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\DOI{10.5802/crchim.217}
\datereceived{2022-06-27}
\daterevised{2022-09-01}
\datererevised{2022-09-22}
\dateaccepted{2022-09-23}
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\dateposted{2023-01-06}
\begin{document}

\begin{noXML}

%\TopicEF{Materials and Clean Processes for Sustainable Energy and
%Environmental Applications}{Mat\'eriaux et proc\'ed\'es propres pour
%des applications \'energ\'etiques et environnementales}

\title{Ultrafiltration operational conditions influence in the
antibacterial activity of native and thermally treated lysozyme}

\author{\firstname{Ariane} \lastname{de Espindola}}
\address{Institut de Science des Mat\'{e}riaux de Mulhouse,
Universit\'{e} de Haute-Alsace, CNRS IS2M UMR 7361, 3 bis rue A.
Werner, 68098 Mulhouse Cedex, France}
\address{Universit\'{e} de Strasbourg, France}
\email[A. de Espindola]{ariane.de-espindola@uha.fr}

\author{\firstname{Simona M.} \lastname{Miron}}
\addressSameAs{1}{Institut de Science des Mat\'{e}riaux de Mulhouse,
Universit\'{e} de Haute-Alsace, CNRS IS2M UMR 7361, 3 bis rue A.
Werner, 68098 Mulhouse Cedex, France}
\addressSameAs{2}{Universit\'{e} de Strasbourg, France}
\email[S. M. Miron]{miron.melani@yahoo.com}

\author{\firstname{Patrick} \lastname{Dutourni\'{e}}}
\addressSameAs{1}{Institut de Science des Mat\'{e}riaux de Mulhouse,
Universit\'{e} de Haute-Alsace, CNRS IS2M UMR 7361, 3 bis rue A.
Werner, 68098 Mulhouse Cedex, France}
\addressSameAs{2}{Universit\'{e} de Strasbourg, France}
\email[P. Dutourni\'{e}]{patrick.dutournie@uha.fr}

\author{\firstname{Arnaud} \lastname{Ponche}\CDRorcid{0000-0002-8993-2896}\IsCorresp}
\addressSameAs{1}{Institut de Science des Mat\'{e}riaux de Mulhouse,
Universit\'{e} de Haute-Alsace, CNRS IS2M UMR 7361, 3 bis rue A.
Werner, 68098 Mulhouse Cedex, France}
\addressSameAs{2}{Universit\'{e} de Strasbourg, France}
\email[A. Ponche]{arnaud.ponche@uha.fr}

\shortrunauthors

\keywords{\kwd{Ultrafiltration}
\kwd{Lysozyme}
\kwd{Antibacterial}
\kwd{Denaturation}
\kwd{Processing}}

\begin{abstract}
To obtain high quality final products, biomolecules can be exposed in
production lines to stress factors such as heating, stirring and
multiple surfaces contact before their commercialization. These factors
can induce stability and efficacy loss. In this work, the antibacterial
activity of lysozyme 
(LSZ),
a protein used as antibiotic which acts by
disrupting the bacterial cell wall, was studied after two operations:
heating and ultrafiltration. Antibacterial activity after extreme
stress factors is expected to decrease due to conformational changes in
the protein molecule. It was observed that ultrafiltration can promote
an activity decrease proportional to the applied transmembrane pressure
of a non-treated 
lysozyme. 
However, when a thermally treated 
lysozyme
(90~\textdegree C) was filtered, no additional effect was observed due to
the pore passage, independently of the applied pressure.
\end{abstract}

\maketitle

{\vspace*{20pt}}

\twocolumngrid

\end{noXML}

\section{Introduction}\label{sec1}

Protein as a therapeutic active ingredient is a good alternative
compared to small molecule drugs due to its specificity. Using proteins
as therapeutic drugs is an alternative to treat some specific illness
without interference on other natural biological processes and proteins
induce less side effects when compared to synthetic drugs \cite{1}.
Thinking about the need of protein consumption, the interest for
protein use as food supplement, vaccines, antibiotics and
biopharmaceutical enzymes increased in the last two 
decades.

