Kaur, Surjit ORCID: 0000-0001-5855-766X (2024) Application of Plant Proteases in Hydrolysis of Dairy Proteins. PhD thesis, Victoria University.

Abstract

Milk proteins are highly valuable nutritional components required for the proper development of infants and toddlers and the maintenance of muscles in the elderly. In addition, functional properties of various commercial milk proteins are widely exploited by the food industry. However, their application in food systems is hindered by several important issues including cow’s milk protein allergy (CMPA) and poor stability during processing among the most prevalent. Actinidin is an important plant protease enzyme that is widely used in dairy systems to improve the properties and processability of milk proteins. The main aim of this research was to establish conditions (temperature, pH, enzyme to substrate ratio, time) required for optimal enzymatic performance on partially or completely hydrolysed reconstituted milk protein preparations to achieve enhanced hypoallergenicity or functionality. Partial hydrolysis, which involves proteases with very high specificity and the cleavage of a limited number of specific peptide bonds in the protein to yield a slightly modified form of the protein. In case of complete hydrolysis, which results in a more extensive hydrolysis of a protein including hydrolysis of multiple peptide bonds and eventually complete conversion into amino acids. Here enhanced hypoallergenicity relates to breakdown of epitope regions into non allergic small peptides or free amino acids. This tailoring of structural characteristics of milk proteins can also lead to improvement in functionality. Thus, allergenicity and functional properties (solubility, heat stability, foaming and emulsification ability) of milk proteins were also assessed as a function of the degree of hydrolysis. The activity of any enzyme is influenced by several important factors including substrate concentration, pH, ionic strength and environment, and temperature. All of these factors play a role in maintaining or disturbing the conformation of enzymes and thus may either stimulate or inhibit enzyme activity. Variations in environmental pH or ionic quality may alter electrostatic interactions among charged amino acid segments and induce conformational changes in the structure of the enzyme. Similarly, the enzyme conformation is held by weak forces which may be perturbed by temperature. Experiments were performed to establish conditions for the optimum hydrolytic activity of commercial proteases (actinidin, bromelain and papain) using milk protein preparations as a substrate. The optimum temperature for activity of plant proteases was determined by executing the enzymatic reaction at different temperatures (15–55 °C) using milk protein concentrate (MPC), whey protein concentrate (WPC) and whey protein isolate (WPI) preparations. Protein solutions were prepared at a constant concentration (5% w/w) by dispersing powder in simulated milk ultrafiltrate (SMUF) followed by continuous overnight mixing for complete hydration. The protease assay mixture at pH 6.8 and at a constant enzyme to substrate (E:S) ratio was incubated at various temperatures and the extent of cleavage of peptide bonds was determined using a spectrophotometric assay using trinitrobenzenesulfonic acid (TNBS). The optimum temperature is defined as the one resulting in the maximum of the degree of hydrolysis under experimental conditions. This study was also performed without pH control to assess the impact of pH change. Actinidin at an E:S ratio of 1:100 resulted in a greater degree of hydrolysis (%DH) of whey proteins. Altering the ratio did not result in substantial change of %DH of MPC. For all three enzymes (actinidin, bromelain and papain), cleavages of proteins were clearly time dependant (p<0.05) while pH, although not controlled, did not change significantly (p>0.05) during the incubation process. The %DH increased with increasing temperature and the maximum %DH was achieved at 60 °C for all three dairy systems. PAGE analysis revealed that actinidin and papain mainly acted on α-lactalbumin and αs-casein in WPI and MPC, respectively. After following these protocols, hydrolysed samples were further assessed for antigenicity by the enzyme-linked immunosorbent assay (ELISA). For this, WPI and MPC substrates were used with the aim to reduce immunoreactivity of hydrolysates of β-lactoglobulin (β-LG) and αs1-casein (αs1-CN) fractions of protein mixtures at 10 and 60 °C when treated with actinidin. Firstly, the %DH was determined by TNBS at an enzyme to substrate ratio of 1:100 (5.21 units of actinidin activity g-1 of protein) at 10 and 60 °C for up to 31 and 5 hours, respectively, for both substrates at uncontrolled pH. The antigenicity was tested using ELISA which confirmed a significant reduction of antigenicity of β-LG and αs1-CN with higher %DH by actinidin, possibly by fragmentation and masking of epitopes. At 60 °C, hydrolysis resulted in a reduction in antigenicity of about 43 and 48% for MPC in the case of β-LG and αs1-CN, respectively, and approximately 54% for WPI (β-LG). Hydrolysates obtained at 10 °C also resulted in a reduction in antigenicity for MPC of β-LG and αs1-CN by about 39 and 42% respectively, but only 14% for WPI (β-LG). Overall, it can be suggested that proteolysis by actinidin can reduce the antigenicity by modification of protein conformation, and cleavage and masking of conformational and linear epitopes of β-LG and αs1-CN to a certain extent in milk protein systems. The impact of selected parameters of milk protein hydrolysates (MPH) of MPC and WPC were assessed to explore the effect on the functional properties. Here 0, 5, 10 and 15% DH was achieved for each substrate which were then reacted with actinidin and evaluated using the TNBS assay. The results revealed that significant changes in the functionality of MPH are associated with %DH. The solubility of MPH increased with an increase in %DH whereas whey proteins attained more than 97% solubility. The PAGE analysis revealed that the most soluble proteins were α-lactalbumin and κ-casein in WPC and MPC respectively, and were therefore more susceptible to the enzymatic action of actinidin. Emulsifying properties showed a decreasing trend with increasing %DH whereas heat stability increased, and the foaming properties of both MPH substrates were improved. These results were further validated using FTIR spectroscopy and zeta potential, however, particle size showed a mixed trend. Actinidin efficiency (kinetic and thermodynamic characteristics) was then compared with other proteases from the same CA1 family including papain and bromelain. The kinetic parameters (Km, kcat, Vmax) of the proteases were assessed from a Lineweaver–Burk plot, by performing activity assays at different concentrations of substrates at concentrations of 20, 40, 60, 80 and 100 mg mL-1. Incubations were performed at 60°C and reactions were triggered by adding 2.6 units of enzyme activity equivalents of actinidin or bromelain or papain to the samples. WPC hydrolysis was characterised with lower Km, higher kcat and higher Vmax as compared to MPC in case of all three enzymes. The values of kcat and Km were used to determine the substrate turnover and binding affinity of each protease. The thermodynamic parameters of these enzymes with MPC and WPC were also determined over a temperature range of 15–60 °C and the results were favourable for the potential application of papain and actinidin in dairy formulations. Overall, actinidin exerted an appreciable and specific enzymatic activity towards the dairy protein substrates tested. Its application was further assessed in milk proteins where limited hydrolysis resulted in modulation of specific functionalities and/or allergenicity. Further manipulation of hydrolysis parameters, processing conditions and pH control could be a promising approach to improving the solubility and further functionality of MPH formulations.

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