Nanoencapsulation of Active Food Ingredients - American

Chapter 9

Controlled Self-Organization of Zein Nanostructures for Encapsulation of Food Ingredients

Downloaded by IOWA STATE UNIV on October 18, 2014 | http://pubs.acs.org Publication Date: March 3, 2009 | doi: 10.1021/bk-2009-1007.ch009

Graciela W. Padua and Qin Wang Department of Food Science and Human Nutrition, University of Illinois at Urbana-Champaign, 382/D AESB, 1304 West Pennsylvania Avenue, Urbana, IL 61801

Micro/nano-encapsulation technologies have the potential to meet food industry challenges concerning the effective delivery of health functional ingredients and controlled release of flavor compounds. The inherent complexity of food systems has translated into an intensive search for novel functional shell materials. Understanding their structure has become critical for the design of effective carriers. Nanotechnology methods may prove useful in the construction of food delivery systems. Zein, the prolamine in corn endosperm, has long being recognized for its coating ability. Zein has a marked amphiphilic character. It is soluble in alcohol-water mixtures. It contains more than 50% nonpolar amino acids arranged in unique spatial disposition consisting of tandem repeats of a-helix segments aligned parallel to each other forming a ribbon or prism. This structure gives rise to well defined hydrophobic and hydrophilic domains at the protein surface. Zein can bind and enrobe lipids, keeping them from deteriorative changes. Zein has been shown to adsorb fatty acids and produce periodic structures, most interestingly, nanoscale layers of cooperatively assembled fatty acid and zein sheets. Other experiments have detected the formation of nanoscale "tubes" of zein formed upon adsorption of the protein on hydrophilic surfaces. Lamellar structures detected by x-ray diffraction, formation of zein "tubes" observed by AFM, and the long rod-like structures observed by SEM may © 2009 American Chemical Society

In Micro/Nanoencapsulation of Active Food Ingredients; Huang, Q., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2009.

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144 be explained in terms of the formation of liquid crystalline phases. Effort has been invested in tracking the self– organization and characterizing resulting tertiary structures formed by zein. The goal is to produce nanostructures of controlled geometry, useful as microencapsulation materials for fatty acids, flavors, oleoresins, vitamins, and peptides.


Introduction Better, widespread information on the impact of diets on health and well being has raised consumers' expectations from the foods they eat. Nutrient and bioactive content, avoidance of food components associated with health risks, and food safety have an increasing impact on consumers' choices. Quality demands are also on the rise, prompted by an increase in trade and availability of wider selections. The food industry has responded by looking into the incorporation of health functional ingredients, food component replacement, and controlled release of flavors and aromas. Major challenges to this effort are effective ingredient delivery without compromising sensory quality, and sustained bioactivity. Micro/nano-encapsulation technologies have the potential to meet these challenges. Encapsulation involves surrounding a core compound with a suitable shell material which carries, protects, and delivers the core in a controlled fashion. For example, in the pharmaceutical industry poly (lactic-co-glycolic acid) (PLGA) is used to encapsulate proteins (i.e. human growth hormone) (1,2). The food industry has seen the micro-encapsulation of antioxidants, flavor compounds, natural extracts, vitamins, probiotic bacteria and others. Conjugated linoleic acid was encapsulated in whey protein concentrate (3). Microcapsules containing short chain fatty acids were produced using gum arabic and maltodextrins as wall materials (4). Desai and Park (5) studied the stabilization of vitamin C in chitosan microspheres cross-linked with tripolyphosphate. Probiotic bacteria (Lactobacillus acidophilus and Bifidobacterium lactis) were encapsulated in calcium-induced alginate-starch shells to enhance the survival of probiotic bacteria in yogurt during storage (6). The variety of shell materials and encapsulation processes reflects the inherent complexity of food systems, which seems to require ad-hoc solutions for every problem. This was translated into an intensive search for novel functional compounds. Understanding their structure has become the basis for design of effective carriers. Nanotechnology methods may prove useful in the construction of food delivery systems (7,8). Graveland-Bikker and de Kruif (9) observed the self-assembly of α-lactalbumin derived peptides into nano-sized tubular structures. These nanostructures promise various applications in food technology.



