the broadband dielectric spectroscopy ( bds ) is considered as a promising method for monitoring biological properties of cells [ 14 ]
. the bds monitoring can give the information on membrane capacitance , cytoplasmic conductivity and permittivity and the cell shape and structure .
the effect of physical and chemical treatments on the cellular membrane can also be detected , including the degree of the disruption of cells walls [ 14 ] .
regarding practical implementations of bds in the industry area , the possibility of monitoring of cell concentration in cultivation and fermentation is worth mentioning , along with the possibility of the use of these data for the estimation of the cell cycle .
these applications are notably strengthened by unique features of the modern bds which can detect , in a single scanning process , up to 15 decades in frequency / time . moreover
, modern bds spectrometers can operate with high resolution in a very broad range of electric capacitances and conductivities , which is particularly important for bio - samples .
nowadays , the temperature and concentrational ( isothermal ) bds behavior of diluted and dense solutions of cells is relatively well evidenced experimentally , although models for the analysis of experimental data are still puzzling [ 14 ] .
this can be associated with extraordinary complexity of  living and active bio - colloids / bio - composites  .
one of notable gaps in experimental bds studies on bio - systems is the practical lack of results related to the impact of hydrostatic pressure .
the latter influences the activation energy whereas the compression changes the density , the free volume and distances between species in a fluid system . on the practical side , significance of the pressure - related insight into properties of bio - systems indicates the boost of high pressure preservation ( hpp ) of foods technology , known also as the  cold pasteurization  , pascalization or bridgemanization [ 810 ] . for basic hpp industrial implementations the high pressure pulse ( 300600  mpa )
this can lead to the reduction of the count of microorganisms from 5 to 8 decades [ 811 ] .
the high microbiological safeness of hpp - treated foods is inherently associated with a set of highly beneficial features [ 610 ] : ( i ) the extension of shelf - life from 1 to 3 to even 90  days , ( ii ) taste , flavor and texture of the fresh product , ( iii ) preservation of vitamins amounts , ( iv ) avoiding of chemical preservatives , ( v ) the method can be applied for already packed food , ( vi ) the application is not limited to fluids , ( vii ) hpp is an environment - friendly method , i.e. it needs much less energy than the thermal pasteurization and there is practically no waste .
these unique advantages of the hpp technology coincides with expectations of the 21st century consumers with regard to high quality and healthy food products .
nowadays , there are over 200 large industry - scale hpp processors with the internal volume of the pressure chamber from 100 to 300  l. their number increase by ca .
notwithstanding , the fundamental biophysical base of hpp - related issues is still very limited .
one of the most important cognitive gaps is related to studies in situ under high pressure .
the comprehensive insight into properties of biosystems and foods in the pressure - temperature ( p - t ) plane can yield a base for a pressure - related modelling of novel foods .
bds offers unique research possibilities for in situ tests under high pressure due to existing state - of - the - art experimental implementations developed during studies on soft matter systems [ 1217 ] .
this report presents results of the first ever in situ high pressure bds investigations for saccharomyces cerevisiae ( baker s yeast ) , the unicellular eukaryotes which are widely used in biotechnological model processes [ 810 ] .
twenty - four hour culture in ypg - broth ( 1  % yeast extract 
difco , 1  % peptone  difco , 2  % glucose  poch ) was centrifuged at 4   c for 10  min at 6000    g , and the sedimented cells were aseptically re - suspended into phosphate - buffered saline ( pbs , ph  7.2 ) and again centrifugated .
the final suspensions of s. cerevisiae were prepared in pbs , reaching cell concentration of about 1010  cfu / ml ( colony forming units / ml , ie .
living cells / ml).electron microscopy.cells after uhp treatment were centrifuged 8  min at 3500    g , washed carefully with distilled water and again centrifuged under the same conditions .
the cells was suspended in 2  % solution of glutaraldehyde and stored over night at room temperature .
the samples were centrifuged for 10  min at 3500    g and the cells were prepared for electron microscopy .
ultrathin sections were observed under a transmission electron microscope jem 100c ( jeol , tokyo , japan ) .
the cells treated at 300 and 400  mpa showed essential ultrastructural changes . about 80  % of cells population , showed cell wall disruption and electron - dense areas of amorphous material packed with crushed microsomes and ribosomes.uv analysis.for the spectroscopic studies , uv - visible 1800 shimadzu double beam spectrophotometer was used to record the spectra .
