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Supercritical impregnation of food packaging films to provide antioxidant properties
Accepted Manuscript Title: Supercritical Impregnation of food packaging films to provide antioxidant properties Authors: C. Cejudo Bastante, L. Casas Cardoso, C. Mantell Serrano, E.J. Mart´ınez de la Ossa PII: DOI: Reference:
S0896-8446(17)30225-5 http://dx.doi.org/doi:10.1016/j.supflu.2017.05.034 SUPFLU 3946
To appear in:
J. of Supercritical Fluids
Received date: Revised date: Accepted date:
28-3-2017 31-5-2017 31-5-2017
Please cite this article as: C.Cejudo Bastante, L.Casas Cardoso, C.Mantell Serrano, E.J.Mart´ınez de la Ossa, Supercritical Impregnation of food packaging films to provide antioxidant properties, The Journal of Supercritical Fluidshttp://dx.doi.org/10.1016/j.supflu.2017.05.034 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Supercritical Impregnation of food packaging films to provide antioxidant properties C. Cejudo Bastante*, L. Casas Cardoso, C. Mantell Serrano, E.J. Martínez de la Ossa Chemical Engineering and Food Technology Department, Wine and Agrifood Research Institute (IVAGRO), University of Cadiz Avda. República Saharaui, s/n, 11510 - Puerto Real, Cádiz (Spain) *corresponding author: Cejudo Bastante, Cristina. [email protected] Graphical abstract
Highlights: Antioxidant films for an alimentary use have been obtained by SSI. SSI is an intricate process and all the variables involved need to be studied. Impregnation yield increased when olive leaf extract was used as active substance.
ABSTRACT Among the different techniques employed to develop active packaging, supercritical solvent impregnation (SSI) of natural extracts is an innovative approach. When developing a method to obtain polyethylene terephthalate/polypropylene (PET/PP) films with antioxidant capacity, several parameters (pressure, temperature, depressurization rate, presence of modifier and time) were studied with caffeic acid as a model substance. The best conditions were applied to an olive leaf extract as a natural extract with antioxidant properties. Antioxidant activity was evaluated by the
DPPH assay and the total loading was calculated to identify the best impregnation conditions. The results revealed that a low level of impregnation provide antioxidant films, both with the standard and the natural extract. Better results were obtained when the natural extract was used. To confirm the presence of the antioxidant particles and to determine their distribution on the matrix, the impregnated samples were studied by Scanning Electron Microscopy (SEM).
Keywords: Olea europea, caffeic acid, supercritical impregnation, supercritical fluids, active packaging, antioxidant films.
1. INTRODUCTION The preservation of food has been widely studied because of its importance in the food industry. Together with edible films and coatings, active packaging is one of the most innovative alternatives for food preservation because it maintains the nutritional and organoleptic quality of food and improves the security parameters. Active packaging is a system that interacts positively with the product and extends its shelf-life due to the action of an active agent included on the packaging material or forming part of the polymer itself [1]. The active agents function by modifying the head-space of the packaging by absorption or release of compounds, or by modifying the composition on the surface of the food [2-4], and they could impart antimicrobial or antioxidant activities to the packaging, amongst other properties. Of the different substances used in active packaging [5], essential oils have been widely studied as active compounds in both coatings and active packaging in recent years due to their antioxidant, antifungal and antimicrobial activity [6, 7]. These properties have been widely demonstrated in polyphenols, which are present in a variety of vegetable sources [8]. Such compounds have also been included in polymer processing to
improve certain properties of these materials and good results have been obtained [9]. Although standards of these compounds have been widely used as active substances [10-12], extracts from natural sources such as plants, spices, seeds and leaves from pruning waste from different plants have also been studied [13-16]. Some of these materials, despite being rich in essential oils and polyphenols, are sometimes discarded by industry. As a consequence, the use of these materials for another purpose gives them added value. In this context, extracts from olive leaves have been used for food packaging applications in recent years [17-20] because compounds like oleuropein, phenolic acids, such as caffeic acid, p-coumaric acid, sinapic acid, ferulic acid and syringic acid, and flavonoids, such quercetin, hesperetin, luteolin and rutin, possess high antioxidant and antimicrobial capacities and also have potential health benefits [21-24]. These compounds are present in a wide variety of plant extracts [25-28]. On the other hand, among the different applications of supercritical fluids (SCFs), such as extraction, micronization and encapsulation [29-32], supercritical solvent impregnation (SSI) has recently been used to develop active packaging as a green alternative to other techniques, which include extrusion, dry pulverization or crystallization. These latter techniques have some drawbacks and these are mainly related to the use of solvents, the energy required to carry out the impregnation process and the low specificity [12, 33, 34]. Furthermore, the high temperatures used to produce plastic films on an industrial scale make it impossible to add the antioxidant substances directly during the formation of the plastic. However, the supercritical region of CO2 (scCO2) is achieved at moderate pressures and temperatures, with a critical temperature (Tc) of 31.06 ºC and a critical pressure (Pc) of 73.8 bar. These values mean that it is possible to develop processes at near ambient temperatures without degradation of the thermolabile substances [6, 35]. Moreover, scCO2 acts as a carrier solvent and swelling agent for the porous matrix [36], which are factors that ensure the success of the impregnation. All of these characteristics make supercritical impregnation a suitable method to impart antioxidant capacity to previously formed films. There are several factors that influence the impregnation process and these include the pressure and temperature, which affect not only the solubility of the active substance in scCO2 but also the
sorption capacity of the polymer. Several studies have focused on the interaction between scCO2 and different polymers and it was concluded in general terms that scCO2 uptake was enhanced by scCO2 density, which improved the solute loading by polymer plasticization and the increase in internal diffusion coefficients. A review of that study was published by Goñi et al. [11]. SSI involves the interaction of three components, namely CO2, the active substance and the porous matrix, and all of the relationships between them, CO2/antioxidant, CO2/matrix and antioxidant/matrix [37]. These influential parameters have also been reported for other kinds of impregnation. For instance, Radziejewska-Kubzdela et al. [38] in a review on vacuum impregnation established that the most influential operational factors were the pressure, the composition, concentration and amount of impregnating solution, temperature, mixing, duration of the treatment and the restoration of atmospheric pressure. To our knowledge, SSI has not previously
been
studied
in
a
multilayer
plastic
comprised
of
polyethylene
terephthalate/polypropylene (PET/PP). For this reason it is necessary to study all of the influential previously reported factors in order to develop a supercritical impregnation method to obtain antioxidant films. Although the most widely studied ranges of pressure and temperature for supercritical impregnation reported in the literature are 90–200 bar and 35–55 ºC, respectively [37], higher pressures and temperatures have been reported. For instance, Champeau et al. [39] in an impregnation study on suture threads made from different polymers, including PET and PP, studied pressures up to 350 bar and concluded that the impregnation yield improves with increasing pressure and temperature. On the other hand, the depressurization rate is an important operational parameter in the impregnation process. As reported by Goñi et al., a fast depressurization may increase polymer loading but it can also cause mechanical damage to the material; nevertheless a slow pressure decrease preserves the polymeric matrix but excessive solute loss in the supercritical fluid could occur depending on the initial pressure. Therefore, this parameter should be optimized for each polymer–solute–solvent system [11]. The selection of the depressurization rate must be in concordance with the affinity of the active substance for the porous matrix. If the materials have a
strong affinity, slow depressurization is appropriate, whereas if they have poor affinity, the active substance can be easily dragged from the matrix by the scCO2 and a high depressurization rate favors entrapment of the substance in the polymer [37]. For this reason, the influence of the antioxidant/polymer affinity should be considered. As far as process time is concerned, different values for supercritical impregnation into different porous matrices have been reported in the literature and these range from several minutes [11, 40-43] to several hours [41]. In view of the above, a wide range of impregnation times should be studied for a proper evaluation of a given system. The aim of the study described here was to identify a method for the impregnation of antioxidants into a PET/PP food film by SSI in order to develop a material that may play a role in extending the shelf-life of food. The caffeic acid present in the majority of plant extracts is proposed as a model to study the parameters for the impregnation of this film. The variables pressure (P) (100, 200, 300 and 400 bar), temperature (T) (35 and 55 ºC) and depressurization rate (DR) (1 bar/min and 100 bar/min) were evaluated. The application of the method to an olive leaf extract was subsequently assessed and the results were compared with those obtained for the model substance. The time parameter was studied over a wide range (5 and 30 min, and 1, 2, 5 and 22 h) to identify the best impregnation conditions.