In the case of a protein function, two important parameters are
followed: stability and efficiency. These factors are correlated
and depend on protein conformation, normally the native state is required
for high efficiency \cite{2}. However, the native state can unfold
into an inactive form under stress conditions such as temperature and
pH changes, elevated pressures, use of solvents, shearing, surface
contact,  etc., and any modification in the structure of the protein can
lead to loss in its efficiency \cite{3}. These structural
modifications can be part of protein process during filtration,
purification, lyophilization, sterilization, storage, transport and
drug application.

Temperature is one of the most important environmental parameters that
directly impact the quality of the pharmaceutical and food products
from the beginning of the formulation/processing until the
administration/consumption \cite{4}. For example, protein drugs need
to be conserved under cold temperatures during storage and
transportation in order to avoid denaturation and physical aggregation
because the protein is thermosentitive  \cite{5}. Removal of
microbial contamination of food product in its final container by heat
(sterilization) is not recommended for protein-based products because
it can lead to product degradation due to irreversible denaturation of
enzymes \cite{6}. 

In recent years, pasteurization has been considered to be adapted to
protein industry in the manufacturing processes. Pasteurization
represents a mild thermal process (compared to sterilization) which can
be divided into 4 different methods as follow: LTLT (Low Temperature,
Long Time---63~\textdegree C for minimum 30~min), HTST (High
Temperature, Shorter Time---72~\textdegree C for minimum 15~s),
flash pasteurization (85--90~\textdegree C for 1--4~s) and UHT
(Ultra High Temperature---135~\textdegree C for minimum 1 second)
\cite{7}. The method chosen for pasteurization takes into account
the composition and characteristics of the compound. As pasteurization
uses temperature, it might lead to impact on the properties of the
product. In order to avoid that, time and temperature have to be
balanced to be enough for decontamination of bacteria while not
affecting the product quality \cite{8}.

An alternative to eliminate microorganisms without using extreme
conditions is to combine more than one method of purification and
sterilization. For example, as presented by Subramanian~\cite{9},
pasteurization could be used in upstream process in order to ease the
downstream process done by ultrafiltration~\cite{9}. Ultrafiltration
is a simple membrane process that can be used concomitantly for the
concentration, purification, separation and microorganisms removal of
protein solutions \cite{10}. The mechanisms responsible for the
separation are size-exclusion and electrostatic effects, the
latter being dependent on membrane material surface \cite{11}. The
size-exclusion or sieving mechanism is dependent on the ratio
between the membrane pore size and the actual size of the studied\unskip\break 
molecule. 

In these conditions, it is interesting to observe the behavior of
proteins that have been subjected to pasteurization (high temperatures)
and ultrafiltration. In the present study, the hydrodynamic properties
and the antibacterial properties are used to elucidate the changes
occurring to 
lysozyme 
(model protein) due to temperature and
filtration. 
Lysozyme
is considered a model protein due to its structure
and functions. Discovered in 1922 by Alexander Fleming, 
lysozyme 
is a basic enzyme with four disulphide bridges and a compact ellipsoid shape
\cite{12}. A key feature of 
lysozyme
is the direct link between its
structure and its antimicrobial activity. Due to the strong link
between conformation and function, any modification of 
the lysozyme 
can temporally or completely change its antimicrobial activity
\cite{13}. 

In literature, works are in agreement and proove that the application of
high temperature (higher than~74~\textdegree C) decreases the
antibacterial activity of 
the lysozyme
\cite{14,15,16,17,18}, but, there is no report about the influence on 
LSZ antibacterial activity caused by the combination
of temperature and shear stress (ultrafiltration process).

\section{Experimental section}\label{sec2}

\subsection{Materials}\label{sec21}

\subsubsection{Materials and solution preparation}\label{sec211}

The chemical reagents used in the present investigation were vitamin B12
(VB12) from Alfa Aesar and hen egg white lysozyme
from Sigma-Aldrich. The
solutions used were obtained by dissolving the desired amount of powder
(LSZ---0.025~mM and VB12---$9.22\times 10^{-3}$ mM)
in deionized water. For the antibacterial assay, \textit{Micrococcus
Lysodeikticus} was purchased from Sigma-Aldrich and prepared 
with 0.01 M phosphate buffer solution. 

