Patent Application: US-6922798-A

Abstract:
the packing and even the excessive packaging of products for human consumption is a current practice in the industrialized countries . as this packaging is made primarily of non - biodegradable polymers , they currently cause environmental problems . these environmental problems are found not only in industrialized countries but also in developing countries . this situation supported the development of various films , more ecological , starting from biodegradable elements containing polysaccharides , proteins and / or lipids . we developed a biodegradable protein film from a casein salt . of dairy origin , casein is abundant and could even be recovered from unsold milk . the process of polymerization is induced by gamma irradiation . indeed , the interaction of hydroxyl radicals with tyrosins present in protein creates a covalent bond . the addition of a plasticizing agent is essential in order to produce a more flexible and less friable film . the presence of glycerol does not inhibit the formation of bityrosine . it protects protein from the denaturation caused by irradiation , increases the deforming capacity and decreases the breaking strength of film . the biodegradation tests , carried out in our laboratories , showed that the film produced by gamma irradiation is accessible to the enzymatic attacks from pseudomonas fragi .

Description:
an explicit description of the material and various techniques used will be presented and illustrated in the following figures . this description does not limit the scope of the invention but rather is provided as an illustration . fig2 : curve of relieving and equation for the calculation of the coefficient of relieving ( peleg , 1979 ). fig3 : f / e ration ( rupture strength versus film thickness ratio ) as a function of the irradiation dose received for the 110 , 180 and 380 alanates , for protein concentrations of 5 . 0 % p / p and 7 . 5 % p / p . fig4 : deformation at rupture according to the amount of irradiation received for the alanates 110 , 180 and 380 with protein concentrations of 5 . 0 % p / p and 7 . 5 % p / p fig5 : rate of formation of bityrosine according to the amount of irradiation received for alanates 110 , 180 and 380 with protein concentrations of 5 . 0 % p / p and 7 . 5 % p / p fig7 : tryptophan dosage according to the amount of irradiation received for alanates 110 , 180 and 380 with protein concentrations of 5 . 0 % p / p and 7 . 5 % p / p fig8 : f / e ration ( ration of the breaking load versus the thickness of film ) according to the amount of received irradiation and glycerol contents for alanate 380 with protein concentrations of 5 . 0 % p / p and 7 . 5 % p / p fig9 : deformation according to the amount of received irradiation and glycerol contents for alanate 380 with protein concentrations of 5 . 0 % p / p and 7 . 5 % p / p fig1 : viscoelasticity according to the amount of received irradiation and glycerol contents for alanate 380 with protein concentrations of 5 . 0 % p / p and 7 . 5 % p / p fig1 : rate of formation of bityrosine according to the amount of received irradiation and glycerol contents for alanate 380 with protein concentrations of 5 . 0 % p / p and 7 . 5 % p / p fig1 : tryptophan proportioning according to the amount of received irradiation and the glycerol contents for alanate 380 with protein concentrations of 5 . 0 % p / p and 7 . 5 % p / p fig1 : test # 1 of the growth of pseudomonas fragi in the presence and absence of a film sample of alanate 380 composed of 5 . 0 % p / p proteins and 2 . 5 % p / p of glycerol , 20 kgy irradiation fig1 : test # 2 of the growth of pseudomonas fragi in the presence and absence of a film sample of alanate 380 composed of 5 . 0 % p / p of proteins and 2 . 5 % p / p of glycerol , 20 kgy irradiation fig1 : test # 3 of the growth of pseudomonas fragi in the presence and absence of a film sample of alanate 380 composed of 5 . 0 % p / p of proteins and 2 . 5 % p / p of glycerol , 20 kgy irradiation in this research project , three caseinates ( see compositions at table 2 ) were initially used , that is , two sodium caseinates ( alanate - 110 and 180 ) and a calcium caseinate ( alanate - 380 , new zealand milk products , inc . ca , usa ). the protein contents are certified higher than 91 . 0 % and this purity was laboratory tested ( leco , fp - 428 , ml , usa ). purity is 94 , 099 %, 94 , 526 % and 93 , 575 % for alanates 110 , 180 and 380 respectively . these results in total nitrogen contents will be retained for the calculation of the protein concentrations (% p / p ) at the time of the formulation of the various compositions of the solutions . table 2______________________________________composition of the three caseinates ( alanates ) used in this research project . alanate alanate alanateelements 110 180 380______________________________________proteins 91 . 1 91 . 1 91 . 8 ( n × 6 . 38 ) % minerals (%) 3 . 6 3 . 5 3 . 8moisture (%) 4 . 1 4 . 0 3 . 9lipids (%) 1 . 1 1 . 1 0 . 7lactose (%) 0 . 1 0 . 1 0 . 1ph ( 5 % at 20 ° c .) 6 . 6 6 . 6 7 . 0______________________________________ the values of the various components of three caseinates come from the product bulletins provided by new zealand milk products , inc ., ca , usa . before the period of irradiation , the caseinates are dissolved in distilled water , previously filtered by inverse osmosis . solubilization is made continuously under magnetic agitation and without heat . two concentrations are used : 5 . 0 % and 7 . 5 % p / p of proteins . a concentration lower than 5 % generates films of inadequate thickness for handling whereas a concentration higher than 7 . 5 %, produced films that were too thick . these two concentrations are selected strictly to validate the film properties , they are otherwise not restrictive . according to the composition of the medium , a quantity of glycerol ( purity ≧ 95 %; a & amp ; c , montreal , canada ) can be added to concentrations of 1 . 0 %, 2 . 5 % and 5 . 0 % p / p . a glycerol concentration higher than 5 . 0 % p / p produces films in a gel state , which are therefore difficult to handle adequately . after complete solubilization of proteins , a 15 minutes vacuum , under magnetic agitation , is applied ; followed immediately by an n 2 o bubbling ( linde , union carbide , toronto , canada ) for a second 15 minutes period under agitation . after the gasing stage , the solution is transferred in screw - top test tubes , under n 2 o flow . the test tubes are sealed with paraffin and are then irradiated . the irradiation is carried out in a co 60 irradiator of the gammacell 220 type ( nordion international inc , located in the canada irradiation center ( c . i . c ), laval , canada ) at an average dosage of 2 , 18 kgy / h for amounts of irradiation of 4 , 8 , 12 , 15 , 20 , 30 and 40 kgy . for the irradiation , the test tubes are placed in a glass beaker located in the center of the irradiation room . in this way , the test tubes are in the zone of 100 ± 5 % of the dose according to the isodose curves of the gammacell 220 irradiator . after each irradiation period , a 20 to 30 minute wait in darkness is allocated so that the longest radicalizing reactions are completed and to avoid photodecomposition of the biphenyls ( lehrer and fasman , 1967 ; prutz , 1983 ). before and after each irradiation , the ph ( corning ph - meter , ps 15 ) and the brix degree of the solutions ( fisher refractometer , 13 - 946 - 70c , no 4754 , montreal , canada ) are checked in order to quickly evaluate any change during the irradiation . the ph measurement ensures the constancy of the ph solutions , before and after each irradiation . the refractometer allows the evaluation of the quantity of soluble solids present in the solutions . its use makes it possible to see any variations of the quantity of solubilized solids before and after irradiation . a homogenisation of the solutions by successive inversions is carried out before each recording of the data in order to prevent the formation of a protein deposit . after verifying the ph and brix degree , five milliliters ( 5 ml ) of the protein solution is pipetted and uniformly deposited in a support of polymethacrylate ( plexiglas ). detailed attention is given in order to avoid the formation of air bubbles . the support has an internal diameter of 8 . 5 cm for a surface of 56 . 7 cm 2 ( see fig1 ). thereafter , the support is kept level as much as possible . a 12 to 14 hour drying period ( that is to say overnight ) at room temperature , is allocated in order to obtain the film . this method of film formation is adapted from the gontard ( 1992 ) and krochta ( 1991 ) research teams . after drying , the film is withdrawn from its support and its thickness is measured using a digimatic indicator ( mitutoyo , japan ). all the films produced in this manner are cut to obtain a sample with a 4 . 0 cm diameter . thereafter , the samples are humidified and balanced during 48 hours , at 25 c ., in a desiccator containing a solution saturated with sodium bromide ( gontard et al ., 1992 ). this handling ensures an atmosphere of 56 % relative humidity ( ganzer and rebenfield , 1987 ) and this water content was measured . the humidified samples are then firmly immobilized between two plexiglas plates exposing a surface of 3 . 2 cm in diameter for the measurement of the mechanical properties . for all tested films , two mechanical properties were determined : breaking load and strain at failure . for certain films containing glycerol , a third mechanical property was evaluated , viscoelasticity . these three properties are measured using a voland texturometer ( stevens - lfra texture analyser , ta - 1000 model , n . y ., usa ) connected to a printer ( texture technologies corp ., l 6512 model , n . y ., usa ). a punch of two millimeter ( 2 mm ) diameter is used for all measurements . some trials were carried out with a 3 mm diameter punch and the readings exceeded the maximum detection limit of the texturometer . the calculation of the results is always according to the punch &# 39 ; s descent speed and the tape speed of the printer paper . before each use , the texturometer is calibrated against a mass standard ( 100 to 1000 g ) and the speed of the printer is verified as a function of time . for the three mechanical properties , the tests were carried out in triplicate . the average obtained , as well as its standard deviation , were represented on a graph . the breaking load and the strain at failure are calculated simultaneously for all the samples . the speed of descent of the punch is 1 . 0 mm / s and the unfolding speed of the printer paper is 50 cm / min . the push of the punch is recorded in grams and is converted into units of force ( n ). the viscoelasticity of a film is measured by the relaxation curve obtained following the application of a force maintained by the punch on the film . the speed of descent of the punch is of 1 . 