2. MATERIAL AND METHODS 2.1. Chemical reagents and raw materials Carbon dioxide (99.99%) was provided Abello-Linde S.A. (Barcelona, Spain). Ethanol was supplied by Panreac (Barcelona, Spain) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) and caffeic acid (>98% HPLC) were supplied by Sigma-Aldrich (Steinheim, Germany). Plastic films for alimentary use were provided by Technopack Univel S.r.L. (Mortara, Italy). The plastic was made of PET/PP (12 μm and 50 μm thickness, respectively), and these were joined by an adhesive. In order ensure that the adhesive did not distort the results, a DPPH assay was also carried out on the film without impregnation and antioxidant activity was not observed.
Olea europea leaves were collected in the region of Jaén (Andalucía, Spain) and were stored in darkness at room temperature prior to use. To obtain the extract, 200 mg of triturated olive leaves were introduced into a 1L-vessel and an enhanced solvent extraction (ESE) was carried out. Chinnarasu et al. [44] studied the extraction of olive leaves using mixtures of 10% and 50% ethanol-CO2, with better results obtained in terms of antioxidant activity and extraction yield on using 50% ethanol-CO2. Thus, the extraction conditions were CO2 + 50% ethanol at 120 bar, 80 °C and a total flow of 10 g/min. The resulting extract was used directly in impregnation runs without any other treatment. 2.2. Supercritical solvent impregnation (SSI) Tests were carried out in high pressure equipment supplied by Thar Technologies (Pittsburgh, PA, USA, model SF500), which includes a vessel (500 mL) with a thermostatic jacket, a high pressure pump, supercritical carbon dioxide (scCO2), and a back pressure regulator (BPR) to control the system pressure. The impregnation procedure was carried out in a lab-scale unit (Figure 1) as follows: known amounts of antioxidant and plastic films were loaded into the vessel. Plastic samples were maintained in a vertical position within the vessel by placing them into a steel support to avoid movement of the films during pressurization and depressurization. The vessel containing the sample was pre-heated to the appropriate temperature by an aluminium jacket. Temperature was controlled by three thermocouples situated on the heater and on the internal and external sides of the vessel. A four-bladed stirrer (Croschopp, Model PM6015) was included in the design in order to enhance the solubilization of the compound in scCO2. A cover was placed on the vessel to seal the contents hermetically. Before starting pressurization, agitation and temperature were switched on. SSI was carried out in a batch mode. Pressurization was carried out by increasing the flow to 10 g/min until appropriate pressure conditions had been achieved. The pressure was then fixed during the required time by a back-pressure regulator (BPR). Depressurization was started once the appropriate time had passed and prior to this step agitation and temperature were switched off.
Plastic samples were subsequently cleaned with a wet napkin in order to remove the excess antioxidant. Temperature thermocouples and the CO2 pump were connected to a monitored controller, which ensured the proper functioning of the system. Each experiment was replicated twice and impregnated films were stored at 4 ºC prior to use in order to avoid possible deterioration. 2.3. Study of SSI parameters using a model substance Different experiments were carried out in order to evaluate the parameters that influenced the SSI process (Table 1). In the first step (Table 1, experiment 1), caffeic acid was used as a model antioxidant to study the influence of pressure (P), temperature (T) and depressurization rate (DR). Different pressure values (100, 200, 300 and 400 bar) were studied provided that the matrix did not suffer any physical damage; temperatures of 35 and 55 °C were employed – i.e., the work was carried out below 100 °C in order to avoid the possible degradation of antioxidant compounds. Regarding the depressurization rate, a high and a low value were considered and these were 1 and 100 bar/min depressurization. An excess of active substance (150 mg of pure caffeic acid), the amount of PET/PP film (600 mg divided into 4 pieces of approximately 8 × 5 cm), the time (22 h) and the agitation rate (a medium agitation speed of 40 rpm) were fixed parameters. Long process times were employed in this study so that time was not considered to be a limiting factor. After this stage the pressure was once again studied in the same range together with the influence of the addition of a modifier (ethanol) to the caffeic acid (Table 1, experiment 2). The addition of ethanol as a co-solvent to increase the solubility of active compounds in scCO2 has previously been reported [37, 43, 45]. However, in the case of impregnation, polyphenols dissolved in ethanol could easily be dragged from the substrate during depressurization rather than remaining on the porous matrix. As a consequence, the addition of ethanol is an interesting parameter to consider. In this step, 150 mg of caffeic acid and 7.5 mL of a solution of 20 mg/mL of caffeic acid in ethanol were introduced into the vessel in order to use the same total amount of caffeic acid. The temperature and depressurization rate applied were those identified previously as the best conditions. The
amount of PET/PP film (600 mg), time (22 h) and agitation (40 rpm) were again kept as fixed parameters. 2.4. Study of SSI parameters using an olive leaf extract In order to ascertain whether the results achieved with a model substance were maintained with a natural extract, the best conditions defined previously in terms of the antioxidant activity were applied to an olive leaf extract. The extract was a mixture of different substances and the solubility in scCO2 could therefore vary. As a consequence, the pressure was studied in the same ranges as above. The extract had a total concentration of 20 mg/mL, so the amount added to the vessel was 7.5 mL in order to compare the results with those obtained previously. In this way, the rest of the parameters (temperature, depressurization rate, agitation and amount of film) were the same as those used before. The time parameter was investigated under the best impregnation conditions previously obtained with the extract (Table 1, experiment 3). A wide time range was studied, with values of 5 and 30 minutes and 1, 2, 5 and 22 hours. 2.5. Analysis of samples The results of the experiments were evaluated by calculating the antioxidant activity, for both active substances and impregnated films, and the impregnation efficiency. 2.5.1. Antioxidant activity of active substances The antioxidant activities were determined using DPPH as a free radical based on the procedure described by Brand-Williams and Scherer [46, 47]. A calibration curve for DPPH in ethanol (Eq. 1) was constructed by linear regression in order to calculate the %DPPH remaining in the reaction (Eq. 2): Eq. 1: Abs = 12.709*CDPPH + 0.002; R2 = 0.998 Eq. 2: % DPPH remaining = CDPPHt/CDPPHo * 100
Where Abs is the absorbance measured at 515 nm, CDPPH is the molar concentration (M), CDPPHt is the molar concentration (M) of unreacted DPPH after a defined time and CDPPHo is the initial molar concentration (M) of DPPH reagent. Ethanolic solutions of caffeic acid in the ranges 5–200 ppm and 400–20,000 ppm for the olive leaf extract were tested. 0.1 mL of each antioxidant solution was added to 3.9 mL of a 6 × 10–5 mol/L solution of DPPH. In order to analyze the reaction kinetics, the decrease in the absorbance was monitored using a spectrophotometer (Shimadzu UVmini-1240 (Sydney, Australia)) at 515 nm every 2 minutes for 2 hours, at which point a steady state had been achieved. The initial DPPH concentration was measured at time zero. Tests were carried out in duplicate. From the kinetic results, the data for the %DPPH remaining in the steady state were plotted against concentration of antioxidant and the Efficient Concentration (EC50) was calculated graphically. EC50 is defined as the concentration of antioxidant required to decrease the initial DPPH concentration by 50% and is expressed as μg antioxidant/mL. The lower the EC50 value, the higher the antioxidant power of the active substance. The Antioxidant Activity Index (AAI), expressed as μg DPPH/μg of dry extract, was also considered. 2.5.2. Antioxidant activity of impregnated films The DPPH protocol was modified slightly in order to evaluate the antioxidant capacity of the impregnated films. An amount of plastic was submerged in 4 mL of 6 × 10–5 mol/L DPPH in ethanol. The absorbance at 515 nm was measured after 2 hours of reaction in darkness at 4 ºC. Comparison of the initial DPPH absorbance (A0) with the 2 h-absorbance measured (Ai) allowed the percentage inhibition (%I) to be calculated (Eq. 3), which represents the amount of DPPH that reacts with a given concentration of antioxidant. Analyses were carried out in duplicate and the results are expressed as %I per 100 mg of plastic (%I/100 mg). Eq. 3: %I = (A0 – Ai)/ A0 * 100 Where %I is the percentage inhibition, A0 is the initial absorbance at 515 nm and Ai is the final absorbance at 515 nm.