\subsubsection{Preparation of thermally denatured 
lysozyme}\label{sec212}

Thermally denatured LSZ
was prepared by heating the solution on a
heating plate at 60, 70, 80 and 90~\textdegree C in a glass vial for 
1~h after the denaturation temperature was reached. The solution was
then cooled down until it reached room temperature and filtered with a
$0.2~\upmu \mathrm{m}$ syringe filter. 

\subsection{Experimental protocol}\label{sec22}

The filtration procedure and the laboratory pilot-plant were
described in previous studies \cite{19,20}. Filtrations were
performed with three tubular mono channel ultrafiltration titania
membranes with a commercial cut-off of 1 kDa (M1, M2 and M3). The
active layer of the membrane is globally negatively charged for pH
higher than 4.1. In this work, all experiments were done with
deionized water (pH around 6.0, ionic conductivity  ${<} 1~\upmu
\mathrm{S}/\mathrm{cm}$). Temperature was maintained constant at
25~\textdegree C using a cooling system composed of a refrigeration
system and heat exchanger. The applied flow rate was 700 L/h (feed
velocity about 5 m/s, turbulent flow $Re > 39{,}000$) and the pressure
varied from 4 to 12 bar. At each pressure, samples from retentate
(solution in the feed tank) and permeate (solution that passed through
the pores) were analyzed. VB12
was used as a neutral molecule to
assess the steric hindrance assuming that the separation is only due to
steric effect. The membrane hydraulic properties (water permeability)
are calculated from pure water filtration tests.

\subsection{Characterization techniques}\label{sec23}

\subsubsection{UV-VIS spectroscopy}\label{sec231}

This technique was used to quantify 
the lysozyme 
after solution
preparations and to determine the rejection rate of ultrafiltration
membrane. Using the Lambert--Beer law and a calibration curve, LSZ
quantification was performed measuring the absorbance in triplicate of
protein solutions at 280 nm (Lambda 750, Perkin Elmer Instrument). On
the basis of the obtained concentration, solution was diluted for
enzymatic tests.

The rejection rate (R) was calculated based upon Equation~(\ref{eq1}) taking into
account the absorbance of the permeate ($A_{\mathrm{perm}}$) and the
retentate ($A_{\mathrm{ret}}$). The absorbance was measured at 360 nm for 
 VB12
and 280 nm for 
LSZ.
{\begin{equation}\label{eq1}
R_{\mathrm{obs}}=(1-(A_{\mathrm{perm}}/A_{\mathrm{ret}}))
\end{equation}}\unskip
where: ${R}_{\mathrm{obs}}$ is the observed rejection rate, 
${A}_{\mathrm{perm}}$ is the absorbance for the permeate and 
${A}_{\mathrm{ret}}$ is the absorbance of the retentate.

\subsubsection{Size-Exclusion High Performance Liquid Chromatography
analysis}\label{sec232}

For this analysis, $100~\upmu\mathrm{L}$ of LSZ were injected into
the SEC-HPLC system (Agilent 1100 Series chain equipped with a UV
detector and a quaternary pump) with a chromatographic  $9.4 \times
250$~mm Zorbax Bio Series GF-250 column (range
400,000--$4000~\mathrm{g}{\cdot} \mathrm{mol}^{-1}$). The mobile phase
was composed of phosphate buffer saline PBS (1 tablet in 200~mL
deionized water), sodium dodecyl sulfate SDS (0.1~wt\%) and sodium
azide $\mathrm{NaN}_{3}$ (0.005~wt\%). All reagents were purchased
from Sigma-Aldrich. The measurements were made at a wavelength of 280
nm, constant temperature of 25~\textdegree C and a flow rate of 
$1.0~\mathrm{mL}{\cdot}\mathrm{min}^{-1}$. 

