Development of Antioxidant Packaging Material

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Development of Antioxidant Packaging Materialby Applying Corn-Zein to LLDPE Filmin Combination with Phenolic CompoundsHye-YeonPark, Sung-Jin Kim, Ki Myong Kim, Young-Sun You, So Yeon Kim, and Jaejoon Han

Abstract: Functional active packaging materials were successfully developed by incorporating antioxidant agents into

corn-zein-laminated linear low-density polyethylene (LLDPE) film. The minimum effective concentrations of the active

compounds (for example, thymol, carvacrol, eugenol) were determined and these compounds were then laminated into

LLDPE films to develop corn-zein-laminated films with antioxidant agents. The release rate of antioxidant agents in

gas and liquid media were determined along with the mechanical and water barrier properties of the films containing

these compounds. Tensile strength and percentage elongation at break were reduced in the corn-zein-laminated LLDPE

films when compared to typical LLDPE film. Furthermore, the ability of the corn-zein-laminated films to repel moisture

decreased by approximately 12.2%, but was improved by incorporating hydrophobic antioxidant compounds in the corn-

zein layer. Examination of release kinetics in the gas and liquid phases verified that antioxidants were effectively released

from the films and inhibited oxidation during testing. Finally, the films were used for fresh ground beef packaging,

and effectively inhibited lipid oxidation and had a positive effect on the color stability of beef patties during storage.

These results indicate that the developed antioxidant films are a novel active packaging material that can be effectively

implemented by the food industry to improve the quality and safety of foods.

Keywords: antioxidant film, corn-zein, diffusion test, ground beef, mechanical property

Practical Application: Zein protein, a by-product of corn processing industry, was laminated into plastic films in combi-

nation with natural phenolic compounds to develop antioxidant packaging films. The films demonstrated their efficient

release patterns of antioxidant compounds, which are suitable for packaging applications and food protection.

IntroductionThe main purpose of food packaging is to maintain the qual-

ity and safety of food during distribution and storage. In recent

years, there have been increasing concerns about food quality and

safety, which have motivated the development of active packaging

(Dallyn and Shorten 1988). Active packaging is an innovative type

of packaging that prevents or slows deterioration in food quality

(Buonocore and others 2003). Oxygen scavengers, carbon diox-

ide scavengers/emitters, moisture absorbers and ethanol generators

are some examples of active packaging. Active packaging also in-

volves the release of antioxidants and/or antimicrobial substances

onto the food surface. Due to environmental and health concerns,

active packaging has been made from eco-friendly and biodegrad-

able food packaging materials combined with natural preservatives

(Appendini and Hotchikiss 2002; Suppakul and others 2003).Polyethylene (PE), the most common and least expensive syn-

thetic polymer, is a widely used plastic material in food packaging.

MS 20120474 Submitted 3/28/2012, Accepted 7/10/2012. Authors Park,S.-J. Kim, and Han are with Dept. of Food Science and Biotec hnology, SungkyunkwanUniv., Suwon 440-746, Republic of Korea. Author K. M. Kim is with Dept. of Foodand Nutrition, Honam Univ., Gwangju 506-714, Republic of Korea. Author Youis with Bio Polymer R&D Center, Korea Bio Material Packaging Assn., Bucheon421-742, Republic of Korea. Author S. Y. Kim is with Boimedical Research Institute,Korea Institute of Science and Technology, Seoul 136-791, Republic of Korea. Directinquiries to author Han (E-mail: [email protected]).

PE is the family name for resins such as low-density polyethy-

lene (LDPE), high-density polyethylene (HDPE), and linear low-

density polyethylene (LLDPE) (Robertson 1993). However, de-

spite the physical and economic advantages of PE films, they cause

environmental problems due to their nondegradability and diffi-

culty in recycling. Therefore, researchers have begun to use nat-

ural polymers as substitutions for synthetic polymers to develop

biodegradable food packaging materials (Peppas 2004).

Biopolymers are biodegradable, nontoxic, recyclable polymers

that are frequently applied as edible coatings or films (Cuq and

others 1998). They are made from natural sources such as polysac-

charides, proteins, and lipids. Zein is the major storage protein of

corn (Zea mays L.) and comprises 45% to 50% of corn protein.

