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Immobilization of green tea extract on polypropylene films to control theantioxidant activity in food packaging

Carol Lopez de Dicastillo, Marıa del Mar Castro-Lopez, Jose ManuelLopez-Vilarino, Marıa Victoria Gonzalez-Rodrıguez

PII: S0963-9969(13)00311-6DOI: doi: 10.1016/j.foodres.2013.05.022Reference: FRIN 4659

To appear in: Food Research International

Received date: 26 February 2013Accepted date: 25 May 2013

Please cite this article as: Lopez de Dicastillo, C., Castro-Lopez, M.M., Lopez-Vilarino,J.M. & Gonzalez-Rodrıguez, M.V., Immobilization of green tea extract on polypropylenefilms to control the antioxidant activity in food packaging, Food Research International(2013), doi: 10.1016/j.foodres.2013.05.022

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Immobilization of green tea extract on polypropylene films to control

the antioxidant activity in food packaging

Carol López de Dicastillo, María del Mar Castro-López, José Manuel López-Vilariño,

María Victoria González-Rodríguez*

Grupo de Polímeros-Centro de Investigacións Tecnolóxicas - Universidade de A

Coruña, Campus de Esteiro s/n 15403, Ferrol – Spain

* Corresponding autor

e-mail: victoria.gonzalez.rodriguez@udc.es

Tel: 34-981 337 400 (3051 / 3485)

Fax: 34-981 337 416

Abbreviations

caffeine (Caff), (+)-catechin (C), (-)-catechin gallate (CG), chemically modified

polypropylene Fusabond PMD511D (MAH511), chemically modified polypropylene

Fusabond PMD203D (MAH203), Differential scanning calorimetry (DSC), (-)-

epicatechin (EC), (-)-epicatechin gallate (ECG), (-)-epigallocatechin (EGC), (-)-

epigallocatechin gallate (EGCG), Ethylene vinyl alcohol (EVOH), gallic acid (GA), (-)-

gallocatechin (GC), (-)-gallocatechin gallate (GCG), green tea extract (GT), maleic

anhydride grafted into polypropylene (PPMAH), material formulated with PP,

chemically modified polypropylenes Fusabond PMD511D and green tea extract

(PPMAH511GT), material formulated with PP, chemically modified polypropylenes

Fusabond PMZ203D and green tea extract (PPMAH203GT), Oxidation induction time

(OIT), Polypropylene (PP), Polypropylene and green tea extract

(PPGT),Thermogravimetric analyses (TGA).

ABSTRACT

In this work, we report the successful immobilization of green tea extract, as a

natural antioxidant, on polypropylene through the incorporation of anhydride maleic

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grafted polypropylene on polymer formulation owing to control active compounds

release and prolong antioxidant activity. The extruded films were thermally

characterized showing that incorporation of green tea extract improved polymer

stability, and presence of grafted polymer did not affect polymer morphology. Green tea

components release profiles depended on the type of food and polymer formulation. The

use of grafted polypropylene changed the ability of the polymer to release green tea

antioxidants; the amount of components released decreased with increasing the degree

of grafted polypropylene. Materials were submitted to sterilization and microwave

heating conditions. The immobilization of the active compounds implied a lower release

during these typical food package treatments, and the available antioxidant components

on the modified materials presented a good correlation with the antiradical activity

toward ABTS•+

radicals, prolonging their antioxidant ability.

Keywords: Maleic anhydride modified polypropylene, natural antioxidants, catechins,

controlled release packaging, green tea extract

1. Introduction

Food products are very susceptible to rancidity caused by oxidation of their lipids

which contain unsaturated fatty acids that can be attacked by oxygen free radicals.

Antioxidants are added to foods to intercept and react with these free radicals at a faster

rate than the lipid substrate. Nevertheless, the current incorporation of antioxidants

throughout the entire food matrix in one large initial dose is not an efficient process due

to oxidation occurring at the surface and high initial doses of antioxidant having

prooxidant effects. Therefore, one emerging technology is the use of an antioxidant

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active packaging, where the antioxidant is incorporated to a packaging material with the

purpose to be delivered to the food surface during commercialization at an appropriate

rate. Most of active packaging developments base their activity on the mass transport

properties of plastic materials (sorption, migration and permeation), and the release of

the active agents depends on several factors, as type of polymer, type of food, etc.