0 mm / s and that of the unfolding of the printer paper is 10 cm / min . in the present case , the deformation is three millimeters ( 3 mm ) and the forces measured at time 0 and 60 seconds are retained for the calculation of the relaxation coefficient y ( 1 min ) ( peleg , 1979 ) according to the equation defined in fig2 . in accordance with this equation , the relaxation coefficient of relieving y ( 1 min ) varies between 1 and 0 . an elastic film will show a low y ( 1 min ) ration since the initial and final forces would then be nearly identical . during relaxation , energy is dissipated thus creating irreversible internal disturbances . a continuously decreasing tension is necessary to maintain the sample in its deformed state ( gontard et al ., 1992 ). a fraction of the irradiated protein solution is kept in the liquid state in order to measure the rates of bityrosine formation and tryptophan loss . in all cases , dosages are performed within 24 hours following the period of irradiation . before carrying out the dosages , a 1 / 100 dilution is performed using a hepes buffer ( a & amp ; c , montreal , canada ) 20 mm , ph 7 . 0 ( davies et al ., 1987a ), in order to avoid a saturation of the apparatus . the bityrosine formation and tryptophan loss measurements are followed by fluorescence ( davies et al ., 1987a ) with the help of a spectrofluorometer ( spectrofluorometer 2070 , varian , ca , usa ). the spectrofluorometer is equipped with a xenon lamp ( 75 w ) and the capacity of the cell is 15 μl . the detectors are photomultipliers for excitation and emission and the detectability threshold 0 . 03 quinine sulfate ppb in a solution of h 2 so 4 0 . 1m ( values reported by varian ). the spectrofluorometer is connected to a hplc ( liquid chromatograph : vista 5500 , varian , ca , usa ) which is connected to an auto - injection system ( auto sampler 9090 , varian , ca , usa ). this entire system is in permanent communication with a computer terminal ( compaq / deskpro 486 / 33m ) which allows for the acquisition and the processing of data ( varian star workstation , copyright 1989 - 1992 , varian associates , inc , ca , usa ). during dosages , no separation column is used but only a fixed flow of one milliliter per minute ( 1 ml / min ). the injection volume is 90 μl and a 100 μl twist is used to receive the injection . the period for data acquisition is fixed at 90 seconds in duration . the rates of bityrosine formation or tryptophan loss are obtained by the calculation of the surface under the curve in arbitrary surface units . as dosages are purely qualitative , we concentrated mainly on the stability and the reproductive capacity of the apparatus . thus , several series of dosages on the three caseinates used and on a tryptophan solution ( sigma , mississauga , canada ) were made at the tryptophan excitation and emission wavelengths . for three different concentrations , the results showed a variance lower than 4 % between dosages and a variation equal or lower than 8 . 5 % between the three concentrations for the same caseinate . the variation between the concentrations would be mainly justified by predosage handling . tests have shown that the hepes buffer ( 20 mm , ph 7 . 0 ), glycerol ( 2 . 5 %) or a mixture of the two do not generate characteristic signals , truly higher than the background noise , for the various wavelengths used for irradiated and non - irradiated solutions . for all the various compositions of the solutions , the rates of bityrosine formation and tryptophan loss were measured in triplicate . the average and the standard deviations were represented on a graph . the rate of bityrosine formation is measured at the excitation and emission wavelengths of 305 nm and 415 nm (± 5 nm ) respectively . these wavelengths were determined using an irradiated tyrosin solution ( 50 ppm ) ( sigma , mississauga , canada ). the casing of the apparatus is adjusted to 1 and an attenuation factor of 4 is applied . as we did not find bityrosine commercial standards , our results could only be interpreted in a qualitative way . the rate of tryptophan loss is followed at the excitation and emission wavelengths of 255 nm and 351 nm (± 5 nm ) respectively . these wavelengths were established using an irradiated tryptophan solution ( 50 ppm ). the casing of the apparatus is 1 and an attenuation factor of 32 is applied . the oxidation of a solution of tryptophan residue by • oh radicals is directly connected to the loss of intensity of the fluorescence signal during its dosage ( davies et al ., 1987a ). as dosage by fluorescence is much more complex with proteins than with only one amino acid ( davies et al ., 1987a ), we did not try to convert the fluorescence intensity into quantities of tryptophan residues , but we only noted experimentally a possible loss of the signal . the biodegradability was verified using pseudomonas fragi because this bacterial species is frequently used in the laboratory and also because the pseudomonas stock is recognized as a bacterium which is able to synthesize a very wide number of enzymes ( tortora , g . j . et al ., 1989 ). the foremost synthesized proteases for the biodegradation of casein are metalloproteases and serine proteases ( alichanidis and andrews , 1977 ; davies , 1987 ; davies et al ., 1987b ). each medium contains 99 ml water with 0 . 85 % p / v of nacl ( anachemia , montreal , canada ) and according to the case , 1 ml of inoculum or a film sample or , both are added . the mediums are incubated at 25 ± 2 ° c . and are continuously under agitation ( 140 ± 5 rpm ). only one type of film was selected in this section . it is composed of 5 . 0 % p / p calcium caseinate with 2 . 5 % p / p glycerol and irradiated at 20 kgy . the samples are prepared according to the methods described in example 1 to 3 . an irradiation dose of 20 kgy is considered a sterilization dose . naturally , a precaution specific to the maintenance of sterility is applied . the inoculation of the mediums is done starting from a mother culture whose time of incubation is 16 to 18 hours . beforehand , the mother culture was inoculated twice in a nutritive bubble ( nutrient broth , difco laboratories , detroit , usa ) in order to adapt the stock and to collect it in an exponential growth phase . one milliliter ( 1 ml ) of this mother culture is taken and diluted until a factor of 1 / 10 4 with a saline solution ( 0 . 85 %- nacl ). three successive centrifugations are made at 3000 rpm for 10 minutes , at 4 ° c . after each centrifugation , nine of the ten milliliters are withdrawn and replaced by physiological [ distilled ?] water , then homogenized with the vortex . the inoculation of the mediums is done after the third centrifugation and dissolution . from these handlings and dilutions , the initial counts of the mediums are roughly 100 ufc / ml . the bacterial counts are done in duplicate on &# 34 ; trypsic soy agar &# 34 ; medium ( tsa , ditco laboratories , detroit , usa ). the counting method used is that advised by the health protection branch ( health and welfare canada , 1979 ). the inoculum is deposited on the surface of the agar by smearing . incubation is done at 23 ° c .± 2 ° c . and the bacterial counts are checked at 24 and 48 hours after setting on the plate . bacterial counts which ranged between 30 and 300 were retained . the results obtained are analyzed statistically by variance analysis and the duncan multiple comparison test with p σ 0 . 05 , whereas the student statistical analysis is used only during the variance analysis and test of comparison per pair with p σ 0 . 05 ( snedecor and cochran , 1978 ). this section will be divided into three main parts . the first part will present the results of a series of experiments on the evaluation of the behavior of the three caseinates used according to the amount of irradiation . after discussing these results , a selection of one of the three caseinates will be made for the continuation of the experimentation . in the second part , we will discuss a second series of experiments on the behavior of the caseinate chosen in the presence of glycerol and according to the amount of irradiation given . finally , in the third part , measurements of the biodegradability will be presented . before claborating on the results , we would like to describe to the reader the physical and visual aspects of the films . at first sight , the non - irradiated films are transparent and the colourless as are the caseinate films without glycerol , irradiated at 4 to 12 kgy . on the other hand , in the absence of a plasticizing agent , the fragility of these films is so great that they cannot be handled without damaging them . these observations apply for the two protein concentrations used ( 5 . 0 % p / p and 7 . 5 % p / p ). the irradiation causes a yellowing of the films formed in the presence of glycerol and this rate of yellowing seems to be proportional to the amount of irradiation received . the presence of glycerol tends to create a certain opacity and it seems to be proportional to the glycerol content . whereas for the same glycerol concentration , protein content affects the aspect of film for the same amount of irradiation . at 2 . 5 % p / p or 5 . 0 % p / p of glycerol , the films produced with 7 . 5 % p / p of proteins are more transparent than those produced with 5 . 0 % p / p . therefore the glycerol / protein ratio seems to be a factor influencing the opacity of films . according to the glycerol and protein contents , the thickness varies from 27 to 64 μm with a variation equal or lower than 8 % ( see table 3 ). coating film must be as thin as possible and preferably its thickness must be equal or lower than 50 μm . a thicker edible film would likely affect the aesthetic properties of the packed product or its components . naturally , glycerol content modifies the texture of the film and with a concentration of 5 . 0 % p / p , its handling requires more delicacy . whatever the composition or the amount of irradiation , no film , once formed , released perceptible odors . table 3______________________________________variation of the thickness of films according to their proteincomposition with or without glycerol on all the irradiation doses .% p / p alanate / thickness dose % p / p glycerol ( μm ) ( kgy ) ______________________________________5 . 0 %- 110 / 0 % 27 ± 2 0 to 25 . 0 %- 180 / 0 % 27 ± 2 0 to 125 . 0 %- 380 / 0 % 28 ± 2 0 to 127 . 5 %- 110 / 0 % 44 ± 2 0 to 127 . 5 %- 180 / 0 % 42 ± 2 0 to 127 . 5 %- 380 / 0 % 41 ± 2 0 to 125 . 0 %- 380 / 1 . 0 % 32 ± 2 0 to 125 . 0 %- 380 / 2 . 5 % 38 ± 3 0 to 405 . 0 %- 380 / 5 . 0 % 44 ± 2 0 to 407 . 5 %- 380 / 2 . 5 % 62 ± 5 0 to 207 . 5 %- 380 / 5 . 0 % 64 ± 5 0 to 40______________________________________ 5 . 0 %- 110 / 0 % means 5 . 0 % p / p of protein 110 with 0 % p / p glycol . in this first part , a comparison of two mechanical properties and characteristics of fluorescence were observed with the aim of selecting one of three caseinates for the continuation of the experiments . initially , the breaking force as a function of the amount of irradiation will be discussed ; followed by strain at failure in function to the dose and finally , the dosages by fluorescence according to the amounts received , will be presented . for these three points of comparisons , two protein concentrations were studied in the absence of a plasticizing agent . as there is a direct relation between the breaking load and the thickness of film , we decided to calculate the ration of one to the other . this was done in order to avoid possible variations of force which are simply due to variations of thickness . this ratio is represented by the symbol f / e and is expressed in n / μm . with a concentration of 5 . 0 % p / p , the f / e ratio varies from 14 . 