It is a widely used biopolymer for producing biodegradable pack-aging materials. The advantage of corn-zein is its ability to form

tough, relatively hydrophobic, and grease-proof films with excel-

lent flexibility and compressibility (Lai and others 1997). However,

corn-zein film has problems relating to its brittleness under dry

conditions, which restricts its use as a free-standing film or a coat-

ing material (Pomes 1971; Shukla and Cheryan 2001). To over-

come this problem, biopolymers are often coated on conventional

synthetic plastic films to develop a multilayered film structure (Atik

and others 2008).

For over 50 y, antioxidants have been added to the food-making

process to delay auto-oxidation (Cuvelier and others 1994). In ac-

tive antioxidant packaging, this consists of introducing antioxidants

C 2012Institute of FoodTechnologists R

doi: 10.1111/j.1750-3841.2012.02906.x Vol. 00, Nr. 0, 2012 Journal of Food Science E1Further reproductionwithout permission is prohibited


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Antioxidant packaging film . . .

into food packaging material instead of adding them directly to

the food (Tovar and others 2005). A wide range of compounds,

most of them synthetic molecules, have been used as antioxidant

agents in packaging (Padgett and others 1998). Currently, the most

frequently and widely used synthetic antioxidants in active food

packaging are butylated hydroxyanisole (BHA) and butylated hy-

droxytoluene (BHT), which are highly stable and efficient, and are

also cost-effective. Although these antioxidants can effectively in-

hibit the lipid oxidation of foods, the use of synthetic antioxidants

is strictly regulated as they are known to cause health risks (Branen1975) and is becoming increasingly unacceptable to consumers be-

cause of their potential toxicity (Chan and others 2007; Yen and

others 2008). Due to safety concerns in the active food packaging

sector, natural compounds have been deemed more appropriate.

Essential oils, extracted from herbs or spices, are regarded as

natural alternatives to synthetic preservatives and the use of these

oils in food meets consumer demands for safe food products (Burt

2004). Many essential oils exhibit antioxidant activity, which is

attributable to their high phenolic compound content. Phenolic

compounds are free radical scavengers, metal chelators, reducing

agents, hydrogen donors, and singlet oxygen quenchers (Proestos

and others 2006). In particular, thymol, carvacrol, and eugenol are

some of the most active phenolic antioxidants found in essentialoils. They are the major phenolic compounds present in thyme,

oregano (thymol and carvacrol), and clove (eugenol) essential oils.

Roberto and Baratta (2000) reported that thymol, eugenol, and

carvacrol exhibited the highest antioxidant activity in a study of

100 pure components of essential oils. In recent reports, eugenol,

menthol, and thymol maintained the quality and safety of sweet

cherries under modified atmospheric conditions (Serrano and

others 2005).

Active packaging acts as an inert barrier between food products

and the external environment, and the incorporation of antiox-

idant agents into polymeric packaging materials is an exciting

development in food packaging (Nern and others 2006). Antiox-

idants are released from packaging films by diffusion, and directlyinhibit lipid rancidity on the food surface, especially in oxidation-

sensitive foods. Antioxidant packaging also has the potential to

extend the shelf life of foods (Anklam and others 1997; Morales-

Aizpuruaand and Tenuta-Filho 2005).

The objective of the present study was to develop multilayer

films based on corn-zein and LLDPE films incorporated with an-

tioxidant agents (thymol, carvacrol, and eugenol). The mechanical

properties and hydrophobic qualities of developed films and the

release rate of antioxidant agents from the film matrix into model

systems were measured. In addition, the effectiveness of the de-

veloped packaging films on color stability and lipid oxidation for

the storage of fresh ground beef was evaluated.

Materials and Methods

Chemicals and reagentsCorn-zein, antioxidant agents (thymol, carvacrol, and eugenol),

and 2,2-diphenyl-1-picrylhydrazyl (DPPH) were purchased from

Sigma-Aldrich Chemicals Co. (St. Louis, Mo., U.S.A.). Polyethy-

lene glycol 400 (PEG 400), a plasticizer of corn-zein film, was

obtained from Junsei Chemicals Co. Ltd. (Tokyo, Japan). LLDPE

film was obtained from Hanjin P&C Co. Ltd. (Seoul, Korea), while

propyl gallate (PG) and ethylenediaminetetraacetic acid (EDTA)

were obtained from DAEJUNG (Siheung, Korea). Thiobarbituric

acid (TBA) was purchased from Alfa Aesar (St. Ward Hill, Mass.,

U.S.A.).