(Miltz, Passy, & Mannecheim, 1995; Vermeiren, Devlieghere, Van beest, De

Kruijf, & Debevere, 1999). Hence, there is an interest in the food industry to develop

polymer packaging devices which can gradually deliver low concentration of

antioxidants in a controlled manner. Some studies have reported the development of

active packaging with synthetic antioxidant, such as Torres-Arreola, Soto-Valedez,

Peralta, Cardenas-Lopez, & Ezquerra-Brauer (2007) that accomplish the delay of

lipid oxidation and of protein denaturation by the incorporation of BHT into low-

density polyethylene. However, the presence of synthetic antioxidants in food is

questioned, owing to the potential risks. Strict statutory controls are then required

(Bruhn, 2000). Natural antioxidants are preferred to artificial substances, especially by

consumers. Moreover, the use of active antioxidant packaging that incorporates natural

antioxidants presents important advantages. The addition of a natural compound to the

package may reduce the need to use synthetic antioxidants in the plastic, reducing the

risk of potential toxicity by migration.

Green tea extract (GT) is a great source of flavonoids with the status of food

additive, presenting considerable interest due to their potential benefits on human

health, as antiviral, antiallergic, anti-inflamatory, antitumor, and antioxidant activities

(Lambert, Sang, Hong, & Yang, 2010; Rietveld, & Wiseman, 2003). The main

compounds responsible for this antioxidant activity are gallic acid (GA) and eight major

catechins: (+)-catechin (C), (-)-epicatechin (EC), (-)-catechin gallate (CG), (-)-

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epicatechin gallate (ECG), (-)-gallocatechin (GC), (-)-epigallocatechin (EGC), (-)-

gallocatechin gallate (GCG), and (-)-epigallocatechin gallate (EGCG) (Poon, 1998;

Zeeb, Nelson, Albert, & Dalluge, 2000). Green tea has been incorporated in food to

extend its shelf life, resulting in food protection and without changing sensorial

properties when it was added at optimum concentrations (Martin-Diana, Rico, &

Barry-Ryan, 2008; Wanasundara & Shahidi, 1998). In addition, several studies have

already shown green tea as a potential source of antioxidants to be used as additives in

plastic to protect them during polymer processing/manufacturing (Dopico-García,

Castro-López, López-Vilariño, González-Rodríguez, Valentao, Andrade, Garcia-

Garabal, & Abad, 2011), and also incorporated in polymers for the developments of

antioxidant active packaging (Colon & Nerin, 2012; López de Dicastillo; Nerin,

Alfaro, Catalá, Gavara, Hernández-Muñoz, 2011; López-de-Dicastillo, Gómez-

Estaca, Catalá, Gavara, Hernandez-Muñoz, 2012).

An interesting approach concerning active polymers that has recently been

developed, is the covalent binding of antioxidant compounds to natural and synthetic

polymers (Arrua, Strumia, & Nazareno, 2010; Goddard & Hotchkiss, 2007; Parisi,

Puoci, Iemma, De Luca, Curcio, Cirillo, Spizzirri, & Picci, 2010). Most of them have

interesting applications, but the important advantage presented in the present research is

that materials were obtained by extrusion, common polymer in the manufacturing

process in food industry. In this work, maleic anhydride grafted into polypropylene

(PPMAH), normally used as compatibilizer between polar and non polar polymers

(Bettini & Agnelli, 2002; Macosko, Jeon, & Hoye, 2005; Pang, Jia, Hu, &

Hourston, 2000), was added on polymer formulation with the purpose to immobilize

antioxidants from green tea by chemical interaction. Despite the increasing demand for

biodegradable materials, polyolefins are still the most common polymers used for food

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packaging. Maleic anhydride modified polyolefins are one of the most important class

of functionalized polyolefins in commercial applications, due to its low cost, high

activity and good processability. The present study was designed to investigate the

feasibility of utilizing two maleic anhydride grafted polypropylenes to control the

release of active compounds and prolong the antioxidant activity of active materials.

Several food products, like the continuously increasing demanded precooked foods

(ready-to-eat products), require a retorting treatment before being commercialized

(Ramesh, 1999). An industrial sterilization of packaged foods is carried out to control

food deterioration. Although sterility can be achieved with certain chemicals, physical

methods are generally more reliable. Heat is one of the most commonly used physical

methods of sterilizing food products (typically 121 ºC during 20 min in an industrial

autoclave, i.e., in the presence of pressurized water vapor)

Another common intense treatment that package materials can suffer is through

microwave oven radiations. Microwave oven has become a modern convenience

appliance in every kitchen in the developed countries to thawing of frozen foods and

warm ready-to-eat foods.

