Properties of Bovine Collagen as Influenced by High-Pressure Processing

Abstract

The physical properties and structure of collagen treated with high-pressure technologies have not yet been investigated in detail. The main goal of this work was to determine whether this modern gentle technology significantly changes the properties of collagen. High pressure in the range of 0-400 MPa was used, and the rheological, mechanical, thermal, and structural properties of collagen were measured. The rheological properties measured in the area of linear viscoelasticity do not statistically significantly change due to the influence of pressure or the duration of pressure exposure. In addition, the mechanical properties measured by compression between two plates are not statistically significantly influenced by pressure value or pressure hold time. The thermal properties T_on and ∆H measured by differential calorimetry depend on pressure value and pressure hold time. Results from amino acids and FTIR analyses show that exposure of collagenous gels to high pressure (400 MPa), regardless of applied time (5 and 10 min), caused only minor changes in the primary and secondary structure and preserved collagenous polymeric integrity. SEM analysis did not show changes in collagen fibril ordering orientation over longer distances after applying 400 MPa of pressure for 10 min.

Keywords: bovine collagen; high-pressure processing; physical properties; structure properties.

Figure 1

CYX 6/103 high-pressure isostatic press made by Žďas joint-stock company with a chamber volume of 2 L.

Figure 2

Combined Kelvin–Voigt–Maxwell model (adapted from Refs. [24,31]).

Figure 3

Experimental data of the storage modulus of elasticity G′ as a function of oscillation frequency and pressure treatment parameters (confidence intervals are marked).

Figure 4

Experimental data and regression curves of the modulus of elasticity G′ as a function of angular velocity and pressure treatment parameters.

Figure 5

Stress-strain data for untreated collagenous gel (A) and gel treated at 400 MPa with a holding time of 10 min (B) (two extreme conditions). The colored lines represent modified Holzapfel–Gasser–Ogden (HGO) models.

Figure 6

A comparison of the HGO models for untreated collagenous gel and gels treated at different pressures with a holding time of 5 (A) or 10 (B) minutes. HGO model parameters are listed in Table 4.

Figure 7

Compression modulus of collagenous gels before and after the treatment at different pressures (200, 300, and 400 MPa) with different holding times (5 and 10 min). Based on the Kruskal–Wallis test and the subsequent Dunn’s multiple comparison tests of moduli of collagen gels in all conditions, it is impossible to reject the null hypothesis (i.e., medians are equal) at the chosen significance level of 0.05 (n = 9).

Figure 8

Peak onset T_on as a function of pressure.

Figure 9

Peak temperature T_peak as a function of pressure.

Figure 10

Peak height H_peak as a function of pressure.

Figure 11

Parameter ΔH as a function of pressure.

Figure 12

Scatter plot of (A) “TOTAL WATER CONTENT,” (B) “AREA RATIO A/I,” and (C) “INTENSITY RATIO AMIDE III/1450” with arithmetical mean and standard deviation for (A) and with medians and interquartile range for (B) and (C). Note that p-values less than or equal to 0.05 (Dunn’s multiple comparison test; n = 3 for (A), n = 20 for (B) and (C)) are displayed for the comparisons of the mean rank of each data set with the mean rank of every other data set.

Figure 13

Comparisons of the infrared spectra before and after application of 400 MPa for 5 and 10 min.

Figure 14

Scatter plot of (A) “AREA 1610,” (B) “AREA 1630,” (C) “AREA 1660,” (D) “AREA 1690,” and (E) “AREA RATIO 1660/1690” with medians and interquartile range. Note that p-values less than or equal to 0.05 (Dunn’s multiple comparison test; n = 20) are displayed for comparisons of the mean rank of each data set with the mean rank of every other data set.

Figure 15

Pressure-related changes in amino acid content (number of amino acid residues per 1000 amino acid units) of collagen after 400 MPa for 10 min. Relative changes were calculated as the difference of the mean value (arithmetical mean, n = 6) of each amino acid content before and after pressure application. Mann–Whitney test was used to compare amino acid composition before and after pressure application (* denotes p-values ≤ 0.05).

Figure 16

Representative SEM images of the two collagenous samples (0 MPa and 400 MPa for 10 min); upper line mag. 5000×, bar 30 μm; bottom line mag. 10,000×, bar 10 μm.