# Properties of Bovine Collagen as Influenced by High-Pressure Processing

^{1}

^{2}

^{3}

^{*}

## Abstract

**:**

_{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.

## 1. Introduction

## 2. Materials and Methods

#### 2.1. Material

#### 2.2. Apparatus

#### 2.3. Pressurizing Samples

#### 2.4. Determination of Dry Matter

#### 2.5. Determination of pH

#### 2.6. Rheological Measurement

#### 2.7. Measurement of Mechanical Properties

_{0}. A correction was made for the increasing diameter of the cylinder due to compression, which is valid under the assumption of the conservation of the cylinder volume:

_{0}

^{2}⋅(H

_{0}/(H

_{0}− x⋅H

_{0}))]

_{0}, and “y” represents the axial deformation of the cylinder. Nine measurements were acquired for each pressurized sample time.

_{1}= 0, the model reduces to the form of Hooke’s model, where E represents the modulus of elasticity (or Young’s modulus when there is uniaxial tension). We have successfully used this approach to model the mechanical behavior of collagen gels in our previous studies (e.g., under uniaxial tension [26]). For the identification of the HGO model and for calculating the associated statistics, the data were processed using MATLAB (The MathWorks, Inc., Natick, MA, USA).

#### 2.8. Measurement of Thermal Properties

_{on}, the temperature at the peak T

_{peak}, the height of the peak H

_{peak}, and the value of the area under the peak (i.e., ΔH), which is proportional to the energy of the reaction caused by heating the sample.

#### 2.9. Determination of Total Water Content

#### 2.10. Infrared Spectrometry (FTIR)

^{−1}via 64 scans at a resolution of 4 cm

^{−1}. Acquired spectra were processed using OMNIC version 9 software (Thermo Scientific, Madison, WI, USA). The areas of the amide I bands were deconvoluted using the same software and statistically evaluated.

#### 2.11. Amino Acids by HPLC-DAD

#### 2.11.1. Chemicals and Solutions

_{2}HPO

_{4}(anhydrous), 10 mM Na

_{2}B

_{4}O

_{7}(decahydrate), 5 mM NaN

_{3}, pH set to 8.2 (with conc. HCl), and filtered through 0.2 µm nylon filter. Mobile phase B: acetonitrile, methanol, water (45:45:10, v/v/v). Injection diluent: 100 mL mobile phase A and 0.4 mL H

_{3}PO

_{4}(85%). Ortho-phthalaldehyde (OPA), 9-fluorenylmethyl chloroformate (FMOC), and the borate buffer (0.4 M in water, pH 10.2) provided by Agilent (in a kit). For hydrolysis, 0.1 M HCl, 6M HCl was purged with N

_{2}for at least 30 min. Needle wash: mobile phase B. Reconstitution solution: 500 µmol/L IS (sarcosine, norvaline) in 0.05 M HCl. Calibration solutions (in 0.05 M HCl): 21 amino acids at 90, 225, and 900 µmol/L containing IS (500 µmol/L) prepared from an Agilent AA standard kit according to instructions and stored at −20 °C. Milli-Q HPLC-grade water (>18 MΩ). All chemicals were HPLC or ACS grade and were purchased from Merck Life Science Ltd. (Prague, Czech Republic), Lach-ner, Ltd. (Neratovice, Czech Republic), Agilent (Santa Clara, CA, USA, or Linde Gas join stock company (Prague, Czech Republic).

#### 2.11.2. Collagen Hydrolysis

_{2}) was added, and the headspace was flushed with N

_{2}, vortexed (IKA

^{®}-Werke GmbH & Co. KG, Staufen, Germany) for 30 s, and put into a laboratory oven (BINDER GmbH, Tuttlingen, Germany) at 110 °C for 20 h, with occasional inversions to mix the contents. After hydrolysis, samples were cooled down to room temperature and vortexed for 30 s. Then, 300 µL of hydrolysate was evaporated in an HPLC vial under N

_{2}at 60 °C for 15 min (BT Lab Systems, Saint Louis, MO, USA). To the dry residue, 1 mL of reconstitution solution was added, vortexed for 1 min, and filtered through a 0.2 µm nylon syringe filter into an HPLC glass vial with a silicone/PTFE screw cap (Chromservis Ltd., Prague, Czech Republic). Each sample was prepared in ten replicates.

#### 2.11.3. HPLC-DAD

^{2}> 0.999 for each amino acid. Wavelengths: 338 nm for OPA derivatives (10 nm bw. 390 nm ref. and 20 nm ref. bw.) and 262 nm for FMOC derivatives (16 nm bw. 324 nm ref. and 8 nm ref. bw.).

