Anova Active packaging by extrusion processing of recyclable and biodegradable polymers

Active packaging by extrusion processing of recyclable and biodegradable polymersM.A.Del Nobile a,b,*,A.Conte a,b ,G.G.Buonocore c ,A.L.Incoronato b ,A.Massaro c ,O.Panza baIstituto per la Ricerca e le Applicazioni Biotecnologiche per la Sicurezza e la Valorizzazione dei Prodotti Tipici e di Qualità,BIOAGROMED,Via Napoli 52,71100Foggia,Italy bDepartment of Food Science,University of Foggia,Via Napoli 25,71100Foggia,Italy cInstitute of Composite and Biomedical Materials,National Research Council,P.le Enrico Fermi,Granatiello,Portici,80055Napoli,Italya r t i c l e i n f o Article history:Received 4September 2008Received in revised form 12December 2008Accepted 19December 2008Available online 31December 2008Keywords:Active packagingBiodegradable material ExtrusionNatural compounds Recyclable materiala b s t r a c tIn this work a preliminary study on the suitability of environmentally friendly active films to be produced by melt extrusion process is presented.In particular,low density polyethylene (LDPE),polylactic acid (PLA)and polycaprolactone (PCL)were used as environmentally friendly polymeric matrices,with the former being recyclable and the latter two biodegradable.Lemon extract,thymol,and lysozyme at three different concentrations were incorporated in each polymeric matrix,respectively,to have a total of nine active films.Results suggest that processing temperatures play a major role in determining the antimi-crobial efficiency of the investigated active films.In particular,antimicrobials incorporated into PLA and LDPE showed retained slight antimicrobial activity,thus having lost some activity due to higher pro-cessing temperature.On the other hand,PCL was processed at a lower temperature,which allowed less degradation of antimicrobial activity.Among the natural compounds under study lysozyme showed the higher thermal stability.Ó2009Elsevier Ltd.All rights reserved.1.IntroductionAntimicrobial food-packaging systems are polymeric materials in which a certain amount of additives with pronounced antimi-crobial properties are embedded into the packaging plastic con-tainer (rigid or flexible)and act with the aim of extending foodstuff shelf life (Appendini and Hotchkiss,2002;Cutter,2002a;Suppakul et al.,2003).Furthermore,consumers are demanding food-packaging materials that are more natural,dis-posable,potentially biodegradable,as well as,recyclable (Cha and Chinnan,2004;Lopez-Rubio et al.,2004).For this reason,there is an urgent need to study and to develop renewable source-based biopolymers able to degrade via a natural composting process.Therefore,it is evident that the interest in active food-packaging systems in which the polymer matrix has low environmental im-pact is growing significantly (Jin and Zhang,2008).As far as the polymer matrix is concerned,an interesting biode-gradable polymer is polylactic acid (PLA),which is made primarily from renewable agricultural resources (corn)following fermenta-tion of starch and condensation of lactic acid.It exhibits mechani-cal and physical properties comparable to other commercially available polymers (Holbert and Taylor,1997;Krishnamurthy et al.,2004).Other biodegradable polymers can be derived from petroleum sources or may be obtained from mixed sources of bio-mass and petroleum.The best-known petroleum source-derived biodegradable polymers are aliphatic polyester or aliphatic–aro-matic co-polyesters.Unlike other petrochemical-based resins,in appropriate environmental conditions,these synthetic polyesters break down rapidly into carbon dioxide,water and humus.Exam-ples of petroleum-based biodegradable polymers include poly(butylene succinate),aliphatic polyesters,poly(e -caprolac-tone),and poly(vinyl alcohol)(Jarerat and Tokiwa,2001).Particu-larly attractive from a packaging point of view is polycaprolactone (PCL),a linear-polyester manufactured