Film preparationCorn-zein coating solutions were prepared according to the

methods described by Mastromatteo and others (2009) with some

modifications. Corn-zein was dissolved in 95% ethanol at 20 g/

100 mL. As a plasticizer, PEG 400 was added to corn-zein solu-

tion at 4 g/100 mL and the solution was then heated at 70 C for

15 min. After cooling, thymol, carvacrol, and eugenol were in-

dividually added to the corn-zein solutions at a designated con-

centration (1.5, 3, or 5%, v/v) and stirred for 15 min. Corn-zein

solutions containing different concentrations of each antioxidantagent were coated onto corona-treated LLDPE films (surface ten-

sion 45 5 dyne/min; thickness 40 0.4 m) using a Nr. 28

coating rod (RDS, Webster, N.Y., U.S.A.) and dried for 15 min

at room temperature. The coated side of the double-layered film

was laminated with another LLDPE film using a heat laminat-

ing machine (GMP Co., Paju, Korea). The final multilayered film

(thickness 95 2.2m) structure is shown in Figure 1.

Measurement of film propertiesFilm thickness. Laminated film thickness was measured with

a digital micrometer (ID-C112X, Mitutoyo, Kawasaki, Japan) at

5 random positions on the film. The mean of the 5 measure-

ments was used to calculate tensile strength (TS) and water vaporpermeability (WVP).

Mechanical properties. Mechanical properties of the films

were determined by ASTM standard method D882-91 (1995)

using an Instron universal testing machine (Model 5566, Instron

Engineering Co., Canton, Mass., U.S.A.). Films were cut into

strips with a test dimension of 25.4 mm 100 mm. Before testing,

all of the films were conditioned for 48 h in a thermo-hygrostat

(LAM-MADE011, Sejong Scientific Co., Bucheon, Korea) held

at 25 C and 50% RH. The equilibrated strip was placed between

grip heads of the machine. The initial grip separation and cross

head speed were set at 50 mm and 500 mm/min, respectively. TS

was expressed in MPa and was calculated by dividing the maximum

load (N) by the initial cross sectional area (m

2

) of the specimen.The percentage elongation at break (%E) was calculated as theratio of the final length at the point of break to the initial length

of a specimen (50 mm) and expressed as a percentage. TS and %Ewere replicated 5 times for each type of film.

Water vapor permeability. WVP was determined gravimet-

rically, based on ASTM standard method E96-95 (1997). Each film

sample was mechanically sealed onto a polymethylmethacrylate

cup containing 30 g of anhydrous calcium chloride. The weights

of the sealed cups were measured using an electronic scale with a

precision of 0.001 g and cups were left in a controlled humidity

chamber adjusted to 30 C and 90% RH. After 24 h, they were

reweighed and WVP was calculated according to the following

Figure 1Structure of zein-laminated LLDPE film with antioxidant agent.

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formula:

WVP

g mn/m2 d kPa= W x/A T(P2 P1) ,

where W is the increase in weight of the cup (g) after 1 d, x isthe average thickness of the film (mm), A is the exposed area ofthe film (1.66 103 m2), and (P2 P1) is the differential vaporpressure across the films (P2 P1 = 1.70 kPa at 30

C). WVP

measurements of each film sample were carried out in triplicate.

Release characteristics in different mediaRelease of antioxidant agent into the atmosphere. The

release test was carried out according to the methods described

in Gamege and others (2009), with some modifications. Corn-

zein-laminated LLDPE films with antioxidant agent were put into

a plastic tube (500 mL volume) and carefully adhered to the in-

ner wall to avoid gaps. The tube was tightly sealed with a cap

and stored at room temperature. Air samples (1 mL) were peri-

odically withdrawn through a rubber septum on the tube using

a syringe to determine the release of antioxidant agent from the

films into the inner atmosphere. The amount of antioxidant in the

gaseous medium was analyzed using the 6890A gas chromatog-

raphy (GC) system (Agilent Technologies, Santa Clara, Calif.,

U.S.A.) equipped with an FID detector and HP5 MS capillary

column (30 m 0.25 mm 0.25 m). Gaseous samples (1 mL)

were injected manually, and the split ratio was 1:60. Helium was

used as the carrier gas at a flow rate of 1 mL/min. The split/splitless

injector temperature and detector temperature were set at 200 C

and 350 C, respectively. The initial column temperature was set

at 60 C and increased to 200 C at a rate of 10 C/min. The

antioxidant compound was identified by comparing its mass spec-

tra with those of authentic compounds in the computer library.

The concentration of the identified compound in the atmosphere

was quantified according to the peak area integrated by the data

analysis program. Tests were conducted 4 times each.

