Antimicrobial, water vapour permeability, mechanical ...

LWT - Food Science and Technology 44 (2011) 2316e2323

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Antimicrobial, water vapour permeability, mechanical and thermal properties of casein based Zataraia multiflora Boiss. Extract containing film Atefeh Broumand, Zahra Emam-Djomeh*, Manouchehr Hamedi, Sayed Hadi Razavi Transfer Properties Lab (TPL), Department of Food Science and Technology, Agricultural Engineering Faculty, University of Tehran, 31587-11167 Karadj, Iran

a r t i c l e i n f o

a b s t r a c t

Article history: Received 8 April 2009 Received in revised form 19 June 2011 Accepted 4 July 2011

Hydrophobic sodium caseinate based edible films were prepared by incorporating stearic and oleic acids. Films were produced with a protein/lipid ratio of 1.05. Antimicrobial films were produced based on the hydrophobic films by adding the essential oil of Zataraia multiflora Boiss. Water vapour permeability, microstructure, antimicrobial, mechanical and thermal properties of these films were studied. Water vapour permeability decreased from 2.18 109 to 7.81 1011 gPa1s1 m1 on the addition of lipid compounds to film. Scanning electron microscopy showed a partial phase separation of lipid compounds and protein. This fact was confirmed by obtaining two endothermic peaks for lipid containing films during Differential scanning calorimeter. The incorporation of fatty acids into the film structure increased the total specific thermal capacity. The presence of lipids and essential oil decreased tensile strength but increased the elongation at break. Films containing essential oil exhibited a large inhibitory effect on Staphylococcus aureus, as compared to Salmonella typhimurium and Escherichia coli O157:H7. Ó 2011 Elsevier Ltd. All rights reserved.

Keywords: Sodium caseinate Emulsionated film Zataraia multiflora Boiss Essential oil Antimicrobial activity Water vapor permeability

1. Introduction With the drive toward renewable materials, it is increasingly common to use packing films based on biodegradable materials. These materials include biopolymers such as casein, gellan, zein, gluten, soy and peanut protein, starch, albumin, gelatin, and collagen (Seydim & Sarikus, 2006). In addition to their biodegradability, these materials are edible, which can serve to improve food appearance, food quality, and food safety (Krochta & De Mulder, 1997; Khwaldia, Perez, Banon, Desobry, & Hardy, 2004; Longares, Monahan, & O’Sullivan, 2004; Kristo, Koutsoumanis, & Biliaderis, 2008). Edible films and coatings can enhance the functional characteristics of food components. They can be used to maintain portions of food. Edible films have been successfully applied for restricting the diffusion of moisture, fats, and oils and also organic compounds such as flavors and pigments (Longares, Monahan, O’Riordan, & O’sullivan, 2005). They can form a barrier against contamination from outside microorganisms (Chick & Hernandez, 2002). Casein film is preferred for food applications due to its high nutritional quality, acceptable sensory characteristics, and its ability to protect food products from the environment (Chen, 2002; Schou et al., 2004). Films formed from milk proteins have good mechanical

* Corresponding author. Tel./fax: þ98 261 2248804. E-mail address: [email protected] (Z. Emam-Djomeh). 0023-6438/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.lwt.2011.07.002