The objective of this work is the incorporation of green tea extract and grafted

polypropylene on polymer formulation to develop an antioxidant active packaging to

improve food protection during longer periods of time after sterilization process,

maintaining antioxidant activity after first release due to humid thermal conditions and

during short strong heating treatment. Consequently, two different food package

treatments were simulated: i) a sterilization of packaged food, to calculate the release

extent of active compounds and their later antioxidant activities; and ii) a microwave

heating, directed to measure the release rate achieved on an ordinary heating.

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2. Methods and Materials

2.1. Chemicals and reagents

Polypropylene PP 070 G2M (PP) was provided by Repsol YPF (Madrid, Spain).

Chemically modified polypropylenes Fusabond PMD511D (MAH511) and PMZ203D

(MAH203) were purchased from DuPontTM

(Barcelona, Spain). Both chemically

modified polypropylenes have medium and high graft level, respectively. Specific graft

levels of both commercial Fusabond modified polypropylene were reported as

confidential by DuPontTM

. Liu & Konlopoulou (2006) reported an estimated

concentration of maleic anhydride in Fusabond PMD511D between 0.25-0.5%. No data

was reported for PMZ203D.

Reagent-grade absolute ethanol, gallic acid, (+)-catechin dihydrate, (-)-epicatechin,

(-)-catechin gallate, (-)-epicatechin gallate, (-)-epigallocatechin, (-)-gallocatechin

gallate, and (-)-epigallocatechin gallate, caffeine (Caff) and 2,2´-azino-bis(3-

ethylbenzothiazoline-6-sulfonic acid) (ABTS) were purchased from Sigma (Madrid,

Spain). Green tea extract was supplied by Plantextrakt (The Nature Network),

Baceiredo S.L. (Vitoria, Spain).

2.2. Materials preparation

The blends with polypropylene, maleic anhydride grafted polypropylene and green

tea extract PP:PPMAH:GT (4:2:1 w:w:w) and PP:GT (6:1 w:w) were carried out using

a miniextruder equipped with twin conical corotating screws and a capacity of 7 cm3

(MiniLab Haake Rheomex CTW5, Thermo Scientific). A screw rotation rate of 40 rpm,

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temperature of 180 ºC, and 1 min of residence time were the parameters used. Materials

PPMAH511GT and PPMAH203GT were formulated with PP, chemically modified

polypropylenes Fusabond PMD511D and PMZ203D, respectively, and green tea

extract. Blend without PPMAH, only PP+GT, was also extruded owing to study the

effect of functionalized modified polypropylene, and they were named PPGT. Blends

without green tea extract were extruded as references (PPMAH511, PPMAH203 and

PP). For all assays, extruded materials were hot-pressed on a pressing plate IQAP LAP

S.L. model PL15-serie1381 (Barcelona), and very thin (approximately 35-45 µm) films

were obtained.

2.3. Chromatographic study of green tea extract

Green tea extract components were quantitatively analyzed by HPLC-PDA-FL to

identify their major compounds using a Waters 2695 (Waters, Mildford, MA, USA)

system with a gradient pump and automatic injector. Chromatographic experiments

were carried out using a stainless steel column packed with SunFireTM

C18 (150 mm x

3.0 mm, 3.5 μm) (Waters) kept at 35 ºC. Detection was performed on a photodiode

array detector (PDA, model 996 UV) set in the range of 200 to 400 nm (277 nm as

output signal), and a fluorescence detector (FL, model 2475) (Waters) with λexcitation 280

nm and λemission 310 nm. Output signals were monitored and processed with a personal

computer operated under the EmpowerTM

software (Waters). A two solvent gradient

elution was performed, with flow rate of 0.5 mL min-1

and injection volume of 20 μL.

Mobile phase was composed by water (A) and methanol (B). The following gradient

elution profile was used: mobile phase composition started at 25% B and maintained for

0.5 min. Then, it was linearly increased to 40% B in 4.5 min, 60% B in 1 min and 100%

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B in 2 min. Finally, it was maintained for 3 min and brought back to the initial

conditions. Each compound was identified by means of retention time and UV or FL

spectrum comparison with corresponding peaks in the standard solution.

Quantification was carried out using a calibration plot of external standards (Dopico-

García, López-Vilariño, Bouza, Abad, González-Soto, & González-Rodríguez,

2004; Dopico-García, Castro-López, López-Vilariño, González-Rodríguez,

Valentao, Andrade, Garcia-Garabal, & Abad, 2011).