Release of antioxidant agent into the liquid state. The

release of antioxidant agent from the films into liquid medium

was measured by 2,2-DPPH radical scavenging assay according

to methods described in Shimada and others (1992), with some

modifications. The films were tightly adhered to the inner wall of

a 30 mL glass vial filled with 25 mL of 0.1 mM DPPH solution.

The vial was wrapped with aluminum foil thoroughly to avoid

light, and then stored in a shaking incubator (Vision Scientific Co.

Ltd., Korea) at 25 C and 70 r pm. The absorbance was measured

with an Ultrospec 3100 Pro UV spectrophotometer (Pharmacia

Biotech Inc., Little Chalfont, U.K.) at 517 nm every 3 h. The test

directly measured the antioxidant activities of the released agent

into solution. DPPH radical scavenging activity was expressed as

milligrams of ascorbic acid equivalents (AAE) per grams of filmsample. Tests were replicated 4 times.

Effects of antioxidant packaging on ground beef pattiesBeef samples and storage conditions. Fresh ground beef

(top round roast) was obtained from a local butcher shop in

Suwon, Korea, and stored under refrigerated conditions (4

1 C). On the day of purchase, the roasts were trimmed of all

separable fat and connective tissue, ground twice, and then ana-

lyzed for fat content according to the methods described in Folch

and others (1957). Beef patties (30 g, 3.8% fat) were prepared us-

ing Petri dishes (6 cm dia, 1.2 cm height) in the laboratory for

each experiment. The raw beef patties were then packaged in 1

of 3 different ways, as follows: (i) over-wrapping with oxygen-

permeable polyvinyl chloride (PVC) film (O2transmission rate =

91.54 cm3/m/m2/24 h at 25 C), (ii) vacuum packaging with

LLDPE film, or (iii) vacuum packaging with developed antioxi-

dant films (0.3% or 3% antioxidant concentration). Samples were

stored at 4 C and the degree of lipid oxidation and the colors of

the patties in each packaging group were measured every 2 d.

Evaluation of lipid oxidation. Lipid oxidation of beef patties

was determined by analysis of 2-thiobarbiutric acid reactive sub-

stances (TBARS) according to the modified distillation methoddescribed in Rhee (1978). To prevent further oxidation, a mixed

solution of PG and EDTA was added to each beef patty before

distillation. After adding 4N HCl solution and distilled water, the

mixture was distilled in a Kjeldahl flask and the distillate was col-

lected in a 50 mL Falcon tube. Then, the distillate was reacted

with 2-TBA in a water bath at 100 C for 35 min to induce color

change. The optical density of the sample was read at 530 nm

with an Ultrospec 3100 pro UV spectrophotometer (Pharmacia

Biotech Inc.). The results of TBARS content were expressed as

milligrams of malondialdehyde equivalents (MDA) per kilogram

of meat sample. Tests were replicated 4 times.

Color changes in beef patty. Color changes were measured

with a CR-400 colorimeter (Minolta Co., Japan). The pattieswere exposed to oxygen for 10 min before color evaluation. The

colorimeter was calibrated using a standard white plate. Measure-

ments were taken 5 times for each sample, and the mean values

were used to determine the color coordinates L (lightness), a

(redness), andb (yellowness). In order to monitor meat browningduring storage, hue angle (H= arctan b/ a; higher values aremore brown) was calculated. Tests were conducted 4 times.

Statistical analysisData analysis was performed using Statistical Analysis System

(SAS) software, version 8.1 (SAS Inst., Cary, N.C., USA). The

General Linear Models Procedure was used for analysis of variance,

with main effect means separated by Student-Newman-Keuls test.Significance was defined asP 0.05.

Results and Discussion

Mechanical properties of filmThe mechanical properties of the corn-zein laminated LLDPE

films were measured by uniaxial tensile tests. The TS and %Eoflaminated films were determined from the attained stressstrain

curves (Table 1).