7 to 17 . 4 for the three caseinates and for irradiation doses . there is no significant difference ( p & gt ; 0 . 05 ) for the f / e ratio , in function of the irradiation dose , between the three caseinates at this concentration . there is only one exception for the 12 kgy dose where there is a significant variation ( p & gt ; 0 . 05 ) between sodium caseinates ( alanate 180 ) and calcium ( alanate 380 ) ( see table 4 and fig3 ). on the other hand , without being clearly dissociated , calcium caseinate ( alanate 380 ) shows a higher f / e ratio than those of the two sodium caseinates for the irradiation doses of 4 , 8 and 12 kgy ( see table 4 ). for the 7 . 5 % concentration , the f / e ratio varies from 14 . 2 to 17 . 4 for the three caseinates for all three irradiation doses . with this concentration , the calcium caseinate ( alanate 380 ) has a relationship f / e significantly higher ( p 0 . 05 ) to the two other sodium caseinates ( alanates 110 and 180 ) during the irradiation between 4 and 12 kgy ( see table 5 and fig3 ). with 0 kgy , there is no significant difference ( p & gt ; 0 . 05 ) between caseinates of sodium ( alanate 110 ) and calcium ( alanate 380 ). the films formed starting from calcium caseinate ( alanate 380 ) require a larger force for rupture compared to those made from the two sodium caseinates ( alanates 110 and 180 ) ( see table 5 ). for the three caseinates , there is no significant difference ( p & gt ; 0 . 05 ) for the f / e ratio between the two protein concentrations used ( 5 . 0 % and 7 . 5 %) and this , according to the amounts of irradiation ( 0 to 12 kgy ). the only exceptions are sodium caseinate ( alanate 110 ) at 12 kgy and calcium caseinate ( alanate 380 ) at 8 kgy where the difference is considered significant ( p σ 0 . 05 ). thus , the irradiation of calcium caseinate ( alanate 380 ) to a concentration of 7 . 5 % protein creates a film more resistant to rupture than the two other proteins . whereas at a concentration of 5 . 0 %, there is no significant difference ( p & gt ; 0 . 05 ) between three caseinates . table 4______________________________________f / e ratio according to the amount of irradiationreceived for alanates 110 , 180 and 380with a concentratian of 5 . 0 % p / p of proteins . f / ex 100 f / ex 100 f / ex 100 ( n / μm ) ( n / μm ) ( n / μm ) dose alanate - alanate - alanate -( kgy ) 110 180 380______________________________________0 16 . 9 ± 0 . 4 . sup . 1 , a 16 . 2 ± 0 . 4 . sup . 3 , a 16 . 8 ± 0 . 9 . sup . 5 , a4 14 . 6 ± 0 . 1 . sup . 2 , b 15 . 0 ± 0 . 3 . sup . 4 , b 16 . 3 ± 1 . 5 . sup . 5 , b8 16 . 0 ± 1 . 6 . sup . 1 , 2 , c 15 . 3 ± 0 . 7 . sup . 4 , c 17 . 4 ± 0 . 5 . sup . 5 , c12 16 . 0 ± 0 . 8 . sup . 1 , 2 , de 14 . 7 ± 0 . 2 . sup . 4 , d 17 . 2 ± 1 . 0 . sup . 5 , e______________________________________ the term f / e expresses the ratio of the breaking load versus the thickness of film . for each line , two averages followed by the same letter are not significantly different between them ( p & gt ; 0 . 05 ). for each column , tow averages followed by the same figure are not significantly different between them ( p & gt ; 0 . 05 ). table 5______________________________________f / e ratio according to the amount of irradiationreceived for alanates 110 , 180 and 380with a concentration of 7 . 5 % p / p of proteins . f / ex 100 f / ex 100 f / ex 100 ( n / μm ) ( n / μm ) ( n / μm ) dose alanate - alanate - alanate -( kgy ) 110 180 380______________________________________0 15 . 6 ± 0 . 9 . sup . 1 , ab 14 . 5 ± 1 . 8 . sup . 3 , a 17 . 4 ± 0 . 4 . sup . 4 , h4 14 . 9 ± 0 . 4 . sup . 1 , 2 , c 14 . 5 ± 0 . 2 . sup . 3 , c 16 . 4 ± 0 . 0 . sup . 5 , d8 14 . 3 ± 0 . 0 . sup . 2 , c 14 . 2 ± 0 . 2 . sup . 3 , c 16 . 3 ± 0 . 2 . sup . 5 , f12 14 . 2 ± 0 . 3 . sup . 2 , g 14 . 3 ± 0 . 5 . sup . 3 , g 16 . 7 ± 0 . 2 . sup . 5 , h______________________________________ the term f / e expresses the ration of the breaking load versus the thickness of film . for each line , two averages followed by the same letter are not significantly different between them ( p & gt ; 0 . 05 ). for each column , two averages followed by the same figure are not significantly different between them ( p & gt ; 0 . 05 ). with a concentration of 5 . 0 % p / p , the deformation is approximately 2 , 3 ± 0 , 1 mm for the three caseinates and for all levels of irradiation . there significant variations ( p & gt ; 0 . 05 ) for the three caseinates as a function of the doses of irradiation except for calcium caseinate between the amounts of 4 and 12 kgy ( see table 6 ). at 7 . 5 % p / p proteins , the deformation is approximately 2 , 6 ± 0 , 2 mm for all three caseinates and for all amounts of irradiation . for the same amount of irradiation , there is no significant difference ( p & gt ; 0 . 05 ) between the three caseinates . on the other hand , for the sodium caseinates ( alanate 110 ) and calcium ( alanate 380 ), there is a significant variation ( p & gt ; 0 . 05 ) between the amounts of 0 and 12 kgy ( see table 7 ). for the two concentrations used , there is no significant difference ( p & gt ; 0 . 05 ) between the three caseinates for the deforming capacity as a function of the amounts of irradiation ( see fig4 ). the deformation is greater by some tenths of millimeters for a concentration of 7 . 5 % compared to 5 . 0 % but this variation is not considered significant ( p & gt ; 0 . 05 ) ( see tables 6 and 7 ). table 6______________________________________strain at failure according to the amount ofirradiation received for alanates 110 , 180 and 380with a concentration of 5 . 0 % protein p / p . deformation deformation deformationdose ( mm ) ( mm ) ( mm )( kgy ) alanate - 110 alanate - 110 alanate - 110______________________________________0 2 . 4 ± 0 . 2 . sup . 1 , a 2 . 3 ± 0 . 2 . sup . 2 , a 2 . 3 ± 0 . 1 . sup . 3 , 4 , a4 2 . 3 ± 0 . 1 . sup . 1 , b 2 . 3 ± 0 . 1 . sup . 2 , b 2 . 2 ± 0 . 1 . sup . 3 , b8 2 . 3 ± 0 . 1 . sup . 1 , c 2 . 3 ± 0 . 1 . sup . 2 , c 2 . 3 ± 0 . 2 . sup . 3 , 4 , c , 12 2 . 4 ± 0 . 3 . sup . 1 , d 2 . 3 ± 0 . 2 . sup . 2 , d 2 . 4 ± 0 . 1 . sup . 4 , d______________________________________ for each line , tow averages followed by the same letter are not significantly different between them ( p & gt ; 0 . 05 ). for each column , two averages followed by the same figure are not significantly different between them ( p & gt ; 0 . 05 ). table 7______________________________________strain at failure according to the amount ofirradiation received for alanates 110 , 180 and 380with a concentration of 7 . 5 % protein p / p . deformation deformation deformationdose ( mm ) ( mm ) ( mm )( kgy ) alanate - 110 alanate - 110 alanate - 110______________________________________0 3 . 0 ± 0 . 5 . sup . 1 , a 2 . 6 ± 0 . 2 . sup . 3 , a 2 . 7 ± 0 . 1 . sup . 4 , a4 2 . 6 ± 0 . 2 . sup . 1 , 2 , b 2 . 5 ± 0 . 1 . sup . 3 , b 2 . 5 ± 0 . 1 . sup . 4 , 5 , b8 2 . 7 ± 0 . 2 . sup . 1 , 2 , c 2 . 7 ± 0 . 1 . sup . 3 , c 2 . 6 ± 0 . 2 . sup . 4 , 5 , c12 2 . 4 ± 0 . 1 . sup . 2 , d 2 . 6 ± 0 . 2 . sup . 3 , d 2 . 3 ± 0 . 2 . sup . 5 , d______________________________________ for each line , tow averages followed by the same letter are not significantly different between them ( p & gt ; 0 . 05 ). for each column , two averages followed by the same figure are not significantly different between them ( p & gt ; 0 . 05 ). viscoelasticity was not evaluated for these films as the deformation applied to measure this parameter is three millimeters ( 3 mm ) and according to fig4 the strain at failure is lower than this value . the real rate of bityrosine formation produced by irradiation is measured by subtracting the average value obtained at 0 kgy from those obtained from the various amounts of irradiation . indeed , a signal of fluorescence is perceived at 0 kgy . the initial presence of bityrosine or the contribution of other neighbouring components can cause this signal of fluorescence . this contribution is the consequence of protein dosages with these multiple functional groupings in the vicinity of to the other . we observed an increase in the formation of bityrosine with an increase in the amount of irradiation for the three caseinates and this , for the two concentrations used . at 5 . 0 % concentration , the rate of bityrosine formation is significantly higher ( p σ 0 . 05 ) for calcium caseinate ( alanate 380 ) compared to the two sodium caseinates ( alanates 110 and 180 ) for irradiation of 4 , 8 and 12 kgy ( see table 8 ). moreover , with 12 kgy , the second sodium caseinate ( alanate 180 ) produced significantly more ( p σ 0 . 05 ) bityrosine than the first ( alanate 110 ). for 7 . 5 % concentration , sodium caseinate ( alanate 110 ) produced significantly more ( p σ 0 . 05 ) bityrosine than the caseinates of sodium ( alanate 180 ) and calcium ( alanate 380 ) and this , at levels of 4 and 12 kgy ( see table 8 and fig5 ), whereas at 8 kgy , no significant difference ( p & gt ; 0 . 05 ) was perceived between the three caseinates ( see table 9 ). there exists a significant difference ( p σ 0 . 05 ) between the two protein concentrations but this difference is not shown for all the levels of irradiation . indeed , the first sodium caseinate ( alanate 110 ) produced significantly ( p σ 0 . 05 ) more bityrosine at 7 . 5 % concentration for the 12 kgy level than at 5 . 0 % concentration . the second sodium caseinate ( alanate 180 ) produced significantly ( p σ 0 . 05 ) more bityrosine at 5 . 0 % concentration with levels of 4 and 8 kgy than at 7 . 5 % concentration . finally , the calcium caseinate ( alanate 380 ) produced significantly ( p σ 0 . 05 ) more bityrosine at 5 . 0 % concentration for levels of 4 , 8 and 12 kgy compared with 7 . 5 % concentration ( see tables 8 and 9 ). table 8______________________________________rate of bityrosine formation according to the amountof irradiation received for alanates 110 , 180 and 380at a concentration of 5 . 0 % p / p of proteins . dose ( kgy ) alanate - 110 alanate - 180 alanate - 380______________________________________4 19354 ± 11421 . sup . 1 , a 20730 ± 762 . sup . 4 , a 28552 ± 1621 . sup . 7 , b8 41071 ± 453 . sup . 2 , c 39651 ± 2095 . sup . 5 , c 66803 ± 2391 . sup . 8 , d12 62999 ± 659 . sup . 3 , c 66271 ± 1287 . sup . 6 , f 82504 ± 1650 . sup . 9 , g______________________________________ there is no unit as these rates are measured by the surface under the curves obtained . for each line , two averages followed by the same letter are not significantly different between them ( p & gt ; 0 . 05 ). for each column , two averages followed by the same figure are not significantly different between them ( p & gt ; 0 . 05 ). table 9______________________________________rate of bityrosine formation according to the amountof irradiation received for alanates 110 , 180 and 380at a concentration of 7 . 5 % p / p of proteins . dose ( kgy ) alanate - 110 alanate - 180 alanate - 380______________________________________4 24429 ± 1307 . sup . 1 , a 17067 ± 547 . sup . 4 , h 17192 ± 707 . sup . 7 , b8 38741 ± 599 . sup . 2 , c 35287 ± 1893 . sup . 5 , c 39344 ± 687 . sup . 8 , c12 69305 ± 795 . sup . 3 , d 64735 ± 769 . sup . 6 , c 61076 ± 607 . sup . 9 , f______________________________________ there is no unit as these rates are measured by the surface under the curves obtained . for each line , two averages followed by the same letter are not significantly different between them ( p & gt ; 0 . 