\subsubsection{Antibacterial activity}\label{sec233}

The antibacterial activity of 
LSZ
was determined by monitoring the
decrease in turbidity of a \textit{Micrococcus Lysodeikticus}
suspension at 450~nm for 10~min with 15~s intervals. In a
96-well microplate, $20~\upmu\mathrm{L}$ of 
LSZ
were put in
contact with $200~\upmu\mathrm{L}$ of bacteria culture 
($0.3~\mathrm{mg}{\cdot}\mathrm{mL}^{-1}$). The solutions were shaken
for 30~s before measurements and incubated at 30~\textdegree C.
The absorbance measurements were repeated 9 times at a wavelength of
450~nm. The reaction rate was estimated by the slope of
$1/A_{450~\mathrm{nm}}$ versus time graph (second order reaction between
\textit{Micrococcus Lysodeikticus} and 
LSZ).
The activity of
LSZ
was determined by Equation~(\ref{eq2}). 
{\begin{equation}\label{eq2}
{A}_{{u}}=\frac{\left(\frac{\text{Slope}}
{[\mathrm{LYS}]}\right)}{[\mathrm{ML}]}
\end{equation}}\unskip
where: $A_{u}$ is 
LSZ
activity, slope is the slope from 
$1/A_{450~\mathrm{nm}}$ versus time graph, [LSZ] and [ML] are the
concentrations of 
LSZ
and \textit{Micrococcus Lysodeikticus} in
the well  in $\mathrm{mg}{\cdot}\mathrm{mL}^{-1}$, respectively.

An activity index was calculated using Equation~(\ref{eq3}) that
normalizes the antibacterial activity of the sample with the one of the
reference (untreated 
LSZ).
{\begin{equation}\label{eq3}
{I}_{{A_u}}=\frac{{A}_{{u}}}{{A}_{{{u}_{\mathrm{reference}}}}}
\end{equation}}\unskip
where: $I_{{A_u}}$ is the activity index, $A_{u}$ is the activity of 
LSZ,
$A_{u_{\mathrm{reference}}}$ is the activity of the
reference.

\paragraph{Statistical tests}\label{sec2331}

Two-samples $t$-test were performed using Origin Pro 2021 with a 95\% 
level of confidence and statistical significance at $p<0.05$ 
to compare the native 
lysozyme 
with the filtered one.

\section{Results and discussion}\label{sec3}

\subsection{Study of membrane influence in native 
lysozyme
antibacterial activity}\label{sec31}

In a previous study \cite{21}, the relationship between membrane
hydraulic properties and 
LSZ
antibacterial activity was evaluated
using different membranes with the same commercial cut-off of 1~kDa.
However, it was showed that size distribution of pores and real cut-off
could be different and membranes with small and bigger pores were
compared. Results indicated that the loss of activity was observed for
the membrane with smaller pores due to the shear forces applied in the
molecule during the filtration. When 
LSZ
passed through small
pores, three-dimensional structure is modified with consequently a loss
of antibacterial action.

Based on these results, in the present investigation, other membranes
were studied according to their permeability and selectivity determined
by water and 
VB12
filtration, respectively. The values are
presented in Table~\ref{tab1}.

\begin{table}[t!] %tab1
\caption{\label{tab1}Membrane performances determined by VB12
filtration and pure water filtration\vspace*{-2pt}}
\begin{tabular}{ccc}
\thead
{Membrane} & \parbox[t]{1.5cm}{\centering Rejection rate (\%)} &
\parbox[t]{2.9cm}{\centering Permeation~flux 
($10^{-14}~\mathrm{m}^3{\cdot}\mathrm{m}^{-2}{\cdot}\mathrm{s}^{-1}$)}
\vspace*{2pt}\\
\endthead
{M1} & 54 & 5.5 \\
{M2} & 56 & 2.9 \\
{M3} & 59 & 5.4 
\botline
\end{tabular}
\vspace*{-2pt}
\end{table}

Regarding the selectivity, membranes M1, M2 and M3 presented similar
values around 50\% and it could be affirmed that they have smaller
pores. To understand which parameter between rejection rate and
permeation flux interferes more in the antibacterial activity, an
enzymatic test was conducted for retentate and permeate solutions. As
the retentate solutions are the ones that did not pass through the pores,
in the previous work \cite{21} no activity loss was observed
and consequently all the retentate results are summarized in 
Figure~\ref{fig1} (transmembrane pressure $=$ 0 bar).

\begin{figure}
\vspace*{-2pt}
\includegraphics{fig01}
\vspace*{-2pt}
\caption{\label{fig1}Evolution of antibacterial activity index
(geometric mean between measures of M1, M2 and M3) for permeate
solutions of LSZ filtered under different applied pressures.}
\vspace*{-2pt}
\end{figure}

For the permeate solutions, the three membranes studied presented
similar results of antibacterial activity decrease and 
Figure~\ref{fig1} shows the average between 27 measures (9 for each
membrane). 