The TS of simple LLDPE film was 31.33 MPa, which was

the highest value among all tested films. When comparing to

the simple LLDPE film, the TS of the corn-zein-laminated film

(LLDPE/zein/LLDPE) decreased (P 0.05) by 28.6% (22.38MPa). The lower TS of the LLDPE/zein/LLDPE film was prob-

ably due to the physical weakness of the zein film layer. Rhim and

others (1997) reported that the TS of simple zein film was 5.52

MPa, which is much lower than LLDPE film. In zein-laminated

film groups, laminated films containing antioxidants had TS values

ranging from 22.28 to 24.27 MPa. Thus, the addition of an an-

tioxidant agent up to 5% (v/v) in the zein layer resulted in a slight

but insignificant increase in TS (P> 0.05). Generally, the TS oflaminated films is dependent on the substrate or base film rather

than the coating or laminated layer (Hong and others 2004). In

this study, zein film was applied to LLDPE film as a coating mate-

rial; LLDPE film was the substrate or base film in these laminated

films. In this study, the TS of zein-laminated LLDPE film was

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22.38 MPa. Earlier studies reported that zein-coated polypropy-

lene film had a TS of 45 MPa (Lee and others 2008), and the TS

of zein-coated carrageenan films ranged from 26.38 to 37.73 MPa

according to the concentration of zein-coating solutions (Rhim

and others 1997). This difference may be attributed to the different

substrate materials of the films.

The value of %E is associated with the elasticity and flex-ibility of the film. Values of %E for simple LLDPE andLLDPE/zein/LLDPE films were estimated to be 776.60% and

667.30%, respectively. Values of %Efor LLDPE/zein/LLDPE filmdecreased by 14% in comparison to LLDPE film, but slightly in-

creased when antioxidant agents were added. Gamage and oth-

ers (2009) reported that the %E values of various essential oil-incorporated films were significantly higher compared to those

of nonessential oil-incorporated films. These results indicate that

antioxidant agents act as adhesives in the protein polymer matrix,

similar to plasticizer. Therefore, the addition of essential oil im-

proved the flexibility and %Eof the films. Based on these results,antioxidants may be considered as potential replacements for plas-

ticizers in protein biopolymer films. In addition, antioxidants can

contribute positively to the mechanical properties of packaging

materials composed of biopolymer.

Water barrier properties of filmsWVP is determined by the chemical arrangement of film-

forming polymers and the morphology of the film (Siripatrawan

and Harte 2010). WVP was calculated according to the aforemen-

tioned formula, and the results are depicted in Table 2. Simple

Table 1 Mechanical properties of zein-laminated LLDPE films

with different antioxidant compounds and concentrations.

Film Tensile Elongationconstruction strength (MPa) at break (%)

LLDPE 31.33 0.98a 776.60 36.19a

LLDPE/zein/LLDPE 22.38 1.80b 667.30 50.88b

LLDPE/zein with thymol1.5%/LLDPE

22.78 2.90b 767.54 63.66a

LLDPE/zein with carvacrol1.5%/LLDPE

24.21 2.29b 746.03 54.78a

LLDPE/zein with eugenol1.5%/LLDPE

23.88 1.81b 786.13 61.11a

LLDPE/zein with thymol 3%/LLDPE 23.02 1.73b 802.20 35.17a

LLDPE/zein with carvacrol3%/LLDPE

23.14 2.05b 744.47 85.85a

LLDPE/zein with eugenol3%/LLDPE

23.58 2.01b 763.87 82.10a

LLDPE/zein with thymol 5%/LLDPE 22.83 1.09b 786.27 74.44a

LLDPE/zein with carvacrol5%/LLDPE

24.27 1.95b 784.73 55.54a

LLDPE/zein with eugenol

5%/LLDPE

22.28 2.54b 784.33 68.44a

a,bWithin a column, different superscripts indicate significant differences ( P 0.05).

Table 2 Water vapor permeability of zein-laminated LLDPE

films with different antioxidant compounds.

Film Water vapor permeabilityconstruction (gmm/m2dkPa)

LLDPE 1.778 0.045a

LLDPE/zein/LLDPE 2.033 0.112b

LLDPE/zein with thymol 3%/LLDPE 1.958 0.105ab

LLDPE/zein with carvacrol 3%/LLDPE 1.913 0.045ab

LLDPE/zei n with eugenol 3%/LLDPE 1.920 0.112ab

a,b Within a column, different superscripts indicate significant differences ( P 0.05).

LLDPE film had a lower WVP (1.778 gmm/m2dkPa) than any

other laminated film, indicating that LLDPE film provides a good

barrier for moisture. In contrast, LLDPE/zein/LLDPE film had

the highest WVP at 2.033 gmm/m2dkPa, and thus performed

the most poorly as a moisture barrier among the film samples

tested in this study. Results showed that simple zein-laminated

films had relatively high WVP compared to LLDPE film, which

was probably due to the hydrophilic groups in zein molecules.