by ring-opening polymerization of e -caprolactone,with a rather low glass transition temperature and melting point (65°C).Its physical prop-erties and commercial availability make it very attractive not only as a substitute material of no-degradable polymers but also as a specific plastic for food and agricultural applications (An et al.,2001;Sanchez et al.,2000).As far as natural antimicrobial compounds are concerned,sev-eral substances such as organic acids,bacteriocins (nisin and lacti-cin),grape seed extracts,spice extracts (thymol,p-cymene and cinnamaldehyde),lemon seed extracts,enzymes (peroxidase and lysozyme),chelating agents (EDTA),metals (silver),or parabens (heptylparaben)have been reported to be able to inhibit or avoid bacterial growth in solution,on culture media,or on a variety of foods (Conte et al.,2007;Cutter,2002b;Devlieghere et al.,2000;Falcone et al.,2005;Han,2000).In the field of food packaging,antimicrobial biodegradable films are generally obtained by using the solvent casting technique:Sebastien et al.(2006)propose a chitosan-loaded PLA film having high inhibitory properties against mycotoxinogen fungal strains.0260-8774/$-see front matter Ó2009Elsevier Ltd.All rights reserved.doi:10.1016/j.jfoodeng.2008.12.022*Corresponding author.Address:Department of Food Science,University of Foggia,Via Napoli 25,71100Foggia,Italy.Tel.:+39881589242;fax:+39881740211.E-mail addresses:ma.delnobile@unifg.it ,delnobil@unina.it (M.A.Del Nobile).Journal of Food Engineering 93(2009)1–6Contents lists available at ScienceDirectJournal of Food Engineeringjournal homepage:www.elsevi e r.c o m /l o c a t e /j f o o d e ngAnother interesting technique used to obtain bioactive materials is the modification of the surface composition of the polymer.Scan-nel et al.(2000)studied the surface immobilization of bacteriocins (such as nisin)on polyethylene/polyamide materials.However, from an industrial point of view,activefilms obtained by using classical polymer technological processes(such as extrusion)are generally preferred.Despite this great interest,in the literature very few works are reported dealing with the development of anti-microbial biodegradablefilms obtained by means of extrusion pro-cesses(Nam et al.,2007).This is mainly due to the fact that most antimicrobial activity deterioration can occur during the extrusion using high temperature,high shear rates and thus,high pressure (Brody et al.,2001).During the process,in fact,the high tempera-ture and pressure in the extruder barrel can affect the chemical stability of the embedded antimicrobial compounds(which gener-ally are heat sensitive and thermally unstable)and reduce their efficacy on food-borne spoilage microorganisms.In this study the effect of manufacturing conditions on the anti-microbial effectiveness of environmentally friendly melt extruded polymericfilms loaded with natural compounds has been ad-dressed by means of in vitro tests.In particular,LDPE,PLA and PCL were used as low environmental impact polymeric matrices, the former being recyclable(Davis et al.,2004)and the latter two biodegradable(Petersen et al.,1999;Sanchez et al.,2000); whereas,lemon extract,thymol and lysozyme were used as anti-microbial compounds.2.Materials and methods2.1.MaterialsIn this study three different polymeric matrices(PLA,LDPE and PCL)and three different natural substances(lysozyme,thymol and lemon extract)were used to produce the tested activefilms.Nature Works TM PLA was purchased from Cargill Dow LLC(USA).LDPE Rib-lene FL30was kindly supplied by Polimeri Europa(Italy).PCL CAPA 6800was purchased from Solvay(UK).Lysozyme and thymol were supplied by Sigma–Aldrich(Milan,Italy).Lemon extract was pur-chased from Spencer Food Industrial(Amsterdam,Netherlands).2.2.Film preparationThe activefilms were prepared through a three-step process. Thefirst step consists of melt blending the polymeric matrix(LDPE, PLA and PCL)with one