INTERACTION OF VANILLIN WITH SOY AND DAIRY PROTEINS IN AQUEOUS MODEL SYSTEMS: A THERMODYNAMIC STUDY ABSTRACT: Interactions of vanillin

with soy, casein, and whey proteins were studied in aqueous model systems using a thermodynamic approach. Vanillin-protein binding was examined at 12 and 4C using 3 proteins sand 6 vanillin concentrations. The results were analyzed using a Klotz plot. Number of binding sites (n) and dissociation constants (Kd) were derived from the plots. The thermodynamic parameters (G, H, and S) for the bindings were calculated. Under identical experimental conditions, whey protein was found to have higher affinity to vanillin than the other 2 proteins. Bindings of vanillin with casein and whey protein were enthalpy driven, while the interaction of vanillin with soy protein was highly entropy driven. The results inferred that conformational changes of soy protein might be important in binding of vanillin. I. INTRODUCTION

Interactions of flavor compounds in food systems with dairy and soy protein have been investigated by numerous researchers in the past 30 years. Due to their complexity, flavorprotein interactions remain a major challenge for food scientists and flavor chemists. Interactions of soy, casein, and whey proteins and their fractions with different volatile compounds, including aldehydes, ketones, alcohols, and methyl esters have been studied (Stevenson and others 1996; McGorrin 1996). Binding phenomena have been studied in headspace and aqueous phases using both instrumental and sensory analyses. Results indicate that the amount of flavorants bound depends mainly on protein type, protein conformation, and type and position of functional groups of the flavorant. The binding is also affected by temperature, pH, and the concentrations of the protein and the flavor compounds (McGorrin 1996). However, it is difficult to compare results and reach conclusions on the protein-binding phenomenon because great differences exist in approaches and experimental conditions in previous studies. Kinsella and coworkes studied the protein-flavor interaction using both thermodynamic and conformational approaches. They investigated interactions among various volatile compounds and proteins, including carbonyls with bovine serum albumin (Damodaran and Kinsella 1980), carbonyls with soy-protein fractions (7s and 11s) (Damodaran and Kinsella 1981a, 1981b), aldehydes and methyl ketone with -lactalbumin, bovine serum albumin, soy-protein concentrate and isolate, and other plant proteins (Franzen and kinsella 1974), and 2-nonanone with soy protein and fractions (-conglycinin and glycinin) (Oneill and Kinsella 1987a), and with -lactoglobulins (Oneill and Kinsella 1987b, 1988). Damodaran and kinsella (1980) studied interactions between carbonyls and bovine serum albumin (BSA) in aqueous phase. Bindings of 2-heptanone, 2-nonanone, and nonanal to BSA at 25C were quantified using gas chromatography. Binding affinity of ketones increased with chain length, suggesting the binding was hydrophobic in nature. Bindings were found to be entropy driven. Damodaran and Kinsella (1981a) determined thermodynamic effects on interactions of carbonyls with native and denatured soy protein using equilibrium dialysis at 5, 25, and 45C. They found that the interaction between carbonyls and soy proteins was relatively weak and reversible. The binding affinity was reported to be the same for 25C and 45C, but binding affinity decreased at 5C. Damodaran and Kinsella (1981b) investigated conformational effects on carbonyls binding onto glycinin and -conglycinin at 25C using equilibrium dialysis.

