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STUDY OF THERMAL AND HYDROLYTIC DENATURATION OF CASEIN ([alpha]S1) USING DIFFERENTIAL SCANNING CALORIMETRY (DSC).

Byline: Z. Farooq

Keywords: Casein, hydrolysis, thermal denaturation, DSC.

INTRODUCTION

Casein, main component of milk protein is of four types [alpha]s1, [alpha]s2, [beta] and k-casein with a ratio of [alpha]s1:[alpha]s2 and [beta]:k as 4:1 and 4:1 respectively (Fox 1989; Elzoghby et al. 2011). Casein is widely used to elucidate various aspects of protein chemistry (Fox 2003). It is also commonly employed as functional ingredient in diversified food, pharmaceutical and nutraceutical formulations (Bryant and McClements 1998; Dalgleish 1997). Casein micelle is a fragile and dynamic structure which can be represented better by an ensemble of interconverting states than by an average state. The content of an essential amino acid has remained at a low level probably because higher levels would be incompatible with the unfolded conformation needed for caseins to form a thermodynamically stable complex with amorphous calcium phosphate (Holt. 2016).

Among caseins, [alpha]S1 casein appears to be the most common allergens for milk-allergic individuals especially its C-terminal, the most hydrophilic region which has been identified as a common binding site for IgE (Docena et al. 1996; Swaisgood. 1992; Pihlanto and Korhonen. 2003). The five genetic variants of bovine [alpha]s1 casein results from deletions and single amino acid substitutions at the C-and N-terminal regions of the molecule (Swaisgood. 1992). Identified antioxidative peptides of casein range from 2-14 amino acids in length containing hydrophobic amino acids at the N-terminus and/or C-terminus and proline, histidine, or tyrosine within the sequence (Li and Li. 2013).

Instead of treating the clinical symptoms of allergy, an alternative approach is to modify the allergenic proteins so that they no longer elicit a response. Various food processing conditions can modify the structure of dairy proteins which might alter their immunodominant epitopes thereby modulating allergenicity (Besler et al. 2001). The resultant effects are likely to be process dependent. Theoretically, allergy can decrease because of the destruction of epitopes or it can increase because of the formation of new epitopes or an improved accessibility of cryptic or hidden epitopes after allergen denaturation (El-Agamy. 2007; Sathe et al. 2005; Heyman. 1999). Moreover, milk protein peptides have quite diversified functions as Food and Drug Administration has approved over 60 peptides as therapeutics (Fosgerau and Hoffmann. 2015). Most bioactive peptides are derived from major milk proteins including significant proportion from [alpha]S1 casein.

Identified bioactive peptides can easily be modified to improve activity as analogues of milk protein-derived bioactive peptides have been investigated (McClean et al. 2014). The [alpha]S1 casein is also a big source of Antimicrobial Peptides (AMPs) and little homology has been revealed between human and cow [alpha]S1 casein. Certain Opioid peptides that bind opiate receptors and exert opiate like effects have also been identified from [alpha]S1 casein (Nielsen et al. 2017).

Among various approaches to denature casein, heat treatment and enzymatic hydrolyses are very likely to impact allergy. Food allergens are generally resistant to extremes of heat by resisting denaturation and degradation which allows the allergen to survive the digestive system long enough to interact with the immune system to either sensitize the individual or to trigger a reaction. Effects of temperature cannot be attributed to changes in charge. More likely, such efforts are attributable to a weakening of hydrophobic interactions at lower temperature, thereby weakening the interactions of caseins in the caseinate particle (Farrell et al. 2013) and allowing some swelling of the particles (Huppertz et al. 2017). Enzymatic hydrolysis to degrade milk proteins is widespread within the food industry with the purpose to reduce allergy thereby obtaining nutritional substitutes for milk allergic children (Host and Halken. 2004).

Porous casein micelle structure is in line with the apparently free diffusion of various compounds like various enzymes in and out of the micelle (Huppertz et al. 2017). Considerable research has been carried out on the functional behaviour of protein hydrolysates (Groleau et al. 2003; Ju et al. 1995; Liu et al. 2001; Otte et al. 1997).

In the peptide self-assembling process, hydrophobic interaction, hydrogen bonding, salt bridges, p-p interaction and cation-p interaction all play important roles. A variety of hydrolyzed milk formulae based on caseins with different degrees of hydrolysis (partial or extensive) are commercially available. The antigenicity of those formulae is profoundly reduced (Chirico et al. 1997). But, even if the majority of the extensively hydrolyzed formulae developed for milk allergic children are well tolerated, their consumption is known to have triggered allergic reactions in several cases (Wroblewska et al. 2004). Even then enzymatic hydrolysis is the most effective and common method for reducing the danger posed by food allergens.