2.5.3. Impregnation efficiency (%AL) The small differences in weight before and after the impregnation process made it necessary to use the DPPH reaction as an indirect method to calculate the mass of impregnated antioxidant. A graphical representation of %I against C (μg/mL) for the pure compound at a fixed time (2 h) was produced, and the equations for both caffeic acid (Eq. 4) and olive leaf extract (Eq. 5) were obtained. The impregnation efficiency was measured as the percentage of antioxidant loading (%AL), which was calculated by taking into account the antioxidant/polymer ratio (w/w) obtained in the DPPH assay and the initial antioxidant/polymer ratio (w/w). Eq. 4: C = –1.7154(%I)2 + 25.38(%I) + 9.6085; R² = 0.998 Eq. 5: C = –0.0163(%I)2 + 2.582(%I) + 0.2498; R² = 0.999 Where C is the concentration that reacts with the DPPH (μg/mL) and %I is the percentage inhibition. (mg antioxidant/mg polymer)reaction Eq. 6: %AL =
*100 (mg antioxidant/mg polymer)initial
2.6. Scanning electron microscopy (SEM) Scanning electron microscopy (SEM) was carried out in order to evaluate the distribution of the antioxidant within the impregnated films. A Quanta 200 scanning electron microscope (FEI, USA) was used on samples under vacuum, with an applied voltage of 20 kV. Prior to imaging, all samples were coated with a layer of gold (15 μm thick) in order to improve the conductivity of the sample. 3. RESULTS AND DISCUSSION 3.1. Antioxidant activity of active substances Regarding the antioxidant power of active substances, the classification described by Scherer and Godoy [46] established four ranges of antioxidant activity based on the antioxidant activity index (AAI): poor antioxidant activity (AAI < 0.5), moderate antioxidant activity (0.5 < AAI < 1.0),
strong antioxidant activity (1.0 < AAI < 2.0) and very strong activity (AAI > 2.0). According to this classification, caffeic acid is described as a ‘very strong antioxidant’, with an AAI of 14.4±1.5 µg DPPH/µg caffeic acid and an EC50 of 1.67±0.3 μg of caffeic acid/mL. As far as the olive leaf extract is concerned, this had a total concentration of 20 mg/mL and was classified in the ‘strong antioxidant’ range based on the classification of Scherer and Godoy [46], with an AAI of 1.06±0.1 µg DPPH/µg antioxidant and an EC50 of 22.3±1.5 μg antioxidant/mL. 3.2. Study of the impregnation efficiency (%AL) The results obtained for pure caffeic acid, 20 mg/mL of caffeic acid in ethanol and 20 mg/mL of an olive leaf extract in ethanol in terms of %AL are provided in Table 2. As can be observed, in the case of pure caffeic acid a low temperature (35 ºC) gave rise to better impregnation efficiency, particularly when combined with fast depressurization. This finding can be explained because, under isobaric conditions, the increase in temperature leads to a decrease in the density of CO2 and therefore the amount of caffeic acid dissolved. Conversely, Murga et al. [48], in a study on the solubility of different polyphenols in scCO2, reported that the solubility of caffeic acid increased with temperature and pressure in the ranges 40–60 ºC and 100–500 bar, respectively, with the best results obtained at 500 bar and 60 ºC. Accordingly, the impregnation efficiency is not only associated with better solubility of the antioxidant in scCO2, but also with the partition coefficient [49]. In general terms, an increase in DR is related to an increase of %AL (Table 2) except at 300 bar, where the data seemed to be independent of the DR. Furthermore, the use of a depressurization rate of 100 bar/min gave more reproducible results than 1 bar/min, which explains the higher standard deviations when a low depressurization rate was used. Dias et al. [40, 41] reached the same conclusions in a study concerning wound dressings. In the case of plastic, the reproducibility could be the result of the faster depressurization dragging away the particles that had been deposited on the surface but not impregnated into the matrix. Although a clear relation between %AL and P was not observed, it must be highlighted that impregnation was not observed at 400 bar, 55 ºC and 1 bar/min. In order to evaluate the experiments statistically, data were submitted to a multifactorial experimental design using the statistical computer package Statgraphics Plus 5.1
(Statpoint Technologies, Inc., USA). The Pareto diagram (Figure 2A) indicated a significant influence of T and DR on impregnation efficiency at 95% significance and showed that T = 35 ºC and DR = 100 bar/min were the best conditions. In all cases, the low values achieved for %AL can be explained in terms of the low solubility of caffeic acid in scCO2 or a low affinity between caffeic acid and the plastic. In an effort to improve these two parameters, the effect of ethanol as a modifier was studied. Ethanol increases the solubility of caffeic acid in scCO2 but the addition also affects the antioxidant-matrix affinity. If we take into account the film composition, the two layers have different structures. PP is obtained from the polymerization of propylene, while PET is formed by a polycondensation reaction between terephthalic acid and ethylene glycol, i.e., it has carbonyl groups conjugated with benzene rings. Given its structure, in the PP layer the antioxidant-matrix chemical reaction is more difficult and it is likely that deposition occurred rather than impregnation. Nevertheless, in the PET layer the carbonyl groups allow the formation of H-bonds with the active substance and thus a proper impregnation occurs. These two impregnation mechanisms were described by Kazarian et al. [50]. When the modifier was added the partition coefficient probably changed in favor of the solid matrix and H-bonding was favored, which resulted in an increase in antioxidant loading. It can be seen from the results in Table 2 that the addition of ethanol to the system increased the %AL for all of the pressures studied. The influence was corroborated by statistical analysis (Figure 2B) at 95% significance, which revealed that the addition of ethanol and the interaction between pressure and ethanol were significant variables. The presence of a modifier improved the %AL values by around 30% at the lowest pressures studied (100 and 200 bar) and by 45% at the highest pressures studied (300–400 bar), when compared with the results obtained under the same conditions (35 ºC and 100 bar/min) using pure caffeic acid for the impregnation assays (Table 2). As far as pressure is concerned, despite having a positive effect on the process this parameter was determined not to have a significant influence. As a consequence, this parameter needs to be studied in more detail. The results obtained for the olive leaf extract under the best conditions of T (35 ºC) and DR (100 bar/min) are shown in Table 2. The best impregnation results were obtained when the olive leaf
extract was used as the active substance and this is thought to be due to the effect of its composition, which includes secoroids, flavonoids and simple phenols [23, 44, 51]. It is likely that the different sizes, polarities and structures of the components played an important role in the impregnation process because these parameters have an influence on the solubility in sCO2. Besides, the different size could be crucial for the inclusion of the compounds within the matrix. In this case, %AL increased above 95% for all pressures studied when compared to the results obtained using pure caffeic acid under the same operation conditions, with a %AL of around 1.2% obtained at 400 bar. 3.2. Study of the antioxidant activity of impregnated films (%I/100 mg of plastic) The graphical representation of %I/100 mg of plastic for the impregnations carried out using caffeic acid revealed the same trend as for %AL (Figure 3). Regarding pressure, a trend was not observed for the data, but better results were generally obtained at T = 35 ºC and DR = 100 bar/min over the whole pressure range. It was expected that a higher %AL would lead to a higher %I for the samples. However, comparison of the data for pure caffeic acid and the olive leaf extract showed that the increase in antioxidant activity was not proportional to the increase in %AL. In the case of pure caffeic acid, despite having a %AL of around 0.034% as the best result, its high antioxidant activity provided films with around 70% for the %I/100 mg of plastic in the best case (400 bar, 35 ºC and 100 bar/min). Under these conditions the films impregnated with olive leaf extract had improved %I/100 mg by 32% while the %AL improved by 95%. This is a consequence of the olive leaf extract having a lower antioxidant capacity than caffeic acid. The %I/100 mg results for the different active substances employed are graphically represented in Figure 4. As can be seen, %I/100 mg results for plastic obtained at lower pressures (100 and 200 bar) were similar for both caffeic acid and olive leaf extracts; however, at higher pressures (300 and 400 bar) the difference in the results obtained was evident and plastic impregnated with olive leaf extract had a higher antioxidant activity. On using the natural extract as the active substance, a growing trend in %I/100 mg of plastic with increasing pressure was observed. This finding indicates that the matrix/antioxidant interactions prevailed over the sCO2/antioxidant ones. The increase in the