05 ). for each column , two averages followed by the same figure are not significantly different between them ( p & gt ; 0 . 05 ). the oxidation of a solution of tryptophan residue by • oh radicals is directly connected to the loss of intensity of the fluorescence signal . fig6 shows the influence of irradiation on a solution of 500 tryptophan ppm . there is no regular and continuous signal loss as function of the level of irradiation during dosage of caseinates . even if a significant difference ( p σ 0 . 05 ) is sometimes perceived between the levels of irradiation for the three caseinates , in no case is there a continuous fall of the signal . this state is perceived for two concentrations ( see tables 10 and 11 and fig6 ). table 10______________________________________tryptophan dosage according to the level ofirradiation received for alanates 110 , 180 and 380with a concentration of 5 . 0 % p / p of proteinsdose ( kgy ) alanate - 110 alanate - 180 alanate - 380______________________________________0 775511 ± 3047 . sup . 1 , a 733889 ± 3222 . sup . 5 , b 764770 ± 2693 . sup . 8 , c4 742878 ± 3570 . sup . 2 , d 712041 ± 9816 . sup . 6 , e 793769 ± 1677 . sup . 9 , f8 710587 ± 2122 . sup . 3 , g 741933 ± 2615 . sup . 5 , h 749959 ± 8149 . sup . 10 , h12 712735 ± 7679 . sup . 3 , i 695793 ± 8417 . sup . 7 , j 778002 ± 10064 . sup . 11 , k______________________________________ there is no unit as these rates are measured by the surface under the curves obtained . for each line , two averages followed by the same letter are not significantly different between them ( p & gt ; 0 . 05 ). for each column , two averages followed by the same figure are not significantly different between them ( p & gt ; 0 . 05 ). table 11______________________________________tryptophan dosage according to the level ofirradiation received for alanates 110 , 180 and 380with a concentration of 5 . 0 % p / p of proteinsdose ( kgy ) alanate - 110 alanate - 180 alanate - 380______________________________________0 1056499 ± 5231 . sup . 1 , a 1022490 ± 8765 . sup . 5 , b 128405 ± 22051 . sup . 8 , c4 1021131 ± 8183 . sup . 2 , d 1074262 ± 5076 . sup . 6 , e 1101364 ± 3365 . sup . 9 , f8 1042411 ± 8518 . sup . 3 , g 1040796 ± 1100961 ± 4713 . sup . 9 , h 14792 . sup . 5 , 7 , g12 988217 ± 7563 . sup . 4 , i 1054722 ± 1108578 ± 13728 . sup . 6 , 7 , j 14058 . sup . 8 , 9 , k______________________________________ here is no unit as these rates are measured by the surface under the curves obtained . for each line , two averages followed by the same letter are not significantly different between them ( p & gt ; 0 . 05 ). for each column , two averages followed by the same figure are not significantly different between them ( p & gt ; 0 . 05 ). the physical chemical behavior of the protein solutions during the irradiation treatment enabled us to select the most adequate protein extract for the manufacture of a film . to accomplish this , we studied : 1 . the rheological parameters : resistance until rupture and the strain at failure 2 . chemical parameters : rates of bityrosine formation and tryptophan loss . the results obtained for the f / e ratio for calcium caseinate ( alanate 380 ), with a concentration of 5 . 0 %, are slightly higher than the two sodium caseinates ( alanates 110 and 180 ) for levels ( 4 , 8 and 12 kgy . on the other hand , at a concentration of 7 . 5 %, the f / e ratio for calcium caseinate ( alanate 380 ) is significantly higher ( p σ 0 . 05 ) than the two sodium caseinates ( alanates 110 and 180 ) at 4 , 8 and 12 kgy ( see tables 4 and 5 and fig3 ). the results of measurements of bityrosine formation showed that with 5 . 0 % concentration , calcium caseinate ( alanate 380 ) showed a significantly higher production of bityrosine ( p σ 0 . 05 ) than the two sodium caseinates ( alanates 110 and 180 ) at 4 , 8 and 12 kgy . with 7 . 5 % concentration , the first sodium caseinate ( alanate 110 ) produced significantly more ( p σ 0 . 05 ) bityrosine at levels of 4 and 12 kgy , whereas at 8 kgy , the three caseinates produced an equivalent quantity of bityrosine ( see tables 8 and 9 and fig5 ). during the irradiation of the proteinic solutions , the observation of the solutions enabled us to note that the viscosity of sodium caseinates ( alanates 110 and 180 ) increases with the amount of irradiation . this state is perceptible at the time of handling when the solutions are treated at 8 and 12 kgy . at the other end , the viscosity of calcium caseinate ( alanate 380 ) is quasi unchanged as a function of irradiation levels . according to tables 6 and 7 and fig4 the protein films formed without a plasticizing agent have a low deformation capacity . then , the presence of a plasticizing agent becomes essential for obtaining a film with a more adequate deformation capacity . therefore , according to the preceding results , we chose calcium caseinate ( alanate 380 ) to continue the tests with a plasticizing agent , that is , glycerol . in this second part , the influence of glycerol as a plasticizing agent was studied . to this end , the three mechanical properties and the dosages of fluorescence were evaluated for purposes of comparison . under certain treatment conditions , it is sometimes difficult if not impossible to obtain films with 5 . 0 % protein and 5 . 0 % glycerol without radiation treatment . fig7 and tables 12 and 13 show that for concentrations of 5 . 0 % and 7 . 5 %, the f / e ratio decreases as glycerol content increases . then , a lesser force is necessary to rupture the film when glycerol content increases . at a concentration of 5 . 0 % protein and 0 % glycerol , the f / e ratio remains high and stable ( 16 . 3 to 17 . 4 ) as a function of the level of irradiation . at 1 . 0 % glycerol , the ratio varies between 11 . 8 and 13 . 9 for levels of 0 to 12 kgy and these two extreme values are considered significantly different ( p δ 0 . 05 ) between them . on the other hand , the f / e ratio increases significantly ( p δ ( 0 . 05 ) at 15 and 20 kgy to reach 16 . 3 and 17 . 2 respectively . at a 2 . 5 % glycerol concentration , the f / e ratio grows significantly ( p δ 0 . 05 ) with the increase in the level of irradiation . it goes from 5 . 6 to 12 . 1 and a maximum is reached at 30 kgy . for 5 . 0 % glycerol concentration the f / e ratio also increases significantly ( p δ 0 . 05 ) as a function of the level of irradiation . it increases from 2 . 7 to 4 . 5 and also reaches its maximum at 30 kgy . ( see table 12 and fig7 ). under these conditions , irradiation contributes to create a more resistant film . for a protein concentration of 7 . 5 % with 0 % of 2 . 5 % glycerol , there is little variation of the f / e ratio for levels varying between 0 and 12 kgy . indeed , the ratio varies from 16 . 3 to 17 . 4 and 10 . 4 to 11 . 2 per 0 % and 2 . 5 % of glycerol respectively . on the other hand , at glycerol 2 . 5 %, the f / e ratio undergoes an increase but it is not significant ( p & gt ; 0 . 05 ) at levels of 15 and 20 kgy . at 5 . 0 % glycerol , the f / e ratio increases significantly ( p δ 0 . 05 ) with the increase in the level of irradiation ; it increases from 4 . 3 to 6 . 3 with a maximum at 30 kgy ( see table 13 and fig5 ). at 5 . 0 % protein the f / e ratio decreases significantly ( p δ 0 . 05 ) with the addition of glycerol whatever its concentration ( 1 . 0 %, 2 . 5 % and 5 . 0 %) and this , for all the tested levels of irradiation . in the presence of 7 . 5 % protein , the same phenomenon is observed for levels between 0 and 20 kgy . at 5 . 0 % glycerol , we observe a significant increase ( p δ 0 . 05 ) of f / e with the increase in the protein contents and this , for all levels of irradiation . at glycerol 2 . 5 %, we observe the same phenomenon save for the samples treated at 15 kgy . table 12______________________________________the f / e ratio according to the level of receivedirradiation and the glycerol contents for alanate 380with a protein concentration of 5 . 0 % p / p . f / e × 100 f / e × 100 f / e × 100 f / e × 100dose ( n x μm ) ( n x μm ) ( n x μm ) ( n x μm )( kgy ) 5 . 0 %/ 0 % 5 . 0 %/ 1 . 0 % 5 . 0 %/ 2 . 5 % 5 . 0 %/ 5 . 0 % ______________________________________ 0 16 . 8 ± 0 . 9 . sup . 1 , a 12 . 5 ± 0 . 3 . sup . 2 , 3 , b 5 . 6 ± 02 . sup . 5 , c -- 4 16 . 3 ± 1 . 5 . sup . 1 , d 13 . 9 ± 1 . 3 . sup . 3 , e 5 . 9 ± 0 . 1 . sup . 5 , f 2 . 7 ± 0 . 2 . sup . 10 , g 8 17 . 4 ± 0 . 5 . sup . 1 , h 12 . 6 ± 1 . 1 . sup . 2 , 3 , i 6 . 9 ± 0 . 1 . sup . 6 , j 3 . 3 ± 0 . 1 . sup . 11 , 12 , k12 17 . 2 ± 1 . 0 . sup . 1 , l 11 . 8 ± 0 . 1 . sup . 2 , m 7 . 0 ± 0 . 2 . sup . 6 , n 3 . 8 ± 0 . 4 . sup . 11 , 13 , o15 -- 16 . 3 ± 0 . 1 . sup . 4 , p 10 . 5 ± 0 . 4 . sup . 7 , q 3 . 6 ± 0 . 3 . sup . 11 , 12 , 13 , r20 -- 17 . 2 ± 0 . 6 . sup . 4 , s 10 . 1 ± 0 . 4 . sup . 8 , t 4 . 0 ± 0 . 3 . sup . 13 , u30 -- -- 12 . 1 ± 0 . 1 . sup . 9 , v 4 . 5 ± 0 . 3 . sup . 14 , w40 -- -- 10 . 7 ± 0 . 3 . sup . 7 , x 3 . 2 ± 0 . 3 . sup . 12 , y______________________________________ the term f / e expresses the ratio of the breaking load versus the thickness of film . the expression 5 . 0 %/ 1 . 0 % means 5 . 0 % protein with 1 . 0 % glycerol . for each line , two averages followed by the same letter are not significantly different between them ( p & gt ; 0 . 05 ). for each column , two averages followed by the same figure are not significantly different between them ( p & gt ; 0 . 05 ). table 13______________________________________the f / e ratio according to the level of receivedirradiation and glycerol contents for alanate 380with a protein concentration of 7 . 5 % p / p . f / e × 100 f / e × 100 f / e × 100dose ( n x μm ) ( n x μm ) ( n x μm )( kgy ) 7 . 5 %/ 0 % 7 . 5 %/ 2 . 5 % 7 . 5 %/ 5 . 0 % ______________________________________ 0 17 . 4 ± 0 . 4 . sup . 1 , a 11 . 0 ± 1 . 0 . sup . 3 , b 4 . 3 ± 0 . 2 . sup . 4 , c 4 16 . 4 ± 0 , 0 . sup . 2 , d 11 . 2 ± 0 . 5 . sup . 3 , e 4 . 4 ± 0 . 2 . sup . 4 , f 8 16 . 3 ± 0 . 2 . sup . 2 , g 10 . 4 ± 1 . 5 . sup . 3 , h 4 . 6 ± 0 . 1 . sup . 4 , i12 16 . 7 ± 0 . 2 . sup . 2 , j 10 . 5 ± 0 . 9 . sup . 3 , k 5 . 8 ± 0 . 2 . sup . 5 , l15 -- 12 . 5 ± 1 . 4 . sup . 3 , m 5 . 2 ± 0 . 2 . sup . 6 , n20 -- 12 . 1 ± 0 . 1 . sup . 3 , o 5 . 7 ± 0 . 15 . sup . 5 , p30 -- -- 6 . 3 ± 0 . 1 . sup . 740 -- -- 5 . 9 ± 0 . 2 . sup . 5______________________________________ the term f / e expresses the ratio of the breaking load versus the thickness of film . the expression 7 . 5 %/ 2 . 5 % means 7 . 5 % protein with 2 . 5 % glycerol . for each line , two averages followed by the same letter are not significantly different between them ( p & gt ; 0 . 05 ). for each column , two averages followed by the same figure are not significantly different between them ( p & gt ; 0 . 05 ). according to fig8 and tables 14 and 15 , the presence of glycerol strongly increases the deforming capacity of films . at 5 . 0 % protein , the amount of irradiation and the addition of 1 . 0 % glycerol do not bring comparatively important changes to the deforming capacity of films in the absence of glycerol . the deformation varies from 2 . 2 mm to 2 . 4 mm and from 2 . 6 mm to 2 . 9 mm for 0 % and 1 . 0 % glycerol respectively . whereas in 2 . 5 % and 5 . 0 % glycerol , the deforming capacity increases significantly ( p δ 0 . 