During denaturation processes, rupture of hydrogen bonds may lead to endothermic reactions, and disruption of hydrophobic bonds may lead to exothermic reactions. Such enthalpy changes can be detected by differential scanning calorimetry (DSC) (Raikos. 2010). DSC is used to investigate heat-induced conformational or structural changes of a broad range of food ingredients in various physico-chemical conditions (Dean et al. 2001; Privalov. 1992).

In current study DSC has been employed to study denaturation of [alpha]-casein. From the calorimetric experiments, the temperature dependence of the partial heat capacity of a protein can be obtained. Analysis of this function could yield fundamental parameters of a thermally and hydrolytically induced transitions like transition temperatures and enthalpies of casein denaturation. Finding of this study can help developing more understanding to engage casein as diversified functional ingredient.

MATERIALS AND METHODS

Materials: Lyophilized bovine [alpha]S1 casein ([greater than or equal to] 70%) was purchased from sigma Aldrich Co. USA). All chemical reagents used were of analytical grade (J.T. Baker Chemical Co., Phillipsburg, NJ, USA; Fisher Chemicals, Colonnade, ON, Canada and Anachemia, Lachine, QC, Canada).

Thermal treatments; Dry heat treatment was applied at the rate of 1400C by putting the sample in the oven for 20, 30 and 60 minutes. For the wet heat treatment, casein sample was put in a plastic tube which was dipped into the water bath at 900C for 30, 45 and 60 minutes intervals. Microwave heat treatment was applied to the casein sample under the power of 7 in a conventional microwave oven for 1, 3 and 5 minutes.

Enzymatic hydrolysis of casein ([alpha]-S1): The following procedures were employed for the enzymatic hydrolysis of casein ([alpha]-S1). Control solutions was prepared under the same conditions but without addition of enzyme. All experiments were performed in triplicate.

Pepsin hydrolysis: Casein ([alpha]-S1) (10 mg) was solubilized in 1mL 0.1M HCl. The solution was then incubated at 37AdegC and the pH was adjusted to pH 2 using NaOH 1M. The hydrolysis reaction was started by the addition of a pepsin (10 mg/mL) at an enzyme: substrate (E:S) ratio of 1:20. The solution was gently stirred for 6h, and then the reaction was stopped by heating at 80 AdegC for 15 min. The pH was then be adjusted to 7.0.

Chymotryptic and tryptic hydrolysis: The protein (10 mg) was solubilized in 1mL of 0.1M sodium phosphate buffer, pH 8.0. The solution was incubated at 37 AdegC and the hydrolysis reaction was started by the addition of a chymotrypsin or trypsin solution (10 mg/mL) at an E:S ratio of 1:100. The solution was gently stirred for 4 h, and then the reaction was stopped by heating at 80 AdegC for 15 min. The pH was then adjusted to pH 7.0.

Simulated gastrointestinal hydrolysis:

Preparation of glycine-HCl solution: A 50 mL 0.2M glycine solution was added to 200 mL flask then 44 mL of 0.2M HCl was added and the volume was raised to the mark on neck by adding water. The pH of the solution was adjusted to the value of 2 by using 2M HCl drops.

Protein hydrolysis: Casein ([alpha]s1) was hydrolyzed first by pepsin subsequently adding trypsin and chymotrypsin enzymes. A 5% w/w of protein sample was added to glycine-HCl solution. The enzymes were added in enzyme/substrate ratio of 1/250 (w/w). The pH was regulated by Titralab (TIM 865, Radiometer Analytical SAS, Villeurbanne, France). The pH was maintained at 2 for digestion by pepsin for two hours. Following the addition of trypsin and chymotrypsin, pH was maintained at 6.5 for 2.5 hrs. During the total period of hydrolysis the temperature of 370C was maintained with constant agitation. The enzymatic reaction was stopped by reducing the pH to 4. The hydrolysate was frozen and proceeded to freeze drying and stored afterwards for further analysis.

Flavorzyme hydrolysis: To 100 mL of 10% protein powder/substrate in a sterile screw-cap bottle, 0.1% enzyme was added and incubated at 600C overnight (20 hrs) in a shaking incubator and deactivated the enzyme at 700C for 30 minutes. The solution was freeze dried and stored at -860C temperature for further evaluation.

Differential Scanning Calorimetry (DSC): Thermal properties of the casein samples were studied by DSC. Each sample (15 uL) of protein solutions (15%) were hermetically sealed in aluminium DSC pans and heated. A scan rate of 50 C/min was used and samples were heated under helium through the range of 20-1400 C. An empty pan was used as reference. Calorimetric measurements were carried out using DSC-Q 200 (TA Instruments, Inc., New Castle, USA) and data was analyzed using a Universal Analysis TA Software. The instrument was calibrated using indium as standard.