The activity loss increased with applied pressure for the membranes
with similar values of rejection rate. The maximum of activity loss was
approximately 60\% for 12 bar. In addition, a linear relationship
between activity index and pressure was obtained in the
studied range of applied pressure ($I_{{A_u}}=-0.038\Delta P$). The
similarity between permeate results in these three membranes indicates
that the parameter of the membrane that plays a significant role in
antibacterial action is the pore size and so, the steric hindrance. 

Taking this into account, membranes with VB12 selectivity bigger than
54\% were used to evaluate the ultrafiltration of 
LSZ
thermally treated to understand if the shear stress in the pores could
change the activity of a LSZ that had already undergone a denaturation
treatment (heating). Consequently, all the following experiments were
conducted with one of the membranes.

\vspace*{-2pt}

\subsection{Study of antibacterial activity of thermally modified LSZ
and hydrodynamic properties} \label{sec32}

\vspace*{-2pt}

Antibacterial activity indexes were obtained for LSZ treated at 60, 70,
80 and 90~\textdegree C and the results are presented in
Figure~\ref{fig2} compared to antibacterial activity of native 
LSZ
at room temperature (reference). Considering the box plot average,
until 70~\textdegree C no relevant change was observed compared with
untreated 
LSZ
(reference). For the treatment at 80 and
90~\textdegree C the antibacterial activity loss was around 20 and
40\% respectively. The increase in activity loss with temperature is in
agreement with the literature \cite{15,14,18,17}.
Furthermore Xing \etal~\cite{16} proved using Raman spectroscopy
and antibacterial activity assay that thermal denaturation is a
three-state mechanism that starts from 74~\textdegree C, which is
consistent with the absence of activity loss for LSZ treated at
70~\textdegree C 
(LSZ 70). 

\begin{figure}
\includegraphics{fig02}
\vspace*{-2pt}
\caption{\label{fig2}Antibacterial activity for 
lysozyme 
treated at different temperatures.}
\vspace*{-2pt}
\end{figure}

The mechanism of thermal denaturation states that during thermal
treatment, 
LSZ
tertiary structure changes prior to the secondary
one. In the first stage (around 74~\textdegree C) the intermolecular
interactions between the side groups are weakened forming an
intermediate tertiary structure called molten state. It is estimated that 
the latter state persists in a 2~\textdegree C interval
being followed by the secondary
structure changes. Taking it into account, in the current work,
temperatures of 70 and 90~\textdegree C were chosen to be compared in
terms of biological activity before and after ultrafiltration.

Some works correlate the biological activity decrease with aggregate
formation: the temperature increase induces soluble aggregate formation
and when the protein is aggregated, the active site is less available
to perform as an enzymatic antibacterial agent \cite{14,15}. To
evaluate protein aggregation and/or changes in conformation, SEC-HPLC
was performed and the chromatograms of 
LSZ
thermally modified are
shown in Figure~\ref{fig3}.

\begin{figure}
\includegraphics{fig03}
\vspace*{3pt}
\caption{\label{fig3}SEC-HPLC chromatograms of
lysozyme 
solutions thermally treated at 60, 70, 80 and 90~\textdegree C.}
\end{figure}

Superimposed chromatograms on Figure~\ref{fig3} show a monomeric peak
without presence of oligomers or aggregates. Absence of soluble
aggregates can be explained by the low concentration used in the
present study (0.025 mM). Therefore, aggregation could not be an
explanation for the activity loss in this case. A slight retention time
shift is observed comparing the higher temperatures with the reference
LSZ, LSZ 60 and LSZ 70 that have similar peaks. This shift for 90 and
80~\textdegree C indicates a modification of the hydrodynamic volume of
LSZ
with increasing temperature. This observation is in
agreement with the literature that situates
the lysozyme
unfolding at
temperatures higher than 70~\textdegree C \cite{16,18,22}.
Thus, antibacterial activity loss can be directly correlated with LSZ
tertiary structure changes that probably modify the environment around
the active site and as a consequence, decrease the antibacterial
activity. 

\subsection{Ultrafiltration of LSZ thermally treated}\label{sec33}

Ultrafiltration was conducted on LSZ pre-treated at 70 and
90~\textdegree C varying the applied pressure from 4 to 12 bar to
assess whether filtration can enhance the loss in biological activity of 
lysozyme. Both permeate and retentate were evaluated according to their
hydrodynamic volume by SEC-HPLC and antibacterial activity.