the yeast samples after high pressure treatment were centrifuged at for 20  min at 6000    g , and the supernatants obtained were scanned in the 240280  nm uv region . the wavelength maximum was observed at 260  nm , which relates to the yeast cell nucleotides and aromatic amino - acids present in the supernatant as the result of the high pressure cell wall disruption .high
pressure treatment.the precise scheme of the high pressure set - up is given ref . .
it enabled changes of pressure up to p  =  500  mpa , monitored via the tensometric pressure meter with precision + 0.1  mpa .
the temperature of the pressure chamber was yielded via a large volume ( 25 liter ) thermostat with external circulation , enabling the temperature stabilization up to 0.02  k. temperature was measured via constantant - copper thermocouples inside the pressure chamber .
pressure was measured with the precission 0.1  mpa using tensometric pressure meter nova swiss .
the sensor was located near but outside the pressure chamber , to avoid any impact of temperature on measurements.the greatest challenge for any in situ measurements under high hydrostatic pressure is the total isolation of the tested sample from the pressurized medium , matched with forceless transmission of the pressure to the sample .
moreover , multi - cycle increasing and decreasing of the applied pressure should be possible .
the authors of the given paper developed the high pressure electric capacitor fulfilling all these conditions .
the flat - parallel measurement capacitor applied in the given research had the gap d  =  1  mm and diameter 2r  =  16  mm ) .
the pressure is transmitted from the pressurized liquid to the sample via the deformation of 50m stretched teflon film .
unique construction of the capacitor is very solid and does not enable changes of any significant part during pressurization .
it is notable that its body is made from invar , hence it is also not sensitive to temperature changes.broadband dielectric spectroscopy under pressure.for bds studies the alpha - a bds novocontrol ( montabaur , germany ) analyzer ( model year 2013 ) , yielding an insight for frequencies f  =  0.001  hz to 10  mhz ( 9 decades ) with 56 digits resolution was used .
generally , bds yields the complex dielectric permittivity ( )  =    '  (  )  +  i( ) ,   =  2f .
the first component can be determined as   '  ( f )  =  c / c0 , where c0 and c are electric capacitances of the measurements capacitor and the capacitor with the tested dielectric sample .
the second component is experimentally defined as   '  '  (  )  =  1/rc0  =    ' /rc , where r denotes the resistivity .
for the so - called static domain , in liquids most often for 1khz  <  f 
<  1mhz ,   '  ( f ) is constant and referred to as dielectric constant (   '  ( f )  =   ) . in this domain
the temperature and pressure evolution of the dielectric constant allow to identify the dominating type of arrangement of permanent dipole moments [ 617 ] .
twenty - four hour culture in ypg - broth ( 1  % yeast extract 
difco , 1  % peptone  difco , 2  % glucose  poch ) was centrifuged at 4   c for 10  min at 6000    g , and the sedimented cells were aseptically re - suspended into phosphate - buffered saline ( pbs , ph  7.2 ) and again centrifugated .
the final suspensions of s. cerevisiae were prepared in pbs , reaching cell concentration of about 1010  cfu / ml ( colony forming units / ml , ie . living cells / ml ) .
cells after uhp treatment were centrifuged 8  min at 3500    g , washed carefully with distilled water and again centrifuged under the same conditions .
the cells was suspended in 2  % solution of glutaraldehyde and stored over night at room temperature .
the samples were centrifuged for 10  min at 3500    g and the cells were prepared for electron microscopy .
ultrathin sections were observed under a transmission electron microscope jem 100c ( jeol , tokyo , japan ) .
about 80  % of cells population , showed cell wall disruption and electron - dense areas of amorphous material packed with crushed microsomes and ribosomes .
for the spectroscopic studies , uv - visible 1800 shimadzu double beam spectrophotometer was used to record the spectra .
the yeast samples after high pressure treatment were centrifuged at for 20  min at 6000    g , and the supernatants obtained were scanned in the 240280  nm uv region . the wavelength maximum was observed at 260  nm , which relates to the yeast cell nucleotides and aromatic amino - acids present in the supernatant as the result of the high pressure cell wall disruption . high pressure treatment .
it enabled changes of pressure up to p  =  500  mpa , monitored via the tensometric pressure meter with precision + 0.1  mpa .
the temperature of the pressure chamber was yielded via a large volume ( 25 liter ) thermostat with external circulation , enabling the temperature stabilization up to 0.02  k. temperature was measured via constantant - copper thermocouples inside the pressure chamber .
pressure was measured with the precission 0.1  mpa using tensometric pressure meter nova swiss .
the sensor was located near but outside the pressure chamber , to avoid any impact of temperature on measurements .
