Study of Some Functional Properties of Casein: Effect of pH and Tryptic Hydrolysis

Abstract

The trypsin was used to hydrolyze commercial casein at varied times and pH range. The functional properties studied were the emulsifying capacity (EC), the emulsifying activity index (EAI), and the emulsion stability (ES). The dispersed phase used was corn oil. The tryptic hydrolysis was beneficial to the solubility and EC of casein in practically all pH values and reaction times. In case of EAI, this same effect was less intense and was observed only in acid region (pH 3.0 to 5.0), while for ES the trypsin action was mainly deleterious in almost all pH range and reaction times.

Keywords:

• Tryptic hydrolysis
• Casein
• Solubility
• Emulsifying capacity
• Emulsifying activity index
• Emulsion stability

INTRODUCTION

One of the most important ingredients in emulsified food products is the protein, used as an emulsifying agent, which has the ability to stabilize emulsions. The interest in replacing artificial ingredients by natural ones has been increasing lately due in part to the increased awareness of consumers. In this way, the use of proteins as functional agents could be very favorable to the food industries.

On the other hand, considering that the action of proteins as emulsifiers is complex and depends on different factors, it is important to study the behavior of proteins under different conditions. Among these factors, the most cited in the literatures are pH, ionic force, protein concentration, temperature, oil type, velocity, and length of mixture. This explains the fact that the available data concerning the emulsifying properties of proteins are quite inconstant and many times conflictive.[1–6]

The modification of protein structure by enzymatic hydrolysis has been used to improve functional properties. However, this result depends on the peptide size, and normally peptides containing more than 20 amino acid residues are needed in order to produce this advantageous effect.[3,7] The most common method involves a partial hydrolysis employing highly specific proteases in order to control the hydrolysis degree and, therefore, the size of produced peptides.[7,8]

The casein, the main milk protein, shows several desired functional properties and, consequently, its use in food industry is of great interest.[9] According to Mulvihill and Fox,[10] the study of emulsifying properties of proteins involves, essentially, the evaluation of their emulsifying capacity (EC), emulsifying activity index (EAI), and emulsion stability (ES). In this article, we focused our interest on the study of the effect of tryptic hydrolysis on the solubility and the emulsifying properties of casein, at different values of pH and hydrolysis time.

MATERIAL AND METHODS

Tryptic Hydrolysis of Casein

The method described by Chobert et al.[11] was used. The casein was solubilized in a buffer solution (0.2 mol/L sodium phosphate and 0.1 mol/L citric acid), pH 8.0, to a protein concentration of 5.0 g/100 mL. Then, the trypsin (bovine pancreas, XIII type, TPCK treated, Sigma Chemical Co., St. Louis, MO, USA), solubilized in the same buffer, was added to obtain a 0.1% enzyme:substrate ratio. The mixture was held in a water bath at 37°C, with stirring for 5, 10, 15, 30, and 60 minutes. In all assays, the hydrolytic reaction was stopped by reducing the pH to 2.0, using hydrochloric acid (HCl). The hydrolysates were then freeze-dryed (Freezone^® 4.5 model, Labconco, Kansas City, MI, USA) and stored at –18°C until the moment of use.

Sample preparation

The casein and its tryptic hydrolysates were solubilized in a buffer solution (0.2 mol/L sodium phosphate and 0.1 mol/L citric acid), at pH 7.0, to a concentration of 0.1 g of protein/100 mL of solution. After 30 minutes in a water bath at 35°C, the solutions were centrifuged (Br4i model, Jouan, S.A., Saint-Herblain, France) at 6500 g for 10 minutes and then filtered (through paper filter Quanty, JP42 model, Curitiba, PR, Brazil). The filtrates were stored at −18°C until the moment of use.