2.4. Thermal Analysis

Thermogravimetric analyses, TGA, were carried out using a thermal analyzer Perkin

Elmer TGA 7 microbalance coupled to a Perkin Elmer 1022 microprocessor. Samples

(ca. 10 mg) were heated in 100 µl platinum sample pans from room temperature to 800

ºC under a nitrogen atmosphere at 10 ºC/min, to determine the degradation temperatures

of new formulation materials.

Differential scanning calorimetry, DSC, measurements (Perkin–Elmer serie 7) were

also performed owing to study the effect of immobilization of green tea components on

the polypropylene morphology and crystallinity. Thermograms were obtained from -20

to 200 ºC with 10 ºC/min heating, cooling to -20 ºC and holding at this temperature for

2 min, and a second heating process to 200 ºC. Melting and crystallization temperatures,

Tm and Tc, and enthalpies, ΔHm and ΔHc, were calculated from the cooling and the

second heating process.

Oxidation induction time (OIT) analyses were conducted to study changes of

polymer stability and antioxidant effectiveness in the new developed blends. The

sample temperature was stabilized for 2 min at 200 ºC under inert atmosphere, which

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was subsequently switched to oxygen atmosphere to start the test. Analyses were carried

out according to EN 728 (European Committee for Standardization, 1997). OIT was

measured as the onset point at which the DSC thermogram suffers as sudden drop

respect to the instrument baseline. OIT measurements were also carried out to materials

after they were exposed to migration conditions (on simulant D1 at 40 ºC during 10

days).

2.5. Release studies

A study of the release of the green tea extract compounds from the films was

performed according to the methodology described by Dopico-García (Dopico-García,

López-Vilariño, & González-Rodríguez, 2003 and 2007). Their specific migration

into two food simulants specified in European law was determined: simulant A (10%

ethanol), assigned for foods that have a hydrophilic character, and simulant D1 (50%

ethanol), assigned for foods that have a lipophilic character (Commission Regulation

(EU) No 10/2011). Double-sided, total immersion migration tests were performed as

follows: rectangular strips film pieces (1 cm x 3 cm) and 5 mL of food simulant were

placed in glass-stoppered tubes with PTFE closures. Release tests were conducted at 40

ºC, and green tea extract compounds were quantified after 10 days of storage. Test

materials were also run simultaneously to check for interferences.

2.6. Study of the influence of different packaging treatments on the release-behavior

2.6.1. Thermal sterilization treatment

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Thermal sterilization is a common system used in food industry to inhibit microbial

growth that affect food spoilage, as Bacillus spores, and consists on heating food

products above 100 ºC. Following European Regulation, these conditions can be

simulated by heating the product at 70 ºC during two hours. Therefore, materials were

immersed in simulant D1 in a relation of 6 dm2 L

-1 in glass-stoppered tubes with PTFE

closures, and exposed to 70 ºC during two hours. Then, samples were cooled to room

temperature. Release of green tea extract components in the simulant was analyzed and

quantified by HPLC-PDA-FL for developed material after sterilization process.

Moreover, films were removed, cleaned, and immersed in 3 mL of ABTS•+

solutions of

known concentration to test the remaining antioxidant activity of materials as radical

scavengers (Sánchez-Moreno, 2002; Conde, Moure, Domínguez, & Parajó, 2011;

López de Dicastillo, Nerín, Alfaro, Catalá, Gavara, & Hernández-Muñoz, 2011;

López de Dicastillo, Gómez-Estaca, Catalá, Gavara, & Hernández-Muñoz, 2012).

The radical cation ABTS•+

on aqueous solution has an intense green color with a

maximum absorption peak at 734 nm, and this absorption disappears when the radical is

neutralized. As a result, ABTS assay measures the antioxidant effectiveness by

monitoring the inhibition/bleaching rate of the radical ABTS•+

. Radicals ABTS•+

are

neutralized either by direct reduction via electron transfers or by radical quenching via

H atom transfer (Prior, Wu, & Schaich, 2005).

2.6.2. Microwave oven heating

In this experiment, a common microwave heating process of pre-cooked packaged

meals was simulated in order to calculate the release rate of antioxidants from the

developed materials into the packaged food product. Following European Legislation,

materials were immersed in simulant D1 in a relation of 6 dm2 L

-1, and submitted to

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microwave conditions, approximately 800 W during 15 min. After this treatment,

samples were cooled to room temperature and different components of green tea release

were analyzed and quantified by HPLC-PDA-FL.