the greatest challenge for any in situ measurements under high hydrostatic pressure is the total isolation of the tested sample from the pressurized medium , matched with forceless transmission of the pressure to the sample .
moreover , multi - cycle increasing and decreasing of the applied pressure should be possible .
the authors of the given paper developed the high pressure electric capacitor fulfilling all these conditions .
the flat - parallel measurement capacitor applied in the given research had the gap d  =  1  mm and diameter 2r  =  16  mm ) .
the pressure is transmitted from the pressurized liquid to the sample via the deformation of 50m stretched teflon film .
unique construction of the capacitor is very solid and does not enable changes of any significant part during pressurization .
it is notable that its body is made from invar , hence it is also not sensitive to temperature changes .
broadband dielectric spectroscopy under pressure . for bds studies the alpha - a bds novocontrol (
montabaur , germany ) analyzer ( model year 2013 ) , yielding an insight for frequencies f  =  0.001  hz to 10  mhz ( 9 decades ) with 56 digits resolution was used .
generally , bds yields the complex dielectric permittivity ( )  =    '  (  )  +  i( ) ,   =  2f .
the first component can be determined as   '  ( f )  =  c / c0 , where c0 and c are electric capacitances of the measurements capacitor and the capacitor with the tested dielectric sample .
the second component is experimentally defined as   '  '  (  )  =  1/rc0  =    ' /rc , where r denotes the resistivity .
for the so - called static domain , in liquids most often for 1khz  <  f 
<  1mhz ,   '  ( f ) is constant and referred to as dielectric constant (   '  ( f )  =   ) . in this domain
the temperature and pressure evolution of the dielectric constant allow to identify the dominating type of arrangement of permanent dipole moments [ 617 ] .
dipole couplings in fluid dielectrics pressure dependences of dielectric constant showing preferable type of dipole 
dipole couplings in fluid dielectrics when lowering the frequency values of   '  ( f ) permanently increase , since the low  frequency electric field can interact with large and inert ions , in dielectric and molecular physics often recalled as  ionic impurities  [ 6 , 17 ] .
the translation of different types of  ionic impurities  present in a fluid dominates the bds output in this region . in the 
dominated region it is convenient to transform experimental data to the complex electric conductivity form ( )  =  i0( )  =    '  (  )  +  i  '  '  (  )  =  0  '  '  (  )  +  0(  '  (  )    1 ) , where the electric constant ( vacuum permittivity , permittivity of free space ) o    8.854pf / m the tradition and practical reasons caused that in dielectrics physics and in soft matter physics for (   ' ,    '  ' ) relative and dimensionless values are used . as visible from the above definition
this is not the case of (   ' ,    '  ' ) .
usually , in liquids and soft matter systems in the region dominated by electric charges motion   '  (  ) is not affected but   '  (  ) increases with decreasing frequency   =  2f in such a way that the slope at the plot log10  '  '  (  ) vs. log10 is equal to   1 .. consequently , in this domain   '  (  ) is independent from changes of the frequency and called dc conductivity ( dc ) and often simply electric conductivity .
electric conductivity can be considered as the metric of translation of  ionic dopants  and the static dielectric permittivity   =    '  ( f )    const gives an insight into polarization and permanent dipoles arrangements .
the imaginary part of dielectric permittivity   '  '  ( f ) is associated with energy losses and offers and insight into relaxation processes , manifesting as peaks in   '  '  ( f ) spectrum . in the low frequency and  ion - dominated  region
one should expect its growing up described by   '  '  ( f )    f with   =  1 dependence , in agreement with the definition of dc electric conductivity above . in 
classical  molecular liquids one should expect that the dc electric conductivity will decrease with increasing pressure . in the simplest case , for describing such process the barus equation , can be used : (p )  =  0p  exp(vap ) , where va denotes the constant activation volume and 0p is the prefactor related the value of (p ) for p  =  0 .
it can be considered as the pressure counterpart of the simple arrhenius relation (t )  =  0  exp(ea / rt ) , where r denotes the gas constant and ea is the activation energy . in a limited range of pressures
, the barus equation can be reduced to the linear dependence (p )  =  0  exp(vap )    0(1    vap  +   )  =  0    ( 0va)p , due to the implementation of the taylor expansion .
such approximation is valid only for relatively small values of the argument ( pressure p in this case ) , if for experimental data notable distortion from such simple relation appears one should use the functional , experimental form ( preferably ) .