Determination of solubility

The soluble protein content was determined according to Lowry et al.[12] as modified by Hartree,[13] using bovine serum albumin (BSA, Sigma Chemical Co., St. Louis, MO, USA) as standard. Filtrate aliquots of 200 µL were used. The absorbance was read at 650 nm in a spectrophotometer (UV-VIS – Cecil, CE2041 model, Cambridge, UK), and the solubility was calculated in terms of percentage of total nitrogen and expressed in g of soluble protein per 100 mL of solution.

Determination of optimum protein concentration

In order to determine the protein concentration to be used in all experiments, casein solutions were prepared in different concentrations varying from 0.025 to 3.0 g/100 mL in a buffer solution (0.2 mol/L sodium phosphate and 0.1 mol/L citric acid) at pH 7.0. In each case, the emulsifying capacity (EC) was determined according to the method described bellow. Then, a graph of the EC as a function of protein concentration was drawn.

Determination of emulsifying capacity (EC)

For determining the emulsifying capacity, the method of Vuillemard et al.[14] was used. Initially, the temperature of the filtrate, prepared as described in the sample preparation item, was adjusted to 10°C in an ice bath. Then, 50 mL of sample were homogenized using a mixer (Fisher, Magiclean, mod. 14057-5, Arno, São Paulo, Brazil) at the highest speed. Corn oil was added continuously (25 mL/min) until the inversion of the emulsion occurred, as indicated by the interruption of the electric current detected by a 120 V lamp. The EC was calculated using Eq. 1:

(1)

where EO and BO are the amount of emulsified oil in the sample and in the blank, respectively. Blank is the buffer solution with no emulsifying agent.

Determination of emulsifying activity index (EAI)

The method of Pearce & Kinsella[15] was used for determining the EAI. For preparing the emulsions, a volume of 30 mL of the protein solution and 10 mL of corn oil were mixed together in the same mixer cited above, at the highest speed for 1 minute. The temperature was maintained at 20°C. Aliquots (1 mL) of the emulsion were diluted (1/100) in a solution containing 0.1% SDS (sodium docecyl sulfate) and 0.1 M NaCl, homogeneized and the absorbance was read at 550 nm (spectrophotometer CECIL, CE 2041 model, UK). The EAI values were calculated using Eq. 2 proposed by Cameron et al.:[16]

(2)

where T is turbidity; θ is the volume fraction of the oil; and C is the initial protein concentration (0.1 g /100 mL). The turbidity was calculated by multiplying the absorbance by 2.203 and by the dilution factor (100) and then dividing this result by the optical path length of the cuvette (0.01 m).

Determination of the emulsion stability (ES)

The method of Chobert et al.[11] was used for determining the emulsion stability. The stock emulsions prepared above were held at 20°C for 24 hours. After stirring, aliquots were diluted in 0.1% SDS and turbidity was measured as described above (EAI, 20°C). The 24 hours-old emulsions were then heated at 80°C for 30 minutes. After the aliquots were cooled to room temperature and stirred, the turbidity was again measured as described above (EAI, 80°C). The ΔEAI% was calculated by the Eq. 3:

(3)

where EAI_max is the maximum value obtained just after emulsion formation; and, EAI_min is the lowest value obtained for the aliquots after 24 hour-storage and 80°C heating. ES values were calculated using Eq. 4:

(4)

Evaluation of pH Effect

For studying the effect of pH on the solubility and on the emulsifying properties, the pH of casein solutions and of its tryptic hydrolysates was adjusted to 3.0, 4.0, 5.0, 6.0, and 8.0, before the centrifugation and filtration steps described in the sample preparation item.

Statistical Analysis

All experiments were replicated 3 times. Analysis of variance was performed for the determination of optimal protein concentration, in order to investigate the presence of significant effects among treatments (p < 0.05). The Duncan test was applied to establish the differences among means.[17] The effect of pH and hydrolysis time on the solubility, EC, EAI, and ES were analysed using split-plot design (in which the main plots were hydrolysis times and the values of pH the subplots). Analysis of variance for each property (p < 0.05) and then the Duncan test was applied to compare means.[17]

RESULTS AND DISCUSSION

Optimum Protein Concentration

The results of optimum protein concentration are shown in Fig. 1. A significant increase of EC was observed up to a value of 0.075 g of protein/100 mL and stayed unaltered until the value of 2.0 g of protein/100 mL. After this point, the EC decreased significantly from one point to another until it reached to the minimum at the highest concentration (3.00 g of protein/100 mL). Thus, 0.1 g% was used in all subsequent analysis in this work.