of the natural substance(i.e.,lysozyme,thy-mol and lemon extract)by using an internal mixer(RheomixÒ600 Haake,Germany)controlled by a measuring drive(Haake Rheo-cordÒ9000).The mixing chamber(volume of50cm3)wasfilled with50g total mass.The rotation speed and mixing time were 20rpm and5min for all tests.The mixing temperature was 155°C,140°C and80°C for PLA,LDPE and PCL,respectively.In the second step a P300P hot press(Collin,Germany)was used to prepare slabs with thickness of1mm.Materials were heated at the same temperature of mixing,pressed at50bar for3min and subsequently cooled to30°C under pressure.The slabs were cut into short pieces in order to obtain a material suitable for extru-sion.Finally,the active antimicrobialfilms were obtained by feed-ing the pellets into a co-rotating laboratory twin-screw extruder (PRISM EUROLAB16,TERMO Electron Corporation)equipped with a sheet die of width10cm.The extruder barrel consisted of seven zones(total length=40cm);each zone is equipped with an inde-pendent temperature control based on resistive heaters.The blends were fed with a single-screw volumetric feeder.Rotation rate of the feeder wasfixed at5rpm,whereas the extruder screw speed was55rpm for all materials.The temperature of the feeding zone,the intermeshing zones and thefinal zone of the extruder were maintained at135–150–140°C,110–135–130°C and80–115–110°C for PLA,LDPE and PCL,respectively.Different amount of antimicrobial compounds were added to PLA,LDPE and PCL in order to obtain the followingfilms:–PLA containing3%,5%and10%(w/w)of lysozyme.–PLA containing7%,10%and15%(w/w)of thymol.–PLA containing3%,5%and7%(w/w)of lemon extract.–LDPE containing3%,5%and10%(w/w)of lysozyme.–LDPE containing7%,10%and15%(w/w)of thymol.–LDPE containing3%,5%and7%(w/w)of lemon extract.–PCL containing3%,5%and10%(w/w)of lysozyme.–PCL containing7%,10%and15%(w/w)of thymol.–PCL containing3%,5%and7%(w/w)of lemon extract.As controls,films made up of PLA(Tg56°C and Tm160°C), LDPE(Tm110°C)and PCL(TgÀ60°C and Tm60°C)without any active compounds were also prepared.2.3.Antimicrobial activity offilms containing lysozymeIn order to determine the antimicrobial activity of the poly-meric material containing lysozyme,Micrococcus lysodeikticus was used as test microorganism,due to its high susceptibility to the active enzyme.As reported by Appendini and Hotchkiss (1997),lysozyme activity can be determined by measuring the de-crease in absorbance of the incubated bacterium in phosphate buf-fer.A suspension of lyophilized M.lysodeikticus cells(Sigma–Aldrich,Milan,Italy)were incubated at room temperature in 610ml of buffer(0.1M,pH6.8),to reach a cell concentration of 107cfu/ml.PCL-based,LDPE-based and PLA-basedfilms were brought in contact with the obtained microbial suspension,respec-tively(volume-to-area ratio2ml/cm2).The absorbance at450nm (Spectophotometer UV1601,Shimadzu,Germany)of the above suspensions,continuously stirred(IKA KS130control,GMBH,Ger-many),was monitored until a constant value was reached.The de-crease in absorbance of a microbial suspension without anyfilm and that of a suspension in contact with lysozyme-freefilm were also measured as controls.All the analyses were carried out twice.2.4.Antimicrobial activity offilms containing lemon extract and thymolIn order to evaluate the effectiveness of thefilms containing lemon extract and thymol,turbidimetric experiments were con-ducted(Falcone et al.,2005;Han et al.,2007).Evidence of micro-bial growth was acquired by reading the absorbance changes at 420nm(Falcone et al.,2005)by using a spectrophotometer(UV 1601,Shimadzu,Germany)at regular intervals.Afive-strain cock-tail of Pseudomonas spp.,isolated from spoiled mozzarella cheese, was used as test microorganism and Plate Count Broth(PCB)(tryp-tone5g/l,glucose1g/l and yeast extract2.5g/l)was used as cul-ture medium(Falcone et al.,2005).Each strain wasfirst grown on plates of Pseudomonas Agar Base(PAB)at25°C