In recent years, the tendency to use biodegradable polymers in food packaging has been increased due to enhanced awareness on the ecological and environmental problems and the contaminations of natural resources caused by non-degradable petrochemical-based polymers. In this regard, two types of biodegradable polymers have been investigated: edible and non-edible biopolymers. Carbohydrates and proteins or their combination with lipids usually are used for producing edible biopolymers (Noshirvani et al., 2018). Cinnamon essential oil (CEO) is a mixture of aldehyde, phenol and terpene active compounds, exhibiting antioxidant and wide spectrum antimicrobial properties against fungal spoilage. CEO is granted by the world health organization (WHO) expert committee as a non-toxic additive and flavoring agent, meanwhile CEO is widely applied in many food formulations thus it fits desirable taste (Ojagh et al., 2010). Mostly, synthetic antimicrobial and antioxidant compounds are directly added into the food products to control the microbial growth. This method may lead to the inactivation or evaporation of active agents and rapid migration into the bulk of the foods. Hence, the antimicrobial activity may be quickly lost by dilution below active concentration and also the flavor of the food may be changed. The incorporation of an antimicrobial compound in a polymer matrix and the production of active packaging potentially cause to gradual release of active agents, which may be promising approach to inhibit the microbial growth. Carboxymethyl cellulose (CMC) is a biodegradable semi-synthetic biopolymer. It is a linear anionic charge polysaccharide due to having many hydrophilic carboxyl groups. CMC produces high viscosity solution and transparent films with favorable properties from consumer point of view. The CMC-based films show good barrier properties against gases and lipophilic compounds, while showing poor inhibition against water vapor permeation, similar to other polysaccharide and protein-based films. In order to fix these defects and improve packaging properties, a number of approaches have been studied such as combination with other compatible biopolymers, using hydrophobic compounds (such as lipid and essential oil), cross linking agents and nanofillers. To our knowledge, there is no study on comparing the effects of nano and macro emulsion of cinnamon essential oil (CEO) on the physicochemical and antifungal properties of the active biopolymer based films. Therefore, the objectives of this work were (i) to produce the emulsified CMC-based films from CEO macroemulsions (ME) and nanoemulsions (NE) prepared by using Ultra-Turrax and Ultrasonication (ii) to compare the effects of these two types of the emulsified films on some physical features of the resulted films (iii) and to study the in-vitroefficiency of these films against Aspergillus niger and mucor racemous.
Macroemulsions (ME) were formed by adding different amounts (0.125, 0.25 or 0.5 gr) of Tween 80 equation with 50% W/W surfactant oil ratio (SOR) to 20 ml of double distilled water and followed by homogenization by Ultra-Turrax (JANKE & KUNKEL, Germany), at 20000 rpm for 1 min. Then, CEO was added at (0.25, 0.50 or 1 gr) equation with 0.25, 0.50 and 1% W/V (CEO/Emulsion) to each solution and mixed again for 2 min. Prepared ME were sonicated at the condition of 20 kHz frequency, 400 W input power and 70S amplitude for 10min by Ultrasonicator (FAPAN, Iran) to obtain nanoemulsions. In order to prevent the increase of emulsions temperature during sonication, the solutions were keeping in ice-water bath. Films were prepared as described by Dashipour et al. (2015), with slight modifications. First, 80 ml of double distilled water was heated in a water bath until temperature increased to about 85 , then, 1.5 g of CMC powder was added to hot water and mixed by magnetic stirrer(800 rpm) at 85 for 60 min. After that, glycerol was added at 0.75 w/w of CMC as a plasticizer and mixing continued for 10 min. Then, the solution temperature reduced to 60 in order to prevent the destruction of CEO active compounds. Having done that, the solution was mixed with 20 ml of macro and nano emulsions solution prepared in the previous section and agitated with magnetic stirrer (500 rpm) at 60 for 30 min to obtain homogeneous solutions. The film forming solutions were degassed with vacuum pump(DV-3E 250, JB,USA), at ambient condition for 5 min, then, 100 ml of the emulsified film forming solutions were poured into an even surface Teflon plates (PTFE) in the dimension of 15 15 cm and dried at 40 for 18 h in cold incubator to cast the films.
The emulsified films based on carboxymethyl cellulose (CMC) containing macroemulsion (ME) and nanoemulsion (NE) of cinnamon essential oil (CEO) were prepared. The atomic force microscopy (AFM) images showed different morphology in the ME and NE films and more compact and uniform microstructure in the NE films than the ME one. Adding CEO led to more flexible films with lower glass transition temperature (Tg) and storage modulus (E'). Low droplet size in NE than ME led to brighter and yellowish films. Antifungal index against A. niger and M.racemous were 14.16% and 20.82 % in the ME films and 18.81% and 25 % in the NE ones. The antifungal activities of cinnamon essential oil (CEO) is related to their major phenolic or aldehyde such as eugenol and cinnamaldehyde constituents, respectively. Eugenol caused to killed microbial cells probably by two important mechanisms; leached out cell content by increasing the permeability of cellular membranes and released proton of hydroxyl groups causing to reduce proton gradient that resulted depletion energy polls in microbial cells. Another effective mechanism of cinnamaldehyde in microbial growth is its reaction with nucleophilic compound in microorganism's structure by higher electrophilic compounds such as carbonyl groups in cinnamaldehyde structure. The disruption of cytoplasmic membranes, the cytoplasm depletion, thedeformation of hyphal tips, the formation of short branches and the collapse of entire hyphaewere the major effect of essential oil on fungus, observed by transmission electron microscopy.
Comparing different physicochemical and antifungal properties of CMC films containing ME and NE of CEO showed significant differences. The decrease of droplet size by sonicator caused to better diffusion of CEO NE from cell membrane of microorganisms, resulting in improved antifungal efficiency. Microscopic images showed smooth and homogenous appearance with lower Sa and Sq of NE films. More interruption among biopolymer chains by nano droplets caused to the production of the films more extensible than ME films. Thermomechanical analysis confirmed more decrease in glass transition temperature (Tg) and storage modulus (Eʹ). This study introduced a new nano active packaging film with some improved functional characteristics.
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