Figure 1 Emulsifying capacity (EC) in function of protein concentration. Each value represents the mean of triple determination. ± Standard error (vertical bars).

According to Pearce & Kinsella,[15] this value refers to the minimum concentration needed to obtain reproducible results for the emulsifying properties and must be established in each case because it depends on the type of protein and oil involved in the emulsifying process. However, in previous studies developed in our laboratory with different proteins, the same value as that of casein for the optimum concentration (0.1 g%) was found for the laboratory-prepared bovine plasma[18] and bovine globin.[19] Thus, the effect of the type of protein involved was not detected in these cases.

Other authors have been checking the effect of protein concentration on emulsifying capacity of casein. Vuillemard et al.[14] and Foley and O'Connell,[20] also found an EC maximum for the sodium caseinate at a concentration of 0.1 g%, but the change of EC in function of protein concentration was different from that of the present work, since the EC increased up to 0.1 g% and then decreased significantly.

Effect of Tryptic Hydrolysis and pH on the Solubility

The solubility of casein was minimum at pH from 3.0 to 5.0, region corresponding to its pI, (Fig. 2), and increased markedly from this value up to pH 7.0 where it reached the maximum. The curve of solubility for bovine plasma was shown by our group to be quite different from that of casein.[18] In fact, the solubility of nonhydrolyzed bovine plasma proteins remained high (between 70% and 80%) and almost unchangeable at all pH values. Only at pH 4.0 the solubility was significantly higher than the other values of pH. It is well known that pH has an effect on protein charge and therefore on its solubility which reaches a minimum at the pI of the protein.[21] However, in case of plasma, which contains different proteins, this effect was not observed since the pH had minimal influence on its solubility.

Figure 2 Effect of the pH and time of tryptic hydrolysis on the solubility of casein. Each value represents the mean of triple determination. ± Standard error (vertical bars).

The effect of pH on the solubility of casein was also investigated by other authors, and in one of these works the results for the sodium caseinate were similar to those of the present study.[22] However, Foley O'Connell[20] reported a higher solubility (around 60%) for the potassium caseinate at pH 3.0. Working with α_s-1 – and β-casein. Cayot et al.[23] observed that the highest values for the solubility were reached at pH 8.0 and 2.0.

The tryptic hydrolysis increased the solubility of casein at almost all pH values and reaction times (Fig. 2). Only at pH 6.0 with a hydrolysis time of 5 hours it was inefficient to improve this property. The positive effect of this treatment was more prominent in the pH region where the solubility of casein was minimum. This figure also shows a similarity between the results achieved after 5 and 10 min of hydrolysis and also among those after 15, 30, and 60 minutes.

According to Das and Kinsella,[24] the enzymatic hydrolysis normally contributes to improve the solubility of proteins since it exposes the charged groups, reduces the molecular size, increases the hydrophilicity, and leads to favorable changes in the molecular configuration. However, in a previous study in our laboratory with bovine plasma, the same enzymatic treatment used here reduced the solubility at almost pH values.[18] We associated this result with the influence of other compounds in the plasma (fat, ions, sugars, etc.), since this study was made using whole plasma and not on its isolated proteins.

The tryptic hydrolysis was also used by Chobert et al.[11] and showed the positive effect like here on the solubility of casein.

