidal oscillation) of MDSC separates total heat flow into its reversing (thermodynamic) and non-reversing (kinetic) components. A mechanical refrigerated cooling system was used to chill the sample up to –90°C. The calorimeter was calibrated according to the instruction provided by TA instruments user manual by checking temperature and enthalpy of fusion of indium and sapphire as standards. Fortified formula powder samples (5–10 mg) equilibrated at different water activities were placed in aluminum pans and were cooled to –90°C at 5°C/min, and equilibrated for 10 minutes. After equilibration it was scanned from –90°C to 90°C at a rate of 5°C/min. Each thermogram was analyzed for the onset, mid, and end of glass transition, and mid values were used. Three replicates were used for the measurement of glass transition temperatures at each water activity/moisture content.

An empirical equation proposed by Gordon and Taylor[32] was used to predict the glass transition temperature of mixtures:

(4)

where T_gm , T_gs , and T_gw are the glass transition temperature of the mixture, solids, and water respectively; X_s and X_w are the mass fraction of solids and water; and k is the Gordon-Taylor parameter. From the thermodynamic standpoint, this parameter is equivalent to the ratio of the change of component mixture specific heat at their T_g . The above equation is also commonly used to predict the vitrification properties of multi-component foods, and other biological mixtures. The model parameters (k and T_gs ) of Eq. (4) were estimated using non-linear regression analysis while considering that the T_gw is –135°C.

RESULTS AND DISCUSSION

Water Sorption and Glass Transition Data

Experimental water sorption and glass transition data for fortified formula is presented in Table 1 and also plotted in Figs. 1 and 2. The constants of BET and GAB models are shown in Table 2. These constants can be used to predict water sorption isotherms at room temperature. The monolayer moisture content values were 6.31 and 9.95% (d.b.) in the BET and GAB models, respectively. The monolayer moisture content estimated by GAB model was higher than the BET monolayer moisture content. The BET monolayer is an effective method for estimating the amount of water bound to specific polar sites in dehydrated systems. The product at its monolayer moisture should be stable against microbial and chemical deterioration.[10] The values of T_gs and k for fortified formula powder were found to be 46.5°C and 2.18, respectively.

Table 1 Water activity and corresponding equilibrium moisture content (EMC, g H₂O/g of dry matter) and initial (T_gi ), mid (T_gm ), and end (T_ge ) glass transition temperatures of fortified formula powder

aw	EMC	T gi	T gm	T ge
0.113	0.0480 (0.00073)	22.8 (2.4)	27.0 (2.47)	34.3 (5.30)
0.216	0.0680 (0.0064)	18.2 (2.52)	22.8 (1.61)	30.8 (2.43)
0.336	0.0900 (0.0013)	13.7 (1.00)	18.10 (1.35)	22.4 (3.00)
0.431	0.100 (0.0041)	12.0 (1.89)	9.23 (0.32)	16.2 (1.87)
0.529	0.129 (0.0023)	2.27 (0.60)	8.63 (1.01)	16.0 (0.06)
0.577	0.152 (0.0016)	0.65 (0.52)	6.87 (0.67)	13.4 (0.68)
0.708	0.193 (0.0069)	−5.75 (1.36)	3.63 (1.46)	11.0 (0.76)
0.877	0.260 (0.0081)	−32.5 (6.05)	−31.1 (2.30)	−24.1 (3.34)
Values in brackets are standard deviations.

Figure 1 Experimental and predicted sorption isotherm data of fortified formula powder.

Figure 2 Variation of glass transition temperature with solid content.

Table 2 BET and GAB model parameters of water adsorption isotherms of fortified formula powder at room temperature

BET			GAB
M b (g/g dry matter)	B	MRE (%)	M g (g/g dry matter)	C	K	MRE (%)
0.0631	17.2	2.5	0.0995	6.26	0.743	5.2

Water activity and glass transition concepts were closely examined for formula powder (Table 3 and Fig. 3). The water activity concept suggests that a product is most stable at its monolayer moisture content, which depends on chemical composition and temperature of the material.[9] The glass transition concept puts forward that a product formulation is stable at or below the corresponding glass transition temperature. Based on the sorption isotherm criterion, the predictions of the glass transition model closely matched the stable temperature. For instance, the sorption isotherm at 23°C indicates that formula powder would be stable at 94.1% solids (corresponds to BET monolayer) (Table 2 and Fig. 3). At this level of solids, the glass transition curve produced using the Gordon and Taylor equation yields a T_g value of 24.7°C. The glass transition temperature of 23°C corresponds to a solid content fraction of 0.936 and a water activity value of 0.225 that is very much within the range of stable water activity. Sablani et al.[25] have reported that based on the sorption isotherm criterion, for most food materials, except sugar rich fruits, the predictions of the glass transition model overestimate the stable temperature range at each level of solids.