for48h and then in PCB broth at the same temperature for another2days. Equal volumes of microbial suspension of each strain were com-bined and,after dilutions in culture medium,the cocktail was inoc-ulated in predefined volumes of PCB broth to perform the turbidimetric measurement.The volumes of PCB broth were calcu-lated on the basis of the total area of each piece offilm(1Â1cm). Thefinal concentration of microbial cells was about105cfu/ml. Each inoculated medium was put in contact with active PCL-based, PLA-based and LDPE-basedfilm(1Â1cm),respectively(volume-to-area ratio2ml/cm2).The decrease in absorbance of a microbial suspension without anyfilm and that of a suspension in contact2M.A.Del Nobile et al./Journal of Food Engineering93(2009)1–6with a compound-free film were also measured as controls.All the prepared samples were incubated at 25°C,continuously stirring at a moderate speed by a stirrer (IKA KS 130control,GMBH,Ger-many).Periodically,1ml aliquot was drawn from each sample to measure the absorbance.All the analyses were carried out twice.2.5.Statistical analysisTo determine whether significant differences (p <0.05)existed among the mean values of the fitting parameters,the one-way analysis of variance (ANOVA)and Duncan’s multiple range test with the option of homogeneous groups were used by means of STATISTICA 7.1for Windows (StatSoft,Inc.,Tulsa,OK,USA).The best fit was evaluated by means of the Root Mean Square Error (RMSE),which is defined as:RMSE ¼ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi1n obs X n obs i ¼1ðy i Ày p i Þ2v u u t where n obs is the number of observations and y i and y p i ,i =l,...,areexperimental and the predicted values,respectively.The lower the value of RMSE ,the better the ability of the model to fit the experi-mental data.3.Results and discussionThe effect of manufacturing conditions on the antimicrobial effectiveness of melt extruded LDPE,PCL and PLA films loaded with natural compounds has been addressed by means of in vitro tests.Results are presented in the following,separately for each antimi-crobial compound under study.3.1.Lemon extract loaded active filmA cocktail of Pseudomonas spp.was used as target microorgan-ism to assess the antimicrobial effectiveness of lemon extract re-leased from the investigated polymeric matrices.The main reason for choosing this microbial group has to be ascribed to the fact that the Pseudomonadaceae are considered food-borne bacteria (Bishop and White,1986).Moreover,several studies in broth model systems have revealed the bacteriostatic or bacterici-dal activity of essential oils against spoilage or pathogenic bacteria (Dorman and Deans,2000;Lambert et al.,2001).Fig.1a shows the pseudomonads cell load plotted as a function of time for the inoc-ulated solution itself (control),for the inoculated solution in con-tact with the active compound-free film (control)and for the lemon extract loaded LDPE film.The curves shown in Fig.1a were obtained by fitting the Gompertz equation as re-parameterized by Corbo et al.(2006)to the experimental data:NA ðt Þ¼NA 0ÀA Áexp Àexp ðl max Á2:71ÞÁkAþ1 þA Áexp Àexp ðl max Á2:71ÞÁk ÀtAþ1ð1Þwhere NA (t )is the normalized absorbance at time t ,NA 0is the initial value of normalized absorbance,A is related to the difference be-tween the maximum normalized absorbance attained at the sta-tionary phase and its initial value,l max is the maximal growth rate,k is the lag time,and t is the time.Results from fitting process are listed in Table 1,along with the RMSE values that indicate the best fit.As can be inferred from data shown in Fig.1a,Eq.