145 Proteins, depending on their amino acid sequence, may acquire distinct spatial conformations and show varied functionalities in response to their environment. Temperature, pH, ionic strength, and the hydrophobic/hydrophilic character of the medium or adsorbing interface affect the 3D structure of individual proteins and the way they interact and associate with each other. Zein, the prolamine in corn endosperm, has long been recognized for its coating ability. Its conventional applications include coating of tablets in the pharmaceutical industry and as a substitute for shellac in candies and confections. New applications include slow release formulations in drug delivery. Zein microspheres were investigated as carriers for ovalbumin and heparin (10,11). Such microspheres were resistant to physical and chemical degradation but degradable by pepsin and pancreatin. Zein also can impart protection as antioxidant. Zein films are reported to have an inherent free radical scavenging activity (12). Zein can also bind and enrobe lipids, keeping them from deteriorative changes. Wang and others (13) reported that zein showed antioxidant activity on methyl linoleate, possibly by binding and physically shielding the lipid. Zein has been shown to adsorb fatty acids and produce periodic structures (14), most interestingly, nanoscale layers of alternating fatty acid and zein sheets. Other experiments have detected the formation of nanoscale "tubes" of zein formed upon adsorption of the protein on hydrophilic surfaces (15). Zein structures have been proposed as encapsulation materials for fatty acids and other lipophilic compounds.

Zein Structure Zein is not soluble in water but in alcohol-water mixtures (16,17). Its amphiphilic character results from the balance between hydrophobic and hydrophilic amino acids in its sequence and their unique spatial disposition (18). Zein amino acid sequence contains more than 50% nonpolar amino acids including leucine, proline, and alanine (19). The high content of nonpolar amino acids is responsible for the hydrophobic nature of zein and its lack of solubility in water. On the other hand, the high content of glutamine makes it insoluble in alcohol because of the formation of intramolecular hydrogen bonds. Zein consists of two groups of polypeptides, according to sodium dodecyi sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (18,20) and MALDI results (21), a chain of 23,000-24,000 molecular weight and a second peptide chain of 26,000-27,000 (22,23). Both peptide chains have sequence homology: N-terminals contain 35 to 37 amino acids, C-terminals have 8 amino acids, and central domains consist of 9 (for the 23,000-24,000 chain) or 10 (for the 26,00027,000) repetitive sequences. The repetitive domains contain blocks of 14 to 25 amino acid residues with an average length of 19-20 (23,24). The secondary structure of zein, as determined by optical rotation, optical rotary dispersion, and



146 circular dichroism (/#), is formed by 50-60% α-helix, mainly existing in the central domains of the peptide chain, 15% of β-sheet, with the rest of the molecule being aperiodic. The tertiary structure of zein consists of asymmetric particles, initially proposed to approximate rods, with axial ratios of 7:1 to 28:1 (/7,25,26) Small-angle x-ray scattering (SAXS) was used to explore the size and shape of zein particles. Matsushima and coworkers (27) reported SAXS measurements on zein in 70% aqueous ethanol over a concentration range of 2-40 mg/mL. They determined Rg and Rc values of 40 and 13.9 Â, respectively, for reduced zein in solutions containing 0.1 or 2% v/v β-mercaptoethanol. For the nonreduced zein, they obtained Rg and Rc values of 49.8 and 19 Â, respectively. From those measurements, they proposed that the reduced zein structural unit has a rectangular prism shape measuring 130 Â for the longest dimension and 34 A for each of the other two dimensions. For the non-reduced zein the longest dimension was determined at 160 Â and 46 Â for the other two lateral dimensions. Along the short dimension, zein was proposed to consist of four molecules. Therefore, the shortest dimension of the molecule was around 12 Â. Matsushima and coworkers (27) assumed a model (Figure 1), related to an earlier model by Argos (18), consisting of 9 (Z19) or 10 (Z22) helical segments folded in an anti-parallel fashion linked by glutamine-rich turns and held in place by hydrogen bonds. They proposed that the helical segments were aligned to form a compact ribbon. A portion of the N-terminus formed an additional helical segment at the end of the ribbon. Figure 2 shows a transmission electron microscope (TEM) image of critical point freeze dried zein showing particle size grains of 200A, in relatively close agreement with the measurements above. The structural model in Figure 1 suggests that the location of zein hydrophobic domains lay along the helix surfaces (front and back). Hydrophilic domains would correspond with the glutamine-rich loops (top and bottom).