this report bases on in situ measurements of (f ) for selected pressures and the discussion of the pressure evolution of dielectric constant (p ) , dc conductivity (p ) and   '  '  ( f ,  p ) spectra .
bds studies has been carried out for high concentration of saccharomyces cerevisiae ( 1010 cells / ml ) to avoid a significant influence of the maxwell  wegner ( mw ) effect   which can lead to the parasitic polarization of the capacitor plates and which can notable bias output results .
the key target of given studies was the preliminary test of the possibility of bds technique as the tool for monitoring the high pressure cold pasteurization - related processes in situ .
second , on the higher level of complexity , one can expect the interruption of cell walls .
the high pressure denaturation in the p - t plane is portrayed by an elliptic - like curve , crossing the p    0.1mpa ( atmospheric pressure ) line near t    80c , related to pasteurization [ 8 , 19 ] . the isothermal 
( i.e. well below the thermal pasteurization limit ) compression , for 5c  <  t 
<  40c , leads to the denaturation at 400  <  p  <  600mpa .
. 2the schematic presentation of proteins denaturation in the pressure  temperature ( p - t ) plane [ 8 , 19 ] .
the form of a protein prior and after the denaturation process is shown the schematic presentation of proteins denaturation in the pressure  temperature ( p - t ) plane [ 8 , 19 ] .
the form of a protein prior and after the denaturation process is shown it is notable that for such path of approaching the denaturation curve one can minimize the influence of the irreversible coagulation , always occurring for the classical ,  thermal  pasteurization .
it is notable that  death curves  of microorganisms portrayed in the p - t , strongly resemble plane by the curves in fig .  2 .
despite this fact , as the key reason of the  killing action  of high pressures on microorganisms is recognized the action on more complex structure , mainly the cell wall .
3the electron microscope of yeast cell before ( ( a ) , p  =  0.1  mpa ) and after ( ( b ) , p  =  400  mpa ) pressurization the electron microscope of yeast cell before ( ( a ) , p  =  0.1  mpa ) and after ( ( b ) , p  =  400  mpa ) pressurization the question arises , how the pressure - induced destruction of cells manifests in the bds monitoring .
the first one is the imaginary part of dielectric permittivity , shown in fig . 
this process is described by   '  '  ( f )    f with the exponent
  =  1 , as shown in fig .  4 . for f 
<  10hz at p  =  0.1mpa and f  <  100hz at p  =  0.1mpa , the impact of the ionic polarizability emerges . on further decreasing of the frequency the evolution of log10  '  ( f ) vs. log10f becomes progressively nonlinear , until reaching the domain described by the exponent s  <  1 for lower frequencies . .
these changes can be related to the so strong impact of permanent  ionic impurities  that under the external electric field from the bds spectrometer causes the parasitic polarization of plates of the capacitor .
it is notable that this phenomenon appear for much higher frequencies for the pressurized yeast which indicates much higher content of  ionic impurities  , presumably associated with free ions of na , k , ca , mg , cl and molecular species related to organic acids .fig .
4the imaginary part of dielectric permittivity in native ( atmospheric pressure ) and pressurized yeast for t  =  25  c isotherm .
the key parts of plots follow the dependence   '  '  ( f )    f 
 , i.e. log10 
   '  '  ( f )        log10 
 f. the frequency f 
 b denotes the crossover to the maxwell  wegner effect - dominated region the imaginary part of dielectric permittivity in native ( atmospheric pressure ) and pressurized yeast for t  =  25  c isotherm .
the key parts of plots follow the dependence   '  '  ( f )    f 
 , i.e. log10 
   '  '  ( f )        log10 
 f. the frequency f 
 b denotes the crossover to the maxwell  wegner effect - dominated region it is also notable that for the pressurized yeast , a primary relaxation process with the peak at ca . 8  mhz emerges .
figures  5 and 6 show the pressure dependence of the static dielectric permittivity and dc electric conductivity as the function of pressure at 5 and 25  c temperatures .
threshold  , near pt  =  200  mpa for t  =  5  c and pt  =  300  mpa for t  =  25  c .
below pt the increase of pressure increases the electric conductivity and above pt further compression decreases the conductivity . for p 
liquids , where the pressurization decreases the electric conductivity , what is coupled with a notable increase of viscosity .
the rise of conductivity (p ) on increasing pressure up to pt indicates the appearance of new free ions in the system , gradually released from a  reservoir of free ions  .
this can be associated only with the break of cell walls and leakage of the intracellular fluid . above the threshold pressure , for p 
>  pt , the evolution of electric conductivity is similar to the behavior observed in 
normal liquids  where number of free ions is constant and does not change during the experiment [ 6 , 15 , 17 ] .