Table 1. Denaturation temperatures and enthalpies of casein at various heat treatments studied by differential scanning calorimeter (DSC).

Variant###Denaturation temperature(0C)###Denaturation enthalpy(J/g)

Casein(native)###103.757###0.079

D-20 min###128.065###9.537

D-30 min###137.477###0.619

D-60 min###109.775###11.05

###122.472###193.10

W-30 min###82.279###0.227

W-45 min###116.425###0.002

###120.173###0.002

W-60 min###74.653###502.00

MW-1 min###103.607###1504.00

MW-3 min###118.430###0.021

MW-5 min###112.040###1505.00

Table 2. Denaturation temperatures and enthalpies of casein for its various enzymatic hydrolysates studied by differential scanning calorimeter (DSC).

Variant/hydrol###Denaturation###Denaturation

ysate###temperature(0C)###enthalpy(J/g)

Casein(native)###103.757###0.079

Pepsin###50.518###816.20

###108.768###144.40

Trypsin###84.115###1324.00

###117.967###120.50

Chymotrypsin###88.518###1374.0

###123.550###176.20

Gastrointestinal###105.187###1219.00

Flavorzyme###109.802###32.18

###134.330###5.170

RESULTS AND DISCUSSION

Thermal denaturation of casein: DSC results of various heat treatments of casein are presented in Table 1 and Fig. 1-3. The denaturation temperature (Td) and enthalpy change (IHd) were determined from the maximal peak temperature and the area of the peak, respectively. Native casein showed thermal transition (IHd) at 0.079 J/g corresponding to the denaturation temperature 103.7570C. At neutral pH, [alpha]S1 casein associates to polymers. The unfolded conformation of casein exposes side chain groups buried in the native structure, and the perturbation process causes an increased reactivity of thiol groups (Paulsson and Dejmek. 1990).

Thermal denaturation of protein involves the disruption of intramolecular bonding and the unfolding and aggregation of protein molecules. Due to the application of various kinds of heat treatments, differences in enthalpies and denaturation temperatures occur. Dry heat treatment to casein for 20 and 30 minutes time showed 9.537 and 0.619 J/g enthalpies corresponding to 128.065 and 137.4770C denaturation temperatures respectively. Dry heat treatment to casein for 60 minutes time showed 11.05 and 193.10 J/g enthalpies corresponding to 109.775 and 122.4720C denaturation temperatures respectively. Similarly, the wet heat treatment for 30 minutes showed 0.227 J/g enthalpy corresponding to 82.2790C denaturation temperature. Wet heat treatment for 45 minutes showed 0.002 and 0.002 J/g enthalpies corresponding to 116.425 and 120.1730C denaturation temperatures respectively. Wet heat treatment for 60 minutes showed enthalpy 502.00 J/g corresponding to 74.6530C denaturation temperature.

For the microwave heat treatments for 1, 3 and 5 minutes exposure, the enthalpies were 1504.00, 0.021 and 1505.00 J/g corresponding to the denaturation temperatures of 103.607, 118.430 and 112.040 0C respectively. These results fall within the range 25-165AdegC temperature corresponding to an enthalpy of 154.34 J/g of the DSC analysis of casein which indicated an endothermic process (Moldoveanu et al. 2013). Furthermore, unfolding of the protein results in an increase in free volume and molecular mobility, and this requires heat to occur. Transitions that involve a process (denaturation and gelatinization) shift to higher temperatures at higher heating rates. This is due to the fact that amount of time at a given temperature decreases as heating rate increases (Thomas and Schmidt. 2010).

Moldoveanu et al. (2013) have noticed two degradation domains of thermal denaturation of casein, an endothermic (two stages) and the exothermic (three stages). The casein is thermally stable between 0-172 AdegC. First degradation stage develops between 41.9AdegC -172.4AdegC. These low temperatures are indicative of the elimination of the physically retained water in casein (4.60%). The thermal stability of casein is expressed by the initial degradation temperature of the second stage, Ti = 172.4AdegC (Moldoveanu et al. 2013). Aggregation of casein micelles is probably caused by restructuring of the micelles induced by changes of the pH and the temperature. Restructuring leads to a progressive change of the balance between repulsive and attractive interaction between the micelles with increasing temperature in favor of the latter and drives aggregation when it reaches a critical value at a critical temperature.

Hydrophobic interactions, hydrogen bonding and complex formation between calcium, phosphate and phosphoserines are all involved in the gelation process (Thomar and Nicolai. 2016).

Moreover, results of the current study are in line with the Thermogravimetric, Differential Scanning Calorimetric (TG-DSC) analysis of casein variants where the weight loss occurred between 30-1300C and it was found that intercalated proteins degrade at relatively higher temperatures (Yu et al. 2010).