(1)satis-factorily describes the trend of the experimental data,suggesting that lemon extract loaded LDPE films do not change,to a great ex-tent,the growth kinetic of the target microorganisms,if compared to the controls.Data listed in Table 1also corroborate the abovehypothesis.In fact,no statistically significant differences were found between the model parameter values.This result is most probably due to the relatively high process temperature used to ex-trude the active LDPE film.The temperature can promote either lemon extract inactivation or antimicrobial compound volatiliza-tion,being both reasons a possible cause of antimicrobial ineffi-ciency of the active films (Brody et al.,2001).Unfortunately,it is not possible to establish which of the above-mentioned factors is more responsible for film ineffectiveness.In fact,the analytical determination of residual concentration of the antimicrobial used after the film formation would not spread light on this concern as lemon extract is a complex mixture of natural compounds,and it is not easy to explain which of its components acts as antimicrobial agent (Baldi et al.,1995;Belletti et al.,2004;Tovar et al.,2005).Fig.1b shows the pseudomonads growth cycle for controls and for lemon extract loaded PLA films.Also in this case,Eq.(1)was satisfactorily fitted to the experimental data;results are also listed in Table 1.As can be seen in Fig.1b,the behaviour of lemon extract loaded PLA films do not differ considerably from that of the two control samples.In fact,no statistically significant difference is observed between the values of model parameters,withtheFig.1.(a)Effectiveness of LDPE-based films with and without lemon extract.(b)Effectiveness of PLA-based films with and without lemon extract.(c)Effectiveness of PCL-based films with and without lemon extract.The curves result from fitting the Eq.(1)to the experimental data.M.A.Del Nobile et al./Journal of Food Engineering 93(2009)1–63exception of A parameter,showing a slightly lower value for PLA7%sample.The similar processing temperatures of PLA and LDPE sup-ports the idea that the extrusion may affect the antimicrobial effi-ciency of lemon extract due to either lemon extract inactivation or antimicrobial compound volatilization.Fig.1c shows the pseudomonads cell load plotted as a function of time for the control samples and for lemon extract loaded PCL films.As above,Eq.(1)was successfully fitted to the experimental data.Also in this case results are listed in Table 1.As shown in Fig.1c,a substantial difference in the growth kinetic can be ob-served between the control samples and the lemon extract loaded films.In particular,the active films seem to prolong the lag phase.Moreover,the lag phase increases as the active compound concen-tration into the polymeric matrix increases.A small decrease in the A value can be also observed in the case of PCL7%sample.The re-sults corroborate the hypothesis that processing temperature plays a major role in determining the antimicrobial effectiveness of the lemon extract loaded plastic film.In fact,PCL is processed at tem-perature considerably lower than that used to process both LDPE and PLA,thus reducing either the risk of compound inactivation or the amount of lemon extract volatilized (Han et al.,2007).3.2.Thymol loaded active filmFig.2a and b shows the pseudomonads growth cycle for thymol loaded LDPE and PLA films,respectively.The LDPE and PLA control samples are also shown in the same figures.Once again,the Eq.(1)was satisfactorily fitted to the experimental data.Results are listed in Table 2.As can be inferred from Fig.2and Table 2,the active films do not markedly change the growth cycle of the selected microbial strains.In fact,differences in the model parameter values are small,and only in few cases they are statistically significant.As for lemon extract,also in this case the processing temperature seems to be the main responsible factor for the antimicrobial inef-ficacy of the above active film,most probably due to either its inac-tivation or volatilization (Brody et al.,2001;Han et al.,2007).For instance,the high volatility of this active compound was also con-sidered the main reason of the reduced effectiveness of thymol fumigation treatments on vegetables by increasing the tempera-ture from 50°C to 70°C (Burt,2004).Fig.2c shows the kinetic of the microbial turbidity for the con-trol samples and for the thymol incorporated in PCL matrix.Eq.