Effect of Tryptic Hydrolysis and pH on Emulsifying Capacity

As shown in Fig. 3, the EC curve for casein as a function of pH was similar to that for solubility. A positive (r  =  0.9647) and significant (p < 0.01) correlation between these two properties was found showing that both of them are similarly affected by the pH. This same result was previously reported by other authors for milk proteins[9,10,22,25] and also by our group for bovine plasma.[18]

Figure 3 Effect of the pH and time of tryptic hydrolysis on the emulsifying capacity of casein. Each value represents the mean of triple determination. ± Standard error (vertical bars).

On the other hand, a difference between the curves of these two parameters can also be observed in Fig. 3, i. e., even in the pH region (3.0 to 5.0), where the solubility is almost zero, casein showed a certain EC.

Our results with bovine globin indicated an increase of EC until pH 5.0 where it reached a maximum. After this point, the EC decreased and at pH 6.0 to 8.0, where goblin shows the lowest solubility, the EC was zero.[19]

In fact, both results are in agreement with the theories. Thus, according to Mangino,[6] the EC measures the capacity of proteins to migrate to the water/oil interface and for doing that proteins must be sufficiently soluble. The pH affects protein charge and hence its solubility, which is minimum in the region of pI. Thus, at pH values close to pI, proteins show low emulsifying properties.[21,26]

Concerning the plasma, the pH had little effect on its EC, which remained high and almost constant at all pH studied, as shown previously by our group.[18]

The influence of the tryptic hydrolysis on the EC was similar to that observed for the solubility, since this treatment improved this property of casein at almost all pH values and reaction times studied. At pH 4.0, 5.0, and 8.0, this benefit was observed in all reaction times, while at other pH values, especially 6.0 and 7.0, a much longer treatment was needed to show the same effect.

Our study with bovine globin showed similar results to those with casein, since the tryptic hydrolysis was advantageous to EC in almost all pH studied. Only at pH 5.0 this treatment showed harmful effect on EC.[19] Contrarily, we showed that the action of pepsin on globin increased the EC only in two pH values (3.0 and 4.0).[27]

Some authors have been considering the beneficial effect of moderate enzymatic hydrolysis on emulsifying properties of proteins and stated that besides improving protein solubility, it can also increase the number of contact points between proteins and the water/oil interface favoring the emulsion formation.[5,11,24]

Contrarily to casein and globin, we showed that the tryptic hydrolysis reduced the EC of plasma at all pH values and reaction times, except at pH 4.0 after 5, 15, and 30 minutes of reaction.[18] We explained this result, at least in part, by the fact that plasma is not an isolated protein but a mixture of proteins and other constituents which may interfere in the behavior of proteins.

Effect of Tryptic Hydrolysis and pH and on Emulsifying Capacity Index

The curve of EAI versus pH (Fig. 4) was also like to that for solubility. The lowest values were found from pH 3.0 to 5.0. After this point, the EAI increased markedly until reach the maximum at pH 6.0.

Figure 4 Effect of the pH and time of tryptic hydrolysis on the emulsifying activity index of casein. Each value represents the mean of triple determination. ± Standard error (vertical bars).

A positive (r  =  0.9895) and significant (p < 0.001) correlation between these two properties was found showing that both of them are similarly affected by the pH. This same result was previously reported by other authors for milk proteins.[9,10,22,25] It is also worth stating that a positive (r  =  0.9513) and significant (p < 0.05) correlation was found between the EC and the EAI of casein.

In Fig. 4, one can also observe that even at the pH region where its solubility was almost zero (pH from 3.0 to 5.0), the casein showed an important value for the EAI. This same behavior was reported by our group for globin, since its EAI was high at pH 7.0 and 8.0 which corresponds to its pI.[19] Our results with plasma indicated an increase of EAI at pH 7.0, although no change in the solubility occurred at any pH value.[18]

In other reports in the literature, the pH also affected the EAI of casein, but the important changes were observed at different pH values than here. Thus, in some studies the EAI of the casein increased at a pH below 4.0,[11,28] and in others, this happened only from pH 7.0.[4,9]

The beneficial action of the trypsin on the EAI of casein was observed only in acid region (pH 3.0 to 5.0), and it was less intense than the results found for the solubility and EC. This positive effect was observed for reaction times equal or higher than 10 minutes, 15 minutes, or 5 minutes at pH 3.0, 4.0, and 5.0, respectively. At pH 5.0, the highest reaction time tested (60 min) was the only one where no change of EAI was observed.