solubility of compounds and the swelling of the polymer with the increase in pressure have been reported previously [36, 45, 52]. It is likely that the high pressures reached not only dissolve a greater quantity of extract, but they could also improve polymer plasticization and internal diffusion coefficients, which could in turn explain the increase in loading on the plastic matrix. This last conclusion is consistent with that reported by Goñi et al. [11] in a study of LLDE films loaded with eugenol. Although %AL had values below 1.2%, it was demonstrated that plastics had a high %I, with values of 112.71%/100 mg of plastic achieved on applying the best impregnation conditions. According to Champeau et al., the % loadings reported in the literature for PET and PP polymers barely achieved %AL values of 5% for pressures below 300 bar and temperatures between 40 and 170 ºC [37], which is consistent the results obtained in this this study. The best impregnation conditions were obtained at a pressure of 400 bar, a temperature of 35 ºC and with a depressurization rate of 100 bar/min. 3.3. Influence of the impregnation time Due to the clearly better results obtained with the olive leaf extract when compared to caffeic acid, the time parameter was studied in conjunction with the olive leaf extract. Up to this point all experiments had been carried out for 22 hours so that time would not be a limiting factor. Nevertheless, it was interesting to study %AL and %I/100 mg of plastic at different times in order to assess the kinetics of the impregnation process. Times of 5 and 30 minutes, and 1, 2, 5 and 22 hours were evaluated under the best conditions studied previously. The results obtained in terms of %AL and %I under the best impregnation conditions are represented in Figure 5. A time of 5 minutes was sufficient to start the impregnation, which indicates that impregnation occurs once the active substance is dissolved. Although a clear trend was not observed with time, the results demonstrate that it is not necessary to extend the process for several hours to improve the impregnation efficiency in future experiments. The study revealed that an extraction time of 1 hour afforded the best results, with 3% and 279.8% for %AL and %I/100 mg of plastic, respectively, compared with the 1.2 %AL and 112.7 %I/100 mg of plastic obtained with a 22 hour process time.
A similar variation in behavior with time was also observed by Sugiura et al. [53] in a study on the impregnation of tranilast into PLA fibers. Despite obtaining a maximum adsorption at 20 minutes, when tranilast diffused better into the fiber swollen by the scCO2, equilibrium was reached after 1 hour due to the simultaneous recrystallization of the fiber during the diffusion. Taking this fact into account, the scCO2 not only had an effect on the solubility and diffusion of the substance, but it also seems to influence the crystallization of the polymer, which was identified as a limiting factor in the impregnation process [37]. As a consequence, the behavior of the three elements had to be studied in each system. 3.4. Scanning electron microscopy (SEM) Comparison of a sample of plastic without treatment (Figure 6A) and a sample of plastic obtained with the best impregnation conditions (Figure 6B) at the same magnification (3000×) in scanning electron microscopy confirmed the presence of the antioxidant on the films and a heterogeneous distribution of these compounds. Microscopical bubbling seemed to appear on impregnated samples, probably due to the phenomenon of swelling during impregnation. However, physical damage was not observed on the sample by the naked eye. The irregular form of the impregnated particles could be observed when the magnification was increased (5000×) (Figure 6C). 4. CONCLUSIONS A method for the SSI of antioxidants into multilayer PET/PP films has been developed. This active packaging has potential uses in the food industry and possible applications in the preservation of food, which would represent a new genre of alimentary films. Among all the techniques employed to obtain antioxidant films, SSI impregnation offers the advantage of providing antioxidant capacity to films that have already been formed, thus providing products that are solvent-free and ready to use. SSI is influenced by a large number of variables and the relationships between them. This situation makes SSI a complex process to optimize. This research was focused on the study of the most influential parameters, i.e., temperature, depressurization rate, time, the use of ethanol as a modifier, and the kind of active substance.
The success of the SSI with natural extracts against standards highlights an interesting alternative for the use of chemical substances in food packaging and provides a way to use by-products from industry due to their high content in antioxidant compounds. This possibility gives the process added value. In the case studied here, olive leaf extract proved to offer remarkable results when compared to caffeic acid, even when a modifier was added to increase the solubility of the compound in scCO2. This information represents the first steps towards a knowledge of the SSI process and provides a starting point for further research.
5. ACKNOWLEDGMENTS This work was supported by the Spanish Government (Project CTQ2014-52427-R).
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Table 1. Summary of the experiments Experiment 1
2
3
Fixed parameters Parameter Agitation Time Amount of plastic Pure caffeic acid Agitation Time Amount of plastic Temperature Depressurization rate Agitation Amount of plastic Temperature Depressurization rate Olive leaf extract
Value 40 rpm 22h 600 mg 150 mg 40 rpm 22h 600 mg 35 °C 100 bar/min 40 rpm 600 mg 35 °C 100 bar/min 20 mg/mL
Variables Parameter Pressure Temperatur Depressurization rate
Value 100, 200, 300 and 400 bar 35 and 55 °C 1 and 100 bar/min
Active substance
Pure caffeic acid (150 mg) Caffeic acid in ethanol (20 mg/mL) Olive leaf extract (20 mg/mL)
Time
5 and 30 min, 1, 2 5 and 22 h
Table 2. Impregnation efficiency (%AL) of different active substances under all conditions studied (n = 2) Pure caffeic acid P (bar) DR (bar/min) 100 𝑋̅ 1 35 0.029 55 0.006 100 35 0.023 55 0.009 Caffeic acid (20 mg/mL ethanol) 100 35 0.035 Olive leaf extract 100 35 0.545 T (ºC)
𝑋̅ = Average value SD = Standard deviation
200
300
SD 0.005 0.005 0.003 0.008
𝑋̅ 0.018 0.014 0.030 0.012
SD 0.003 0.001 0.001 0.003
𝑋̅ 0.026 0.016 0.028 0.016
400 SD 0.002 0.014 0.002 0.003
𝑋̅ 0.024 ND 0.034 0.018
SD 0.001 0.007 0.023
0.001
0.043
0.010
0.053
0.003
0.057
0.006
0.044
0.721
0.113
0.790
0.109
1.209
0.104
A
B
Figure 2. Pareto diagram (p = 0.05) of %AL for the two systems considered in the study A) Effect of pressure, temperature and depressurization rate with pure caffeic acid and B) effect of modifier and P on impregnation efficiency
90
%I/100 mg plastic
80 70 60 50
100 bar
40
200 bar
30
300 bar
20
400 bar
10 0 35 ºC
55 ºC 1 bar/min
35 ºC
55 ºC
100 bar/min DR and T conditions
Figure 3. Antioxidant activity of impregnated films using pure caffeic acid
120.0
%I/100 mg plastic
100.0 80.0 60.0
Pure caffeic acid Olive leaf extract
40.0 20.0 0.0 100
200
300
400
P (bar)
Figure 4. Antioxidant activity at the pressures studied at T = 35 ºC and DR = 100 bar/min
3.5
A 3 2.5
% AL
2 1.5 1 0.5 0 5 min
30 min
1h
2h
5h
22h
2h
5h
22h
Time
350
B % I/100 mg plastic
300 250 200 150 100 50 0 5 min
30 min
1h Time
Figure 5. Study of time parameter regarding %AL (A) and % I/100 mg of plastic (B) on olive leaf extract impregnation (P = 400bar, T = 35 ºC, DR = 100 bar/min)
S0896-8446(17)30225-5 http://dx.doi.org/doi:10.1016/j.supflu.2017.05.034 SUPFLU 3946
To appear in:
J. of Supercritical Fluids
Received date: Revised date: Accepted date:
28-3-2017 31-5-2017 31-5-2017
Please cite this article as: C.Cejudo Bastante, L.Casas Cardoso, C.Mantell Serrano, E.J.Mart´ınez de la Ossa, Supercritical Impregnation of food packaging films to provide antioxidant properties, The Journal of Supercritical Fluidshttp://dx.doi.org/10.1016/j.supflu.2017.05.034 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Supercritical Impregnation of food packaging films to provide antioxidant properties C. Cejudo Bastante*, L. Casas Cardoso, C. Mantell Serrano, E.J. Martínez de la Ossa Chemical Engineering and Food Technology Department, Wine and Agrifood Research Institute (IVAGRO), University of Cadiz Avda. República Saharaui, s/n, 11510 - Puerto Real, Cádiz (Spain) *corresponding author: Cejudo Bastante, Cristina. [email protected] Graphical abstract
Highlights: Antioxidant films for an alimentary use have been obtained by SSI. SSI is an intricate process and all the variables involved need to be studied. Impregnation yield increased when olive leaf extract was used as active substance.