These hydrophilic groups are formed from the amino acids that

compose corn-zein protein, and that they weakened the water bar-rier properties of corn-zein-laminated film. The WVP of other

laminated films containing phenolic antioxidant agents were es-

timated between 1.913 and 1.958 gmm/m2dkPa. Regardless

of the type of antioxidant agent used at 3% concentration (v/v),

films containing antioxidants had improved water barrier prop-

erties when compared to simple zein-laminated film due to the

strong hydrophobicity of the added antioxidant agents, which in-

terrupts the penetration of water molecules. Thymol, carvacrol,

and eugenol, which were used as antioxidant agents in this study,

are hydrophobic and are major compounds of naturally occurring

essential oils in thyme, oregano, and cloves. According to Sanchez-

Gonzalez and others (2011), the same behavior was observed when

essential oils (bergamot, lemon, or tea tree oil) were incorporatedinto chitosan/hydropropylmethylcellulose composite films. They

reported that WVP values of composite films showed a decrease

in line with the increase in the concentration of hydrophobic an-

tioxidant agent. The results of this study were in accordance with

these observations.

Release into the atmosphereGC analysis was used to measure changes in the concentration

of antioxidants released from the developed films into the atmo-

sphere (Figure 2). Overall, the maximum concentration of antiox-

idants in the headspace was correlated with the concentrations ofantioxidants added to corn-zein film solution, and the released

concentrations of antioxidants reached a maximum within 2 d in

all experimental groups. Films containing higher concentrations

of antioxidants also had faster diffusion rates of antioxidants into

the air yet still maintaining higher concentrations of antioxidants

than films with lower concentrations.

GC analyses of headspace gas composition revealed that the

concentrations of diffused antioxidants in the container reached

saturation after approximately 1 to 2 d, after which the amount

of antioxidants was reduced or maintained. Groups containing

thymol and carvacrol displayed a similar quantitative trend. There

were no significant changes in the concentration of antioxidants

in these groups after day 2, indicating that all antioxidants escaped

from these films within 2 d.

The diffused antioxidants of zein-5% eugenol-laminated film

displayed the highest concentration and the longest holding time

in the gas state. In this film, concentration of eugenol remained at

a high level for approximately 3 d and decreased over the following

2 d. From day 5 to day 7, eugenol levels inside the headspace re-

mained constant. The maximum value of this group was observed

up to day 3, and then it gradually reduced over last time pe-

riod. This might suggest that eugenol leaked out of the container

through the lid or septum of the container, even though the con-

tainer was sealed carefully. However, LLDPE/zein/LLDPE films

containing 1.5% and 3% eugenol showed a similar pattern, even

though they contained different initial amounts of antioxidants.



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Release into the liquid stateDPPH radical assays were performed with some modifications,

to evaluate the effectiveness of the antioxidant agents released

from the films into the liquid medium. DPPH radical scavenging

activities of the samples consistently increased with storage time

in all of the experimental groups, suggesting that there was a

continuous release of antioxidant agents (Figure 3).

There were slight changes in the radical scavenging effects of

LLDPE film and zein-laminated film even though they did not

contain any antioxidant agent. These changes might have been

Figure 2Concentration changes of thymol (A), carvacrol (B), and eugenol(C) released by antioxidant films into the atmosphere over 1 wk.

caused by the instability of DPPH radicals resulting in a grad-

ual extinction of radicals over time. By contrast, zein-laminated

films containing antioxidant (thymol, carvacrol, or eugenol) dis-

played considerable changes in the activity of released antioxidants.

Antioxidant film with eugenol had the maximum DPPH radical

scavenging activity of all the tested films. In addition, the reac-

tion mechanism of DPPH radicals and eugenol molecules released

from the film was terminated much sooner than thymol and car-

vacrol, at a concentration one-tenth that of the others. After 21 h,

absorbance at 517 nm of eugenol-containing film dropped below0.1, which is a value that was too small to be measured correctly

by a UV spectrophotometer with the optimum measurable ab-

sorbance range of 0.1 to 2.0. Therefore absorbance of this film

was not evaluated after 21 h. The DPPH radical scavenging activi-

ties of thymol and carvacrol were similar, although thymol activity

was not significantly higher than that of carvacrol, perhaps because

they are isomers with the same molecular formula and a similar

chemical nature.