Their model was based on the assumption of reversible and non-cooperative binding, and they concluded that soy glycinin has almost no affinity for 2-nonanone. The study of binding properties of 2-nonanone with soy protein and fractions (-conglycinin and glycinin) (Oneill and Kinsella 1987a) was a follow-up study to the previous study that investigated conformational effects on carbonyls binding onto these 2 soy-protein fractions (Damodaran and Kinsella 1981b).binding affinity of 2-nonanone onto pure soy protein, glycinin, -conglycinin, and subunits of glycinin were determined using equilibrium dialysis. Glycinin was shown to have lower affinity for 2-nonanone than -conglycinin. However, the number of binding sites and binding constants were different from the finding of Damodaran and Kinsella (1981b). They explained that the differences in results was due to differences in the composition of soy proteins used, and more importantly, an inappropriate absorption coefficient was used for estimating protein concentration in their previous studies. Wilson and coworkers used a thermodynamic approach for studying flavor interactions with soy protein. Using headspace analysis, Aspelund and Wilson (1983) investigated the adsorption of off-flavor compounds onto soy-protein isolate in the dry state at 80 to 100C. Compounds studied were aldehydes, ketones, methyl esters, hydrocarbons, and alcohols. The results indicated that functional groups of the flavor compounds play important roles in binding to soyprotein isolate in the dry state. Binding of volatiles onto soy protein in an aqueous phase was also investigated, including binding with soy-protein isolate (Wilson 1985), glycinin, and conglycinin (OKeefe and others 1991). Unequal positive cooperative bindings were found for ketones, aldehydes, and methyl esters. OKeefe and others (1991) reported that glycinin has a higher affinity than -conglycinin for binding carbonyl compounds, including 2-nonanone. These results disagree with those of Kinsellas group (Damodaran and Kinsella 1981b; ONeill and Kinsella 1987a). However, differences in experimental conditions make it very difficult to compare results from different studies. Bindings of 2-nonanone and nonanal by whey proteins (-lactoglobulins and -lactalbumin, bovine serum albumin, and whey-protein concentrate) were studied by Jasinski and Kilara (1985). The equilibrium dialysis approach used was based on procedures of Kinsellas group, with slight modifications (Jasinski and Kilara 1985). Quantification methods other than the most commonly used GC and HPLC have also been used for binding studies. Using fluorescence spectrometry, Dufour and Haertle (1990) studied the effect of chemical modification (esterification and reductive alkylation) on binding properties of terpenes with -lactoglobulins. The brief review above indicates the diversity of binding study designs, approaches, and results obtained. Large variations exist among numbers of binding sites, properties, and thermodynamic data (G, H, and S) in the literature (Wilson 1985; OKeefe and others 1991; Stevenson and others 1996). Direct comparisons of casein, whey, and soy proteins cannot be made due to differences in experimental conditions and analytical procedures used by different researchers. Therefore, the objective of the current project was to compare interactions of casein, whey, and soy proteins with vanillin under identical experimental conditions. II. MATERIALS AND METHODS Vanillin

Six standard vanillin solutions were used for generating a standard curve. Two g of vanillin crystals (Fisher Scientific, St. Louis, Mo., U.S.A.) were dissolved into 200 mL HPLC-grade water (Fisher Scientific). Diluting 5, 10, 20, 30, 40, and 50 mL of the above solution into 100 mL each resulted in 500, 1000, 2000, 3000, 4000, and 5000 mg/L stock vanillin solutions, respectively. Then 1.25 mL of each of these vanillin stock solutions was diluted to 50 mL with water, which resulted in 12.5, 25, 50, 75, 100, and 125 mg/L vanillin standard solutions that were injected into the HPLC for generating the standard curve. Proteins

Sodium caseinate (ALNATE 180) and whey-protein isolate (ALACEN 895) were obtained from the New Zealand Milk Products (North America) Inc. (Santa Rosa, Calif., U.S.A.), and soyprotein isolate (500E) was kindly provided by Protein Technologies International (St. Louis, Mo., U.S.A.). Five g of each protein were mixed with 250 mL HPLC-grade water in a glass jar using an Osterizer Galaxies 10-speed blender (Oster Direct/Sunbeam Co., McMinnville, Tenn., U.S.A.) for 10 s at medium speed. The 2% (w/v) protein mixtures were transferred into 400-mL glass beakers. The protein solutions were placed in a water bath and heated at 81.5C for 25 s, then cooled down to 4C in an ice bath. The cooled protein solutions were then aged at 4C in the refrigerator for 12 to 20 h to allow complete hydration of the protein and were then ready to be used for the binding studies. Micropartition System (MPS)

Operating procedures for the MPS have been described by Li and others (1997). After the binding equilibrium was reached, 1mL of each vanillin-protein solution was transferred into the MPS system (Millipore/Amicon, Bedford, Mass., U.S.A.) and centrifuged through an MPS membrane at 13,000 x g for 30 min using a Beckman Model J2-21 centrifuge (Beckman Instrument, Inc., Schaumburg, I11., U.S.A). Based on Fukushima (1991), average molecular weights for components of whey, casein, and soy proteins are 4,000 + Da, 19,000 + Da, and 18,000 + Da, respectively. Since vanillin has a molecular weight of 152, YM membranes with 1,000 Da molecular weight cut-off (MWCO) (Millipore/Amicon) were used for the separation. Free vanillin was collected in the filtrate and injected into the HPLC system. Before injection, the filtrates were further filtered through a 0.2 m syringe filter (4mm nylon white, Alltech Assoc., Inc., Deerfield, I11., U.S.A.) to remove small peptides that could be precipitated by the mobile phase and clog the HPLC column. Determination of binding properties