Other authors described this same effect not only in low pH values (2.0 or 3.0) but also at higher pH values, like 6.0 or 10.0.[11,28]

Effect of Tryptic Hydrolysis and pH on the Emulsion Stability

The ES of casein increased markedly from pH 6.0 and still more from pH 7.0 reaching its maximum at pH 8.0 (Fig. 5). Contrarily to EC and EAI, no significant correlation was observed between ES and the solubility. However, it is worth stating that at pH region (6.0 to 8.0) where the solubility was higher, the ES also reached its maximum.

Figure 5 Effect of the pH and time of tryptic hydrolysis on the stability of the emulsion of casein. Each value represents the mean of triple determination. ± Standard error (vertical bars).

Data from our laboratory showed that in the case of globin a sharp increase of its ES occurred from pH 5.0 to pH 6.0 where it reached its maximum. Then, the ES decreased until the minimum value at pH 7.0 and 8.0 region, where the solubility of this protein shows the lowest value.[19] For the plasma, we showed that it presented very low ES values situated near zero at all pH range. No significant difference was observed all along the pH range.[18]

The effect of pH on the stability of the emulsions shows a certain complexity. According to Mangino,[6] at pH values near the pI, proteins are able to form more viscous and joined interfacial films improving the ES. However, in some cases, like we showed here for casein and previously for globin,[19] the ES is higher at pH values far from its pI. This same author stated that sometimes the higher production of charged molecules during the preparation of the emulsions, associated to a higher energy input by the equipment used, would increase the repulsion forces and improve the ES of the emulsions.

Other authors obtained similar results as those here for the effect of pH on the ES of casein, since the values found at pH 7.0 and 8.0 were higher than those at pH 4.0.[4,11,22,28]

The tryptic treatment had no effect on the ES of the casein at pH 5.0 and 6.0 after 5 minutes of reaction, while at other pH values, mostly at pH 8.0, it reduced the ES for all reaction times tested (Fig. 5).

The influence of the tryptic hydrolysis on the ES of globin and plasma was minimum, as we showed before. Thus, our results for globin indicated that the tryptic hydrolysis influenced its ES only at pH 7.0 (the pI region of this protein) and after 60 minutes of reaction. For the action of pepsin, we showed that this treatment had no beneficial effect on the ES of globin, in all pH region and reaction times studied.[27] In the case of plasma, its ES remained unchangeable after this same treatment at all pH range studied (3.0 to 8.0).[18]

The action of enzymatic hydrolysis in improving the stability of emulsions is associated to the increase of the solubility and of the hydrolysis degree.[24] However, according to these same authors, a very high solubility is not desired after the emulsion is formed, since some desorption and loss of protein interfacial film could occur reducing the stability of the emulsions. This could, at least in part, explain some of the results obtained by our group. Moreover, other factors could be interfering with the ES of these proteins, such as the volumes of aqueous and oil phases, the properties of the protein film, the size and the conformation of the protein molecules.[24]

According to the results of other authors, the tryptic hydrolysis may affect positively or negatively the ES of casein depending on the reaction conditions employed.[11,28]

CONCLUSIONS

The pH affected similarly the solubility, the EC and the EAI of casein, and near the pI these properties showed their minimum values. Contrarily, no correlation was observed between the curves of ES and solubility in function of pH. The tryptic hydrolysis was advantageous to the solubility and EC at all pH values and reaction times, to EAI in acid region, while this treatment reduced ES in almost all studied conditions.

ACKNOWLEDGEMENT

The authors thank CNPq and FAPEMIG, in Brazil, for the support of this work.