ABSTRACT Among the different techniques employed to develop active packaging, supercritical solvent impregnation (SSI) of natural extracts is an innovative approach. When developing a method to obtain polyethylene terephthalate/polypropylene (PET/PP) films with antioxidant capacity, several parameters (pressure, temperature, depressurization rate, presence of modifier and time) were studied with caffeic acid as a model substance. The best conditions were applied to an olive leaf extract as a natural extract with antioxidant properties. Antioxidant activity was evaluated by the
DPPH assay and the total loading was calculated to identify the best impregnation conditions. The results revealed that a low level of impregnation provide antioxidant films, both with the standard and the natural extract. Better results were obtained when the natural extract was used. To confirm the presence of the antioxidant particles and to determine their distribution on the matrix, the impregnated samples were studied by Scanning Electron Microscopy (SEM).
Keywords: Olea europea, caffeic acid, supercritical impregnation, supercritical fluids, active packaging, antioxidant films.
1. INTRODUCTION The preservation of food has been widely studied because of its importance in the food industry. Together with edible films and coatings, active packaging is one of the most innovative alternatives for food preservation because it maintains the nutritional and organoleptic quality of food and improves the security parameters. Active packaging is a system that interacts positively with the product and extends its shelf-life due to the action of an active agent included on the packaging material or forming part of the polymer itself [1]. The active agents function by modifying the head-space of the packaging by absorption or release of compounds, or by modifying the composition on the surface of the food [2-4], and they could impart antimicrobial or antioxidant activities to the packaging, amongst other properties. Of the different substances used in active packaging [5], essential oils have been widely studied as active compounds in both coatings and active packaging in recent years due to their antioxidant, antifungal and antimicrobial activity [6, 7]. These properties have been widely demonstrated in polyphenols, which are present in a variety of vegetable sources [8]. Such compounds have also been included in polymer processing to
improve certain properties of these materials and good results have been obtained [9]. Although standards of these compounds have been widely used as active substances [10-12], extracts from natural sources such as plants, spices, seeds and leaves from pruning waste from different plants have also been studied [13-16]. Some of these materials, despite being rich in essential oils and polyphenols, are sometimes discarded by industry. As a consequence, the use of these materials for another purpose gives them added value. In this context, extracts from olive leaves have been used for food packaging applications in recent years [17-20] because compounds like oleuropein, phenolic acids, such as caffeic acid, p-coumaric acid, sinapic acid, ferulic acid and syringic acid, and flavonoids, such quercetin, hesperetin, luteolin and rutin, possess high antioxidant and antimicrobial capacities and also have potential health benefits [21-24]. These compounds are present in a wide variety of plant extracts [25-28]. On the other hand, among the different applications of supercritical fluids (SCFs), such as extraction, micronization and encapsulation [29-32], supercritical solvent impregnation (SSI) has recently been used to develop active packaging as a green alternative to other techniques, which include extrusion, dry pulverization or crystallization. These latter techniques have some drawbacks and these are mainly related to the use of solvents, the energy required to carry out the impregnation process and the low specificity [12, 33, 34]. Furthermore, the high temperatures used to produce plastic films on an industrial scale make it impossible to add the antioxidant substances directly during the formation of the plastic. However, the supercritical region of CO2 (scCO2) is achieved at moderate pressures and temperatures, with a critical temperature (Tc) of 31.06 ºC and a critical pressure (Pc) of 73.8 bar. These values mean that it is possible to develop processes at near ambient temperatures without degradation of the thermolabile substances [6, 35]. Moreover, scCO2 acts as a carrier solvent and swelling agent for the porous matrix [36], which are factors that ensure the success of the impregnation. All of these characteristics make supercritical impregnation a suitable method to impart antioxidant capacity to previously formed films. There are several factors that influence the impregnation process and these include the pressure and temperature, which affect not only the solubility of the active substance in scCO2 but also the
sorption capacity of the polymer. Several studies have focused on the interaction between scCO2 and different polymers and it was concluded in general terms that scCO2 uptake was enhanced by scCO2 density, which improved the solute loading by polymer plasticization and the increase in internal diffusion coefficients. A review of that study was published by Goñi et al. [11]. SSI involves the interaction of three components, namely CO2, the active substance and the porous matrix, and all of the relationships between them, CO2/antioxidant, CO2/matrix and antioxidant/matrix [37]. These influential parameters have also been reported for other kinds of impregnation. For instance, Radziejewska-Kubzdela et al. [38] in a review on vacuum impregnation established that the most influential operational factors were the pressure, the composition, concentration and amount of impregnating solution, temperature, mixing, duration of the treatment and the restoration of atmospheric pressure. To our knowledge, SSI has not previously
been
studied
in
a
multilayer
plastic
comprised
of
polyethylene
terephthalate/polypropylene (PET/PP). For this reason it is necessary to study all of the influential previously reported factors in order to develop a supercritical impregnation method to obtain antioxidant films. Although the most widely studied ranges of pressure and temperature for supercritical impregnation reported in the literature are 90–200 bar and 35–55 ºC, respectively [37], higher pressures and temperatures have been reported. For instance, Champeau et al. [39] in an impregnation study on suture threads made from different polymers, including PET and PP, studied pressures up to 350 bar and concluded that the impregnation yield improves with increasing pressure and temperature. On the other hand, the depressurization rate is an important operational parameter in the impregnation process. As reported by Goñi et al., a fast depressurization may increase polymer loading but it can also cause mechanical damage to the material; nevertheless a slow pressure decrease preserves the polymeric matrix but excessive solute loss in the supercritical fluid could occur depending on the initial pressure. Therefore, this parameter should be optimized for each polymer–solute–solvent system [11]. The selection of the depressurization rate must be in concordance with the affinity of the active substance for the porous matrix. If the materials have a
strong affinity, slow depressurization is appropriate, whereas if they have poor affinity, the active substance can be easily dragged from the matrix by the scCO2 and a high depressurization rate favors entrapment of the substance in the polymer [37]. For this reason, the influence of the antioxidant/polymer affinity should be considered. As far as process time is concerned, different values for supercritical impregnation into different porous matrices have been reported in the literature and these range from several minutes [11, 40-43] to several hours [41]. In view of the above, a wide range of impregnation times should be studied for a proper evaluation of a given system. The aim of the study described here was to identify a method for the impregnation of antioxidants into a PET/PP food film by SSI in order to develop a material that may play a role in extending the shelf-life of food. The caffeic acid present in the majority of plant extracts is proposed as a model to study the parameters for the impregnation of this film. The variables pressure (P) (100, 200, 300 and 400 bar), temperature (T) (35 and 55 ºC) and depressurization rate (DR) (1 bar/min and 100 bar/min) were evaluated. The application of the method to an olive leaf extract was subsequently assessed and the results were compared with those obtained for the model substance. The time parameter was studied over a wide range (5 and 30 min, and 1, 2, 5 and 22 h) to identify the best impregnation conditions.
