Materials and methods

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7.

Enzyme
High pressure processing
Experimental design
Proteolytic activity determination
Milk-clotting activity determination
Rheological assays
Three dimensional microstructure of the
coagulation process and rennet-induced
gels obtained by confocal scanning laser
Microscopy
8. Image analysis

1. Enzyme
A commercial powder calf rennet containing 94
g/100 g of chymosin and 6 g/100 g of pepsin
was used in the assays.
The activity of the enzyme was 1700 IMCU per
gram of powder.
2. High Pressure Processing
This equipment has a cylindrical chamber of 15
mm in diameter and 35 mm in height, with 10
mL of capacity.
The equipment works at pressures of up to 350
MPa and ethanol was used as the pressurizing
fluid.
The come up time to reach 350 MPa was 3 min

3. Experimental Design
Aliquots of 2 mL of enzyme solution prepared in sodium
acetate buffer were vacuum-packed in sterile plastic
polyolefin bags at 4 C.
The samples were subjected to the HPP at room temperature
(25 C) to evaluate the simultaneous effect of two independent
variables: isostatic pressure (X1) and process time (X2) on the
enzyme.
4. Proteolytic Activity Determination
The proteolytic activity was based on using sodium caseinate
as substrate.
The activity was determined spectrophotometrically by
measurement of the release of peptides.
The analyses were carried out immediately after processing
(time 0 h) and after 24 h.
The samples were stored under refrigeration (4 C) during this
period.
A non-processed sample was also prepared for a comparative
evaluation.

5. Milk-clotting activity determination

The milk-clotting activity (MCA) using a reconstituted
skim milk powder as substrate. One milk-clotting activity
unit (MCA) was defined as the amount of enzyme
required to clot 1 mL of substrate in 40 min at 35 C.
MCA assays were carried out immediately after
processing (time 0 h) and after 24 h. The samples were
stored under refrigeration (4 C) during this period.
Furthermore, a non-processed enzyme was also
prepared for comparative evaluation
6. Rheological assays
This assaywas carried out for the native (non-processed)
enzyme and the HPP enzyme under optimum pressure
and time conditions, considering the results of the
response surface for the greater milk-clotting activity
(280 MPa for 20 min).

7. Three dimensional microstructure of the coagulation

process and rennet-induced gels obtained by confocal
scanning laser microscopy
Confocal scanning laser microscopy (CLSM) is able to
penetrate deeply but noninvasively through the sample
to obtain a large number of sequential, thin optical
sections that may then be assembled by imageanalysis software to produce three dimensional (3D)
reconstructions and projections.
8. Image analysis
Image J analysis was used to quantify the porosity and
total number of pores from the CLSM micrographs
The average pore areawas calculated as the total pore
area with respect to the total sample area (0.018
mm2), divided by the total number of pores.