this domain is described by the barus equation as discussed above and shown in fig . 
these threshold pressures are also clearly visible in behavior of the dielectric constant   '  ( p )  =  (p ) . up to p  =  pt
the linear increase of   '  ( p ) is very weak and in fact close to (p )    const , what suggests the virtual lack of the preferable arrangement of permanent dipole moment . for p 
>  pt dielectric constant   '  ( p ) strongly increases indicating the creation of a notable parallel arrangement of permanent dipole moments .
it is worth stressing that  threshold pressures  values manifest clearly both for (p ) and (p ) evolutions.fig .
5the pressure evolution of the real part of dielectric permittivity and the dc conductivity in yeast at t  =  5  cfig .
6the pressure application time ( p  =  400  mpa ) evolution of the real part of dielectric permittivity and the dc conductivity in yeast at t  =  25  c . the pressure evolution of the real part of dielectric permittivity and the dc conductivity in yeast at t  =  5  c the pressure application time ( p  =  400  mpa ) evolution of the real part of dielectric permittivity and the dc conductivity in yeast at t  =  25  c .
figure  7 shows the impact of time on the pressurized yeast , monitored by the changes of (p ) and (p ) .
after 1  hour of high pressure acting , the electric conductivity increases and the dielectric constant decreases  by ca .
these results indicate that despite different nature of processes monitored by dielectric constant and electric conductivity , they are strongly coupled.fig . 7the time evolution of the dc electric conductivity and the static dielectric permittivity in the pressurized yeast at t  =  5  c the time evolution of the dc electric conductivity and the static dielectric permittivity in the pressurized yeast at t  =  5  c the increase of the electric conductivity can be linked to the leakage of intracellular liquid , appearing due to the pressure - induced breaks of the cellular walls .
one of methods to estimate the amount of the intracellular , naturally turbid , liquid , are spectrophotometric measurements of absorbance of clear supernatant at 260  nm after the removal of cells and cells debris by centrifugation .
figure  9 shows the clearly correlation between the normalized absorbance and the dc conductivity on increasing pressure in tested samples.fig .
8the uv absorbance of supernatant leaking out from cells of s. cerevisiae after pressurizing from 0.1 to 500  mpafig .
9the correlation between absorbance at 260  nm of the supernatant after pressurizing of saccharomyces cerevisiae and electric dc conductivity the uv absorbance of supernatant leaking out from cells of s. cerevisiae after pressurizing from 0.1 to 500  mpa the correlation between absorbance at 260  nm of the supernatant after pressurizing of saccharomyces cerevisiae and electric dc conductivity
results presented show that the evolution of the static dielectric permittivity and particularly the dc electric conductivity can be considered as notable and sensitive tools for testing characteristics of pressurized yeast cells .
it is notable that bds experiments under pressure can yield high resolution results , hardly available by other physical method implemented for such challenging conditions .
microbiological studies of pressurized solutions of yeast and other model cells indicated that conditions related to the pasteurization , i.e. the reduction of the number of microorganisms by at least five decades (  5 log - units  ) , needs at least 400  mpa for minimum 3  minutes [ 811 ] .
moreover ,  deeper  pasteurization takes place for pressurization under higher temperature . this paper lead to the conclusion that the break of cell walls may take place for lower pressure at lower temperatures ( 5  c ) , already at 200300  mpa .
the effective process needs also more time than 35  minutes used in hpp technology .
all these may suggest that the action of pressure leading to the  cold pasteurization 
phenomenon for commercially most often used pressures p  =  400600  mpa is associated not only with the break of cell walls but also the influence of intracellular structures and pressure - induced denaturation .
 results of broadband dielectric spectroscopy ( bds ) in saccharomyces cerevisiae ( baker s yeast ) , in situ as the function of pressure are presented . 
 they show a clear evidence of a threshold to the new pattern of the pressure evolution of the static dielectric permittivity and dc electric conductivity already for pt    200mpa at t  =  5oc and pt    300mpa at t  =  25oc . 
 bds monitoring versus pressure tests up to p  =  400mpa revealed particularly notable changes of properties after 30  minutes of compressing . finally , the correlation between the amount of the spectrophotometric maximum absorbance and the dc electric conductivity was found . 
 all these indicate significance of bds as the tool for testing of pressure properties of cells assemblies , model foods etc . , in situ under high pressures . 
Introduction
Experimental
Results and Discussion
Conclusions