properties and act as excellent lipid, oxygen, and aroma barriers. However, they do not have good moisture barrier properties, due to their hydrophilic nature (Gounga, Xu, & Wang, 2007; Khwaldia et al., 2004; Seydim & Sarikus, 2006). Lipid-based films have excellent moisture barrier properties but lack sufficient mechanical strength (Longares et al., 2005). Conversely, films formed from proteins and carbohydrates have good tensile properties but poor moisture barrier properties (Galielta, Di Gioia, Guilbert, & Cuq, 1998; Longares et al., 2005). To take advantage of the attributes of both types of materials, current research has focused on producing composite films of lipids and carbohydrates (Longares et al., 2005) or proteins (Avena-Bustillos & Krochta, 1993; Banerjee & Chen, 1995; Chick & Hernandez, 2002; Longares et al., 2005). Casein proteins mainly consist of five fractions including as1, as2, b, k, and d-casein, and their sizes vary from 11.5 to 25 kDa. One of the most important molecular features of casein is that it is a random coil (Banerjee, Chen, & Wu, 1996; Chen, 1995; Chen, 2002; Chick & Hernandez, 2002; Khwaldia et al., 2004). Casein is a good emulsifier, and it can easily form stable casein-lipid emulsions. Since these emulsions are good gas and moisture barriers, they are more suitable as food films and coatings than lipid films alone (Chick & Ustunol, 1998; Chen, 2002; O’Regan & Mulvihill, 2010). Caseins are amphiphilic and are very surface-active. These properties allow casein to stabilize at the watereoil interface (AvenaBustillos & Krochta, 1993). Kamper and Fennma (1984) reported that the water vapour permeability of emulsion-based films varied

A. Broumand et al. / LWT - Food Science and Technology 44 (2011) 2316e2323

considerably with film composition, and it appears that lipids are the most effective barrier against moisture (Avena-Bustillos & Krochta, 1993; Chick & Hernandez, 2002; Fabra, Talens, & Amparo, 2008). The amount and type of lipids used are critical determining factors of the water vapour permeability value of films formed from protein-lipid emulsions (Chick & Hernandez, 2002). Antimicrobial packaging is a form of active packaging that can extend the shelf-life of a product (Cha & Chinnan, 2004; Seydim & Sarikus, 2006). There have been an increasing number of reports on the production of edible films and coating with antimicrobial compounds (Rojas-Graü et al., 2007; Ponce, Roura, del Valle, & Moreira, 2008; Sayanjali, Ghanbarzade, & Ghiassifar, 2011). These films could extend the shelf-life and safety of food products, due to their inhibitory effects on pathogenic and spoilage microorganisms. This method is one of the most effective means of preserving food quality. Antimicrobial compounds mixed with film components could gradually penetrate through the surface of food (Pranato, Salokhe, & Rakshit, 2005). The use of plant essential oils and their components has been considered in food products, due to their inhibitory effects; they can prevent and control the growth of food-borne pathogens and food spoilage microorganisms. Previous research has shown that essential oils can be used as natural food preservatives (Atarés, Bonilla, & Chiralt, 2010; Burt, 2004; Carson & Reily, 1995; Holley & Patel, 2005; Lanciotti et al., 2004; Misaghi & Akhondzadeh Basti, 2007; Oussallah, Caillet, Salmieri, Saucier, & Lacroix, 2004). The growth of microorganisms on food surfaces is a very important problem because it can lead to spoilage and contamination. In particular, the contamination of ready-to-eat products is a very serious health concern (Pranato et al., 2005; Rojas-Graü et al., 2007). In general, essential oils containing higher concentrations of phenolic compounds, such as carvacrol, eugenol (2-methoxy-4-(2-propenyl) phenol), and thymol, have the most powerful effect against foodborne pathogens (Burt, 2004; Seydim & Sarikus, 2006). These compounds show a wide spectrum of biological effects, including antioxidant and antimicrobial properties. However, they can impact the sensorial properties (such as: odour, colour and taste) of products. Studies have shown that these properties are exhibited at the cytoplasmic membrane through the disruption of the proton motive force, electron flow, active transport, and/or coagulation of cell contents (Burt, 2004). Some plant essential oils that are incorporated into packaging materials can control microbial contamination, such as growth of Escherichia coli O157:H7 and Pseudomonas spp. in beef muscle (Oussallah et al., 2004; Seydim & Sarikus, 2006). Zataria multiflora Bioss is a plant belonging to the laminanceae family that grows only in Iran, Pakistan, and Afghanistan. The local name is Avishan Shirazi, and it is traditionally used as an antiseptic, anesthetic, and antispasmodic (Misaghi & Akhondzadeh Basti, 2007). In addition, this plant is used extensively as a flavor ingredient in a wide variety of food in Iran. The main constituents of the essential oils of this plant are phenolic compounds such as carvacrol and thymol (Ali, Saleem, Ali, & Ahmad, 2000; Shafiee & Javidnia, 1997). The aim of this study is to analyze the effect of fatty acids and essential oil (from Zataria multiflora) incorporation in casein based films on the characteristics such as water vapour permeation, antimicrobial activity, mechanical, optical and thermal properties. 2. Materials and methods 2.1. Materials 2.1.1. Films The primary film components were the powder form of sodium caseinate, stearic acid, oleic acid, and glycerol (as a plasticizer). The sodium caseinate composition was 85.3% protein, 1.2% fat, and 1.8%