#### 2.12. Scanning Electron Microscopy (SEM) and the Characterization of the Orientation of the Collagen Fibrils

#### 2.13. Statistical Analysis

#### 2.14. Rheological Measurement

_{i}= G

_{i}⋅τ

_{i}

_{i}= 0). The usual four-parameter Maxwell model (n = 2, η

_{i}= 0) represents an excellent approximation of the storage modulus G′ but cannot describe the loss modulus G′′ due to the asymptotic properties at high frequencies (i.e., all Maxwellian G′′ terms approach zero at ω → ∞).

_{i}for n = 1 (or a six-parameter model for n = 2). The parameters of the combined models could be adjusted to describe the plateau and the growing region (i.e., “G”).

_{1}, G

_{2}, µ

_{1}, and µ

_{2}were found using non-linear regressions of the G′ data using DataFit software version 6.1.10 (Oakdale Engineering, Pittsburgh, PA, USA) and Equation (3) simplified for η

_{1}= η

_{2}= 0 and n = 2. Parameters of the Maxwell model G

_{1,2}and μ

_{1,2}were correlated relative to pressure P

_{I}and holding time D

_{I}. Relationships were examined using DataFit statistical software version 6.1.10. Parameters were correlated using linear relationships, as follows:

_{1}= a

_{1}+ b1⋅DI + c1⋅PI

_{2}= d

_{2}+ e2⋅DI + f2⋅PI

_{f}= the number of experimental points minus the number of model parameters: 7 − 3 = 4; r

_{crit, 4}= 0.811. Data on critical values of correlation coefficients were taken from a publication by Štěpánek [32].

#### 2.15. Measurement of Mechanical Properties

#### 2.16. Measurement of Thermal Properties

## 3. Results

#### 3.1. Dry Matter Content and pH

#### 3.2. Rheological Properties

_{1,2}= 0. The figure shows that the elastic modulus G′ grows with increasing angular velocity of oscillations. It is also evident that the simplified model we used does an excellent job of describing the experimental data for the individual parameters of pressure treatment.

#### 3.2.1. The Results of Evaluating the Parameters of the Simplified Maxwell Model (Equation (3))

_{1}. G

_{2}, τ

_{1}, and τ

_{2}. Numerical values for these parameters, including calculated values µ

_{1}and µ

_{2}, are presented in Table 1. This table shows the values of the correlation coefficients determined for the experimental data and valid for the individual samples (i.e., individual treatment methods). The total data for five repeated measurements of each sample were 55 (11 frequencies × 5 repetitions). The number of parameters of the model (i.e., Equation (3)) was 4. Therefore, the number of degrees of freedom is 55 − 4 = 51. For this value, r

_{crit,50}= 0.273. By comparing this value with the r data in Table 1, it is clear that Equation (3) almost perfectly describes the individual measured data for a given pressure-treated sample; this is also evident in Figure 4.

#### 3.2.2. Results of Parameter Evaluation of Correlation Equations (5) and (6)

_{1}and d

_{2}, the other parameters are statistically insignificant since p > 0.05 demonstrates the independence of G

_{1}and G

_{2}from pressure holding times. For Equation (5), the value of the multiple correlation coefficient was r = 0.447. If we compare this value with the required value of the correlation coefficient of 0.811, it is evident that the G

_{1}parameter does not depend on pressure or holding time. For Equation (6), the value of the multiple correlation coefficient was r = 0.488. If we compare this value with the required value of the correlation coefficient, 0.811, the G

_{2}parameter also does not depend on pressure or holding time.

#### 3.3. Mechanical Properties

#### 3.4. Thermal Properties

_{on}collagen sample is statistically demonstrably dependent on the pressure and the duration. The same conclusion can be drawn regarding parameter ΔH.

_{peak}shows a statistically significant dependence on the pressure but is independent of holding time. On the other hand, parameter H

_{peak}does not depend on the pressure but statistically significantly depends on the holding time.

#### 3.5. Determination of Total Water Content

#### 3.6. Infrared Spectrometry

^{−1}belongs to amide A, incorporating N-H stretching and several modes of OH groups (i.e., free OH groups, intramolecular, and intermolecular H-bridges of the OH groups) [34,35]. The band at ~3075 cm

^{−1}is a mutual band of C-H vibrations in sp

_{2}hybridization and stretching vibration of the N-H bonds in secondary amides (amide B). The stretching vibrations of C=O coupled with N–H bending vibrations seen in the amide I and amide II bands arise from N–H bending vibrations coupled with C–N stretching vibrations. Another demonstration of the triple helical collagen structure can be seen in amide III (at ~1205, 1235, and 1280 cm

^{−1}) together with a band at 1338 cm

^{−1}[36,37].