Zein Adsorption to Hydrophobic and Hydrophilic Surfaces Zein adsorption to hydrophobic and hydrophilic surfaces was investigated by surface plasmon resonance (SPR) (15). Hydrophilic and hydrophobic surfaces were generated by monolayers of a carboxylic acid terminated thiol, 11mercaptoundecanoic acid (COOH(CH )i SH) and a methyl-terminated alkanethiol, 1-octanethiol (CH (CH ) SH), respectively, fixed on gold-coated glass slides. Figure 3 shows the rate of zein adsorption at various bulk concentration levels (Cb). SPR experiments indicated that zein was adsorbed to I- octanethiol (hydrophobic) as well as to 11-mercaptoundecanoic acid (hydrophilic) surfaces. However, initial adsorption rate was higher for zein on II- mercaptoundecanoic acid than on 1-octanethiol suggesting that a different adsorption mechanism for each case. 2

3

2

0

7



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Figure L Diagram ofzein tertiary structure according to Matsushima et al. (1997).

Figure 2. TEM image of critical point freeze dried zein showing particle size grains of200A. Large particles are starch. In the same experiment, flushing off loosely adsorbed zein allowed the observation of a monolayer, which was thicker for zein on 11-mercaptoundecanoic acid than for zein on 1-octanethiol. Figure 3 shows the desorption effect caused by flushing the SPR cell with 75% 2-propanol (at 1200 sec). Zein adsorption decreased from to a lower value, r f l u s h e d , which was similar for all curves in each figure. Flushing could have removed loosely bound zein adsorbed above surface saturation. The consistency of rflushed values suggested that each corresponded to its monolayer. r f l u s h e d was higher for hydrophilic (0.54 mg/m ) than for hydrophobic (0.11 mg/m ) surfaces. The difference in monolayer values was explained in terms of footprint size, which according to the above structural model would be larger for zein on hydrophobic than on hydrophilic surfaces. Matsushima et al (1997) considered that the zein molecule measured 170 χ 46 χ 12 2

2


148 3

2

Â . The 170 χ 46 Â faces corresponding to the exterior of α-helix segments are largely hydrophobic. The 170 χ 12 Λ faces containing glutamine loops or turns are hydrophilic. Zein may have used different faces of its molecule to adsorb on hydrophobic or hydrophilic surfaces, as shown in Figure 4. It was suggested that zein may be induced into anisotropic behavior by controlling the polar character of solvent media or adsorbing surfaces. 2


Topography of Zein Deposits The topography of zein deposits after SPR experiments was examined by atomic force microscopy (AFM). Images of zein adsorbed on 11mercaptoundecanoic acid and 1-octanethiol SAMs fixed on gold-coated slides are shown in Figures 5 and 6, respectively. Section analyses for both images are also presented. Figure 5, zein deposited on 11-mercaptoundecanoic acid, shows a surface populated by distinct tubular structures, 35 nm high. The approximate diameter of those cylinders is 200 nm. Surface roughness was calculated at 13.7 nm. By comparison, in Figure 6, zein adsorbed on a hydrophobic surface appears uniform, nearly featureless, and has a lower calculated roughness of 2.2 nm.

Zein Structured Solids Zein molecular structure allows it to readily stack and form films on hard surfaces. The formation offree-standingzein films for environmental packaging and other applications has been the focus of intensive research (28-31). Zein films are brittle, necessitating the addition of plasticizers to impart ductility. Zein films were prepared by solubilizing zein and fatty acids (0.5 - 1 g fatty acid/g zein) in aqueous ethanol (70% v/v) followed by the addition of water. The aqueous environment promoted co-precipitation of zein-fatty acid aggregates which were collected as a soft solid (32). The hydrated resin was very ductile and stretchable. Films were drawn from this soft mass and allowed to dry at room conditions. The study of zein plasticization provided theframeworkfor the investigation of the interaction between zein and fatty acids, which will be essential to the development of microencapsulation systems. The structure of zein-oleic acid films was investigated by x-ray diffraction (14). Wide-angle (WAXS) diffraction patterns showed

Nanoencapsulation of Active Food Ingredients - American

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