(1)has been successfully fitted to the experimental data;results areTable 1Parameter values obtained by fitting Eq.(1)to the experimental data on polymeric films containing lemon extract,along with the relative RMSE values.Each mean value was reported along with its standard deviation.Lemon extractNA 0Al max kRMSE PCL 3%0.97A ±0.0333.5F ±0.5620.4ABC ±18.415.0B ±5.40.345% 1.0A ±0.0334.2F ±0.6 3.7A ±0.229.6D ±0.08 1.27% 1.1A ±0.0131.7E ±1.215.8AB ±39.859.4E ±3.120.63Lemon-free film 1.1A ±0.00833.5F ±0.1136.8C ±0.7320.7C ±0.070.58Control 0.99A ±0.00736.1G ±0.1334.0BC ±5.120.6C ±0.930.63PLA 3%0.88A ±0.2624.2B ±0.301.7A ±0.127.7A ±1.3 1.85%0.92A ±0.7124.5B ±0.821.9A ±0.437.6A ±3.80.547%0.87A ±0.3721.8A ±0.421.7A ±0.227.5A ±2.20.28Lemon-free film 0.78A ±0.624.4B ±0.711.4A ±0.177.2A ±2.30.46Control 0.95A ±0.4424.0B ±0.551.6A ±0.318.6A ±3.20.38LDPE 3%0.95A ±0.3328.2D ±0.402.7A ±0.4610.7AB ±2.20.805%0.98A ±0.2725.8C ±0.353.3A ±1.214.3B ±3.20.257%0.91A ±0.1926.5C ±0.222.3A ±0.1610.5AB ±0.960.13Lemon-free film 0.92A ±0.6726.0C ±0.793.0A ±1.010.2AB ±4.30.45Control 0.91A ±0.5228.4D ±0.603.1A ±0.4810.0AB ±2.00.34Data in column with different letters are significantly different (p <0.05).Fig. 2.(a)Effectiveness of LDPE-based films with and without thymol.(b)Effectiveness of PLA-based films with and without thymol.(c)Effectiveness of PCL-based films with and without thymol.The curves result from fitting the Eq.(1)to the experimental data.Table 2Parameter values obtained by fitting Eq.(1)to the experimental data on polymeric films containing thymol,along with the relative RMSE values.Each mean value was reported along with its standard deviation.Thymol NA 0A l maxkRMSE PCL7%0.98A ±0.0327.5F ±0.61 2.0A ±0.4911.0BC ±2.20.4810% 1.0A ±0.0417.8A ±0.43 1.0A ±0.08 5.3A ±0.26 2.215%1.0A ±0.0322.0C ±0.46 1.1A ±0.05 6.1AB ±0.280.76Thymol-free film 0.98A ±0.0328.0FG ±0.771.8A ±0.4010.2ABC ±1.91.0Control 1.0A ±0.0327.5F ±0.61 2.2A ±0.9110.4ABC ±2.70.57PLA 7%1.0A ±0.0328.4FG ±0.702.3A ±0.7211.4BC ±2.30.4710% 1.0A ±0.0328.8G ±0.61 6.8A ±18.313.0C ±3.70.2415%0.9A ±0.0326.0E ±0.52 3.7A ±9.110.6ABC ±3.50.45Thymol-free film 0.83B ±0.0320.6B ±0.4 1.7A ±0.4711.2BC ±2.50.68Control 0.98A ±0.0327.6F ±0.69 2.1A ±0.7210.4ABC ±2.40.57LDPE 7%1.0A ±0.0421.0B ±0.41 5.1A ±7.18.6ABC ±4.22.210% 1.0A ±0.0424.0D ±0.57 1.4A ±0.087.8ABC ±0.4 1.815%1.0A ±0.0323.4D ±0.50 3.4A ±5.311.9C ±3.10.97Thymol-free film 1.0A ±0.0420.7B ±0.511.5A ±0.299.0ABC ±1.63.1Control1.0A ±0.0418.0A ±0.417.51A ±8.7011.8C ±5.61.5Data in column with different letters are significantly different (p <0.05).4M.A.Del Nobile et al./Journal of Food Engineering 93(2009)1–6also listed in Table 2.The kinetic data provided further support to the hypothesis that the low processing temperature used for pro-ducing active PCL films does not compromise completely the thy-mol antimicrobial effectiveness.In fact,for both PCL10%and PCL15%a statistically significant decrease in the A value has been observed,if compared to the film without antimicrobial agent.However,the efficacy of these active films was found substantially different from that of the same polymeric matrix loaded with lem-on extract,most probably due to the superior resistance to inacti-vation or volatilization of the citrus compound.3.3.Lysozyme loaded active filmFig.3a and b shows the M.lysodeikticus viable cell concentration plotted as function of time for lysozyme incorporated in LDPE and PLA samples,respectively.For the sake of comparison,the control samples are also shown in the same figures.It is well known that lysozyme is effective in increasing the death kinetic of M.lys-odeikticus (Appendini and Hotchkiss,1997).Therefore,a possibleantimicrobial inefficacy of films loaded with this compound could be attributed to the manufacturing process.Considering that mon-itoring the death of M.lysodeikticus lysozyme antimicrobial activity was assessed,Eq.