Binding studies were conducted at 12 and 4C. For each temperature, a randomized complete block design (Cochran and Cox 1957) was used with 3 proteins (casein, whey, and soy proteins) and 6 vanillin concentrations (10, 20, 40, 60, 80, and 100 mg/L). The experiments were conducted in 3 replications. For each protein, 25 mL of the aged protein solution was pipette into 6 50-mL Erlenmeyer flasks, and 0.5 mL of each vanillin stock solution (500 to 5000 mg/L) was added to separate flasks. This resulted in approximately 10, 20, 40, 60, 80, and 100 mg/L vanillin in the protein solutions. A control sample, 10 mg/L vanillin in water, was prepared for each replication. The flasks were sealed with parafilm immediately after adding the vanillin. The solutions were mixed for 30 s at high speed using a stir bar on a Corning Hot Plate-Stirrer PC-351 (Corning Glass Works, Corning, N.Y., U.S.A.). After mixing, each solution was transferred into a 12-mL

glass bottle containing a mini stir bar. The bottles were completely filled and tightly sealed with 3 layers of parafilm. Samples were placed into either a 12C cooler for > 48 h or a 4C cooler for > 72 h to allow binding equilibrium to be reached. Times to reach binding equilibrium for the 3 proteins were determined in preliminary tests as follows. Casein, whey, and soy protein solutions (2% w/v) were flavored with 50 mg/L vanillin and stored at 4 or 12C. The amount of free vanillin was measured over time (0.5 to 126 h). Equilibrium curves were generated by plotting the amount of free vanillin over time to determine time for reaching equilibrium at different temperatures. After binding equilibrium was reached,1 mL of each sample was transferred into the MPS (Millipore/Amicon) to separate the free vanillin from the protein solution. The concentration of the free vanillin in each sample was then determined by HPLC. For each protein, the binding data were plotted as a Klotz plot, which is one of the most common plotting techniques in binding studies (Price and Dwek 1979). In the Klotz plot, the number of moles of ligand (A) bound per mole of protein (P) is defined as r, the reciprocal of which is plotted on the ordinate, while the abscissa is the reciprocal of the concentration of unbound ligand. Numbers of binding sites (n) and dissociation constants (Kd) were derived from the plots (Klotz and others 1946; Price and Dwek 1979) because the number of binding sites is the intercept on the ordinate and the slope of the line is Kd/n. Binding equilibrium constants (K) were calculated using K=1/Kd. Thermodynamic properties were calculated using the binding equilibrium constant (K). The Gibbs free energy (G) for each protein at different temperatures was calculated using the equation:

Where R was the gas constant, T was the absolute temperature in degrees of Kelvin, and K is the binding constant. The enthalpy of binding (H) was determined using the Vant Hoff Equation: ( )

where K1 and K2 were the binding constants at 4 and 12C, T was the absolute temperature in degrees of Kelvin, and R was the gas constant. The entropy of binding (S) was determined using the equation:

(Price and Dwek 1979; OKeefe and others 1991). HPLC methodology

The HPLC method for quantification of vanillin was developed by Rouseff (1985). A similar method developed by Guarino and Brown (1985) has been evaluated in an AOAC collaborative study. The method used in the current project was developed based on the above methods (Li and others 1997). The HPLC system (Perkin-Elmer Corp., Norwalk, Conn., U.S.A.) included a Series 410 pump and solvent system and a LC 90 UV spectrophotometric detector, which was set at 254 nm. A

Rheodyne 20 L injection loop was used for injection. The column used was a Supelcosil LC 18 column (25 cm x 4.6 mm i.d., with 5 m particle size) with a 2-cm Supelguard LC18 guard column (Supelco, Inc., Bellefonte, Pa., U.S.A.) HPLC data were collected and processed using Dynamax HPLC Data Processing Software (Rainin Instrument Co., Inc., Woburn, Mass., U.S.A.). The mobile phase used was acidic water: methanol = 75:25, where acidic water was made of glacial acetic acid (A.C.S. reagent, Fisher Scientific) in HPLC-grade water (10:800). HPLC conditions included a flow rate of 1 mL/min, a column temperature at 35C, an equilibrium time of 2 min, and a 15-min run time. III. RESULTS AND DISCUSSION

Figure 1 shows the standard curve for vanillin from 12.5 to 125 mg/L. Table 1 shows average molecular weights of proteins used to determine the molarity of proteins, which were calculated based on molecular weight and percentage of each protein fractions (Fukushima 1991; Rosenthal 1991).

Table 1 Molecular weight of casein, whey, and soy proteins Average %in Molecular % in calculated total milk Weight casein molecular protein x 1000 weight Casein* s1 s2 80 34 8 25 9 4 43 10 31 11 5 23 23 24.1 19 21 22801 Whey protein* -Lactalbumin -Lactoglobulin Blood serum albumin Immunoglobulins Protease-peptone % in whey protein 20 4 20 9 45 1 5 2 10 4 20 % in soy protein Soy protein** 2s -Conglycinin 7s -Conglycinin 11s Glycinin 15s15 34 42 9 18-33 180-210 300-350 600 236760
The binding of protein with vanillin at 4 and 12C are presented as Klotz plots (Fig. 2 and 3, respectively). These Klotz plots show that the bindings were all linear, which indicates that vanillin was bound to all 3 proteins noncooperatively. Numbers of binding sites and dissociation constants (Kd) were obtained from the plots (Table 2).