2.7. Statistical analysis

Data resulted from thermal and release studies were analyzed by a one-way analysis

of variance (ANOVA) test, using the SPSS computer program (SPSS Inc., Chigaco, IL).

Significant differences in pairs of mean values were evaluated by the Tukey’s test at

a significant level of p<0.05. Different letters (a-d and x-y) indicate significant

differences among the diverse data from the materials tested. Moreover, data was also

presented as the mean ± standard deviation.

3. Results and Discussion

3.1. Thermal Characterization

Table 1 summarizes most relevant data obtained from thermal analysis. TGA

revealed the incorporation of green tea extract improved slightly the thermal stability.

On Table 1 are exposed the degradation peaks expressed as temperature of the

minimum at the derivative weight loss with. The influence of the incorporation of

maleic anhydride grafted composites was considerably important, presenting highest

degradation temperatures the samples with PPMAH and GT.

As Fig. 1 shows, there is a broad degradation band in the thermogram of green tea,

including a peak at 100 ºC related to the evaporation of water from the extract. The

catechins from the extract are partially glycosylated, and a “caramelization” of these

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sugars takes place probably due to the heating, giving rise to the broad degradation band

that occurs between 200-400 ºC owing to the degradation of the extract, blends with

green tea presented small degradations bands (shown better in the insert in Fig. 1)

before polymer degradation peak. By other side, incorporation of maleic anhydride

polypropylene did not imply any additional degradation process (shown in PPMAH511

thermogram in Fig. 1).

Materials were also thermally analyzed by DSC owing to study the effect of the

different composition on the polymer crystallinity of the new materials. Results

obtained from cooling and second heating processes are also included in Table 1.

Differences were not extremely relevant, and incorporation of PPMAH and GT had

similar effect. Comparing to thermal properties values of the reference material PP,

during second heating process samples with PPMAH and GT presented lower melting

point (minimum of the endotherm) and melting enthalpy, probably due to the fact that

functionalized groups interrupted chain polymer crystallization. Furthermore, as much

to PPGT as to materials with grafted polypropylene, an increase on Tc and decrease on

ΔHc was shown, owing to the presence of new linkages that altered crystallization

process of polypropylene matrix. Normally, reduction on melt and crystallization

capacities of blends is attributed to the increase of the interaction between polymer

phases that disturbs their crystallization. Higher value of Tc also indicated that the

movement of polymer chains were more restricted, definitely due to maleic anhydride

interaction and incorporation of green tea extract (Bettini & Agnelli, 2002; Pracella,

Haque & Alvarez, 2010; Pang, Jia, Hu, & Hourston, 2000).

OIT measures the amount of time a polymer needs to oxidize at an elevated

temperature. This gives an insight into the efficiency of antioxidants in the polymer

matrix. Therefore, results showed that the incorporation of green tea extract increased

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considerably OIT values for all materials, and maleic anhydride group presence in the

blend implied a little increase in the protection (Table 1), indicating antioxidant

immobilized into the polymer matrix is available to protect the polymer (Al-Malaika,

2003). OIT measurements were also performed after the materials were exposed to

migration assays. Results showed OIT values slightly decreased but maintained high

values (48.0, 53.6 and 55.1 min for PPGT, PPMAH511GT and PPMAH203GT,

respectively). In general, the antioxidant effectiveness of the samples were not

practically modified by antioxidant release occurred after migration. This behavior is

necessary for active packages that will be heated after storage step, as pre-cooked

packaged meals for microwave heating.

Moreover, as indicated in Table 1, statistical analysis of data also confirmed the

differences in the values achieved for each material accordingly which have already

been commented.

3.2. Chromatographic analysis of green tea extract

Green tea is known for being an important source of phenolic compounds, especially

flavonoids, which are one of the most effective antioxidant constituents (Harbowy &

Balentine, 1997). Hence, it is important to quantify phenolic content as to assess its

contribution to antioxidant activity. The identification of all compounds was confirmed

by injection of pure standards (shown in Fig. 2). Table 2 exposes the quantification data

obtained of different catechins, gallic acid and caffeine present on 1 g of green tea.

The content of green tea catechins together with gallic acid was found to be

approximately 62% of its weight. Seven catechins were determined being

epigallocatechin gallate, epicatechin gallate, and gallocatechin gallate, the most

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abundant in green tea extract, constituting up to 80% of the content of green tea in

catechins. Therefore, the largest percentage of catechins present in green tea exists as

gallate forms, which are also the more polar catechins. Gallic acid was only found to be

in less than 2% of the total content.