Table 3 Comparing sorption isotherm and glass transition models using monolayer a_w as the basis for stability

Water activity model					Glass transition model
Sorption isotherm model	a w at mono-layer moisture	T (°C)	Solid content at monolayer moisture content (fraction)	T g from GT equation (°C)	T g (°C)	Solid content (fraction)	a w at corresponding solid fraction
BET	0.194	23	0.941	24.7	23	0.936	0.225

Figure 3 Variation of glass transition temperature with water activity and moisture content.

Stability of Vitamin C

The initial concentration of vitamin C in fortified formula powder was 690 ± 55 mg/100 g of dry formula. After 73 days of storage, vitamin C contents of samples decreased to 273 and 8 mg/100 g of dry formula at water activity of 0.11 and 0.87, respectively. The change in concentration of vitamin C with storage time at different water activities is shown in Fig. 4. The destruction of vitamin C occurred at all the water activities; however, the rate of degradation was lower at reduced level of water activity. The degradation of vitamin C was modeled using zero and first order kinetics. The values of R² varied from 0.78 to 0.98 in case of for first order model and it varied from 0.91 to 0.99 for zero-order model. Hence the reaction rate constants of degradation of vitamin C were used from zero-order kinetic model (Fig. 5). The rate constants ranged from 5.8 to 10 mgd⁻¹ depending on water activities.

Figure 4 Loss of vitamin C in fortified formula stored at room temperature at different water activities.

Figure 5 Zero-order plot of vitamin C loss in fortified formula stored at room temperature at different water activities.

There have been studies reported that degradation of vitamin C followed zero-order,[3] first order,[2,5] or second-order reaction.[4] The reaction rate constant calculated at different moisture levels were plotted as a function of water activity and the distance from the glass transition temperature (i.e. T-T_g ) (Fig. 6). The reaction rate decreased significantly (P < 0.5) with decreasing water activity and T-T_g . However, at very low water activity (a_w <0.2; corresponding to the storage temperature below glass transition), the reaction rate did not approach to zero. Overall, the reactions rates correlated very well with both water activity and the glass transition temperatures. The reaction rate constant was minimal around monolayer moisture content and as water activity increased the reaction rate increased. The condition of monolayer moisture content also corresponded with the glassy state of the formula powder. That is at monolayer moisture content the glass transition temperature of formula powder was above the storage temperature of 23°C. The formula powder at monolayer moisture was in the glassy state when stored at the room temperature. Both water activity and glass transition concepts predicted similar conditions of moisture/solids level and temperature for safe storage. In the literature, the water activity and glass transition data have been compared for stability of aspartame and thiamin in model food systems, i.e., polyvinylpyrrolidone of different molecular weight.[15] Bell and Hageman[15] observed that aspartame degradation depended on the water activity rather than upon the state of the system. While both a_w and T_g appeared to affect thiamin stability in solids with glass transition considerations having larger effect on the reaction.[33] The article suggests that a_w or T_g criteria alone can be used to evaluate stability of vitamin C in fortified formula powder.

Figure 6 Vitamin C degradation in fortified formula stored at room temperature as affected by (a) water activity and (b) distance from glass transition temperature.

CONCLUSIONS

Vitamin C in fortified formula decreased more rapidly with increasing water activity and distance from glass transition temperature. The loss of vitamin C in fortified formula at different moisture levels was described by zero-order reaction. Both a_w and T_g affected the stability of vitamin C. In the storage and processing of fortified food products, both the water activity and glass transition should be recognized as important criteria for gaining a better understanding of vitamin stability. Research on the stability of other nutrients in different food systems as affected by water activity and glass transition would provide additional insight into the factors influencing their degradation during storage.

NOMENCLATURE

a w	=	Water activity (fraction)
B	=	Constant related to net heat of sorption in Eq. 1
C	=	Constant related to first layer heat of sorption in Eq. 2
i	=	ith experimental data
K	=	Constant related to heat of sorption of multi-layer in Eq. 2
M w	=	Moisture content (fraction, gH2O/g dry matter, d.b.)
M b	=	Monolayer moisture content in BET equation (g/g dry matter)
M g	=	Monolayer moisture content in GAB equation (g/g dry matter)
k	=	Gordon-Taylor constant parameter
T	=	Temperature (K)
X w	=	Moisture content (fraction, gH2O/g sample, w.b.)