Recipient Organization
UNIV OF HAWAII
3190 MAILE WAY
HONOLULU,HI 96822
Performing Department
(N/A)
Non Technical Summary
Within the food industry, a wide range of products exists in the form of emulsions, such as milk and mayonnaise. An emulsion is a mixture where typically two immiscible substances, like water and lipids (oils and fats), are stably mixed together by emulsifiers. Solid lipid nanoparticles (SLNs) play a crucial role as carriers with emulsion-based systems. They consist of nano-sized solid lipid particles dispersed in a liquid aqueous medium. SLNs are utilized to transport drugs or other active ingredients in medicines and cosmetics owing to their capacity to shield these active compounds and regulate their release in the body. The pharmaceutical industry, in particular, focuses on potential applications in drug delivery, clinical medicine, and research. In recent years, researchers in the food industry have also been ventured into exploring emulsion-based delivery mechanisms for bioactive compounds derived from fruits and vegetables. Many bioactive compounds are chemically unstable and insoluble in water, leading to rapid degradation and limited absorption. SLNs offer a remedy to these challenges by effectively encapsulating and transporting bioactive compounds, thereby enhancing their stability and controlling their release within the body.However, SLNs encounter a significant challenge with alterations in their crystalline structure during storage, potentially resulting in the leakage of encapsulated compounds. The undesired change in lipid crystalline structure occurs due to the intrinsic tendency of substances to transition from thermodynamically unstable states to stable ones. Cooling SLNs at lower temperatures can hinder this unfacorable alteration, thus preventing instability and leakage. This indicates that cooling and stoaring SLNs at sub-zero temperatures can potentially resolve the undesirable transition in lipid crystalline structure. However, in general, subjecting SLNs or emulsions at sub-zero temperatures is not preferred due to the instability induced by ice formation.Supercooling technology can present a solution to this predicament by maintaining SLNs in a supercooled state. Supercooling involves lowering the temperature of a liquid below its freezing point without ice formation. In particular, research has been conducted to maintain the supercooled state using magnetic fields to maintain the quality of food or biomaterials.The proposed research aims to (1) optimize supercooling conditions for stable SLNs using magnetic fields, (2) investigate their physicochemical properties and thermal behavior, and (3) encapsulate bioactive substances into SLNs and analyze their delivery efficacy. The first objective will be achieved through experimental optimization of the magnetic field-based supercooling technique for SLNs. Parameters such as magnetic field strength, cooling temperature, and duration are systematically varied to identify conditions for stable SLN formation. The second objective involves a comprehensive physicochemical and thermal behavior analysis. The physicochemical analysis includes particle size, particle distribution, and zeta potential, which represents the electrostatic potential of the particles, to gauge the SLN stability. Thermal behavior analysis includes melting point, freezing point, and solid fat content to access the crystalline structure of solid lipids in SLNs and the extent of transition from liquid to solid phase. The final objective entails encapsulating β-carotene, a representative fat-soluble bioactive substance, in SLNs and then evaluating the ability of supercooled SLNs to deliver β-carotene through in vitro digestion by simulating the conditions of the digestive system.In summary, employing SLNs as a carrier for bioactive substances in the food sector holds immense promise. By surmounting existing challenges such as crystalline structure changes and controlled release of compounds, SLNs stand poised to transform the landscape of functional food production, making substantial contributions to its expanding market segment. Ongoing exploration and creativity on SLNs using the developed supercooling technology are poised to yield novel food products with enhanced nutritional value and prolonged shelf life, with highly anticipated effects in allied industries like pharmaceuticals, biotechnology, and cosmetics.