3.3. Green tea migration studies

Green tea components released on food simulants quantified through HPLC-PDA-FL

are exposed on Table 3. Statistical analysis shown through differences among data pairs

is also compiled in Table 3. The effect of the type of food was clearly noticeable,

presenting considerably higher release into simulant D1. The extent of the release at

equilibrium (after a long exposure time) depends on the compatibility of the migrant

with both the food simulant and the polymeric film. The higher the solubility in the

simulant, the higher the release. Gallic acid also presented considerable release extent

probably related with a degradation process suffered by the tea sample during extrusion.

All these results agree with previous studies where this green tea extract was already

incorporated on ethylene vinyl alcohol (EVOH) (López de Dicastillo et al., 2011).

Fig. 2 shows, as an example, the HPLC-PDA profile of the release of different GT

components from PPMAH203GT blend on simulant D1 (chromatogram B). The

identification of all compounds was confirmed by injection of pure standards, shown in

Fig. 2-A.

It is also obvious that large differences in the rate and amount of migration were

found depending on the type of polypropylene used, grafted vs. non grafted films. The

use of grafted PP has notably changed the ability of the polymer to release green tea

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extract into the food simulants; the amount of components released decreased with

increasing the degree of grafted PP.

3.4. Green tea release by food package treatments

3.4.1. Green tea release from sterilization process

In this section, materials were exposed to the conditions of a sterilization process;

green tea components released into simulant D1 were quantified. Afterward, their

radical scavenging activities were measured owing to determine their antioxidant

activity after sterilization. As Fig. 3 shows, there was a significant amount of green tea

components released. Certainly, the conditions disrupt the self-association of different

catechins with polymer matrix, leading even to disrupt the immobilization between

green tea components and PPMAH. Thus, the thermally activated molecular structure

certainly become more plasticized by the combined presence of heat and water which,

in turn, gave rise to sufficient segmental molecular mobility, increasing the release.

The aim of achieving a smaller level of migration was reached, being dependent on

maleic anhydride content on polymer formulation. In most of the cases, release extent

for PPGT materials were approximately two times higher than the values obtained for

PPMAH203GT, which formulation is presumably related to the best green tea

immobilization. As it was expected, values for PPMAH511GT were intermediate

between PPMAH203GT and PPGT.

After release studies, films were exposed to a solution of ABTS•+

radicals. These

assay measured the ability of materials for trapping free radicals by donating hydrogen

atoms or electrons, producing, in consequence, the bleaching of the colored radical

solutions. As Fig. 4 shows, it was found that the order of radical scavenging activity of

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the materials developed was in accordance with the antioxidant concentration remained

in the blend. According with the results obtained from release studies shown in Fig. 3,

the material PPGT exhibited the highest level of GT components release, implying a

low inhibition rate of radical ABTS•+

(Fig. 4). This clearly indicates that the linkage of

green tea components with PPMAH increased its protective antioxidant ability in the

blend. The addition of maleic anhydride polypropylene reduced the release, allowing

the availability of the antioxidants during latter storage step of food.

As it can be seen from Fig. 4, materials with PPMAH presented similar radical

scavenger activity during the first two days, but after the antioxidant activity rate of

PPMAH511GT started to diminish, while PPMAH203GT continued active.

3.4.2 GT release from microwave heating process

In Fig. 3 is also exposed the release extent of different compounds of green tea

extract into a food packaged with these materials when there is a heating in the

microwave. As mentioned in the case of sterilization, microwave conditions implied

higher mobility of polymer chains, obtaining high release values. As expected, it is

interesting to point out that the release was dependent with the concentration of maleic

anhydride present in the blend. As higher it was the concentration of grafted PPMAH in

the polymer formulation, lower release rate occurred. Slowing down the release of some

active agents can be interesting when high concentration of active agents can imply

changes on organoleptical properties of the food product.

On both aggressive treatments suffered by package materials, it was possible to

measure the release of an important amount of green tea antioxidants. Nevertheless, as

Fig. 3 shows, concentration of the antioxidant EGCG was not detected on simulant D1

after both treatments, and in the case of the flavonoid EGC, it was also not found in

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simulant after microwave heating, probably due to their low stability under these

conditions. It has been already extensively reported the epimerization of tea catechins

when they are exposed to heating or different processes, as microwave reactor.

Specially, catechins with a 2,3-cis configuration, such as EGCG, has been considered as

stereochemically less stable than those possessing a 2,3-trans configuration, which lead

to their higher epimerization rate (Unnadkat & Elias, 2012; Wang & Helliwell, 2000;

Wang, Zhou, & Wem, 2006).