Generally, the release of an active compound from the swelled

polymeric network occurs in 2 steps. First, water molecules pene-

trate into the polymeric matrix, causing the matrix to swell. Then,

active compounds diffuse through the widened mesh of the poly-

meric network (Del Nobile and others 2008). Therefore, releaseof an active compound is affected by the structural properties of

the polymer or polymeric network and the diffusion rate of the

active compound. However, in this study, the structural proper-

ties of the films were likely not factors because the antioxidant

active films had the same structure as the LLDPE/zein/LLDPE

film. Moreover, the 3 antioxidant compounds added to the de-

veloped films have similar molecular weights and water solubility,

and therefore differences in diffusion rates were also negligible.

Consequently, the factor that influenced DPPH radical scaveng-

ing activities the most was the antioxidant ability of the antioxidant

agent. Yanishilieva and others (1999) reported that thymol is a bet-

ter antioxidant than carvacrol due to the greater steric hindrance

of the phenolic g roup in thymol. According to Mastelic and others(2008), eugenol exhibited outstanding DPPH radical scavenging

activity, followed by thymol and carvacrol. This study reported

similar results.

TBA valuesIn the diffusion test of antioxidants in a liquid state, zein-

laminated film with 0.3% eugenol showed the greatest antioxidant

Figure3Diffusionofantioxidantcompoundsfromthefilmtoliquidmediumover 30 h.



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properties. Therefore, this film formulation was used for raw beef

patty packaging in this study. TBA value was measured in or-

der to examine the preventive effects of antioxidant-containing

films on lipid oxidation of meat during storage (Figure 4). In

all experimental groups, TBARS contents increased consistently

throughout storage. The control group, over-wrapped with the

oxygen-permeable PVC film, had a higher TBA value (P 0.05)than all of the vacuum-packaged g roups. In contrast, vacuum

packaging effectively protected beef patties from the beginning of

storage, with a TBA value lower than 2 mg MDA/kg over thecourse of 14 d. There were few differences in TBA values among

the vacuum-packaged groups, and vacuum-packaged patties with

zein-laminated film with 3.0% eugenol had the lowest TBA val-

ues. Even though vacuum packaging alone inhibited lipid oxida-

tion effectively, the developed antioxidant films demonstrated a

synergistic effect in combination with vacuum treatment against

lipid oxidation, and completely suppressed lipid oxidation during

the 14 d. Specifically, TBA value of vacuum only packaging

increased 86.1% after 14 d, but TBA values of vacuum packaging

with 0.3% eugenol-containing film and vacuum packaging with

3% eugenol-containing film increased 40.9% and 27.7%, respec-

tively, after 14 d. This result implied that the antioxidant films

Figure4Effects of packaging material on theTBARSvalues of beef pattiesafter 14 d.

improved the efficacy of vacuum packaging and stabilized raw

beef patties against oxidation. The antioxidant property of pre-

pared film containing 3% eugenol was similar to that of the film

containing 0.3% eugenol, which suggests that eugenol is a strong

antioxidant and 0.3% concentration was sufficient to completely

retard lipid oxidation in beef patties.

The inhibitory effect of eugenol on lipid oxidation is induced

by the phenolic g roup, which contains an electron-repelling group

in the ortho-position. This phenolic group is capable of reducing

both lipid radicals and ferric ion (Dorman and others 2000). Ogataand others (1997) suggested that eugenol prevents lipid oxidation

by trapping active oxygen species, such as O2 or hydroxyl radicals.

Lipid oxidation has been associated with off-flavors and off-

taste in meat and meat products. According to Insausti and others

(2001), humans detect off-flavors or off-taste in meat when TBA

values are greater than or equal to 5 mg MDA/kg. In this study,

TBA values above 5 mg MDA/kg meat were only observed in

the PVC-wrapped control group. In the other vacuum-packaged

groups, TBA values remained below 2 mg MDA/kg at all times

during storage.

Color changes

Color values including L (lightness), a (redness), and b(yellowness) of the oxygen-permeable control group and the

vacuum-packaged groups using antioxidant active films were mea-

sured and used to calculate the hue index. The color of meat is

one of the most important factors in customer selection because

color is an indicator of freshness (Mancini and Hunt 2005). Color

changes during storage at 4 C are presented in Table 3.

Treatment and storage time had no significant effects onL andb values of beef patties. In the control group, beef patty samplessuffered considerable decreases ina values, in contrast to the othergroups which exhibited relatively higha values. Vacuum packag-ing and the addition of eugenol made it possible to maintain initial

a levels over time. Allen and Conforth (2010) also observed that

after 14 d, the a

value of beef patties mixed with 0.05% eugenolwas significantly more reddish, and therefore more desirable to

consumers, than the control group. Control samples had signif-

icantly higher (P 0.05) hue angle values (more reddish andless yellow) in comparison with the hue angle values of vacuum-

packaged samples, regardless of eugenol existence within films.