The superposed chromatograms of the permeate and retentate for LSZ 70
are presented in Figure~\ref{fig4}. Reference in this case is the LSZ treated at
70~\textdegree C before the filtration. Both permeate and retentate of
LSZ 70 are characterized by one main asymmetric peak with tailing,
corresponding to the non-fragmented 
lysozyme 
monomer. A second peak
appears for the permeate at around 12~min, which can be associated
with the presence of impurities. No soluble aggregates can be observed
due to filtration (permeate sample) or temperature treatment as
mentioned previously. After filtration however, there is an apparent
slight shift in the retention time to higher values (around 0.07~min), 
which suggests that the molecule has a smaller hydrodynamic
volume. This slight shift suggests
a change in the hydrodynamic volume
different from thermal denaturation at 80 and 90~\textdegree C.
Smaller retention times were observed for these two cases, meaning that
there was an increase in LSZ hydrodynamic volume.

\begin{figure}
{\vspace*{-2pt}}
\includegraphics{fig04}
{\vspace*{-2pt}}
\caption{\label{fig4}Normalized response of the permeate, retentate and
reference (untreated  LSZ sample) for  LSZ 70.}
{\vspace*{-2pt}}
\end{figure}

The difference observed in SEC-HPLC chromatograms for ultrafiltration
(Figure~\ref{fig4}) and thermal (Figure~\ref{fig3})  treatments
indicates two different denaturation mechanisms, where the LSZ treated
at 70~\textdegree C had its structure changed after ultrafiltration
and not due to heating. 

In the current experimental conditions, protein adsorption in membrane
pores was studied by an \mbox{indirect} method: the membrane properties
(hydraulic permeability and selectivity) and the results are presented
in Table~\ref{tab2}.

\begin{table*}
\caption{\label{tab2}Membrane properties before and after filtration of
LSZ 70 and LSZ 90}
\begin{tabular}{lcc}
\thead
& $R_{\mathrm{obs}}$ (\%) & Hydraulic permeability 
($10^{-14}~\mathrm{m}^{3}{\cdot}\mathrm{m}^{-2}$) \\
 \endthead
\multicolumn{3}{c}{{Filtration of LSZ 70}}  \\
Initial properties (VB12 filtration) & 57 & 5.5 \\
LSZ 70 & 98 & 4.8 \\
After filtration (VB12 filtration) & 72 & 4.7\vspace*{4pt} \\
\multicolumn{3}{c}{Filtration of LSZ 90}  \\
Initial properties (VB12 filtration) & 47 & 5.0 \\
LSZ 90 & 95 & 2.9 \\
After filtration (VB12 filtration) & 75 & 3.1
\botline
\end{tabular}
\end{table*}

The permeability loss after filtration of 
LSZ 70
is lower (14\%) than for 
LSZ 90
(38\%). Membrane selectivity increases by 15\% for 
LSZ 70
versus 28\% for
LSZ 90.
Hence, it seems that 
LSZ
is adsorbed onto the membrane in a
greater amount if it is pretreated at 90~\textdegree C, rather than
70~\textdegree C. When high temperatures are applied (superior to
75~\textdegree C), 
lysozyme 
is unfolded, exposing the hydrophobic
groups to the aqueous environment thus facilitating adsorption onto
surfaces \cite{23}. In addition, as observed by HPLC
(Figure~\ref{fig3}), LSZ 90 was supposed to have a bigger hydraulic
volume when compared to native and LSZ 70, which can explain this
different behavior in membrane properties. 