4. Conclusions

The immobilization of green tea components was successfully performed through the

incorporation of maleic anhydride grafted polymer. The interactions between

antioxidant substances of green tea and functionalized grafted polypropylene explain the

reduction of the release levels of green tea components during storage of films in

contact with food simulants. Immobilization of green tea components was proportional

to the concentration of maleic anhydride groups, achieving highest level of

immobilization in the material PPMAH203GT

According to thermal properties studies, thermogravimetric results revealed a slight

improvement on thermal stability provided by green tea extract incorporation. OIT

results also showed an increase in antioxidant effectiveness caused by the presence of

green tea and maleic modified polypropylene in the blend formulations. Furthermore,

no significant differences between studied composites were provided by DSC analysis.

Considering real food packaging applications, GT loaded MAH-grafted-PP materials

have proved to be effective systems for controlled release of the antioxidant during

longer periods of time, being green tea antioxidants also available in the film

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formulation to protect it from aging. In this system, the release of the active compounds

during the first days is reduced, remaining the additive in the polymer intended both for

a greater protection of the polymer and food for later stages in which its use will be

required.

Results showed microwave and sterilization heating implied lower release of GT

compounds to food in the case of materials with PPMAH due to the immobilization of

the active compounds. Regarding sterilization, this formulation avoids the fast

migration typical in a high temperature process. In addition, results showed up the

available green tea components on the modified materials presented a good correlation

with the antiradical activity toward ABTS•+

radicals, prolonging their antioxidant

ability.

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Figure Captions

Figure 1. Derivative of the weight loss of polypropylene, maleic anhydride grafted

polypropylene blends and green tea extract obtained by TGA. Insert: magnified image

during a short temperature range.

Figure 2. Chromatograms by PDA detection of: A) pure standards, B) release of

material formulated with PP, chemically modified polypropylenes Fusabond PMZ203D

and green tea extract (PPMAH203GT) in simulant D1.

(Abbreviations: caffeine (Caff), (+)-catechin (C), (-)-catechin gallate (CG), (-)-

epicatechin (EC), (-)-epicatechin gallate (ECG), (-)-epigallocatechin (EGC), (-)-

epigallocatechin gallate (EGCG), (-)-gallocatechin gallate (GCG))

Figure 3. Antioxidant components released when polymeric materials were exposed to

sterilization conditions (EST) and microwave heating (MW). (a, b and c indicate

significant differences among the values of each antioxidant migrated from different

materials exposed at the same conditions) (x and y indicate significant differences

among the values of each antioxidant migrated from the same material exposed at

different treatments). (Abbreviations: material formulated with PP, chemically modified

polypropylenes Fusabond PMD511D and green tea extract (PPMAH511GT); material

formulated with PP, chemically modified polypropylenes Fusabond PMZ203D and

green tea extract (PPMAH203GT); Polypropylene and green tea extract (PPGT); (+)-

catechin (C); (-)-catechin gallate (CG); (-)-epicatechin (EC); (-)-epicatechin gallate

(ECG); (-)-epigallocatechin (EGC); gallic acid (GA); (-)-gallocatechin gallate (GCG)).

Figure 4. Radical scavenging activity of ABTS•+

radical of developed materials.

(Abbreviations: material formulated with PP, chemically modified polypropylenes

Fusabond PMD511D and green tea extract (PPMAH511GT); material formulated with

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PP, chemically modified polypropylenes Fusabond PMZ203D and green tea extract

(PPMAH203GT); Polypropylene and green tea extract (PPGT),

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Figure 1

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Figure 2

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Figure 3

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Figure 4

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Table 1. Thermal properties information of developed materials obtained from Oxidation induction time,

Thermogravimetric and Differential scanning calorimetry analyses.

OIT TGA Cooling process Second Heating process

SAMPLE time (min) (ºC) Tc ( ºC) ΔHccorreg (J g-1

) Tm ( ºC) ΔHmcorreg(J g-1

)

PP 4.2 ± 0.1 a 473. 7 112.7 ± 0.1 a 30.4 ± 0.3 d 165.6 ± 0.5 d 24.9 ± 0.2 d

PPMAH511 8.2 ± 0.1 b 470.5 115.9 ± 0.1 bc 29.1 ± 1.2 bc 163.7 ± 0.1 c 23.8 ± 0.9 bc

PPMAH203 7.2 ± 0.3 b 472.7 116.0 ± 0.2 bc 28.1 ± 0.3 ab 162.9 ± 0.2 ab 22.4 ± 0.4 a