2. MATERIAL AND METHODS 2.1. Chemical reagents and raw materials Carbon dioxide (99.99%) was provided Abello-Linde S.A. (Barcelona, Spain). Ethanol was supplied by Panreac (Barcelona, Spain) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) and caffeic acid (>98% HPLC) were supplied by Sigma-Aldrich (Steinheim, Germany). Plastic films for alimentary use were provided by Technopack Univel S.r.L. (Mortara, Italy). The plastic was made of PET/PP (12 μm and 50 μm thickness, respectively), and these were joined by an adhesive. In order ensure that the adhesive did not distort the results, a DPPH assay was also carried out on the film without impregnation and antioxidant activity was not observed.
Olea europea leaves were collected in the region of Jaén (Andalucía, Spain) and were stored in darkness at room temperature prior to use. To obtain the extract, 200 mg of triturated olive leaves were introduced into a 1L-vessel and an enhanced solvent extraction (ESE) was carried out. Chinnarasu et al. [44] studied the extraction of olive leaves using mixtures of 10% and 50% ethanol-CO2, with better results obtained in terms of antioxidant activity and extraction yield on using 50% ethanol-CO2. Thus, the extraction conditions were CO2 + 50% ethanol at 120 bar, 80 °C and a total flow of 10 g/min. The resulting extract was used directly in impregnation runs without any other treatment. 2.2. Supercritical solvent impregnation (SSI) Tests were carried out in high pressure equipment supplied by Thar Technologies (Pittsburgh, PA, USA, model SF500), which includes a vessel (500 mL) with a thermostatic jacket, a high pressure pump, supercritical carbon dioxide (scCO2), and a back pressure regulator (BPR) to control the system pressure. The impregnation procedure was carried out in a lab-scale unit (Figure 1) as follows: known amounts of antioxidant and plastic films were loaded into the vessel. Plastic samples were maintained in a vertical position within the vessel by placing them into a steel support to avoid movement of the films during pressurization and depressurization. The vessel containing the sample was pre-heated to the appropriate temperature by an aluminium jacket. Temperature was controlled by three thermocouples situated on the heater and on the internal and external sides of the vessel. A four-bladed stirrer (Croschopp, Model PM6015) was included in the design in order to enhance the solubilization of the compound in scCO2. A cover was placed on the vessel to seal the contents hermetically. Before starting pressurization, agitation and temperature were switched on. SSI was carried out in a batch mode. Pressurization was carried out by increasing the flow to 10 g/min until appropriate pressure conditions had been achieved. The pressure was then fixed during the required time by a back-pressure regulator (BPR). Depressurization was started once the appropriate time had passed and prior to this step agitation and temperature were switched off.
Plastic samples were subsequently cleaned with a wet napkin in order to remove the excess antioxidant. Temperature thermocouples and the CO2 pump were connected to a monitored controller, which ensured the proper functioning of the system. Each experiment was replicated twice and impregnated films were stored at 4 ºC prior to use in order to avoid possible deterioration. 2.3. Study of SSI parameters using a model substance Different experiments were carried out in order to evaluate the parameters that influenced the SSI process (Table 1). In the first step (Table 1, experiment 1), caffeic acid was used as a model antioxidant to study the influence of pressure (P), temperature (T) and depressurization rate (DR). Different pressure values (100, 200, 300 and 400 bar) were studied provided that the matrix did not suffer any physical damage; temperatures of 35 and 55 °C were employed – i.e., the work was carried out below 100 °C in order to avoid the possible degradation of antioxidant compounds. Regarding the depressurization rate, a high and a low value were considered and these were 1 and 100 bar/min depressurization. An excess of active substance (150 mg of pure caffeic acid), the amount of PET/PP film (600 mg divided into 4 pieces of approximately 8 × 5 cm), the time (22 h) and the agitation rate (a medium agitation speed of 40 rpm) were fixed parameters. Long process times were employed in this study so that time was not considered to be a limiting factor. After this stage the pressure was once again studied in the same range together with the influence of the addition of a modifier (ethanol) to the caffeic acid (Table 1, experiment 2). The addition of ethanol as a co-solvent to increase the solubility of active compounds in scCO2 has previously been reported [37, 43, 45]. However, in the case of impregnation, polyphenols dissolved in ethanol could easily be dragged from the substrate during depressurization rather than remaining on the porous matrix. As a consequence, the addition of ethanol is an interesting parameter to consider. In this step, 150 mg of caffeic acid and 7.5 mL of a solution of 20 mg/mL of caffeic acid in ethanol were introduced into the vessel in order to use the same total amount of caffeic acid. The temperature and depressurization rate applied were those identified previously as the best conditions. The
amount of PET/PP film (600 mg), time (22 h) and agitation (40 rpm) were again kept as fixed parameters. 2.4. Study of SSI parameters using an olive leaf extract In order to ascertain whether the results achieved with a model substance were maintained with a natural extract, the best conditions defined previously in terms of the antioxidant activity were applied to an olive leaf extract. The extract was a mixture of different substances and the solubility in scCO2 could therefore vary. As a consequence, the pressure was studied in the same ranges as above. The extract had a total concentration of 20 mg/mL, so the amount added to the vessel was 7.5 mL in order to compare the results with those obtained previously. In this way, the rest of the parameters (temperature, depressurization rate, agitation and amount of film) were the same as those used before. The time parameter was investigated under the best impregnation conditions previously obtained with the extract (Table 1, experiment 3). A wide time range was studied, with values of 5 and 30 minutes and 1, 2, 5 and 22 hours. 2.5. Analysis of samples The results of the experiments were evaluated by calculating the antioxidant activity, for both active substances and impregnated films, and the impregnation efficiency. 2.5.1. Antioxidant activity of active substances The antioxidant activities were determined using DPPH as a free radical based on the procedure described by Brand-Williams and Scherer [46, 47]. A calibration curve for DPPH in ethanol (Eq. 1) was constructed by linear regression in order to calculate the %DPPH remaining in the reaction (Eq. 2): Eq. 1: Abs = 12.709*CDPPH + 0.002; R2 = 0.998 Eq. 2: % DPPH remaining = CDPPHt/CDPPHo * 100
Where Abs is the absorbance measured at 515 nm, CDPPH is the molar concentration (M), CDPPHt is the molar concentration (M) of unreacted DPPH after a defined time and CDPPHo is the initial molar concentration (M) of DPPH reagent. Ethanolic solutions of caffeic acid in the ranges 5–200 ppm and 400–20,000 ppm for the olive leaf extract were tested. 0.1 mL of each antioxidant solution was added to 3.9 mL of a 6 × 10–5 mol/L solution of DPPH. In order to analyze the reaction kinetics, the decrease in the absorbance was monitored using a spectrophotometer (Shimadzu UVmini-1240 (Sydney, Australia)) at 515 nm every 2 minutes for 2 hours, at which point a steady state had been achieved. The initial DPPH concentration was measured at time zero. Tests were carried out in duplicate. From the kinetic results, the data for the %DPPH remaining in the steady state were plotted against concentration of antioxidant and the Efficient Concentration (EC50) was calculated graphically. EC50 is defined as the concentration of antioxidant required to decrease the initial DPPH concentration by 50% and is expressed as μg antioxidant/mL. The lower the EC50 value, the higher the antioxidant power of the active substance. The Antioxidant Activity Index (AAI), expressed as μg DPPH/μg of dry extract, was also considered. 2.5.2. Antioxidant activity of impregnated films The DPPH protocol was modified slightly in order to evaluate the antioxidant capacity of the impregnated films. An amount of plastic was submerged in 4 mL of 6 × 10–5 mol/L DPPH in ethanol. The absorbance at 515 nm was measured after 2 hours of reaction in darkness at 4 ºC. Comparison of the initial DPPH absorbance (A0) with the 2 h-absorbance measured (Ai) allowed the percentage inhibition (%I) to be calculated (Eq. 3), which represents the amount of DPPH that reacts with a given concentration of antioxidant. Analyses were carried out in duplicate and the results are expressed as %I per 100 mg of plastic (%I/100 mg). Eq. 3: %I = (A0 – Ai)/ A0 * 100 Where %I is the percentage inhibition, A0 is the initial absorbance at 515 nm and Ai is the final absorbance at 515 nm.