05 ) with the increase in the level of irradiation . the deformation varies from 5 . 7 mm to 7 . 4 mm for 2 . 5 % glycerol and from 7 . 6 mm to 10 . 8 mm for glycerol 5 . 0 %. a maximum is reached between 20 and 30 kgy for these two concentrations of glycerol ( see table 14 and fig9 ). therefore , irradiation contributes to improve the deforming capacity of films . the difference between the deformation and the three concentrations of glycerol is considered significant ( p δ 0 . 05 ) at all levels of irradiation . for a 7 . 5 % concentration of protein , the presence of 2 . 5 % of glycerol does not bring a significant different ( p & gt ; 0 . 05 ) in the deformation as a function of the levels of irradiation ( 0 to 20 kgy ). the deformation varies from 3 . 8 mm to 4 . 2 mm between levels of 0 and 20 kgy . on the other hand , the difference in deformation between 0 % and 2 . 5 % of glycerol is considered significant ( p δ 0 . 05 ) for levels 0 to 12 kgy . with the addition of 5 . 0 % of glycerol , the deformation is significantly higher ( p δ 0 . 05 ) than in the presence of 2 . 5 % or the in absence of glycerol . the deformation values vary from 7 . 7 mm to 11 . 6 mm as a function of the levels of irradiation to a maximum value towards 15 to 20 kgy ( see table 15 and fig9 ). at 2 . 5 % glycerol , the deformation to 5 . 0 % of proteins is significantly higher ( p δ 0 . 05 ) than at 7 . 5 % of proteins for levels of 0 to 20 kgy ( see tables 14 and 15 ). for a 5 . 0 % glycerol concentration , there is a significant difference ( p δ 0 . 05 ) of the deformation between the two protein concentrations . the deformation is higher at protein 7 . 5 % compared to 5 . 0 % for levels of 4 , 8 , 15 , 20 and 40 kgy . it is significantly higher ( p δ 0 . 05 ) than for levels of 8 , 15 and 40 kgy . at 12 and 30 kgy , the deformation with 7 . 5 % protein is lower than that obtained at 5 .% ( see tables 14 and 15 ). however , this difference is significantly lower ( p δ 0 . 05 ) only at 12 kgy . therefore , deformation as a function of the level of irradiation is higher in the presence of 5 . 0 % glycerol and this , for the two protein concentrations . the effect of the level of irradiation on deformation is more evident in the presence of 7 . 5 % of proteins and 5 . 0 % of glycerol . in the presence of 2 . 5 % glycerol and 5 . 0 % protein , the deformation undergoes a slight increase as a function of the level amount but it is considered significant ( p δ 0 . 05 ). table 14______________________________________strain at failure as a function of the level of receivedirradiation and glycerol contents for alanate 380at a protein concentration of 5 . 0 % p / p . defor - defor - defor - defor - mation mation mation mationdose ( mm ) ( mm ) ( mm ) ( mm )( kgy ) 5 . 0 %/ 0 % 5 . 0 %/ 1 . 0 % 5 . 0 %/ 2 . 5 % 5 . 0 %/ 5 . 0 % ______________________________________ 0 2 . 3 ± 0 . 1 . sup . 1 , 2 , a 2 . 6 ± 0 . 1 . sup . 3 , a 5 . 7 ± 0 . 4 . sup . 6 , b -- 4 2 . 2 ± 0 . 1 . sup . 1 , c 2 . 9 ± 0 . 1 . sup . 4 , d 6 . 1 ± 0 . 2 . sup . 6 , 7 , e 8 . 5 ± 04 . sup . 10 , f 8 2 . 3 ± 0 . 2 . sup . 1 , 2 , g 2 . 8 ± 0 . 1 . sup . 3 , 4 , 5 , h 6 . 6 ± 0 . 1 . sup . 7 , 8 , i 9 . 5 ± 0 . 1 . sup . 11 , j12 2 . 4 ± 0 . 1 . sup . 2 , k 2 . 6 ± 0 . 1 . sup . 3 , 5 , k 7 . 0 ± 0 . 3 . sup . 8 , 9 , l 10 . 3 ± 0 . 3 . sup . 12 , 13 , m15 -- 2 . 9 ± 0 . 2 . sup . 4 , 5 , n 6 . 2 ± 0 . 2 . sup . 6 , 7 , o 9 . 6 ± 0 . 6 . sup . 11 , 12 , p20 -- 2 . 9 ± 0 . 2 . sup . 4 , q 7 . 3 ± 0 . 3 . sup . 9 , r 10 . 8 ± 0 . 7 . sup . 13 , s30 -- -- 7 . 4 ± 0 . 5 . sup . 9 , t 10 . 7 ± 0 . 2 . sup . 13 , u40 -- -- 5 . 9 ± 0 . 2 . sup . 6 , v 7 . 6 ± 0 . 3 . sup . 14 , w______________________________________ the expression 5 . 0 %/ 1 . 0 % means protein 5 . 0 % with glycerol 1 . 0 %. for each line , two averages followed by the same letter are not significantly different between them ( p & gt ; 0 . 05 ). for each column , two averages followed by the same figure are not significantly different between them ( p & gt ; 0 . 05 ). table 15______________________________________strain at failure as a function of the amount of receivedirradiation and glycerol contents for alanate 380with a protein concentration of 7 . 5 % p / p . defor - defor - defor - mation mation mationdose ( mm ) ( mm ) ( mm )( kgy ) 7 . 5 %/ 0 % 7 . 5 %/ 2 . 5 % 7 . 5 %/ 5 . 0 % ______________________________________ 0 2 . 7 ± 0 . 1 . sup . 1 , a 4 . 1 ± 0 . 4 . sup . 3 , b 7 . 7 ± 0 . 2 . sup . 4 , c 4 2 . 5 ± 0 . 1 . sup . 1 , 2 , d 3 . 8 ± 0 . 3 . sup . 3 , e 9 . 4 ± 0 . 5 . sup . 5 , 6 , f 8 2 . 6 ± 0 . 2 . sup . 1 , 2 , g 4 . 0 ± 0 . 3 . sup . 3 , h 11 . 0 ± 0 . 1 . sup . 7 , i12 2 . 3 ± 0 . 2 . sup . 2 , j 4 . 0 ± 0 . 3 . sup . 3 , k 9 . 1 ± 0 . 3 . sup . 5 , l15 -- 3 . 9 ± 0 . 4 . sup . 3 , m 11 . 6 ± 0 . 6 . sup . 7 , n20 -- 4 . 2 ± 0 . 2 . sup . 3 , o 11 . 2 ± 0 . 6 . sup . 7 , p30 -- -- 10 . 0 ± 0 . 5 . sup . 640 -- -- 8 . 3 ± 0 . 3 . sup . 4______________________________________ the expression7 . 5 %/ 2 . 5 % means protein 7 . 5 % with glycerol 2 . 5 %. for each line , two averages followed by the same letter are not significantly different between them ( p & gt ; 0 . 05 ). for each column , two averages followed by the same figure are not significantly different between them ( p & gt ; 0 . 05 ). a characteristic sought in a film is its elasticity , i . e ., a film having a low relaxation coefficient . according to relaxation results , films irradiated with calcium caseinate ( alanate 380 ) are viscoelastic products with a relaxation coefficient varying between 0 . 57 to 0 . 69 according to the composition of the films ( see table 16 and fig1 ). only films able to deform with more than three millimeters ( 3 . 0 mm ) can be studied as viscoelasticity is measured following a sustained deformation of 3 . 0 mm . in the present case , three compositions of mediums were retained , that is , 5 . 0 % protein with 2 . 5 % and 5 . 0 % glycerol and 7 . 5 % proteins with 5 . 0 % glycerol . for a concentration of 5 . 0 % proteins with 2 . 5 % and 5 . 0 % glycerol , irradiation tends to produce a more elastic film since the relaxation coefficients decrease significantly ( p δ 0 . 05 ) with the increase in the level of irradiation . the values of coefficients vary from 0 . 66 to 0 . 61 and 0 . 63 to 0 . 57 for glycerol concentrations of 2 . 5 % and 5 . 0 % respectively . a minimum relaxation coefficient is obtained between 30 and 40 kgy for these two glycerol concentrations ( see table 16 and fig1 ). the difference between the two glycerol concentrations is significant ( p δ 0 . 05 ) at all levels of irradiation except for levels of 15 and 20 kgy . thus , the addition of 5 . 0 % glycerol produces a more elastic film with a weaker relaxation coefficient . with a protein concentration of 7 . 5 % and 5 . 0 % glycerol , irradiation tends to lower significantly ( p δ 0 . 05 ) the relaxation coefficient except when one irradiates at 8 kgy where a maximum value is reached . the values of coefficients vary from 0 . 67 to 0 . 63 according to the amounts of irradiation . then , irradiation contributes to produce a more elastic film with a minimal value following a treatment located between 20 and 40 kgy ( see table 16 and fig1 ). at 5 . 0 % glycerol , the relaxation coefficients at 5 . 0 % protein are significantly lower ( p δ 0 . 05 ) than those obtained 7 . 5 % proteins for all levels of irradiation . table 16______________________________________relaxation coefficients as a function to the level of receivedirradiation and glycerol contents for alanate 380 withprotein concentrations of 5 . 0 % p / p and 7 . 5 % p / pdose ( kgy ) 5 . 0 %/ 2 . 5 % 5 . 0 %/ 5 . 0 % 7 . 5 %/ 5 . 0 % ______________________________________ 0 0 . 66 ± 0 . 01 . sup . 1 , 2 -- 0 . 67 ± 0 . 01 . sup . 10 4 0 . 67 ± 0 . 00 . sup . 1 , a 0 . 63 ± 0 . 00 . sup . 7 , b 0 . 66 ± 0 . 01 . sup . 11 , 12 , c 8 0 . 66 ± 0 . 01 . sup . 2 , 3 , d 0 . 61 ± 0 . 01 . sup . 7 , 8 , e 0 . 69 ± 0 . 01 . sup . 13 , f12 0 . 65 ± 0 . 01 . sup . 3 , 4 , g 0 . 62 ± 0 . 01 . sup . 7 , 8 , h 0 . 66 ± 0 . 01 . sup . 10 , 11 , f15 0 . 64 ± 0 . 00 . sup . 4 , 5 , j 0 . 63 ± 0 . 01 . sup . 7 , j 0 . 65 ± 0 . 01 . sup . 12 , 14 , k20 0 . 63 ± 0 . 01 . sup . 5 , l 0 . 60 ± 0 . 02 . sup . 8 , l 0 . 64 ± 0 . 01 . sup . 15 , m30 0 . 61 ± 0 . 01 . sup . 6 , n 0 . 57 ± 0 . 01 . sup . 9 , o 0 . 64 ± 0 . 01 . sup . 15 , p40 0 . 61 ± 0 . 01 . sup . 6 , q 0 . 57 ± 0 . 02 . sup . 9 , r 0 . 63 ± 0 . 00 . sup . 15______________________________________ the expression 5 . 0 %/ 2 . 5 % means protein 5 . 0 % with glycerol 2 . 5 %. for each line , two averages followed by the same letter are not significantly different between them ( p & gt ; 0 . 05 ). for each column , two averages followed by the same figure are not significantly different between them ( p & gt ; 0 . 05 ). the statistical evaluation with glycerol 5 . 0 % is made only for one concentration of 5 . 0 % and protein 7 . 5 %. the average value obtained with 0 kgy was subtracted from those obtained from the various levels of irradiation with the aim of obtaining the real rate of bityrosine formation . the rate of bityrosine formation increases proportionally with the increase in the level of irradiation for the two protein concentrations ( see tables 17 and 18 and fig1 ). the rate of bityrosine formation increases by 23 , 000 to 350 , 000 ( in arbitrary units of area ) for amounts varying from 4 to 40 kgy for the two protein concentrations . for a 5 . 0 % protein concentration , the rate of bityrosine formation is significantly higher ( p δ 0 . 05 ) in the presence of glycerol ( 1 . 0 %, 2 . 5 % and 5 . 0 %) compared to its absence ( 0 %) for levels of 15 and 20 kgy ( see table 17 ). at 7 . 5 % protein , the presence of glycerol ( 2 . 5 % and 5 . 0 %) significantly increases ( p δ 0 . 05 ) the rate of bityrosine formation for levels of 4 to 20 kgy ( see table 17 and fig1 ). moreover , we note than in the presence of 2 . 5 % glycerol , the rate of bityrosine formation in the sample of protein containing 7 . 5 % doubled compared to the sample containing protein containing 5 . 0 % ( 43513 vs . 20503 ) and when this one is irradiated at 4 kgy ( see tables 17 and 18 ). we also note an increase of approximately 10 % of the bityrosine content when the protein samples ( 5 . 0 % and 7 . 5 %) are irradiated at 20 kgy in absence and in the presence of glycerol 2 . 5 %. on the other hand , there is no relation between the rate of bityrosine formation and the protein concentration in the presence of 5 . 0 % glycerol . as glycerol alone in the buffer does not absorb and does not emit at the of excitation and emission wavelengths used , it seems to favour the formation of bityrosine . there exists a linear relation between the rate of bityrosine formation and the levels of irradiation for the various protein - glycerol mixtures that were tested . table 17__________________________________________________________________________rate of bityrosine formation according to the level ofreceived irradiation and glycerol contents for alanate 380with a protein concentration of 5 . 0 % p / p . dose ( kgy ) 5 . 0 %/ 0 % 5 . 0 %/ 1 . 0 % 5 . 0 %/ 2 . 5 % 5 . 0 %/ 5 . 0 % __________________________________________________________________________ 4 28552 ± 1621 . sup . 1 , a 21836 ± 1484 . sup . 6 , bc 20503 ± 941 . sup . 11 , b 23126 ± 1482 . sup . 18 , c 8 66803 ± 2391 . sup . 