Fabrication, characterization and antimicrobial activities of thymol-loaded zein nanoparticles stabilized by sodium caseinate–chitosan hydrochloride double layers
Introduction
Foodborne pathogens are the leading cause of foodborne illness in the United States. They are responsible for a number of multi-state and expensive outbreaks related to food products including fresh produces (e.g. cantaloupe, tomato and lettuce) and meat products (e.g. beef jerky and ham). These outbreaks have negative impacts on consumer confidence on food safety. On the other hand, consumers more appeal to fresh and minimally processed products, and the products with natural ingredients and improved microbiological safety.
Many essential oils (EOs) have shown a broad spectrum of biological activities, including growth inhibition against bacteria, fungi and yeasts (Bakkali, Averbeck, Averbeck, & Idaomar, 2008). Thymol (5-methyl-2-iso-propylphenol), a natural antimicrobial agent from thyme and thyme oil, showed significant antimicrobial activity against both gram-positive and gram-negative bacteria (Wattanasatcha, Rengpipat, & Wanichwecharungruang, 2012). Although the exact mechanism is not completely understood, essential oils containing phenolics are thought to disrupt the membrane of microorganisms (Di Pasqua, Hoskins, Betts, & Mauriello, 2006). Moreover, thymol is a generally-recognized-as-safe (GRAS) food additive according to the United States Food and Drug Administration (FDA). However, thymol has relatively poor water solubility, strong impact on food flavor, and may interact with various food constituents, such as protein and fat. These properties may alter its antimicrobial efficacy and limit the application of thymol as a food antimicrobial agent (Marques, 2010).
Nano-/micro-encapsulation technology has been recently applied to improve the physicochemical properties of food ingredients (Lai and Guo, 2011, Luo et al., 2012, Luo et al., 2011, Patel et al., 2010, Shah et al., 2012, Wattanasatcha et al., 2012, Wu et al., 2012, Zhong et al., 2009). Shah et al. (2012) incorporated thymol into whey protein isolate-maltodextrin conjugate capsules, and demonstrated that the efficiency and stability of the nanodispersions were affected by the emulsion composition. Recently, thymol was encapsulated into water dispersible submicron-sized ethylcellulose/methylcellulose spheres, attaining better antimicrobial activity than methylparaben, a conventional food preservative (Wattanasatcha et al., 2012). Zein, a corn prolamine protein, is a GRAS food-grade ingredient. It has three quarters of lipophilic and one quarter of hydrophilic amino acid residues. Because of its high hydrophobicity, zein has been investigated in food and pharmaceutical industries for encapsulation and sustained release of hydrophobic bioactives, such as fish oil (Zhong et al., 2009), α-tocopherol (Luo et al., 2011), vitamin D (Luo et al., 2012), curcumin (Patel, Hu, Tiwari, & Velikov, 2010) and 5-fluorouracil (Lai & Guo, 2011). Our group has successfully encapsulated thymol into zein nanoparticles using a liquid–liquid dispersion method (Wu et al., 2012). However, zein, as a protein, has an isoelectric point at around 6.2 (Patel et al., 2010), which may result in poor physical stability and redispersibility of freeze-dried zein nanoparticles at a neutral pH in aqueous systems, limiting its application in delivery systems in food and pharmaceutical industries.
The synthetic and natural polymeric materials, including small-molecule surfactants (Surh, Gu, Decker, & McClements, 2005), phospholipids (Klinkesorn, Sophanodora, Chinachoti, McClements, & Decker, 2005), proteins (Hong & McClements, 2007) and polysaccharides (Calero, Muñoz, Cox, Heuer, & Guerrero, 2013) are used to stabilize nanoparticles in different systems. Sodium caseinate (SC), a protein isolated from milk, is particularly attractive as an emulsifier because it is nontoxic, natural, bland taste and widely available (McClements, 2005). SC acted as an effective stabilizer to prevent the aggregation of zein nanoparticles in solutions at neutral pH under high ionic strength conditions (Patel et al., 2010, Patel et al., 2010). Li et al. fabricated antimicrobial films based on zein colloidal nanoparticles coated with SC as a stabilizer (Li, Yin, Yang, Tang, & Wei, 2012). The results showed that SC costing was able to improve the zein nanoparticle stability and avoid the flocculation during film formation.
In addition, layer-by-layer electrostatic deposition of polyelectrolytes onto oppositely charged surfaces was reported to effectively generate oil-in-water emulsions with multi-component interfacial coatings (Calero et al., 2013, Mun et al., 2006). The formed relatively thick and highly charged double-layer interfaces may increase the electrostatic and steric repulsion between emulsion droplets, which may increase the particle stability against the environmental stresses. However, high energy processing, such as high speed or high pressure homogenization, was required for preparing the emulsions (Hong and McClements, 2007, Hu et al., 2011). To the best of our knowledge, few studies have been conducted to investigate the utilization of layer-by-layer electrostatic deposition technique to coat another polyelectrolyte layer onto SC stabilized zein nanoparticles and the investigation of compositions of double layers of stabilizers on its physical stability and encapsulation efficiency.
Chitosan coating has shown potential in food preservation due to the inherent antimicrobial properties (Alvarez, Ponce, & Moreira, 2013). Recently, water-soluble chitosan hydrochloride (CHC) has been reported as a model positively charged polyelectrolyte (Seyfarth, Schliemann, Elsner, & Hipler, 2008), which may overcome the low water solubility of chitosan at neutral pH. As a continuation of our research in nano-encapsulation to develop novel applications of food ingredients, this study was conducted to investigate the possible application of CHC coating to improve the antimicrobial and physicochemical properties of SC stabilized thymol-loaded zein nanoparticles.
Section snippets
Materials
Zein with a minimum protein content of 97% was provided by Showa Sangyo (Tokyo, Japan). Thymol and sodium caseinate were obtained from Sigma–Aldrich Chemical Co., Ltd (St. Louis, MO, USA). Chitosan hydrochloride (deacetylation degree of 80–90% and molecular weight of 100 KDa, Batch Code: HK120808061) was purchased from Jinke Biochemical Co. Ltd. (Wenzhou, Zhejiang, China). All other reagents were of analytical grade and used without further purification. Water purified with a Milli-Q system was
Influence of mass ratio of zein to SC
A series of nanoparticle dispersions containing a constant zein concentration (4 mg/ml) was produced with different concentrations of SC (0.4–5 mg/ml). The pH, particle size and zeta potential of nanoparticles were measured (Table 1). In the absence of SC, the particle size and zeta potential of zein nanoparticles were 118.30 nm and +28.10 mV, respectively, which were consistent with the previously published findings (Patel et al., 2010). SC particles in the solution had a negative charge and their
Conclusion
The thymol-loaded zein nanoparticles were successfully prepared with sodium caseinate and chitosan hydrochloride as electrosteric stabilizers using a simple and low-energy liquid–liquid dispersion method. They had excellent redispersibility in aqueous systems at neutral pH. The average particle size and surface electrical characteristics could be controlled through the mass ratios between zein to sodium caseinate, and sodium caseinate to chitosan hydrochloride. A combination of sodium caseinate
Acknowledgements
This research was partially supported by a special fund for agro-scientific research in the public interest (No. 201203069), grants from National High Technology Research and Development Program of China (Grant Nos. 2013AA102202; 2013AA102207), grants from the Chinese Science Foundation Grant for Post-doctoral Researcher (Grant Nos. 2012M511098; 2013T60450), grants from SJTU 985-III disciplines platform and talent fund (Grant Nos. TS0414115001; TS0320215001), grants from Maryland Agricultural
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