Modification of the interfacial properties of sodium caseinate using a commercial peptidase preparation from Geobacillus stearothermophilus

Highlights

• A model of the interactions of caseinate hydrolysates at interphases is described.

• Interfacial properties are linked to the viscosity, hydrophobicity and peptide size.

• The peptides form specific, higher ordered structures with different interfacial interactions.

• The foam and emulsion stability is improved at low degrees of hydrolysis.

Abstract

Sodium caseinate was hydrolyzed with the commercial enzyme preparation Sternzym BP 25201, containing a thermolysin-like peptidase from Geobacillus stearothermophilus as the only peptidase. The hydrolysis was carried out at 65 °C with an enzyme activity of 1 nkat mL⁻¹ or 15 nkat mL⁻¹, leading to various degrees of hydrolysis (DH) ranging between 0.1 and 8.5%. The hydrolysates obtained were analyzed in a multi-scale approach, covering the hydrolysate properties (viscosity, hydrophobicity, peptide composition) and their interaction at oil–water (emulsion) and air–water (foam) interphases. The viscosity and surface hydrophobicity generally decreased with an increasing DH. Longer, more hydrophobic peptides, which self-assembled into network-like supramolecular particles, were detected up to a DH of 2.2%. Compared to untreated sodium caseinate, these structures could increase the half-life of emulsions (+400%) and foams (+31%). This was most probably caused by an increase in particle size (45.2-fold). By contrast, a higher DH led to a less hydrophobic product and smaller, spherical-shaped supramolecular structures. Foams and emulsions prepared with those hydrolysates were not stable and phase separated within minutes (for example, emulsion half-life = 5 min; foam half-life = 4.6 min at DH of 8.5%).

Introduction

Milk proteins are increasingly being used in the food industry due to their technofunctionality (Kilara & Panyam, 2003). The technofunctionality of proteins includes the water-holding capacity, oil- and fat-binding properties, gelling properties and the ability to stabilize oil–water (emulsion) or gas–water (foam) interphases (Zayas, 1997). The strong amphiphilic nature of milk proteins enables them particularly to adsorb onto the interphase (Caessens, Gruppen, Visser, Aken, & Voragen, 1997). The proteins have to diffuse from the bulk liquid phase to the close proximity of the interphase and, subsequently, adsorb, based on flip-flop mechanism, for the stabilization of an interphase (Germain & Aguilera, 2014). The proteins undergo conformational changes here during the adsorption at the interphase and expose their hidden hydrophobic groups to the non-aqueous phase (oil or gas), while hydrophilic groups orient to the continuous liquid phase – a fact that is also known as surface denaturation (McClements, 2004). In addition, the proteins might also aggregate and form a network, called the lamella (Ewert et al., 2016). The oil or gas phase is stabilized according to the size (steric hindrance) or charge distribution (electrostatic repulsion) of the proteins in the lamella (Germain & Aguilera, 2014).

In addition to the native food proteins used commonly, modified (chemically or enzymatically) proteins can also be applied (Celus, Brijs, & Delcour, 2007). These modifications can improve the technofunctional properties, such as the attachment onto the interphase (Kilara & Panyam, 2003). An example of these modifications is the enzymatic protein hydrolysis. During the hydrolysis, proteins are broken down into peptides of different sizes and free amino acids, as a result of cleavage of the peptide bonds (Sinha, Radha, Prakash, & Kaul, 2007). Possible consequences of a hydrolysis are: a decreased molecular weight, an increased number of ionizable groups, an exposure of hydrophobic groups from the inner protein molecule, an increased solubility, a decreased viscosity and, consequently, changes regarding the environmental interactions at the interphase (Panyam & Kilara, 2004; Tavano, 2013). In contrast to the chemical hydrolyses (conditions for acid hydrolysis: 2 M HCl, 110 °C, 24 h incubation), the enzymatic process usually avoids side reactions which might lead to toxic substances such as lysino-alanine and a decrease of the nutritional value of the protein source (Merz et al., 2015a; Sinha et al., 2007; Tavano, 2013). A further benefit of the enzymatic hydrolysis is that it provides a more uniform product, can be performed under milder conditions and, thus, avoid the extreme environments required for chemical treatments (Sun, 2011; Tavano, 2013). For the enzymatic hydrolysis peptidases can be applied. Consistent with the International Union of Biochemistry and Molecular Biology (IUBMB), all enzymes, which act on a peptide bond are called peptidases (Ewert, Glück, Strasdeit, Fischer, & Stressler, 2018). The specificity of the peptidase used for hydrolysis and the resulting degree of hydrolysis (DH) of the protein hydrolysate are the major parameters for the technofunctional behavior obtained (Althouse, Dinakar, & Kilara, 1995; Kunst, 2002; van der Ven, Gruppen, de Bont, & Voragen, 2002). The hydrolysis product has to be investigated in a multi-scale approach for evaluation, taking into account the molecular structure of the product, the microscopic effects (e.g. droplet size) at the interphase and the macroscopic effects (e.g. overrun, stability) of the emulsion or foam (Germain & Aguilera, 2014). Regarding the enzyme used to produce technofunctional protein hydrolysates, our group recently suggested the application of thermolysin (EC 3.4.24.27) (Ewert et al., 2016), due to its near neutral optimum pH (∼7.0), high optimum temperature (70–80 °C) and stability (stable ≥ 50 °C), as well as specificity to hydrophobic amino acids (preferred at P1’: Leu, Phe, Ile, Val) (Rawlings & Salvesen, 2013). Compared to thermolysin from Bacillus thermoproteolyticus, the thermolysin-like peptidase (TLP) from Geobacillus stearothermophillus ssp. (also called stearolysin, boilysin) offers an improved temperature stability. Takii, Taguchi, Shimoto, and Suzuki (1987), for example, showed that TLP from Geobacillus stearothermophillus KP1236 was stable for 18 h at 60 °C and pH 6.0–8.6, while this treatment inactivated Bacillus thermoproteolyticus thermolysin greatly (residual activity: 6, 34 and 7% at pH 7.0, 7.5 and 8.0, respectively). Our own investigations prior to this study using the commercial TLP preparation Sternzym BP 25201 (Stern-Wywiol Gruppe, Ahrensburg, Germany) confirmed the characteristics described in the literature (see supplementary data, Table S1).

To the best of our knowledge, no studies are available so far describing the effect of a TLP hydrolysis on the interfacial properties of sodium caseinate. As already suggested in the literature (Althouse et al., 1995; Kunst, 2002; van der Ven et al., 2002), we hypothesized that a low DH might lead to an improved emulsifying and foaming behavior of the hydrolysate, while a high DH might diminish these properties. Therefore, the objective of the current study was to investigate the foaming and emulsifying properties of caseinate, hydrolyzed with the technical TLP preparation Sternzym BP 25201, in a multi-scale approach and to further investigate the mechanism behind the changes in the interfacial behavior due to the applied hydrolysis.