Animal Health Component
40%
Research Effort Categories
Basic
30%
Applied
40%
Developmental
30%
Goals / Objectives
The long-term goal of this study is to optimize novel manufacturing technologies (referred to as supercooling technology) that enhance the quality and nutritional benefits of foods. The overall objective of this project is to optimize magnetic field-based supercooling technology for bioactive compound delivery systems, called SLNs to improve their physicochemical stability and ability to carry bioactive compounds into the body. The key hypothesis is that the supercooling process involved in the formulation of SLNs will lead to enhanced physical, chemical, and nutritional properties compared to control SLNs produced under typical conditions. The rapid cooling steps facilitated by subzero supercooling technology would effectively mitigate common limitations of SLNs, such as the expulsion of bioactive compounds due to lipid crystal transformation. It is anticipated that the crystallinity and polymorphic transitions fully manipulated using the developed supercooling technology will enhance the properties of SLNs by minimizing particle size and preventing the expulsion of bioactive compounds.Our specific objectives based on the central hypothesis are as follows:1. Test and optimize supercooling treatment of emulsion-based SLNs formulated with select solid lipids matrixes and emulsifiers2. Characterize and determine the physicochemical stability and crystalline structure in formulated SLNs3. Encapsulate bioactive compounds and assess the in vitro digestibility of lipids and the in vitro bioaccessibility of bioactive compounds in SLNs
Project Methods
Task 1. Test and optimize supercooling treatment of emulsion-based SLNs formulated with select solid lipids matrixes and emulsifiersTask 1 involves the preparation of emulsions for SLN fabrication and formulating the SLNs utilizing supercooling treatment with finding optimized conditions. For the preparation of emulsions, solid lipid matrices will include hard fats which have melting points above 40? and intermediate hard fats which have intermediate-high melting points between 10 to 40?. Emulsifiers that will be tested are phosphatidylcholines (PCs), including soy PC, hydrogenated PC, and distearoylphosphatidylcholine (DSPC).Fabrication of SLNs.Emulsions for SLN formulation will be fabricated using an ultrasonication method. Firstly, the aqueous phase will be prepared by dissolving the emulsifiers (2 wt%) in Milli-Q water (93 wt%) at 75? for 1 hr while the solid lipid matrix (5 wt%) will be completely melted at 75? to eliminate fat crystal memory. Then, the melted solid lipid will be dropped into the aqueous phase during stirring at 500 rpm for 10 min. All samples will be sonicated with 6 cycles of 30s on/off at 60% amplitude. The temperature of the produced hot oil-in-water emulsion will be kept at 505? before creating SLNs to prevent lipid recrystallization.Magnetic Field-Assisted Supercooling The control unit available in the PI's laboratory is equipped with the capability to handle loads of up to 10 Amps at 60 V for the OMF lines, operating within the frequency range of 5 Hz to 10 kHz. A chamber has a total volume of approximately 1 liter (180 × 90 × 65 mm³), tailored to facilitate supercool preservation. Our target supercooling temperature range in this study will span from -5 to -15°C, providing a controlled environment for the preservation and study of samples. To validate and optimize the system function, testing with diverse environmental and operational parameters will be conducted.Task 2. Characterize and determine the physicochemical stability and crystalline structure in formulated SLNsTo determine the effect of magnetic field-assisted supercooling in SLNs, physical, chemical, and crystalline properties will be performed using a series of analytical methods. In particular, these analytical methods will be used to focus on the comparison of supercooled and frozen-thawed SLNs. The physicochemical properties will be determined by mean particle diameter (Z-average), polydispersity index, ζ-potential (surface charge), and differential scanning calorimetry (DSC). The crystalline characteristics will be confirmed by X-ray diffraction (XRD) and nuclear magnetic resonance (NMR) while the morphology of SLNs will be examined using transmission electron microscope (TEM).Characterization of physicochemical stability in SLNsZ-average, ζ-potential, and polydispersity of SLNs can serve as valuable indicators to evaluate the stability of a colloidal system and can be used to observe any changes or destabilization over time.. For Z-average and polydispersity index measurement, refractive