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ash. Oleic and stearic acids were used as lipid part. Glycerol, a naturally occurring carbon back-boned polyhydric alcohol, is a plasticizer of sodium caseinate. Glycerol is highly soluble in water, is insoluble in most organic solvents, and is chemically stable. Span80 and span85 were used as emulsifiers for producing stable lipid-casein emulsions. All the chemicals were procured from Merck Chemicals Co. (Darmstadt, Germany). 2.1.2. Essential oil Essential oil of Avishan Shirazi (Zataria multiflora Boiss) which was obtained by hydro distillation (Golmakani & Rezaei, 2007) was provided by the Department of Food Science, Technology and Engineering at the University of Tehran, Iran. 2.2. Methods 2.2.1. Films preparation Emulsion solutions were prepared by first mixing 5 g (4.2 g protein) sodium caseinate with 100 g distilled water and 1.5 g glycerol (gly/pro ¼ 0.36) and, while glycerol was added to the distilled water before adding sodium caseinate. The sodium caseinate powder was slowly added to the mixture (at 60e65 C) with constant agitation (550 rpm). The mixture was stirred and heated (by Heater Stirrer: IKAÒ RCT basic, Staufen, Germany) for 1 h at 80 3 C in order to form di-sulphidic bounds in casein structure which may improve the mechanical properties of produced films (Boyd, Mitchell, Irons, Musselwhite, & Sherman, 1973). Film without lipids or fatty acids was cast from this solution. To produce the emulsionated film, 3.32 g oleic acid (83%) and 0.68 g (17%) stearic acid were weighed and then added to the mixture. We chose these amounts because in our experimental they were the best ratio of tow fatty acids. This ratio is ideal because adding the higher amount of stearic acid causes a phase separation before film formation .The ratio of protein to fatty acid was 1.05. Before blending stearic acid with the solution, this fatty acid was melted by heating it to 50 C. 1.6 g of emulsifiers (40% of lipid part) (span 80 ¼ 0.13 g (8%), span 85 ¼ 1.47 (92%) for achieving HLB (hydrophil-lipophil balance) ¼ 2), which are composed of 40% fatty acid weight, were added to the mixture, disregarding the effect of casein on HLB. The final film dispersion weighted 106.5 g. The dispersions were homogenized at 20,000 rpm for 3 min in an ultra-turrax T-25 homogenizer (IKA T25-digital ultra turrax, Staufen, Germany) with an S25N-25F probe. The prepared film solution was casted (35 ml) in a silicon mold (dimensions: 85 mm 230 mm). A vacuum was then applied to remove bubbles that could become pinholes after drying. Antimicrobial films were produced based on the emulsion-filmproducing method, but we also added fatty acids, plant essential oils, at defined quantities to the formulation. The amount of essential oil was estimated using the Broth Dilution Susceptibility (BDS) test (Barnon & Finegold, 1990). The purpose of this method is testing decreeing accentuations of the antimicrobial agent(s), which usually are prepared in serial tow fold dilutions, and placed in tubes of a broth medium that will support the growth of the test microorganism. Mueller-Hinton agar and Mueller-Hinton broth obtained from MST- England were used in this method. To perform BDS test, a standard inoculum of the microorganism (organisms 1 106 ml, a 1:500 dilution of a suspension of turbidity equal to a McFarland standard 1), was added to an equal volume of each concentration of antimicrobial agent and to a tube of the growth medium without antimicrobial agent, which has been served as a growth control. An un-inoculated tube of medium has been incubated to serve as a negative growth control. After overnight incubation, the tubes were cultured on the plats containing Mueller Hinton agar and were incubated at 37 C for overnight (Barnon & Finegold, 1990). The amount of minimum bactericidal concentration (MBC) for Staph.

