Nanoencapsulation of limonene in amylose structure with Thermo-mechanical (Ultrasound) stress

In recent years, production of nutraceuticals by adding bioactive compounds and nutrients has been grown substantially. These compounds are generally sensitive to environmental or gastrointestinal conditions and their bioavailability is limited due to destructive reactions. One of the common methods to reduce or prevent these kind of problems, is microencapsulation of valuable compounds in some materials which can protect them against environmental conditions, and enabling them to controlled release from trapped compounds at specific time and place. Orange peel oil, contains some important bioactive compounds such as limonene that is used in a variety of beverages, foods, cosmetics, pharmaceuticals and chemicals. D-limonene is the main constituent of orange peel oil, because it makes an 80-95% fraction of the orange peel oil volatile compounds, depending on fruit variety. In addition to its technological characteristics (flavor), D limonene can stop or delay the initiation of cancer. It can also be used as a safe alternative to antimicrobial compounds. Nevertheless, technological limitations (hydrophobic structure, high reactivity, sensitivity to oxidation and volatility) often avoid suitable use of this compound as a dietary supplement. Polysaccharides are among of the basic materials which are applied more in this field. Several factors such as cheap and easy access, having active groups interacting with hydrophobic and hydrophilic compounds, biodegradation, biocompatibility and relatively high thermal resistance, have turned them to be superior to lipid and protein carriers. One of the most important polysaccharide compounds existing in nature, is starch. It can be used as a carrier in encapsulation processes with different purposes, having advantages such as inexpensive, non-toxic, capable of recrystallization, the ability to form film and complex and resistant to various degrees of enzymatic hydrolysis. Spatial configuration of amylose is changed in the presence of ligands such as iodine and linear alcohols, resulting in a left-handed helix which can trap ligands within or between curvatures derived from glucose connections. One of the major structures which is created in the interaction of amylose and lipophilic substances, is known as V-amylose structure. V-amylose is a left-handed helix with an inner hole which ligands can be placed within it. The aim of this study was to determine the effectiveness of amylose in nanoencapsulation of limonene as a bioactive compound with desirable sensory characteristics using a thermo-mechanical stress.
Based on the analysis of pure limonene samples (Sigma-Aldrich) as well as samples used in this study, more than 92% of examined sample comprised of D-limonene. In order to prepare amylose nanoparticles containing limonene, 0.1 molar solution of potassium hydroxide (Merck, Germany) was prepared in deionized water and then high amylose corn starch (HACS) (Sigma-Aldrich (St. Louis, MO, USA) with 70% amylose was added to it in the ratios of 2: 4% while stirring continuously for 30 minutes at 80°C. Limonene was then used in the ratios of 5: 10% of HACS was added to the suspension and stirring continued for 1 minute. Initial suspension has been processed by using ultrasound system (Model UP100- Hescheler Company, Germany) with 100 W power and frequency of 30 kHz for 9 and 18 minutes. The viscosity of amylose suspensions containing nanoparticles with different formulations was measured by using a capillary viscometer (Schott-Gerate-Capillary-Viscometer-525-00- Germany). Size and zeta potential was measured by using dynamic light scattering (DLS) and Nanotrac Flex In-situ Particle Size Analyzer devices and Microtrac ZETA-check determined. The morphology of nanoparticles was studied using a scanning electron microscopy (TESCAN-Vega3- Czech Republic). Microencapsulation efficiency and loading efficiency were determined by using spectrophotometry.
In all formulations, particle sizewere less than 50 nm. Starch granules were exposed to cavitation stress by applying the ultrasonic process .The constant formation of bubbles creates a mechanical impact with high energy on starch granules during bursting. Fast impingement of fluid to granule surfaces, hitting particles to each other as well as resistant of the granules against fluid stream cause breaking of starch particles into nanoparticle scales. The highest amount of zeta potential was related to the sample which had the highest starch and limonene concentration. Amylose concentration had the main effect on zeta potential changes. Electrostatic charges can be the main reasons for the higher zeta potential in samples with 4% amylose concentration. More increasing in surface active agents of amylose, namely ionized hydroxyl groups of glucose molecules leads to increasing in surface charge, and results in zeta potential. The most impact on solutions viscosity is related to amylose concentration. Generally, increasing the amylose concentration leads to increasing the solution viscosity, in other side, with ultrasound treatment, the amount of this index was reduced and the solution became more fluent. Microencapsulation and loading efficiency values ranged between 28-82% and 0.38-1.63% respectively. The limonene concentration had the most impact on the efficiency in various formulations. At similar treatments with %4 amylose concentration and 9 min sonication period, by increasing the amount of limonene from %5 to 10, microencapsulation and loading efficiency were increased from %31 to %82 (%62 growth) and from 0.52 to 1.41 (%63 growth) respectively.