\subsection{Study of antibacterial activity of thermally modified
LSZ}\label{sec34}

Two parameters of ultrafiltration can be correlated with the
antibacterial activity; the time-effect of the turbulent flow in the
feed solution that is stirred during the test period (velocity of 700
L/h---$Re \approx 39{,}720$) and the effect of shear stress in the pores
(permeate flow for different transmembrane pressure). In this sense,
retentate samples of LSZ 70 and LSZ 90
were studied and compared with LSZ native. All of the retentate samples
presented a constant behavior during the test period, which means that
stirring (turbulent flow) and surface contact with stainless steel
material in tubing and feed tank did not induce any modification in
lysozyme 
antibacterial activity.

\begin{figure}
\includegraphics{fig05}
\caption{\label{fig5}LSZ 70 antibacterial activity for permeate
solutions as a function of applied pressure, \S~represents the
significant difference for $p<0.05$ with respect to 
LSZ reference (black line).}
{\vspace*{-2pt}}
\end{figure}

Figure~\ref{fig5} shows permeate behavior according to the applied
pressure for LSZ 70. The antibacterial activity of filtrated LSZ 70
decreases linearly with the increase of pressure
($I_{{A_u}}=-0.035\Delta P$) as observed before for filtration at room
temperature. The slopes of the linear curves obtained at 25 and
70~\textdegree C are close {(0.035 versus 0.038)} indicating a probably
identical denaturation mechanism. The difference of activity is
statistically significant for 8, 10 and 12 bar compared to the
reference. The loss for 8 bar is 23\%, 37\% for 10 bar and 42\% for 12
bar. The decrease in activity can be correlated with the slight change in
the LSZ
hydrodynamic volume (Figure~\ref{fig4}) to smaller size. In this case,
the modifications could be in the amino acid environment (cleavages of
bonds) \cite{24,25}. The results show that there is indeed a
change in the structure of 
the lysozyme 
(structure is directly linked
with the antibacterial activity) due to filtration and not due to the
temperature treatment before filtration. \looseness=-1

\begin{figure}
\includegraphics{fig06}
{\vspace*{-2pt}}
\caption{\label{fig6}LSZ 90 antibacterial activity for permeate
solutions as a function of applied pressure, 
LSZ reference (black line).}
{\vspace*{-2pt}}
\end{figure}

LSZ 90 shows first a decrease of antibacterial activity due to the
temperature treatment (retentate sample Figure~\ref{fig6}). Following
the thermal treatment, 
lysozyme 
loses approximately 40\% of its
activity. \mbox{Filtration} of LSZ 90
does not further affect the antibacterial activity of the solution
(Figure~\ref{fig6}, permeate samples). The decrease in antibacterial
activity is around 40\% (same value as the retentate) regardless of the
applied pressure. In the current case, the inactivation of the
\textit{Micrococcus Lysodeikticus} obtained 
with LSZ 90 is 
comparable to the one obtained with LSZ 70 filtered
at around 10 bar (around 37\%). However,
there are differences in the hydrodynamic properties which confirm that
different stress \mbox{factors} imprint different denaturation routes 
\cite{26}. According to Ibrahim \etal \cite{27} surpassing the
 denaturation temperature (72~\textdegree C) cleaves the \mbox{disulfide}
bonds (characteristic of lysozyme's structure) and exposes the
tryptophan residues to the polar environment, which induces substantial
conformational changes.


Overall, the changes in activity and in hydrodynamic
properties suggest that filtrated 
lysozyme 
is further denatured by the
filtration process, probably due to a combination of pore size, shear stress
and protein-membrane interactions \cite{28}. Nevertheless, the
denaturation by filtration is produced to a different extent depending
on the temperature used for the pre-treatment and the operational
conditions.

\section{Conclusion}\label{sec4}

The study focused on the hydrodynamic properties and on the
antibacterial activity against \textit{Micrococcus Lysodeikticus} of
lysozyme
denatured by heat and by a combination of heat and
ultrafiltration operation. It was showed that antibacterial
activity of heat-treated 
LSZ
decreases in function of hydrodynamic volume
modification as reported by SEC-HPLC. Depending on the temperature used
for the pretreatment of 
LSZ,
filtration imposes different
degrees of change. By using a temperature of 70~\textdegree C, after
filtration, the solution suffers a decrease in antibacterial
activity similar to non-heated 
LSZ.
The loss in antibacterial
activity can be linearly correlated with an increase in pressure (in
the range studied). On the other hand, by completely denaturing
lysozyme
(thermally treated at 90~\textdegree C), the antibacterial
activity remains unchanged after filtration (same activity in the
retentate and permeate) regardless of the applied pressure. Thus, the
present study highlights the behavior of a model protein
(LSZ)
denatured by temperature or a combination of temperature and shear
stress by using ultrafiltration.

\section*{Conflicts of interest}

Authors have no conflict of interest to declare.

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