^{−1}reflects stretching vibrations of amino acids, NH

_{2}bonds (amide A), and OH bonds in free and interstitial water. Crosslinking causes free −NH

_{2}groups to change into −NH- groups, water bonded to collagen is lost [4], and as a consequence, the integral absorbance of amide A decreases.

^{−1}) to the 1450 cm

^{−1}band [39] (see Figure 12C).

^{−1}) were expressed as percentages of the total area amide I band and statistically analyzed (Figure 14).

#### 3.7. Amino Acids Analysis

#### 3.8. Scanning Electron Microscopy and the Characterization of Collagen Fibril Orientation

## 4. Discussion

_{m}and denaturation energy ΔH. The T

_{m}parameter increases with increasing pressure following a non-linear equation (p is pressure in MPa, T

_{m}in °C), as follows:

_{m}= 41.4 + 4.7 ⋅10

^{−2}· p − 6.6⋅10

^{−5}⋅ p

^{2}

_{m}can be compared with the data T

_{peak}in Figure 9. Both the T

_{peak}valid for the denaturation of frog collagen and the T

_{peak}valid for bovine collagen increase with increasing pressure.

^{−1}ranged from 0.97 to 1.15; these values correspond to collagen, while a ratio around 0.76 is typical for gelatin [44]. No statistically significant differences were found (Figure 12C). Collagen’s integrity, in gels, was not damaged even after 400 MPa for 5 and 10 min. Collagen’s triple helix structure, represented by the main band at 1660, can be used as a marker of collagen change. No statistically significant differences were determined in this area (Figure 14C). Changes in other parameters range from 1 to 2%. The 1610 spectral band (Figure 14A) can be assigned to the spectral manifestation of aromatic amino acids, which may be more spectroscopically active in disintegrated states of collagen (i.e., gelatin) [45]. Band 1630 represents a denatured state of a collagen left-handed 3–10 helix (Figure 14B), and band 1690 (Figure 14D) represents β-turn and antiparallel β-sheet structures [40]. Beta sheets consist of β-strands (chains are typically 3–10 amino acids long) that are connected laterally by hydrogen bonds, thus forming a twisted, pleated sheet. Two sub-bands in the amide I spectral peak, 1660 and 1690 cm

^{−1}, are of particular interest (Figure 14E).

## 5. Conclusions

#### 5.1. Rheological Properties

_{1}and G

_{2}using Equations (5) and (6). It has been shown that these parameters do not depend statistically conclusively on the pressure or hold times in the range of the values used for these process parameters. The G

_{1}value can be characterized by the constant a

_{1}= 9804.4 Pa. The G

_{2}value can be characterized by the constant d

_{2}= 2246.5 Pa.

#### 5.2. Mechanical Properties

#### 5.3. Thermal Properties

_{on}collagen sample was statistically demonstrably dependent on the pressure and holding time. The ΔH parameter showed the same dependence. This parameter represents the area under the peak of the curve, which characterizes the energy required for the ongoing reaction caused by heating the collagen sample.

_{peak}parameter shows a statistically significant dependence on the pressure but is independent of holding time. The H

_{peak}parameter, on the other hand, does not depend on pressure but statistically significantly depends on holding time.

#### 5.4. Overall Rating, Limitations, and Practical Implications

_{on}and ΔH measured using differential calorimetry depend on both pressure and hold time.

## Author Contributions

## Funding

## Institutional Review Board Statement

## Data Availability Statement

## Acknowledgments

## Conflicts of Interest

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**Figure 1.**CYX 6/103 high-pressure isostatic press made by Žďas joint-stock company with a chamber volume of 2 L.