14.4 36 69 150-1000 4-20 82430

Table 2. Binding properties of casein, whey, and soy proteins at 12 and 4C K G H S n (M-1) (cal/mol) (cal/mol) (cal/Kmol) 12C (285K) Casein 0.66 352.66 -3321.61 Casein -1264.20 -32.70 Whey 0.67 1713.04 -4216.65 Whey -8495.76 -15.01 Soy 3.81 683.50 -3696.33 Soy 7423.97 39.02 4C (277K) Casein 1.06 672.00 -3583.24 Whey 1.16 2642.04 -4336.76 Soy 10.92 468.06 -3384.18

Changes in free energy, entropy, and enthalpy were calculated using the Hughs-Klotz Eq. (Price and Dwek 1979). Numbers of binding sites for casein and whey protein were very similar. Soy protein had more binding sites than the other 2 proteins. Our n and K values for binding of vanillin to soy-protein isolate at 12C (3.81 and 683.5 M-1, respectively) were close to the values of 2-nonanone binding onto native soy protein at 25C (5.5 and 570 M-1, respectively), which were reported by ONeill and Kinsella (1987a). Our n and K values for vanillin binding to whey protein were 0.67 and 1713.04 M-1 at 12C and 1.16 and 2642.04 M-1 at 4C (Table 2). Pelletier and others (1998) studied the binding of 29 compounds with -Lactoglobulin, the main component of whey protein. They reported n values of 0.9 and 0.8 and binding constant values of 280 and 1339 M-1 for methyl and ethyl benzoate, respectively. The binding data reported were often derived using compounds with a single representative functional group, such as a simple aldehyde, ketone, alcohol, methyl ester, and alkane. Therefore, it is difficult to compare the results we obtained for vanillin, which has several functional groups, with these literature data. Decreasing temperature from 12 to 4C increased the number of binding sites for vanillin on all proteins, especially soy protein. Equilibrium constants for casein and whey protein increased with decreasing temperature, especially for whey protein. Kinsella and Damodaran (1980) and Damodaran and Kinsella (1981b) reported that for interactions of ketones with soy protein, decreasing temperature from 25 to 5C resulted in increasing numbers of binding sites and binding constants. They concluded that the changes in binding site and affinity were attributed to possible changes in the tertiary and quaternary structures of the proteins at low temperatures. Negative free energy (G) for all 3 proteins indicated that binding of vanillin onto each protein was thermodynamically favorable, therefore, spontaneous. Free energy values for the association of vanillin with whey protein at 4C and 12C were 750.52 and 895.04 calories less than those of casein, and 952.58 and 520.33 calories less than those of soy, respectively. The differences in G between soy protein and casein were 199.06 and 374.72 calories at 4 and 12C, respectively (Table 2). The free energy values indicated that the affinity of vanillin for whey protein was higher than that of the other 2 proteins (ONeill 1996). Our results agree with the findings of Hansen and Booker (1996) who examined the binding of vanillin, benzaldehyde, citral, and d-limonene with sodium caseinate and whey-protein concentrate. They reported that whey protein exhibited greater degrees of binding to these flavor compounds than casein did. This could be because whey protein is more heat sensitive than casein; therefore, it unfolds at pasteurization temperature resulting in more binding.

The H and S values (Table 2) indicated that the interactions of vanillin with ca sein and whey were enthalpy driven, while the interaction between vanillin and soy protein was entropy driven (Price and Dwek 1979). Our results for H for soy protein was highly positive (7423.97 cal/mol, Table 2), which means the interaction was not favorable because it was endothermic. However, the entropy change was also very high 39.02 cal/K*mol), which resulted in negative G values causing binding to be spontaneous. Considering that vanillin possesses both a hydroxyl and a carbonyl group, this finding is in agreement with results reported by Aspelund and Wilson (1983), who found that the interactions of soy-protein isolate with carbonyl compounds (hexanal and 2-hexanone) and 1-hexanol were also highly entropy driven. A high entropy value often indicates a high-degree of protein unfolding, increasing the randomness of the system (Price and Dwek 1979). Therefore, possible conformational changes of soy protein may play a key role in its interaction with vanillin. IV. CONCLUSIONS

Our direct comparison of interactions of vanillin with casein, whey, and soy proteins indicated differences in binding principles among proteins. Whey protein has a higher affinity to vanillin than the other 2 proteins, which means it can bind more vanillin than the other 2. Bindings of vanillin with casein and whey protein were enthalpy driven, which may be due to the interactions of carbonyl and hydroxyl groups of vanillin with the proteins. Interaction of soy protein and vanillin was highly entropy driven, which means that conformational changes of soy protein may be important in binding of vanillin. Therefore, any process that causes denaturation of soy protein, such as heating, may increase binding of vanillin to soy protein.