(1)has been modified accordingly.Moreover,the same expression has been re-parameterized in such a way to intro-duce a new parameter directly related to the antimicrobial effec-tiveness of lysozyme loaded film,t *,which is defined as the time at which the NA (t )decreases by 70%of its initial value:NA ðt Þ¼0:3þA Áexp Àexp ðl max Á2:71ÞÁk Àt ÃAþ1ÀA Áexp Àexp ðl max Á2:71ÞÁk Àtþ1ð2Þwhere A is related to the difference between the initial value of nor-malized absorbance and its minimum value attained at the station-ary phase,l max is the maximal death rate,k is the lag time,and t is the time.As can be inferred from the data shown in Fig.3,Eq.(2)satisfactorily describes the trend of the experimental data.The re-sults from fitting process are listed in Table 3.Data provided sug-gest that lysozyme can withstand either the LDPE or PLA manufacturing process.In fact,both LDPE and PLA-based active films show markedly different behaviours,if compared to the respective controls.The data listed in Table 3also corroborate the above observation,being the t *value of the active films at least one order of magnitude lower than that obtained for control sam-ples.It is noteworthy that a slight difference has been observed be-tween LDPE and PLA films in terms of antimicrobial effectiveness.In particular,the results suggest that LDPE is slightly more efficient in promoting M.lysodeikticus death,if compared to PLA-based active film.Moreover,a small increase in film antimicrobial effectiveness with the increase of the active compound concentration has been also observed for both LDPE and PLA polymeric systems.Contrary to what was observed for both lemon extract and thymol loaded films,lysozyme loaded LDPE and PLA films exert an antimicrobial action against M.lysodeikticus .This may be due to either a higher thermal stability of lysozyme or to the fact that lysozyme is a non-volatile compound,because of its high molecular weight (Nam et al.,2007);therefore,it is reasonable to assume that its residual concentration after the film formation is not affected by the processing conditions,as in the case of lemon extract and thymol.Fig.3c shows the M.lysodeikticus concentration plotted as a function of time for control samples and for lysozyme loaded PCL film.As above,Eq.(2)was successfully fitted to the experimental data;results are also listed in Table 3.As can be inferred from data shown in Fig.3c and listed in Table 3,the lysozyme loaded PCL film strongly enhanced the rate of destruction of the selected microor-ganism.In this case a t *value two order of magnitude lower than that measured for the control samples has been observed.As above,a small decrease in the t *value has been found with the in-crease of lysozyme concentration.However,these differences are not statistically significant.The results obtained for lysozyme loaded films further support the idea that extrusion processing strongly influences the efficiency of the investigated active films.In fact,being lysozyme a non-volatile compound,it is reasonable to assume that the lower effectiveness of PE and PLA antimicrobial films is strictly related to the thermal inactivation of lysozyme,if compared to active PCL films (Nam et al.,2007).4.ConclusionsThe results provided in this work showed that antimicrobial films might be supplied commercially for packaging applications.In particular,PCL-based material incorporated with lysozyme seems to be the most interesting polymeric matrix.ThepromisingFig. 3.(a)Effectiveness of LDPE-based films with and without lysozyme.(b)Effectiveness of PLA-based films with and without lysozyme.(c)Effectiveness of PCL-based films with and without lysozyme.The curves result from fitting the Eq.(2)to the experimental data.M.A.Del Nobile et al./Journal of Food Engineering 93(2009)1–65。