High Pressure Homogenization of Porcine Pepsin Protease: Effects on Enzyme Activity, Stability, Milk Coagulation Profile and Gel Development

Abstract

This study investigated the effect of high pressure homogenization (HPH) (up to 190 MPa) on porcine pepsin (proteolytic and milk-clotting activities), and the consequences of using the processed enzyme in milk coagulation and gel formation (rheological profile, proteolysis, syneresis, and microstructure). Although the proteolytic activity (PA) was not altered immediately after the HPH process, it reduced during enzyme storage, with a 5% decrease after 60 days of storage for samples obtained with the enzyme processed at 50, 100 and 150 MPa. HPH increased the milk-clotting activity (MCA) of the enzyme processed at 150 MPa, being 15% higher than the MCA of non-processed samples after 60 days of storage. The enzyme processed at 150 MPa produced faster aggregation and a more consistent milk gel (G' value 92% higher after 90 minutes) when compared with the non-processed enzyme. In addition, the gels produced with the enzyme processed at 150 MPa showed greater syneresis after 40 minutes of coagulation (forming a more compact protein network) and lower porosity (evidenced by confocal microscopy). These effects on the milk gel can be associated with the increment in MCA and reduction in PA caused by the effects of HPH on pepsin during storage. According to the results, HPH stands out as a process capable of changing the proteolytic characteristics of porcine pepsin, with improvements on the milk coagulation step and gel characteristics. Therefore, the porcine pepsin submitted to HPH process can be a suitable alternative for the production of cheese.

Fig 1. Relative proteolytic activity of the porcine pepsin subjected to the high pressure homogenization process.

Different letters (a-b) mean significant difference evaluated by the Tukey test (p<0.05) between the non-processed and processed porcine pepsin samples. Evaluation is considered for each group of time (0, 7, 14, 30 and 60 days) separately.

Fig 2. Relative milk-clotting activity of the porcine pepsin subjected to the high pressure homogenization process.

Different letters (a-b) mean significant difference evaluated by the Tukey test (p<0.05) between the non-processed and processed porcine pepsin samples. Evaluation is considered for each group of time (0, 7, 14, 30 and 60 days) separately.

Fig 3. Evaluation of milk coagulation subjected to porcine pepsin processed by to high pressure homogenization at different storage time: (a1) immediately after processing (0h) (a2) log scale, and (b1) after 60 days storage at 4°C (b2) log scale.

** Values are means of replicates (n = 6).

Fig 4. Capillary electrophoregram of the porcine pepsin-induced gels using (A) non-processed and (B) enzymes processed by high pressure homogenization at 150 MPa, throughout a 24 hour coagulation period at 35°C.

Peak identification: 1: para-κ-CN; 2: α_s2-CN; 3: α_s2-CN; 4: α_s2-CN (three phosphorylation states of the α_s2-CN monomer were achieved); 5: α_s1-CN; 6: α_s0-CN (two phosphorylation states of the α_s1-CN monomer were achieved); 7: κ-CN; 8: β-CN^B; 9: β-CN^A1; 10: β-CN^A2 (two genetic variants of β-CN: A1 and A2); 11: γ₂-CN; 12: αs₁-I-CN 8P; 13: αs₁-I-CN 9P.

Fig 5. Evaluation of milk coagulation using a porcine pepsin enzyme subjected to high pressure homogenization (processed at 150 MPa—green lines) and a non-processed control (red lines) immediately after processing, throughout a 24 hours period at 35°C: (A) G’; (B) G”; (C) Loss tangent.

** Values are means of replicates (n = 6).

Fig 6. CLSM micrographs of gels obtained using a porcine pepsin enzyme processed at 150 MPa and non-processed porcine pepsin (A1-D1: control at a height of 5 μm; A2-D2: control at a height of 20 μm; E1-H1: 150 MPa at a height of 5 μm; E2-H2: 150 MPa at a height of 20 μm) throughout a 24 hour period at 35°C.

The fast-green FCF stained protein appears red and the serum phase appears black in these images. Each set of images is presented with two views: the X–Y (a height of 5 and 20 μm from the bottom—the surface of the coverglass) and the Z–Y (right) projections. For each sample, 20 adjacent planes were acquired with the separation between the planes kept constant at 0.75 μm, giving a total observation depth of 15 μm. The scale bars are 20 μm in length.

Fig 7. Gel porosity (A), number of pores (B) and average pore area (C) of gels obtained using porcine pepsin enzyme processed at 150 MPa and non-processed porcine pepsin.

The results are expressed as the mean ± standard deviation (n = 6).