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aureus, E. coli O157:H7and S. typhimurium obtained as 0.250 mg/g, 0.500 mg/g and 0.500 mg/g, respectively. Since the presence of essential oil in the protein matrix and the presence of emulsifier in the mixture will decrease the antimicrobial activity of the essential oil, the amount of essential oil was increased to 0.750 mg/g (1.5 times more than MBC). Drying was carried out at 40 2 C and 50 5% relative humidity (RH) for 13.5 h until reaching constant moisture content. Obviously, the amount of essential oil residue could not be less then MIC, because the microorganisms were inhibited by exiting amount into the antimicrobial film. Once the films were peeled from the casting surface, they were stored at 20 C and 50 5% RH until testing. 2.2.2. Culture preparation S. typhimurium (ATCC14028) and E. coli O157:H7 (ATCC35218) were provided by the Faculty of Veterinary Medicine in the Department of Microbiology at the University of Tehran. Staph. aureus (PTCC 1431) was provided by the Iranian Research Organization for Science and Technology (Tehran, Iran). They were maintained on brain heart agar (BHA), McConkey agar, and nutrient broth stored at 4 C, respectively (all media were obtained from Merck Chemicals Co., Darmstadt, Germany). For use in experiments, the bacteria were grown separately in these media and incubated for 18 h at 37 C. 2.2.3. Film microstructure Film microstructure was determined by scanning electron microscopy (SEM) (Philips-XL30, Rotterdam, Netherlands). Samples were prepared using standard techniques, mounted on aluminum tubs, and sputter coated with gold (100 Å) (Gounga et al., 2007). The micrographs were collected using an accelerating voltage of 25e30 kV. 2.2.4. Film thickness Film thickness was measured using a micrometer (Mitutoyo No. 293-766, Tokyo, Japan). Reported values were the average of 20 different samples measured randomly over the test area, with the film equilibrated at 23 2 C and 50 5% RH. 2.2.5. Mechanical properties The tensile strength (TS) and elongation (E) of films were evaluated according to the ASTM standard D-882-3, using a testometeric machine model M350-10ct (Type DBB MTCl, Testometric Co., Rochdale, England). TS and E tests were determined at 50 5% RH. The samples were 25 mm wide and at least 80 mm in length. The initial grip separation of the instrument was 50 mm, and crosshead speed was 10 mm/s. TS and E values were reported in Pascals (Pa), and the percentage change in the value of E was determined by the current length over the original length. Young’s Modulus (YM) was calculated by dividing stress to strain for the linear part of curve. The reported values are the average of at least 12 repetitions. (Avena-Bustillos & Krochta, 1993; Chick & Hernandez, 2002; Cho & Rhee, 2004; Longares et al., 2005). 2.2.6. Water vapour permeability (WVP) WVP tests were conducted using a modified ASTM (1995) method. Each film sample was sealed over a circular opening of 0.01075 m2 in a permeation cell that was stored at 25 C in a desiccator. To maintain a 75% RH gradient across the film, anhydrous calcium chloride (0% RH) was placed inside, and a sodiumchloride-saturated solution (75% RH) was used in the desiccator. This difference in RH corresponds to a driving force of 1753.55 Pa, expressed as the water vapour partial pressure. Top side of film was facing towards the high or low RH environment during the test. Water vapour transport was determined from the weight gain of

the permeation cell. Changes in the weight of the cell were recorded to the nearest 0.0001 g and plotted as a function of time (several film samples in several desiccators were used to keep the continuity of test). The slope of each line was calculated by linear regression (r2 > 0.99), and the water vapour transmission rate (WVTR) was calculated from the slope of the straight line (g s 1) divided by the test area (m2). After the permeation tests, the film thickness was measured, and WVP (g Pa1 s1 m1) was calculated as:

WVP ¼ ½WVTR=SðR1 R2 Þd

(1)

where S is the saturation vapour pressure of water, 3179 Pa at 25 C, R1 is the RH in the desiccator, R2 is the RH in the permeation cell, and d is the thickness of the film (in mm) (Garcia, Pinotti, Martino, & Zaritzky, 2004). 2.2.7. Thermal characteristics The thermal behavior was determined by differential scanning calorimetry (DSC) using a 2010 Modulated DSC (TA Instrument, New Castle, Del., USA). Samples of 2e4 mg were sealed in standard aluminum dishes, using a sealed empty aluminum dish as the reference sample. Experiments were conducted from 50 to 200 C, with a heating rate of 10 C/min, on all of the film samples including sodium caseinate and emulsified and antimicrobial films. All of the samples were measured three times to ensure that the results were consistent. 2.2.8. Evaluation of antimicrobial characteristics of antimicrobial films Disk diffusion method was used to examine the antimicrobial characteristics of the films (Sayanjali et al., 2011). Disks with a diameter of w6 mm were cut out from the antimicrobial films using a sterile punch. These disks were then placed on plates that containing (Muller Hinton agar, solid state medium). The medium had been previously inoculated by microbial suspension with an optical density of 0.5 MF. There were a total of four disks on each plate, and they were incubated at 37 C for 18 h (Barnon & Finegold, 1990). Following incubation, the diameter of the halo formed around each disk was measured. For each plate, one disk was used as the control for the microorganism, and another disk was used as the control from the essential-oil-free film. 2.2.9. Statistical analysis All experiments were repeated three times (except mechanical properties), and data sets were subjected to analysis of variance (ANOVA) and the Duncan’s multiple range test using Minitab software (Minitab 15; Minitab Inc., Minneapolis, USA). In all cases, a value of p < 0.05 was considered significant. The data were expressed as mean S.D. 3. Results and discussion Films were produced with a protein/lipid ratio of 1.05. A total of 17% (0.68 g) of the lipid was stearic acid, and the remaining 83% (3.32 g) was oleic acid. The films without lipids were smooth, transparent in appearance, and relatively flexible. The incorporation of lipids (stearic and oleic acids) and essential oil resulted in films with a frosted appearance (Fig. 1), thicker (0.2 0.03 mm comparing to 0.14 0.01 mm, Table 1), while maintaining the smoothness and imparting greater flexibility. 3.1. Microstructure Scanning electron microscopy (SEM) was used to evaluate the top and bottom surfaces of the film. The top surface of the film without lipids showed a smooth and flat appearance (Fig. 2-A). The


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The existence of holes might be related to the volatility of the essential oil. In fact, these spaces were filled by essential oil that had evaporated from the film surface the same results were reported by Sanchez-Gonzalez, Pastor, Vargas, Chiralt, Gonzalez-Martinez, Chafer (2011). Consequently, the film structure will become wider, and holes will be produced during the drying process. SEM images of the bottom surface of film C (Fig. 2-H) also showed a rough surface with many holes. The cross-section of film C (Fig. 2-I) can be divided into three separate parts. Similar to the morphology of film B, the middle part consisted of a lipid-protein region, and this middle region is sandwiched by two lipid parts (Fig. 2-I). There are numerous holes on the film surface, which would increase the permeability. Consequently, we conclude that the presence of essential oil in the film structure will lead to poor film formation. 3.2. Mechanical properties

Fig. 1. Comparison of film appearance: control film (film A), hydrophobic film (film B), and antimicrobial film (film C).