2 , d 76436 ± 1006 . sup . 7 , e 66439 ± 1805 . sup . 12 , d 81304 ± 2395 . sup . 19 , f12 82504 ± 1650 . sup . 3 , g 79531 ± 1964 . sup . 8 , h 75782 ± 1206 . sup . 13 , i 85465 ± 1392 . sup . 20 , j15 89584 ± 1817 . sup . 4 , k 113309 ± 2249 . sup . 9 , l 134112 ± 1328 . sup . 14 , m 125394 ± 1551 . sup . 21 , n20 129044 ± 931 . sup . 5 , n 146654 ± 1511 . sup . 10 , p 163519 ± 1126 . sup . 15 , q 158853 ± 2892 . sup . 22 , r30 -- -- 254378 ± 1440 . sup . 16 , s 256610 ± 2398 . sup . 23 , s40 -- -- 348299 ± 4022 . sup . 17 , t 336676 ± 2254 . sup . 24 , u__________________________________________________________________________ there is not unit as these rates are measured by the surface under the curves obtained . expression 5 . 0 %/ 2 . 5 % means protein 5 . 0 % with glycerol 2 . 5 %. for each line , two averages followed by the same letter are not significantly different between them ( p & gt ; 0 . 05 ). for each column , two averages followed by the same figure are not significantly different between them ( p & gt ; 0 . 05 ). table 18______________________________________rate of bityrosine formation according to the level ofreceived irradiation and glycerol contents for alanate 380with protein concentration of 7 . 5 % p / p . dose ( kgy ) 7 . 5 %/ 0 % 7 . 5 %/ 2 . 5 % 7 . 5 %/ 5 . 0 % ______________________________________ 4 17192 ± 1621 . sup . 1 , a 43513 ± 1328 . sup . 6 , b 20803 ± 928 . sup . 11 , e 8 39344 ± 687 . sup . 2 , d 97240 ± 4388 . sup . 7 , e 57961 ± 897 . sup . 12 , f12 61076 ± 607 . sup . 3 , g 103811 ± 1653 . sup . 8 , h 92734 ± 1901 . sup . 13 , i15 955871 ± 1252 . sup . d , j 128415 ± 1232 . sup . 9 , k 117110 ± 1398 . sup . 14 , l20 1392491 ± 1697 . sup . 5 , m 184986 ± 1581 . sup . 10 , n 151133 ± 1653 . sup . 15 , o30 -- -- 290277 ± 1848 . sup . 1640 -- -- 3627561 ± 1564 . sup . 17______________________________________ there is no unit for these rates are measured by the surface under the curves obtained . expression 7 . 5 %/ 2 . 5 % means 7 . 5 % protein with 2 . 5 % glycerol . for each line , two averages followed by the same letter are not significantly different between them ( p & gt ; 0 . 05 ). for each column , two averages followed by the same figure are not significantly different between them ( p & gt ; 0 . 05 ). in the presence of glycerol , the rate of tryptophan loss due to the irradiation treatment is significant ( p δ 0 . 05 ), that is , a fall of the fluorescence signal is perceived with the increase in the level of irradiation ( see tables 19 and 20 and fig1 ). at 5 . 0 % protein concentration , the decrease of the signal is significant ( p δ 0 . 05 ) in the presence of glycerol ( 1 . 0 %, 2 . 5 % and 5 . 0 %) compared to its absence for levels varying from 4 to 20 kgy . the signal varies approximately from 760 , 000 to 560 , 000 between 0 and 20 kgy and decreases approximately to 470 , 000 for the level of 40 kgy when the dosages are done on mediums in the presence of glycerol ( 1 . 0 %, 2 . 5 % and 5 . 0 %). however , there is not regular and continuous signal loss as a function of the level of irradiation to 0 % glycerol . the average signal obtained between 0 and 20 kgy is 778 , 000 for a protein concentration of 5 . 0 %. even if sometimes a significant different ( p δ 0 . 05 ) to 0 % glycerol is perceived between the levels of irradiation , there is no constant fall of the signal . thus , the perceived signal with 0 % glycerol is significantly higher ( p δ 0 . 05 ) than the one perceived in the presence of glycerol for levels of irradiation higher or equivalent to 4 kgy ( see table 19 and fig1 ). with amounts higher or equivalents to 15 kgy , the presence of glycerol favours a reduction in the signal obtained in the 5 . 0 % proteins solutions . this fall of signal seems to be more important with the increase in glycerol content . it becomes a significant ( p δ 0 . 05 ), however , between 5 . 0 % and 2 . 5 % glycerol for levels of 30 and 40 kgy ( see table 19 and fig1 ). at 7 . 5 % proteins , the loss of the signal in the presence of glycerol is significant ( p δ 0 . 05 ) at 12 , 15 and 20 kgy in the presence of 2 . 5 % and 5 . 0 % glycerol compared to its absence . in the presence of glycerol ( 2 . 5 % or 5 . 0 %), the signal varies approximately 1 , 115 , 000 to 900 , 000 between 0 and 20 kgy and decreases up to 814 , 000 for the level of 40 kgy at 5 . 0 % glycerol . on the other hand , in the absence of glycerol , there is no regular and continuous signal loss during irradiation . an average signal of 1 , 122 , 000 is obtained for levels of irradiation of 0 to 20 kgy . thus , the signal obtained in the absence of glycerol is significantly higher ( p δ 0 . 05 ) than that obtained in its presence for levels of irradiation higher than 8 kgy ( see table 20 and fig1 ). for an irradiation level equivalent or higher than 12 kgy , the signal perceived at 7 . 5 % proteins is significantly lower ( pδ0 . 05 ) in presence of 5 . 0 % glycerol than 2 . 5 % glycerol ( see table 20 and fig1 ). table 19__________________________________________________________________________tryptophan dosage as a function of the level of receivedirradiation and glycerol contents for alanate 380with a protein concentration of 5 . 0 % p / p . dose ( kgy ) 5 . 0 %/ 0 % 5 . 0 %/ 1 . 0 % 5 . 0 %/ 2 . 5 % 5 . 0 %/ 5 . 0 % __________________________________________________________________________ 0 764770 ± 2693 . sup . 1 , a 796315 ± 3894 . sup . 6 , b 782666 ± 23326 . sup . 10 , ab 700887 ± 4245 . sup . 18 , c 4 793769 ± 1677 . sup . 2 , d 710132 ± 2361 . sup . 7 , c 714711 ± 6221 . sup . 11 , e 739892 ± 10315 . sup . 19 , f 8 749959 ± 8149 . sup . 3 , g 677805 ± 7018 . sup . 8 , h 686849 ± 3743 . sup . 12 , h 684373 ± 7521 . sup . 20 , h12 778002 ± 10064 . sup . 4 , i 669722 ± 10182 . sup . 8 , j 623320 ± 4653 . sup . 13 , k 623178 ± 2164 . sup . 21 , k15 814920 ± 2447 . sup . 5 , l 585219 ± 2866 . sup . 9 , m 585219 ± 2866 . sup . 14 , m 583052 ± 1559 . sup . 22 , m20 765954 ± 9612 . sup . 1 , n 579666 ± 7450 . sup . 9 , o 561248 ± 11830 . sup . 15 , p 546316 ± 3320 . sup . 23 , p30 -- -- 531297 ± 5928 . sup . 16 , q 494688 ± 2136 . sup . 24 , r40 471847 ± 4943 . sup . 17 , s 466259 ± 3208 . sup . 25 , t__________________________________________________________________________ there is no unit as these rates are measured by the surface under the curves obtained . the expression 5 . 0 %/ 2 . 5 % means 5 . 0 % protein with 2 . 5 % glycerol . for each line , two averages followed by the same letter are not significantly different between them ( p & gt ; 0 . 05 ). for each column , two averages followed by the same figure are not significantly different between them ( p & gt ; 0 . 05 ). table 20__________________________________________________________________________tryptophan dosage as a function of the level of receivedirradiation and glycerol contents for alanate 380with a protein concentration of 7 . 5 % p / p . dose ( kgy ) 7 . 5 %/ 0 % 7 . 5 %/ 2 . 5 % 7 . 5 %/ 5 . 0 % __________________________________________________________________________ 0 1128405 ± 22051 . sup . 1 , a 1140330 ± 12521 . sup . 4 , ab 1179255 ± 31338 . sup . 9 , b 4 1101364 ± 3365 . sup . 2 , c 1146510 ± 20464 . sup . 4 , d 1131341 ± 8699 . sup . 10 , d 8 1100961 ± 4713 . sup . 2 , e 10081601 ± 16486 . sup . 5 , ef 1070098 ± 14308 . sup . 11 , f12 1108578 ± 14058 . sup . 1 , 2 , m 1006871 ± 7153 . sup . 6 , h 980093 ± 10770 . sup . 12 , i15 1158964 ± 5372 . sup . 3 , j 972919 ± 5338 . sup . 7 , k 935709 ± 8417 . sup . 13 , l20 1133910 ± 21926 . sup . 1 , 3 , m 934142 ± 7591 . sup . 8 , n 876339 ± 9557 . sup . 14 , o30 -- -- 847660 ± 3469 . sup . 1540 814091 ± 11282 . sup . 16__________________________________________________________________________ there is no unit as these rates are measured by the surface under the curves obtained . the expression 7 . 5 %/ 2 . 5 % means 7 . 5 % protein with 2 . 5 % glycerol . for each line , two averages followed by the same letter are not significantly different between them ( p & gt ; 0 . 05 ). for each column , two averages followed by the same figure are not significantly different between them ( p & gt ; 0 . 05 ). in this section , the results of the biodegradability of a type of film will be presented . the tests were repeated in triplicate with three recoveries . the results of the first test show that when p . fragi is in contact with film , the bacterial growth is fast and a maximum of population ( approximately 107 ufc / ml of medium ) is reached after approximately 80 hours of agitation ( see table 21 and fig1 ). the population tends to decrease very slowly as a function of time and this one remained higher than 10 6 ufc / ml after 1127 hours of experimentation . on the other hand , in the absence of film , the population remained appreciably the same at 20 ufc / ml for the first 100 hours . thereafter , it decreased and remained & lt ; 5 ufc / ml until the end . in presence of single film , the bacterial population is & lt ; 5 ufc / ml from beginning to end of the experimentation . table 21______________________________________results of test # 1 countings of pseudomonas fragi in thepresence and / or in the absence of a film sample of alanate380 composed of 5 . 0 % p / p proteins and 2 . 5 % p / p glycerolirradiated at 20 kgy . p . fragi p . fragi filmtime with film only only ( h ) ( ufc / ml ) ( ufc / ml ) ( ufc / ml ) ______________________________________ 0 23 ± 12 15 ± 12 & lt ; 5 4 20 ± 17 -- -- 8 18 ± 12 -- -- 9 -- -- & lt ; 5 10 53 ± 45 -- -- 21 100 ± 18 -- -- 27 370 ± 120 27 ± 20 & lt ; 5 53 -- -- & lt ; 5 54 12 × 10 . sup . 4 ± 61 × 10 . sup . 3 -- -- 76 89 × 10 . sup . 5 ± 18 × 10 . sup . 5 -- -- 102 12 × 10 . sup . 6 ± 24 × 10 . sup . 5 13 ± 13 & lt ; 5 125 12 × 10 . sup . 6 ± 23 × 10 . sup . 5 -- -- 146 10 . 4 × 10 . sup . 6 ± 16 × 10 . sup . 5 -- -- 148 -- & lt ; 5 & lt ; 5 267 92 × 10 . sup . 5 ± 27 × 10 . sup . 5 -- -- 287 -- & lt ; 5 & lt ; 5 293 93 × 10 . sup . 5 ± 22 × 10 . sup . 5 -- -- 413 77 × 10 . sup . 5 ± 24 × 10 . sup . 5 -- -- 414 -- & lt ; 5 & lt ; 5 624 46 × 10 . sup . 5 ± 26 × 10 . sup . 5 -- -- 646 -- & lt ; 5 & lt ; 51127 36 × 10 . sup . 5 ± 87 × 10 . sup . 4 & lt ; 5 & lt ; 5______________________________________ in the second test , the bacterial growth is rapid in the presence of film and a maximum ( approximately 107 ufc / ml ) is reached after approximately 60 hours of agitation ( see table 22 and fig1 ), the maximum population was reached more quickly but the initial population was 70 ufc / ml compared to 20 ufc / ml for the first time . the population only decreased very slightly as a function of time and remains at 106 fc / ml after about a hundred hours of agitation . thereafter , the population decreased regularly as a function of time to read approximately 10 3 ufc / ml after 1102 hours ( see table 22 and fig1 ). in presence of film only , the bacterial calculation is & lt ; 5 ufc / ml over the duration of the experimentation . table 22______________________________________results of test # 2 counting the in presenceand / or absence of a film sample of alanate 380composed of 5 . 0 % p / p proteins and 2 . 5 % p / pglycerol , irradiation at 20 kgy . p . fragi p . fragi filmtime with film only only ( h ) ( ufc / ml ) ( ufc / ml ) ( ufc / ml ) ______________________________________ 0 67 ± 45 87 ± 27 & lt ; 5 4 83 ± 22 -- -- 21 7400 ± 5300 -- -- 24 -- 180 ± 44 & lt ; 5 34 81 × 10 . sup . 