indices of 1.33 and 1.45 will be used for water and oil, respectively. The sample averages will be calculated based on data obtained from 10 runs for Z-average and polydispersity index measurements, and 30 runs for ζ-potential measurements.Determination of Physical State using DSCDSC will be employed to determine the physical state of fabricated SLNs (e.g., solid or liquid) and crystal structures of SLNs. Samples (5-10 mg) will be placed in a hermetic aluminum pan and sealed. A sealed empty pan will be used as a reference for the samples. To observe thermal events related to the heating and cooling of SLNs, experiments will be conducted under three different heating and cooling conditions, in accordance with the Objective 2.Crystallinity and Lipid Modification Analysis by XRDThe crystallization of the triglycerides (solid lipid matrix) in the SLNs will be studied using an X-ray diffractometer. The X-ray diffractograms of SLNs will be obtained at a scan rate of 2°/min in a 2θ range from 16 to 32°. This analysis can be used to assess the amorphous or crystalline phases formed in the samples. Solid Fat Content Measurement by NMRThe solid fat content in SLNs will be determined using low-resolution pulsed nuclear magnetic resonance (p-NMR) performed in a Mq-one SFC Analyzer. Samples will be transferred into NMR glass tubes and then the tubes will be placed into the p-NMR analyzer to obtain solid fat content values directly. Morphology of SLNs using Electron MicroscopyThe morphology (particle size and shape) will be examined using a Transmission electron microscopy (Hitachi HT7700) available from the Biological Electron Microscope Facility.Task 3. Encapsulate bioactive compounds and assess the in vitro digestibility of lipids and the in vitro bioaccessibility of bioactive compounds in SLNsDifferent SLNs with magnetic field treatment will be formulated using different solid lipid matrices and emulsifiers based on Task 1 and Task 2. In particular, the physicochemical properties of fabricated β-carotene-loaded SLNs will be analyzed and compared with unloaded SLNs based on the results of Task 2. The β-carotene content in SLNs will be determined by encapsulation efficiency analysis as part of the physicochemical properties. Additionally, in vitro lipid digestibility and in vitro bioaccessibility of β-carotene will be analyzed to assess their ability as a delivery system. These experiments proposed in this study will be conducted by mimicking the digestion model of humans and will provide the effects of types of solid lipid matrices and emulsifiers and the relationship between lipid digestibility and β-carotene bioaccessibility of SLNs in terms of nutritional aspect.Formulation of β-carotene encapsulated SLNsEmulsions for β-carotene-loaded SLNs will be fabricated using an ultrasonication method. The emulsion will be subject to a magnetic field below its freezing point while maintaining a supercooled state and forming SLNs. The conditions (e.g., frequency, storage temperature) of magnetic field treatment will be determined based on Task 1. The control sample will be prepared under the identical conditions (e.g., temperature), but without magnetic field treatment. Also, SLNs made with the typical formulation method, referred to as normal SLNs, will also be fabricated by crystallizing the lipid particles at room temperature.In vitro Digestion Model for β-carotene Bioaccessibility β-carotene encapsulated SLNs will be subjected to a simulated in vitro digestion model taking place in the mouth, stomach and small intestine. β-carotene will be extracted with hexane (0.1% butylated hydroxytoluene w/v). The content of β-carotene will be quantified using an Agilent 1100 Series HPLC equipped with a photodiode array detector and a YMC Carotenoid S-3 C-30 Column (2.0 x 150 mm, 3 µm particle size). The bioaccessibility of the samples will be compared against normal SLNs and different SLN compositions (i.e., solid lipid matrices and emulsifiers) to compare the effect of magnetic field treatment and SLN compositions on bioaccessibility.In vitro Simulated Small Intestine Digestion for LipolysisThe pH-stat digestion model simulated small intestine will be used to measure the lipolysis by analyzing the amount of free fatty acids released from SLNs. The team utilizes the principle of titrating free fatty acids generated from the hydrolysis of triglycerides, a constituent of the solid lipid matrix, using NaOH to measure the extent of lipid hydrolysis. Lipolysis will be conducted by adding pancreatin and lipase. The mixture will be agitating at 37?. The volume of used NaOH will be recorded every minute to calculate the percentage of released free fatty acids.