2.5.3. Impregnation efficiency (%AL) The small differences in weight before and after the impregnation process made it necessary to use the DPPH reaction as an indirect method to calculate the mass of impregnated antioxidant. A graphical representation of %I against C (μg/mL) for the pure compound at a fixed time (2 h) was produced, and the equations for both caffeic acid (Eq. 4) and olive leaf extract (Eq. 5) were obtained. The impregnation efficiency was measured as the percentage of antioxidant loading (%AL), which was calculated by taking into account the antioxidant/polymer ratio (w/w) obtained in the DPPH assay and the initial antioxidant/polymer ratio (w/w). Eq. 4: C = –1.7154(%I)2 + 25.38(%I) + 9.6085; R² = 0.998 Eq. 5: C = –0.0163(%I)2 + 2.582(%I) + 0.2498; R² = 0.999 Where C is the concentration that reacts with the DPPH (μg/mL) and %I is the percentage inhibition. (mg antioxidant/mg polymer)reaction Eq. 6: %AL =
*100 (mg antioxidant/mg polymer)initial
2.6. Scanning electron microscopy (SEM) Scanning electron microscopy (SEM) was carried out in order to evaluate the distribution of the antioxidant within the impregnated films. A Quanta 200 scanning electron microscope (FEI, USA) was used on samples under vacuum, with an applied voltage of 20 kV. Prior to imaging, all samples were coated with a layer of gold (15 μm thick) in order to improve the conductivity of the sample. 3. RESULTS AND DISCUSSION 3.1. Antioxidant activity of active substances Regarding the antioxidant power of active substances, the classification described by Scherer and Godoy [46] established four ranges of antioxidant activity based on the antioxidant activity index (AAI): poor antioxidant activity (AAI < 0.5), moderate antioxidant activity (0.5 < AAI < 1.0),
strong antioxidant activity (1.0 < AAI < 2.0) and very strong activity (AAI > 2.0). According to this classification, caffeic acid is described as a ‘very strong antioxidant’, with an AAI of 14.4±1.5 µg DPPH/µg caffeic acid and an EC50 of 1.67±0.3 μg of caffeic acid/mL. As far as the olive leaf extract is concerned, this had a total concentration of 20 mg/mL and was classified in the ‘strong antioxidant’ range based on the classification of Scherer and Godoy [46], with an AAI of 1.06±0.1 µg DPPH/µg antioxidant and an EC50 of 22.3±1.5 μg antioxidant/mL. 3.2. Study of the impregnation efficiency (%AL) The results obtained for pure caffeic acid, 20 mg/mL of caffeic acid in ethanol and 20 mg/mL of an olive leaf extract in ethanol in terms of %AL are provided in Table 2. As can be observed, in the case of pure caffeic acid a low temperature (35 ºC) gave rise to better impregnation efficiency, particularly when combined with fast depressurization. This finding can be explained because, under isobaric conditions, the increase in temperature leads to a decrease in the density of CO2 and therefore the amount of caffeic acid dissolved. Conversely, Murga et al. [48], in a study on the solubility of different polyphenols in scCO2, reported that the solubility of caffeic acid increased with temperature and pressure in the ranges 40–60 ºC and 100–500 bar, respectively, with the best results obtained at 500 bar and 60 ºC. Accordingly, the impregnation efficiency is not only associated with better solubility of the antioxidant in scCO2, but also with the partition coefficient [49]. In general terms, an increase in DR is related to an increase of %AL (Table 2) except at 300 bar, where the data seemed to be independent of the DR. Furthermore, the use of a depressurization rate of 100 bar/min gave more reproducible results than 1 bar/min, which explains the higher standard deviations when a low depressurization rate was used. Dias et al. [40, 41] reached the same conclusions in a study concerning wound dressings. In the case of plastic, the reproducibility could be the result of the faster depressurization dragging away the particles that had been deposited on the surface but not impregnated into the matrix. Although a clear relation between %AL and P was not observed, it must be highlighted that impregnation was not observed at 400 bar, 55 ºC and 1 bar/min. In order to evaluate the experiments statistically, data were submitted to a multifactorial experimental design using the statistical computer package Statgraphics Plus 5.1
(Statpoint Technologies, Inc., USA). The Pareto diagram (Figure 2A) indicated a significant influence of T and DR on impregnation efficiency at 95% significance and showed that T = 35 ºC and DR = 100 bar/min were the best conditions. In all cases, the low values achieved for %AL can be explained in terms of the low solubility of caffeic acid in scCO2 or a low affinity between caffeic acid and the plastic. In an effort to improve these two parameters, the effect of ethanol as a modifier was studied. Ethanol increases the solubility of caffeic acid in scCO2 but the addition also affects the antioxidant-matrix affinity. If we take into account the film composition, the two layers have different structures. PP is obtained from the polymerization of propylene, while PET is formed by a polycondensation reaction between terephthalic acid and ethylene glycol, i.e., it has carbonyl groups conjugated with benzene rings. Given its structure, in the PP layer the antioxidant-matrix chemical reaction is more difficult and it is likely that deposition occurred rather than impregnation. Nevertheless, in the PET layer the carbonyl groups allow the formation of H-bonds with the active substance and thus a proper impregnation occurs. These two impregnation mechanisms were described by Kazarian et al. [50]. When the modifier was added the partition coefficient probably changed in favor of the solid matrix and H-bonding was favored, which resulted in an increase in antioxidant loading. It can be seen from the results in Table 2 that the addition of ethanol to the system increased the %AL for all of the pressures studied. The influence was corroborated by statistical analysis (Figure 2B) at 95% significance, which revealed that the addition of ethanol and the interaction between pressure and ethanol were significant variables. The presence of a modifier improved the %AL values by around 30% at the lowest pressures studied (100 and 200 bar) and by 45% at the highest pressures studied (300–400 bar), when compared with the results obtained under the same conditions (35 ºC and 100 bar/min) using pure caffeic acid for the impregnation assays (Table 2). As far as pressure is concerned, despite having a positive effect on the process this parameter was determined not to have a significant influence. As a consequence, this parameter needs to be studied in more detail. The results obtained for the olive leaf extract under the best conditions of T (35 ºC) and DR (100 bar/min) are shown in Table 2. The best impregnation results were obtained when the olive leaf
extract was used as the active substance and this is thought to be due to the effect of its composition, which includes secoroids, flavonoids and simple phenols [23, 44, 51]. It is likely that the different sizes, polarities and structures of the components played an important role in the impregnation process because these parameters have an influence on the solubility in sCO2. Besides, the different size could be crucial for the inclusion of the compounds within the matrix. In this case, %AL increased above 95% for all pressures studied when compared to the results obtained using pure caffeic acid under the same operation conditions, with a %AL of around 1.2% obtained at 400 bar. 3.2. Study of the antioxidant activity of impregnated films (%I/100 mg of plastic) The graphical representation of %I/100 mg of plastic for the impregnations carried out using caffeic acid revealed the same trend as for %AL (Figure 3). Regarding pressure, a trend was not observed for the data, but better results were generally obtained at T = 35 ºC and DR = 100 bar/min over the whole pressure range. It was expected that a higher %AL would lead to a higher %I for the samples. However, comparison of the data for pure caffeic acid and the olive leaf extract showed that the increase in antioxidant activity was not proportional to the increase in %AL. In the case of pure caffeic acid, despite having a %AL of around 0.034% as the best result, its high antioxidant activity provided films with around 70% for the %I/100 mg of plastic in the best case (400 bar, 35 ºC and 100 bar/min). Under these conditions the films impregnated with olive leaf extract had improved %I/100 mg by 32% while the %AL improved by 95%. This is a consequence of the olive leaf extract having a lower antioxidant capacity than caffeic acid. The %I/100 mg results for the different active substances employed are graphically represented in Figure 4. As can be seen, %I/100 mg results for plastic obtained at lower pressures (100 and 200 bar) were similar for both caffeic acid and olive leaf extracts; however, at higher pressures (300 and 400 bar) the difference in the results obtained was evident and plastic impregnated with olive leaf extract had a higher antioxidant activity. On using the natural extract as the active substance, a growing trend in %I/100 mg of plastic with increasing pressure was observed. This finding indicates that the matrix/antioxidant interactions prevailed over the sCO2/antioxidant ones. The increase in the