4 ± 62 × 10 . sup . 4 -- -- 47 48 × 10 . sup . 5 ± 12 × 10 . sup . 5 -- -- 53 66 × 10 . sup . 5 ± 67 × 10 . sup . 4 -- -- 70 -- -- & lt ; 5 71 62 × 10 . sup . 5 ± 14 × 10 . sup . 5 -- -- 93 64 × 10 . sup . 5 ± 64 × 10 . sup . 4 -- -- 96 -- 83 × 10 . sup . 4 ± 14 × 10 . sup . 4 -- 120 -- 91 × 10 . sup . 4 ± 13 × 10 . sup . 4 -- 189 47 × 10 . sup . 5 ± 10 × 10 . sup . 5 -- -- 190 -- -- & lt ; 5192 -- 80 × 10 . sup . 4 ± 69 × 10 . sup . 3720 31 × 10 . sup . 5 ± 21 × 10 . sup . 5 -- & lt ; 5746 -- 43 × 10 . sup . 3 ± 30 × 10 . sup . 3 -- 1102 99 × 10 . sup . 4 ± 26 × 10 . sup . 4 800 ± 730 & lt ; 5______________________________________ for the third test , bacterial growth is very fast in the presence of film and is maximum ( approximately 107 ufc / ml ) after about sixty hours of agitation . the initial population was approximately 130 ufc / ml and the maximum was reached within a time similar to the second test . in this test , the population also tends to decrease according to time but after 1200 hours of agitation , this population tends to increase slightly thereafter ( see table 23 and fig1 ). in absence of film , the population remains stable ( approximately 110 ufc / ml ) for first period the 24 hours and increases quickly to 10 6 ufc / ml after a hundred hours of agitation . then , the population decreases by one logarithmic unit and remains stable for the interval from 500 to 1500 hours . thereafter , the population falls quickly to 2800 ufc / ml after 1779 hours of agitation ( see fig1 ). in presence of film only , the population is & lt ; 5 ufc / ml throughout the experiment . the experiments of biodegradability were stopped after obtaining the stability of the p . fragi population in the mediums in the presence of film and when the population had distinctly decreased , in the mediums in the absence of film ( tests 2 and 3 ). for the three tests , the film was not entirely biodegraded at the time of stopping the experimentation . various attempts were made in order to follow the rate of biodegradation of film by p . fragi as a function of time by soluble nitrogen dosage . in no case were we able to could recover film after complete immersion in the mediums without losing a significant quantity of it . indeed , the film has a tendency to disorganize once immersed in water , so that it loses its initial structure . then , a recovery by filtration and / or evaporation was not effective to isolate the film from the mediums during the biodegradation tests . table 23______________________________________results of test # 3 countings of pseudomonas fragiin the presence and / or absence of a film sample ofalanate 380 composed of 5 . 0 % p / p proteins and 2 . 5 % p / pglycerol , irradiated at 20 kgy . p . fragi p . fragi filmtime with film only only ( h ) ( upc / mi ) ( ufc / ml ) ( ufc / ml ) ______________________________________ 0 130 ± 56 130 ± 39 & lt ; 5 4 170 ± 47 103 ± 29 -- 24 21 × 10 . sup . 3 ± 2700 88 ± 22 & lt ; 5 30 85 × 10 . sup . 3 ± 31 × 10 . sup . 3 160 ± 18 -- 50 47 × 10 . sup . 5 ± 14 × 10 . sup . 5 30 × 10 . sup . 3 ± 20 × 10 . sup . 3 & lt ; 5 73 87 × 10 . sup . 5 ± 98 × 10 . sup . 4 75 × 10 . sup . 4 ± 57 × 10 . sup . 4 -- 98 78 × 10 . sup . 5 ± 10 × 10 . sup . 5 98 × 10 . sup . 4 ± 59 × 10 . sup . 4 -- 173 56 × 10 . sup . 5 ± 85 × 10 . sup . 4 10 × 10 . sup . 5 ± 45 × 10 . sup . 4 & lt ; 5 291 61 × 10 . sup . 5 ± 25 × 10 . sup . 5 83 × 10 . sup . 4 ± 55 × 10 . sup . 4 -- 503 38 × 10 . sup . 5 ± 11 × 10 . sup . 3 82 × 10 . sup . 3 ± 23 × 10 . sup . 3 & lt ; 5 634 29 × 10 . sup . 5 ± 33 × 10 . sup . 4 -- & lt ; 51083 26 × 10 . sup . 5 ± 49 × 10 . sup . 4 -- & lt ; 51152 -- 10 × 10 . sup . 4 ± 16 × 10 . sup . 3 -- 1465 33 × 10 . sup . 5 ± 11 × 10 . sup . 5 82 × 10 . sup . 3 ± 19 × 10 . sup . 3 & lt ; 51779 74 × 10 . sup . 5 ± 21 × 10 . sup . 5 2800 ± 480 & lt ; 5______________________________________ of the three caseinates used , calcium caseinate ( alanate 380 ) has a behavior to irradiation which differs from that of the two sodium caseinates ( alanates 110 and 180 ). various measurements of the rheological and physical chemical properties showed that : the breaking load ( f / e ratio ) of calcium caseinate ( alanate 380 ) is higher compared to the two sodium caseinates ( alanates 110 and 180 ). at 5 . 0 % p / p and 7 . 5 % p / p , the calcium caseinate ( alanate 380 ) has a higher f / e ratio than the two sodium caseinates ( alanates 110 and 180 ) for levels of 4 , 8 and 12 kgy . however , there is no direct relation between protein concentration and film resistance . indeed , for a concentration of 5 . 0 % p / p , the f / e ratio is higher than the one at 7 . 5 % p / p for the three caseinates . the only exceptions are for the two sodium caseinates ( alanates 110 and 180 ) irradiated at 4 kgy where the ratio is very slightly lower ( see tables 4 and 5 ). therefore , all things considered , a film produced with a greater quantity of proteins does not form obligatorily a more resistant film after irradiation . at 5 . 0 % protein , irradiation up to 12 kgy of the second sodium caseinate ( alanate 180 ) generates a significant reduction ( p δ 0 . 05 ) in the f / e ratio . this phenomenon is observed for the first sodium caseinate ( alanate 110 ) at a level of 4 kgy only . irradiation up to 12 kgy of calcium caseinate ( alanate 380 ) does not have a significant effect ( p & gt ; 0 . 05 ) on the f / e ratio ( see table 4 ). in the absence of glycerol , irradiation generates a significant reduction ( p δ 0 . 05 ) in the f / e ratio for the sodium caseinates ( alanate 110 ) and the calcium caseinates ( alanate 380 ) at a concentration of 7 . 5 %. irradiation up to 12 kgy does not have a significant effect ( p & gt ; 0 . 05 ) on the f / e ratio for the second sodium caseinate ( alanate 180 ) at 7 . 5 % ( see table 5 ). thus , irradiation up to 12 kgy does not generate a more resistant film for these three caseinates in the absence of plasticizing agents . at a 5 . 0 % concentration , the calcium caseinate ( alanate 380 ) produced a rate of bityrosine formation significantly higher ( p δ 0 . 05 ) than the two sodium caseinates ( alanates 110 and 180 ) when this caseinate is treated at levels of 4 , 8 and 12 kgy . on the other hand , at 7 . 5 % protein , the sodium caseinate ( alanate 110 ) formed significantly more ( p δ 0 . 05 ) bityrosine that the sodium caseinates ( alanates 180 ) and the calcium caseinates ( alanate 380 ) at levels of 4 and 12 kgy . at 8 kgy , the three caseinates formed a similar proportion of bityrosine ( see tables 8 and 9 ). however , the sodium caseinates ( alanate 180 ) and the calcium caseinates ( alanate 380 ) produced significantly more ( p δ 0 . 05 ) bityrosine at a 5 . 0 % protein concentration than at 7 . 5 % for levels of 4 , 8 and 12 kgy . the sodium caseinate ( alanate 110 ) produced more bityrosine at 5 . 0 % protein than at 7 . 5 % for levels of 8 kgy only ( see tables 8 and 9 ). then , would it be possible that the 5 . 0 % concentration would represent a zone where the rate of bityrosine formation would be a maximum and that the 7 . 5 % concentration would represent a point of saturation ? a much more thorough study should be made to validate this assumption . the cohesion force of a film , among others , is connected to its polymeric and chemical structure ( kester and fennema , 1986 ). the rate of bityrosine formation represents an important factor in the process of polymerization induced by the hydroxyl radicals ( davies , 1987 and davies et al ., 1987a ). thus , at a concentration of 5 . 0 % protein , the calcium caseinate ( alanate 380 ) shows , at the same time , a f / e ratio and a rate of bityrosine formation that is higher than the two sodium caseinates ( alanates 110 and 180 ). on the other hand , such a relation is not observed with a 7 . 5 % protein concentration for the three caseinates . on the other hand , irradiation up to 12 kgy has little or no significant effect ( p & gt ; 0 . 05 ) on the strain at failure of the three caseinates used for the two tested concentrations ( see tables 6 and 7 ). likewise , there was no occurrence of a regular and continuous tryptophan loss as a function of the level of irradiation during dosage by fluorescence . this situation was noticed for the three caseinates and at the two concentrations used ( see tables 10 and 11 ). in the absence of glycerol , it is possible that the gamma irradiation generates a proteinic denaturation which exposes the hydrophobic pockets on the surface of the protein . then , the relative stability of the signal fluorescence which is perceived during the tryptophan dosage , would be more likely explained by a greater quantity of tryptophan having migrated on the surface rather than the formation of new residues by the irradiation . compared to the results obtained in the absence of glycerol , for a treatment from 0 to 12 kgy , at a concentration of 5 . 0 % protein , the presence of 1 . 0 % glycerol significantly lowers ( p δ 0 . 05 ) the breaking load , increases the deformation ( approximately 0 . 4 mm ) and does not affect the rate of bityrosine formation ( see tables 12 , 14 and 17 ). on the other hand , for levels of 15 and 20 kgy , the load breaking shows f / e ratios which are comparable to those obtained in the absence of glycerol for a 0 treatment at 12 kgy . the deformation is increased ( approximately 0 . 6 mm ) compared to the results obtained in the absence of glycerol for the amounts varying between 0 and 12 kgy ( see tables 12 and 14 ). the rate of bityrosine formation is significantly higher p δ 0 . 05 ) compared to the absence of glycerol for levels of 15 and 20 kgy ( see table 17 ). at 2 . 5 % and 5 . 0 % glycerol with 5 . 0 % protein , the f / e ratio , the deformation , the viscoelasticity and the rate of bityrosine formation increase significantly ( p δ 0 . 05 ) with the increase in the level of irradiation ( see tables 12 , 14 , 16 and 17 ). in the presence of 2 . 5 % glycerol , irradiation made it possible to increase the f / e ratio by a factor of 2 . 2 and to increase the deformation by a factor of 1 . 3 , while at 5 . 0 % glycerol , irradiation made it possible to increase the f / e ratio by a factor of 1 . 6 and the deformation by a factor of 1 . 4 ( see tables 12 and 14 ). the viscoelasticity of films with 5 . 0 % glycerol is higher than that with 2 . 5 %. however , the handling of films with 5 . 0 % glycerol remains much more difficult ( see table 16 ). the fact of adding a greater quantity of glycerol ( 2 . 5 and 5 . 0 %) in the medium considerably reduced the resistance of film but improves greatly its deforming capacity . the glycerol does not seem to act like a radicalizing inhibitor . by its presence , it even seems to encourage bityrosine formation as a function of the level of irradiation . indeed , the presence of glycerol ( 1 . 0 %, 2 . 5 % or 5 . 0 %) significantly improves ( p δ 0 . 05 ) the rate of bityrosine formation for levels of irradiation equivalent or higher than 15 kgy for a 5 . 0 % concentration of protein ( see table 17 ). on the other hand , the reasons justifying the beneficial effect that the presence of glycerol produces on the rate of bityrosine formation are not shown . in the presence of 2 . 5 % glycerol and of 7 . 5 % proteins , irradiation ( 0 - 12 kgy ) generates a significant reduction ( p δ 0 . 