**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 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.

**Table 1.**Numerical values of the parameters of Equation (4) depending on the pressure treatment parameters.

Pressure Holding Time (min) | Pressure (MPa) | G_{1} (Pa) | G_{2} (Pa) | τ_{1} (s) | τ_{2} (s) | µ_{1} (Pa∙s) | µ_{2} (Pa∙s) | r^{2} (−) | r (−) |
---|---|---|---|---|---|---|---|---|---|

0 | 0 | 9105 | 2089 | 5.40 | 0.20 | 49,206 | 11,292 | 0.588 | 0.767 |

5 | 200 | 12,111 | 2856 | 5.03 | 0.19 | 60,973 | 14,379 | 0.700 | 0.837 |

10 | 200 | 10,931 | 2516 | 5.20 | 0.20 | 568,41 | 13,083 | 0.229 | 0.479 |

5 | 300 | 10,006 | 2300 | 5.04 | 0.20 | 50,393 | 11,586 | 0.628 | 0.793 |

10 | 300 | 10,464 | 2453 | 4.98 | 0.20 | 52,116 | 12,217 | 0.519 | 0.720 |

5 | 400 | 11,465 | 2625 | 5.16 | 0.20 | 59,112 | 13,534 | 0.793 | 0.891 |

10 | 400 | 10,519 | 2518 | 5.94 | 0.21 | 62,468 | 14,951 | 0.610 | 0.781 |

Parameter | Value | Standard Deviation | t-Parameter | p | Evaluation |
---|---|---|---|---|---|

a_{1} (Pa) | 9804.4 | 955.1 | 10.3 | 0.0005 | significant |

b_{1} (Pa/min) | 28.7 | 145.7 | 0.2 | 0.854 | insignificant |

c_{1} (−) | 2.6 | 3.9 | 0.7 | 0.545 | insignificant |

Parameter | Value | Standard Deviation | t-Parameter | p | Evaluation |
---|---|---|---|---|---|

d_{2} (Pa) | 2246.5 | 230.9 | 9.7 | 0.0006 | significant |

e_{2} (Pa/min) | 11.9 | 35.2 | 0.3 | 0.752 | insignificant |

f_{2} (−) | 0.6 | 0.95 | 0.6 | 0.557 | insignificant |

Pressure (MPa) | Holding Time (min) | E (kPa) | k_{1} (kPa) | r^{2} (−) |
---|---|---|---|---|

0 | 0 | 15.63 ± 0.69 | −2.01 ± 0.63 | 0.945 |

200 | 5 | 17.95 ± 0.73 | −2.51 ± 0.64 | 0.949 |

10 | 16.84 ± 0.45 | −1.89 ± 0.40 | 0.980 | |

300 | 5 | 17.54 ± 0.83 | −2.95 ± 0.72 | 0.919 |

10 | 17.04 ± 0.69 | −3.03 ± 0.60 | 0.941 | |

400 | 5 | 18.01 ± 0.58 | −3.06 ± 0.50 | 0.964 |

10 | 18.17 ± 0.52 | −3.52 ± 0.45 | 0.968 |

**Table 5.**Results of an analysis of variance of thermal properties of high-pressure-treated collagen.

Thermal Properties/ Parameters of UHP | T_{on} (°C) | T_{peak} (°C) | H_{peak} (J/g∙ K) | ΔH (J/g) | ||||
---|---|---|---|---|---|---|---|---|

p | Statistical Significance | p | Statistical Significance | p | Statistical Significance | p | Statistical Significance | |

Pressure (MPa) | 0.00210 | yes | 0.00358 | yes | 0.05608 | no | 0.00045 | yes |

Holding time (min) | 0.02155 | yes | 0.14404 | no | 0.04463 | yes | 0.00399 | yes |

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**MDPI and ACS Style**

Houška, M.; Landfeld, A.; Novotná, P.; Strohalm, J.; Šupová, M.; Suchý, T.; Chlup, H.; Skočilas, J.; Štípek, J.; Žaloudková, M.;
et al. Properties of Bovine Collagen as Influenced by High-Pressure Processing. *Polymers* **2023**, *15*, 2472.
https://doi.org/10.3390/polym15112472

**AMA Style**

Houška M, Landfeld A, Novotná P, Strohalm J, Šupová M, Suchý T, Chlup H, Skočilas J, Štípek J, Žaloudková M,
et al. Properties of Bovine Collagen as Influenced by High-Pressure Processing. *Polymers*. 2023; 15(11):2472.
https://doi.org/10.3390/polym15112472

**Chicago/Turabian Style**

Houška, Milan, Aleš Landfeld, Pavla Novotná, Jan Strohalm, Monika Šupová, Tomáš Suchý, Hynek Chlup, Jan Skočilas, Jan Štípek, Margit Žaloudková,
and et al. 2023. "Properties of Bovine Collagen as Influenced by High-Pressure Processing" *Polymers* 15, no. 11: 2472.
https://doi.org/10.3390/polym15112472