PPGT 48.9 ± 2.6 c 477.1 116.5 ± 0.1 d 29.8 ± 0.1 c 163.8 ± 0.2 c 24.1 ± 0.2 cd

PPMAH511GT 55.7 ± 2.1 d 483.2 115.8 ± 0.2 b 28.7 ± 0.5 bc 163.1 ± 0.9 bc 24.3 ± 0.4 cd

PPMAH203GT 55.6 ± 2.0 d 481.3 116.1 ± 0.2 c 27.5 ± 0.8 a 162.3 ± 0.1 a 23.0 ± 0.7 ab * Different letters (a-d) indicate significant differences among the diverse materials in every thermal property.

PPGT, material formulated with PP and green tea extract.

PPMAH511, material formulated with PP, chemically modified polypropylenes Fusabond PMD511D.

PPMAH203, material formulated with PP, chemically modified polypropylenes Fusabond PMZ203D.

PPMAH511GT, material formulated with PP, chemically modified polypropylenes Fusabond PMD511D, green tea extract.

PPMAH203GT, material formulated with PP, chemically modified polypropylenes Fusabond PMZ203D, green tea extract.

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Table 2. Green tea extract composition

Component mg component g-1

green tea

gallic acid 13.7 ± 0.2

(-)-epigallocatechin 49.4 ± 0.3

(+)-catechin 17.4 ± 1.2

(-)-epigallocatechin 303.0 ± 3.1

(-)-epicatechin 38.7 ± 1.8

(-)-gallocatechin gallate 73.0 ± 5.4

(-)-epicatechin gallate 107.0 ± 0.2

(-)-catechin gallate 15.0 ± 0.5

Caffeine 94.5 ± 1.9

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Table 3. Release data of green tea extract compounds migrated on food simulants A and

D1 (mg components g-1

material).

Simulant PPMAH511GT PPMAH203GT PPGT

GA A 0.68 ± 0.35 a,x 0.41 ± 0.35 a,x 0.82 ± 0.35 a,x

D1 5.47 ± 0.46 a,y 4.80 ± 0.60 a,y 8.70 ± 1.12 b,y

C A 1.42 ± 0.31 b,x 0.25 ± 0.09 a,x 1.95 ± 0.17 c,x

D1 3.05 ± 0.71 a,y 2.26 ± 0.30 a,y 3.37 ± 0.66 a,y

EGCG A 7.30 ± 0.10 a,x 6.00 ± 1.70 a,x 7.15 ± 0.60 a,x

D1 25.60 ± 0.87 b,y 17.15 ± 0.65 a,y 26.60 ± 4.44 b,y

EC A 1.03 ± 0.29 b,x 0.20 ± 0.09 a,x 0.98 ± 0.15 b,x

D1 1.13 ± 0.23 a,x 1.08 ± 0.15 a,y 1.19 ± 0.05 a,x

GCG A 5.51 ± 0.94 a,x -**

8.84 ± 3.50 a,x

D1 21.40 ± 5.65 b,y 11.5 ± 2.79 a 25.9 ± 4.16 b,y

ECG A 2.08 ± 1.23 a,x 1.42 ± 0.96 a,x 13.4 ± 1.80 b,y

D1 6.49 ± 1.15 b,y 2.48 ± 0.91 a,x 7.57 ± 1.26 b,x

CG A 2.77 ± 0.49 a,x 3.51 ± 1.30 a,x 5.36 ± 0.64 b,x

D1 5.86 ± 2.08 ab,x 4.71 ± 0.54 a,x 8.68 ± 2.34 b,x * Different letters (a-c) indicate significant differences of migration among the diverse materials

in each simulant. Different letters (x-y) indicate significant differences among the simulants in

each material.

** Data was not quantitatively possible to integrate.

Abbreviations: (+)-catechin (C), (-)-catechin gallate (CG), (-)-epicatechin (EC), (-)-epicatechin

gallate (ECG), (-)-epigallocatechin gallate (EGCG), gallic acid (GA), (-)-gallocatechin gallate

(GCG).

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Highlights.

Polypropylene-anhydride maleic grafted polypropylene-green tea for food active

package

Maleic anhydride grafted into polypropylene changed polymer release ability

No changes or slight improvements induced by new formulations on blend

effectiveness

Maleic anhydride polypropylene: prolonging antioxidant activity after sterilization

Maleic anhydride polypropylene decreases antioxidant release due to microwave

heating