05 ) in the f / e ratio , a significant increase ( p δ 0 . 05 ) in the deforming capacity and the rate of bityrosine formation compared to the results obtained in the absence of glycerol ( see tables 13 , 15 and 18 ). nevertheless , a radiative treatment up to 20 kgy does not have significant consequences ( p & gt ; 0 . 05 ) on the f / e ratio and the deformation of films formed with 7 . 5 % protein and 2 . 5 % glycerol ( see tables 13 and 14 ). at 5 . 0 % glycerol and 7 . 5 % protein , the f / e ratio , the deformation , the viscoelasticity and the rate of bityrosine formation increase significantly ( p δ 0 . 05 ) with the increase in the level of irradiation ( see tables 13 , 15 and 18 ). in the presence of 5 . 0 % glycerol , irradiation made it possible to increase the f / e ratio and the deformation by a factor of 1 . 5 ( see tables 13 and 15 ). the presence of glycerol also contributes to greatly reduce the resistance of film but greatly improves its deformation . in the presence of 5 . 0 % protein , the addition of glycerol does not inhibit the formation of bityrosine . quite to the contrary , the formation of bityrosine , as a function of the levels of irradiation ( 4 to 20 kgy ), is significantly higher ( p δ 0 . 05 ) ( see table 18 ). in the presence of 2 . 5 % glycerol , the f / e ratios at 5 . 0 % protein are lower than those at 7 . 5 % for levels of irradiation from 0 to 20 kgy . on the other hand , the deformations at proteins 5 . 0 % are significantly higher ( p δ 0 . 05 ) than those at 7 . 5 % for same the treatments ( see tables 12 to 15 ). at 5 . 0 % glycerol , the f / e ratios at 5 . 0 % protein are significantly lower ( p δ 0 . 05 ) than those at 7 . 5 % for levels which vary from 0 to 40 kgy , while the deformations at 7 . 5 % proteins are higher than those at 5 . 0 % for levels of 4 , 8 , 15 , 20 and 40 kgy . at 12 and 30 kgy , the deformations at 7 . 5 % protein are lower than those at 5 . 0 % ( see tables 12 to 15 ). the viscoelasticity of films with 5 . 0 % glycerol is significantly higher ( p δ 0 . 05 ) with proteins 5 . 0 % than at 7 . 5 % ( see table 16 ). the addition of 2 . 5 % glycerol with 7 . 5 % protein significantly increases ( p δ 0 . 05 ) the rate of bityrosine formation between the two protein concentrations except for the 15 kgy level . nevertheless , there is no direct relation between the protein concentration and the formation of bityrosine . thus , the rate of bityrosine formation is not proportional to the quantity of proteins present in the medium for the same level of irradiation ( see tables 17 and 18 ). a maximum f / e ratio is obtained at 30 kgy for the two protein concentrations with 2 . 5 % and / or 5 . 0 % glycerol . the deformation is highest between 20 and 30 kgy in the presence of 5 . 0 % protein and 2 . 5 % or 5 . 0 % glycerol . at 7 . 5 % protein with 5 . 0 % glycerol , the deformation is highest between 15 and 20 kgy . finally , viscoelasticity is at its maximum between 30 and 40 kgy for the two protein concentrations with 2 . 5 % and / or 5 . 0 % glycerol ( see tables 12 to 16 ). thus , a level of irradiation between 20 and 30 kgy seems to be an area where the tested mechanical properties are highest for these two concentrations of proteins and glycerol . the glycerol / protein ratio seems to be an important factor on the influence of irradiation on the mechanical , physical and chemical properties for the two protein concentrations with 2 . 5 % or 5 . 0 % glycerol . thus , a ratio of 0 . 5 ( 2 . 5 % glycerol / 5 . 0 % protein ) shows the strongest increase in the f / e ratio as a function of the irradiation levels whereas the weakest is perceived for a ratio of 0 . 33 ( 2 . 5 % glycerol / 7 . 0 % protein ). the strongest capacity of deformation during irradiation was noticed for a ratio of 0 . 67 ( 5 . 0 % glycerol / 7 . 5 % protein ) and the weakest was obtained for a ratio of 0 . 33 . finally , the progression obtained for viscoelasticity is appreciably the same one for the 0 . 5 ; 0 . 67 and 1 . 0 ratios . thus , the glycerol / protein ratios located between 0 . 5 and 0 . 67 seem to show the strongest variations of the rheological properties to irradiation . therefore , a radiation treatment is beneficial for the resistance of a film , its deforming capacity and bityrosine formation for the two protein concentrations with 2 . 5 % or 5 . 0 % glycerol . during the process of polymerization , all the polymeric chains are inter - connected and gathered in a gigantic network . if the number of points of contact is not too high , the network shows an appreciable elastic capacity . this recoverable deformation would be due to the presence of flexible junctions ( wunderlich , 1981 ). thus , a period of irradiation or an inadequate quantity of glycerol would concretely affect the structure of the protein network which , inevitably , would deteriorate the rheological properties of the film . the oxidation of a tryptophan solution by the hydroxyl radicals is directly connected to the loss of intensity of the fluorescence signal ( davies et al ., 1987a and see fig6 ). in absence of glycerol , there is no tryptophan loss during dosages of the irradiated caseinate solutions . at the opposite side , a loss of the signal is perceived when glycerol is present in the treated mediums . at 5 . 0 % protein , the loss of the signal in the presence of glycerol is significant ( p δ 0 . 05 ) from 4 to 20 kgy whereas at 7 . 5 % protein , it is significant ( p δ 0 . 05 ) for levels of 12 , 15 and 20 kgy ( see tables 19 and 20 ). the presence of glycerol tends to privilege the native or folded up state of a globular protein rather than a denatured state ( gekko and timasheff , 1981 ). thus , only the tryptophan located on the surface of the protein will be affected during irradiation . all in all , the loss of intensity of the signal at 40 kgy compared to 0 kgy varies from 30 to 40 % for the two protein concentrations . in the presence of 5 . 0 % proteins and 5 . 0 % glycerol , a better protection against the tryptophan loss is observed compared to 2 . 5 % or 1 . 0 % glycerol , whereas between 2 . 5 % and 1 . 0 % glycerol , the proportion of signal loss as a function of the level of irradiation is roughly the same . on the other hand , at 7 . 5 % protein , the loss of signal intensity as a function of the level of irradiation in the presence of 5 . 0 % glycerol is proportionally higher than when only 2 . 5 % glycerol is present ( see tables 19 and 20 ). the addition of 2 . 5 % glycerol concentration shows a greater resistance to tryptophan loss compared to a solution containing 5 . 0 % protein levels of irradiation varying from 0 to 20 kgy . at 5 . 0 % glycerol , the reverse situation arises ; however , for the 30 and 40 kgy levels , the loss becomes slightly higher with 5 . 0 % protein than 7 . 5 % ( see tables 19 and 20 ). essentially , there is no direct relation between the loss of the signal and the glycerol content as a function of the level of irradiation for the two studied protein concentrations . it is difficult to establish a glycerol / protein ratio for which protection against tryptophan loss as a function of the level of irradiation would be maximized . for a 5 . 0 % protein concentration a ratio of 1 . 0 is most adequate whereas for a 7 . 5 % protein concentration , a ratio of 0 . 33 is more adequate . the presence of glycerol strongly modifies the physical and chemical properties of calcium caseinate films ( alanate 380 ). it tends to decrease the force at rupture , it increases the strain at failure , it improves viscoelasticity , it does not inhibit the formation of bityrosine and it protects protein from radiation denaturation . generally , maximum bacterial growth is quickly reached when p . fragi is in the presence of the film . for the three tests , a maximum of approximately 107 ufc / ml is reached within a time of 60 to 80 hours after the removal of the mediums . a downward trend of the population is noticed after the maximum is reached except for the third test , where the population tends to increase after 1200 hours of agitation . for the last two tests in the absence of film , the bacterial population requires a latency time of approximately 24 hours before starting to grow . a maximum of 106 ufc / ml is reached after about one hundred hours of agitation and in both cases , the population decreases in an obvious way thereafter . with the single presence of film , the population remained & lt ; 5 ufc / ml for the duration of the experimentation and on the three tests . other films containing caseinate were manufactured by taking the same protocols as in the preceding examples with the flowing modifications . alanate 380 was solubilized at a rate of 5 % p / p in a tris - hcl buffer 1 mm with ph 8 . 0 . the added plasticizing agents were propylene glycol ( pg ) and triethylene glycol ( teg ) at 0 , 2 . 5 % and 5 % p / p concentrations . the average flow of irradiation was 1 . 5 kgy / h for levels of 8 , 16 , 32 , 64 , 96 and 128 kgy . calcium chloride was added after the irradiation at 0 , 0 . 125 and 0 . 25 % p / p concentrations . the best films obtained were made of 5 % caseinate / 2 . 5 % pg and 5 % caseinate / 2 . 5 % teg ( amounts lower than 32 kgy ), the first having a higher breaking load and the second being more viscoelastic . calcium seems to increase the cohesion force of film without affecting the strain at failure . one can add polysaccharides to calcium caseinate films / plasticizing agents . for example , the addition of carboxymethyl cellulose ( cmc ) gives a rigid film ( total composition 5 % alanate 380 / 2 . 5 % glycerol / 0 . 25 % cmc ). this film is made more viscoelastic if one adds a plasticizing agent supplement like 2 . 5 % sorbitol . the cmc are added after irradiation to avoid a precipitation . the resistance and viscoelasticity properties of a caseinate film can thus be modified at will by the addition of other components ( calcium , polysaccharides and plasticizing agents ( polyethylene , propylene and triethylene glycols , glycerol and sorbitol ). the best mechanical properties are obtained with ratios of 0 . 5 to 0 . 67 plasticizing agent / protein to levels of approximately 30 kgy . when one adds the peg as a plasticizing agent , concentrations lower than 1 % are preferred to avoid the formation of heterogeneous films . the addition of cacl 2 ( approximately 0 . 125 % w / w ) to the solution with three components ( above ) increases the formation of bityrosine and the breaking load . the caseinate films are formed at irradiation levels equal or higher than 16 kgy . the maximum force of films is obtained at 64 kgy . with higher amounts , protein degradation seems to overcome the formation of bityrosine . at 64 kgy , the presence of cacl 2 has little influence on the breaking load in the presence of absence of mannitol or sorbitol . peg decreases the breaking load in the presence of cacl 2 . peg seems to inhibit the formation of electrostatic bonds and between salts . sorbitol is the preferred plasticizing agent since it increases viscoelasticity the most . one of the preferred formulations is 5 % alanate 380 / 2 . 5 % sorbitol / 0 . 25 % cmc / 0 . 125 % cacl 2 , combining force of cohesion and viscoelasticity . the amount of optimal irradiation is located between 32 and 64 kgy . adams , d . m . ; 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