solubility of compounds and the swelling of the polymer with the increase in pressure have been reported previously [36, 45, 52]. It is likely that the high pressures reached not only dissolve a greater quantity of extract, but they could also improve polymer plasticization and internal diffusion coefficients, which could in turn explain the increase in loading on the plastic matrix. This last conclusion is consistent with that reported by Goñi et al. [11] in a study of LLDE films loaded with eugenol. Although %AL had values below 1.2%, it was demonstrated that plastics had a high %I, with values of 112.71%/100 mg of plastic achieved on applying the best impregnation conditions. According to Champeau et al., the % loadings reported in the literature for PET and PP polymers barely achieved %AL values of 5% for pressures below 300 bar and temperatures between 40 and 170 ºC [37], which is consistent the results obtained in this this study. The best impregnation conditions were obtained at a pressure of 400 bar, a temperature of 35 ºC and with a depressurization rate of 100 bar/min. 3.3. Influence of the impregnation time Due to the clearly better results obtained with the olive leaf extract when compared to caffeic acid, the time parameter was studied in conjunction with the olive leaf extract. Up to this point all experiments had been carried out for 22 hours so that time would not be a limiting factor. Nevertheless, it was interesting to study %AL and %I/100 mg of plastic at different times in order to assess the kinetics of the impregnation process. Times of 5 and 30 minutes, and 1, 2, 5 and 22 hours were evaluated under the best conditions studied previously. The results obtained in terms of %AL and %I under the best impregnation conditions are represented in Figure 5. A time of 5 minutes was sufficient to start the impregnation, which indicates that impregnation occurs once the active substance is dissolved. Although a clear trend was not observed with time, the results demonstrate that it is not necessary to extend the process for several hours to improve the impregnation efficiency in future experiments. The study revealed that an extraction time of 1 hour afforded the best results, with 3% and 279.8% for %AL and %I/100 mg of plastic, respectively, compared with the 1.2 %AL and 112.7 %I/100 mg of plastic obtained with a 22 hour process time.
A similar variation in behavior with time was also observed by Sugiura et al. [53] in a study on the impregnation of tranilast into PLA fibers. Despite obtaining a maximum adsorption at 20 minutes, when tranilast diffused better into the fiber swollen by the scCO2, equilibrium was reached after 1 hour due to the simultaneous recrystallization of the fiber during the diffusion. Taking this fact into account, the scCO2 not only had an effect on the solubility and diffusion of the substance, but it also seems to influence the crystallization of the polymer, which was identified as a limiting factor in the impregnation process [37]. As a consequence, the behavior of the three elements had to be studied in each system. 3.4. Scanning electron microscopy (SEM) Comparison of a sample of plastic without treatment (Figure 6A) and a sample of plastic obtained with the best impregnation conditions (Figure 6B) at the same magnification (3000×) in scanning electron microscopy confirmed the presence of the antioxidant on the films and a heterogeneous distribution of these compounds. Microscopical bubbling seemed to appear on impregnated samples, probably due to the phenomenon of swelling during impregnation. However, physical damage was not observed on the sample by the naked eye. The irregular form of the impregnated particles could be observed when the magnification was increased (5000×) (Figure 6C). 4. CONCLUSIONS A method for the SSI of antioxidants into multilayer PET/PP films has been developed. This active packaging has potential uses in the food industry and possible applications in the preservation of food, which would represent a new genre of alimentary films. Among all the techniques employed to obtain antioxidant films, SSI impregnation offers the advantage of providing antioxidant capacity to films that have already been formed, thus providing products that are solvent-free and ready to use. SSI is influenced by a large number of variables and the relationships between them. This situation makes SSI a complex process to optimize. This research was focused on the study of the most influential parameters, i.e., temperature, depressurization rate, time, the use of ethanol as a modifier, and the kind of active substance.
The success of the SSI with natural extracts against standards highlights an interesting alternative for the use of chemical substances in food packaging and provides a way to use by-products from industry due to their high content in antioxidant compounds. This possibility gives the process added value. In the case studied here, olive leaf extract proved to offer remarkable results when compared to caffeic acid, even when a modifier was added to increase the solubility of the compound in scCO2. This information represents the first steps towards a knowledge of the SSI process and provides a starting point for further research.
5. ACKNOWLEDGMENTS This work was supported by the Spanish Government (Project CTQ2014-52427-R).
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Table 1. Summary of the experiments Experiment 1
2
3
Fixed parameters Parameter Agitation Time Amount of plastic Pure caffeic acid Agitation Time Amount of plastic Temperature Depressurization rate Agitation Amount of plastic Temperature Depressurization rate Olive leaf extract
Value 40 rpm 22h 600 mg 150 mg 40 rpm 22h 600 mg 35 °C 100 bar/min 40 rpm 600 mg 35 °C 100 bar/min 20 mg/mL
Variables Parameter Pressure Temperatur Depressurization rate
Value 100, 200, 300 and 400 bar 35 and 55 °C 1 and 100 bar/min
Active substance
Pure caffeic acid (150 mg) Caffeic acid in ethanol (20 mg/mL) Olive leaf extract (20 mg/mL)
Time
5 and 30 min, 1, 2 5 and 22 h
Table 2. Impregnation efficiency (%AL) of different active substances under all conditions studied (n = 2) Pure caffeic acid P (bar) DR (bar/min) 100 𝑋̅ 1 35 0.029 55 0.006 100 35 0.023 55 0.009 Caffeic acid (20 mg/mL ethanol) 100 35 0.035 Olive leaf extract 100 35 0.545 T (ºC)
𝑋̅ = Average value SD = Standard deviation
200
300
SD 0.005 0.005 0.003 0.008
𝑋̅ 0.018 0.014 0.030 0.012
SD 0.003 0.001 0.001 0.003
𝑋̅ 0.026 0.016 0.028 0.016
400 SD 0.002 0.014 0.002 0.003
𝑋̅ 0.024 ND 0.034 0.018
SD 0.001 0.007 0.023
0.001
0.043
0.010
0.053
0.003
0.057
0.006
0.044
0.721
0.113
0.790
0.109
1.209
0.104
A
B
Figure 2. Pareto diagram (p = 0.05) of %AL for the two systems considered in the study A) Effect of pressure, temperature and depressurization rate with pure caffeic acid and B) effect of modifier and P on impregnation efficiency
90
%I/100 mg plastic
80 70 60 50
100 bar
40
200 bar
30
300 bar
20
400 bar
10 0 35 ºC
55 ºC 1 bar/min
35 ºC
55 ºC
100 bar/min DR and T conditions
Figure 3. Antioxidant activity of impregnated films using pure caffeic acid
120.0
%I/100 mg plastic
100.0 80.0 60.0
Pure caffeic acid Olive leaf extract
40.0 20.0 0.0 100
200
300
400
P (bar)
Figure 4. Antioxidant activity at the pressures studied at T = 35 ºC and DR = 100 bar/min
3.5
A 3 2.5
% AL
2 1.5 1 0.5 0 5 min
30 min
1h
2h
5h
22h
2h
5h
22h
Time
350
B % I/100 mg plastic
300 250 200 150 100 50 0 5 min
30 min
1h Time
Figure 5. Study of time parameter regarding %AL (A) and % I/100 mg of plastic (B) on olive leaf extract impregnation (P = 400bar, T = 35 ºC, DR = 100 bar/min)