bottom surface was similar to the top surface; there were no apparent differences in their physical aspects, only a series of pinholes were observed (Fig. 2-B). SEM micrographs of the crosssection of the film showed a homogenous structure. Even though the surface that was facing the dish during the drying process was relatively smooth and compact, the top surface had a slightly porous structure. Structural differences between the top and bottom surfaces may have been caused by different rates of water evaporation during drying (Fig. 2-C). The microstructure of the top surface of films containing lipids (stearic and oleic acids) showed the appearance of lipid droplets of w10 mm in size, dispersed throughout the surface, which could possibly lead to the formation of an uneven surface (Fig. 2-D). The droplets appeared to be equal in size, due to the homogenization process. When comparing SEM images of the top and bottom surfaces (Fig. 2-E) of lipid-containing films, a separate phase of the lipid (stearic acid) was dispersed near the top surface. In fact, a layer of stearic acid was found on the top surface of the film. Cross-sections of the films showed a three-layer structure. Additionally, no defect was found throughout the entire cross-section of the film. Both surfaces appear to contain stearic acid (the surface exposed to air and the other surface), but there appears to be more stearic acid on the surface exposed to air. We attribute this observation to lower fatty acid incorporation with the aqueous solution. Since the bottom surface is in contact with the smooth dish surface, the stearic acid on this surface remains smooth, even after the drying process. There appears to be a layer of crystallized stearic acid on the top and bottom surfaces of the film (Fig. 2-F). SEM images of the film surface containing essential oil confirmed the presence of stearic acid (Fig. 2-G). However, many holes were observed on the surface of this film, which were probably due to the presence of essential oil in the film formulation.

Tensile strength (TS) (the maximum tensile stress that a film can sustain) is a measure of a film’s mechanical strength. For a caseinbased edible film, the TS should increase (p < 0.05) with protein content and decrease with plasticizer content (Chen, 2002). Since proteineprotein interactions would probably diminish in the film containing lipids, it was expected that a film containing lipids would have a lower TS and higher elongation (E), as compared to a film without lipids (sodium caseinate with glycerol) Ozdemir & Floros, 2008. Our results showed that the composite film has a lower tensile strength and higher elongation as compared to a film without lipids. The tensile strength varied from 9.27 to 2.23 MPa. The incorporation of lipid portions of stearic and oleic acids significantly affected the interactions that contribute to network formation of the film, which led to a reduction in the tensile strength (P < 0.05) (Table 1). It should be mentioned that the actual amount of stearic acid that remains in the matrix was not determined. In order to compare our results with literature values, we only compared literature values obtained under the same conditions as those used in our tests (Table 1). As presented in Table 1, the addition of essential oil led to a reduction in film properties. The comparison of the tensile strength of the three films, i.e., the control (A), lipid (B), and antimicrobial films (C), showed that film C had the lowest tensile strength and elongation values. Differences in tensile strength were insignificant, but differences in E% were significantly lower, as compared with film B. This indicates that the presence of essential oil can also enhance the effects of oleic and stearic acids, by weakening the specific interactions such as proteineprotein interactions. Young’s modulus (YM) values were compared in the three films. The highest and lowest YM values were observed in films A and B, respectively (Table 1). 3.3. Water vapour permeability (WVP) Water vapour transmission rates of all films produced in this study are shown in Fig. 3. The water vapour permeability values of

Table 1 Comparison of the tensile strength (TS) and elongation (E) of edible films produced in this study, (n ¼ 12, p < 0.05) (Ca: Calcium, Cas: Caseinate, Gly: Glycerol, Na: Sodium, EO: Essential Oil, Ol: Oleic acid, St: Stearic acid, TS: Tensile Strength, E: Elnogation, YM: Yang’s module). Film

Plasticizer to protein ration

Hydrophobic additive

Protein to lipid ratio

Film thickness (mm)

Cross-head speed (mm/s)

TS (MPa)

E (%)

YM

A: Na-CaseGly B: Na-CaseGly C: Na-CaseGly- EO

0.36 0.36 0.36

e Ol 83%- St 17% Ol 83%- St 17%

e 1.05 1.05 (without EO)

0.14 0.01a 0.20 0.03b 0.20 0.03b

10 10 10

9.27 0.29a 2.23 0.20b 1.94 0.15b

322 21a 745 48b 212 41c

1.99 0.17a 0.31 0.05b 0.95 0.19c

There is no significance difference between the numbers with the same letter at (p