A significant increase in the hue angle value of a control sample

Table 3Color value of beef patties packaged with the developed film.

Time of storage (d)

Color value Type of packaging 0 4 7 14

L PVC wrap 36.44 2.57Aa 36.01 1.93Aa 36.77 2.22Aa 36.88 1.52Aa

vacuum packaging only 38.09 3.10ABa 36.81 3.31ABa 35.77 2.34Aa 36.19 2.44ABa

vacuum packaging with antioxidant film (0.3% eugenol) 36.77 3.71ABa

34.59 2.27Ba

36.00 2.25Aa

34.75 2.00Ba

vacuum packaging with antioxidant film (3% eugenol) 34.96 1.87Ba 34.06 1.87Ba 34.76 1.37Aa 35.36 1.60ABa

a PVC wrap 13.57 2.07Aa 9.60 1.36Ab 10.05 1.94Ab 9.35 1.17Ab

vacuum packaging only 12.19 0.97Ba 12.99 1.06Bab 13.94 1.10Bb 12.86 1.94Bab

vacuum packaging with antioxidant film (0.3% eugenol) 12.01 1.01Ba 13.80 1.80Bb 13.64 1.42Bb 12.94 1.40Bab

vacuum packaging with antioxidant film (3% eugenol) 12.63 1.19ABa 13.52 0.69Ba 13.68 1.23Ba 11.26 2.08Cb

b PVC wrap 4.49 1.17Aa 4.62 0.75Aa 3.43 0.82Ab 3.32 0.78Ab

vacuum packaging only 3.61 1.50Ba 3.73 1.22Ba 3.52 0.57Aa 3.85 1.68Aa

vacuum packaging with antioxidant film (0.3% eugenol) 2.80 0.81Ba 3.25 0.72Bab 3.12 0.65Aab 3.81 1.13Ab

vacuum packaging with antioxidant film (3% eugenol) 2.98 0.60Ba 3.00 0.67Ba 3.36 0.90Aa 2.79 1.01Aa

Hue angle PVC wrap 18.09 2.05Aa 24.30 2.85Ab 25.27 3.14Abc 27.20 2.78Ac

vacuum packaging only 14.54 2.66Ba 15.23 1.70Ba 14.58 1.19Ba 16.19 2.55Ba

vacuum packaging with antioxidant film (0.3% eugenol) 13.06 1.66Ba 13.39 1.57Ca 12.83 1.67Ba 15.42 2.03Bb

vacuum packaging with antioxidant film (3% eugenol) 13.24 2.14Ba 13.52 1.62Ca 13.40 1.69Ba 14.33 2.52Ba

AtoC: Within a column, different letters indicate significant differences (P 0.05).a,b : Within a row, different letters indicate significant differences (P 0.05).



7/7

E:Food

Engineering

&


indicated discoloration. The control group became brownish (de-

creased redness and increased yellowness) with an increase in stor-

age time. Color change in the control group might be associated

with lipid oxidation and the formation of metmyoglobin (Djenane

and others 2002; Nern and others 2006). Beef patties vacuum-

packaged with antioxidant films showed lower changes in hue

angle values. In these groups, the films acted as an oxygen gas bar-

rier preventing the direct contact of meat with oxygen. Moreover,

the strong antioxidant property of eugenol released from the films

contributed to protection of the beef patties from lipid oxidation.These data suggest that diffusion of eugenol from the packaging

delays oxidation of beef color pigments.

ConclusionWe developed zein-laminated LLDPE films with natural an-

tioxidants and measured their physical and functional properties.

In addition, diffusion modes of antioxidants in the gas and liquid

states were estimated. Zein-laminated films with antioxidants had

reduced TS and %Evalues compared to LLDPE films. These filmsalso had higher WVP than LLDPE film. The antioxidant film was

tested in fresh beef patty packaging. Antioxidant films effectively

retarded lipid oxidation and inhibited color change in beef during

storage. These results suggest that zein-laminated films contain-ing antioxidants are suitable for packaging applications and food

protection.

AcknowledgmentThis work was supported by the Natl. Research Foundation

of Korea (NRF) grant funded by the Korea government (MEST)

(Nr. 2011-0014710).

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Development of Antioxidant Packaging Material