Hydrolytic denaturation of casein: When the casein is hydrolysed/digested, it is degraded into an array of peptide fragments that have different biological effects as compared to the native/parent protein. The peptides of [alpha]-S1casein have been reported to have an array of activities like antimicrobial, angiotensin-converting enzyme (ACE) inhibition, dipeptidyl peptidase IV (DPP-IV) inhibition, opioid agonist and antagonist activities, immunomodulation, mineral binding and antioxidative functions (Nielsen et al. 2017). The resultant peptides are absorbed into the blood stream and function systematically as ACE-inhibitory peptides have the potential to lower blood pressure. The fragments of [alpha]S1 casein have been detected in the blood of an adult human for a period of 8 hours after consumption of milk or yogurt (Chabance et al. 1998). DSC results of various enzymatic hydrolysates of casein are presented in Table 2 and Fig. 4.

Extensive intraandinter-subunit disulfide bonds provide greater stability against thermal denaturation. Therefore, enzymatic hydrolyses provide significant differences in the thermal transition behaviour of casein.

Hydrolysis with the pepsin showed enthalpies 816.20 and 144.40 J/g corresponding to 50.518 and 108.768 0C, respectively. Similarly hydrolysis of casein with trypsin enzyme showed 1324.00 and 120.50 J/g enthalpies corresponding to 84.115 and 117.967 0C denaturation temperatures respectively. The hydrolysate of chymotrypsin showed 1374.0 and 176.20 J/g enthalpies corresponding to 88.518 and 123.550 0C denaturation temperatures respectively. The hydrolysate from simulated gastrointestinal hydrolysis showed thermal transition (IHd) at 1219.00 J/g corresponding to the denaturation temperature 105.1870C. The hydrolysate from flavorzyme showed thermal transition (IHd) at 32.18 and 5.170 J/g corresponding to the denaturation temperature 109.802 and 134.330 0C, respectively. Variation in thermal transitions might be due to variation in degree of hydrolysis as presence of proline can increase peptide resistance to hydrolysis by digestive enzymes (Yang and Russel. 1992).

Low hydrolytic activity could be related to high number of proline residues which are likely to survive of a longer period of time in the digestive system and more likely exert in vivo bioactivity (Nielsen et al. 2017). The results of the current study are further supported by the findings of Nielsen et al. 2017, wherein [alpha]S1 casein has been reported to have high peptide density regions with full or partial peptide sequences. The peptides range from 2-14 amino acids in length where half of the unique sequences have a proline at the second position from the N-terminus, indicating a possible structural pattern for DPP-IV-inhibitory peptides.

The increased entropy of amino acid residues in the unfolded state is balanced by the decreased entropy of water clusters induced by apolar groups. Classically and historically, the intermolecular forces prevailing in protein interactions are ionic +/- bonds, hydrogen bonds and Van der Waals forces. The latter should properly be termed dispersion forces and would include electrostatic-and dipole-or induced dipole-dipole interactions, thereby encompassing the cation- and -forces involved more recently in protein-protein interaction (Waters. 2002 and Dalkas et al. 2014). The stabilizing effect of disulfide bridges causes restriction of the mobility of the unfolded peptide backbone (Paulsson and Dejmek. 1990). Ionic bonds are simple +/- interactions between oppositely charged residues such as lysine, arginine or histidine (all+) with glutamate or aspartate (both-) and require no further explanation.

Hydrogen bonds are more complex and there is still debate as to their exact nature and the extent to which they possess an electrostatic or covalent character (Hubbard et al. 2010 and Martin and Derewenda. 1999). Casein enzyme hydrolysate (CEH) aggregates differ markedly and may represent viable vehicles to sequester hydrophobic insoluble organic molecules (Liu and Guo. 2008). Moreover, multiple clusters of hydrophobic residues are identified as potential reaction sites in the hydrophobic tails and trains of the casein proteins (Horne. 2017). Only one hydrophobic essential amino acid with a low disorder propensity, Leucine occurs at moderate frequency in [alpha]S1 casein. Threonine in [alpha]S1 casein occur less commonly on average than in the other orthologues of bovine casein (Holt. 2016).

Conclusion: Application of various heat treatments denatured casein ([alpha]S1) at various degrees. Enzymatic hydrolysis caused epitope formation at various points. Denatured and hydrolyzed molecule of casein produced various enthalpies of denaturation corresponding to different thermal and hydrolytic treatments. Dry heat for 60 minutes and wet heat for 45 minutes produced two points of enthalpies of denaturation against respective temperatures while rest of each heat treatment produced only at one point enthalpy of denaturation. Likewise, all the enzymes hydrolyzed casein well wherein each enzyme produced two points of enthalpies of denaturation against their respective temperatures except gastrointestinal enzyme which produced only one point of denaturation.

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