Sponsoring Institution
National Institute of Food and Agriculture
Project Status
Funding Source
Reporting Frequency
Accession No.
Grant No.
Project No.
Proposal No.
Multistate No.
Program Code
Project Start Date
Sep 1, 2009
Project End Date
Aug 31, 2013
Grant Year
Project Director
Coupland, J. N.
Recipient Organization
Performing Department
Food Science
Non Technical Summary
There are many biologically-active, lipophilic ingredients (BLI) added to foods that are both chemically labile and expensive (e.g., oil-soluble vitamins, flavors, colors, phytochemicals). Although present in small quantities, these ingredients are essential to the perceived sensory and nutritional quality of the food. To prevent their degradation during manufacture and storage and ensure their bioavailability after consumption they are often encapsulated, frequently in liquid oil emulsions. Similar challenges exist for certain drugs and the pharmaceutical industry has recently developed alternative delivery systems, solid lipid nanoparticles (SLN, fine droplets of crystalline lipid), that offer significant advantages in certain applications. In the present work we will adapt the technology from pharmaceutical science to foods and manufacture emulsion-based delivery systems varying in droplet size (micro-scale and nano-scale) and lipid crystallinity (liquid, alpha-polymorph, and beta-polymorphs). We will characterize the structure of the particles by light scattering, calorimetry, X-ray diffraction and atomic force microscopy and the distribution of the solute molecules by electron paramagnetic resonance. We will load the particles with model BLI (beta-carotene and limonene) and compare their chemical stability and functionality. Finally we will apply our findings in real foods (a salad dressing and a carbonated beverage). This project addresses the fundamental relationship between the structure of lipid droplets (i.e., droplet size and lipid crystallinity) and their functionality as a delivery system in foods. Our fundamental findings will be immediately applied to the enrichment of real foods, allowing rapid translation into the industry.
Animal Health Component
Research Effort Categories

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
Knowledge Area
501 - New and Improved Food Processing Technologies;

Subject Of Investigation
5010 - Food;

Field Of Science
2000 - Chemistry;
Goals / Objectives
This work is collaboration between multiple investigators at Penn State and UMass. The UMass group will produce and characterize nanoparticles systems and study the behavior of b-carotene while the PSU group will localize the solutes in the particles and study applications with limonene. We will effectively integrate the two research teams by regular conference calls and student exchanges between the sites to learn techniques and share experiences. Key meetings of the entire research team will be held after 9 months when we will finalize our formulations for the detailed quantitative analysis of SLN structure, 18 months in when we will decide the formulations for functionality testing (Task 2), and 24 months when we will decide the real food trials (Task 3) Task 1: Production and Characterization of Emulsion-based Delivery Systems Our hypotheses are (1) emulsified lipids can be crystallized in a stable alpha form by modifying the cooling rate and by the presence of certain emulsifiers, (2) Emulsifiers can stabilize the dispersion and delay the polymorphic transition, (3) BLI do not co-crystallize with the lipid. Our first experiment will be a screening experiment when we merely report the number of days before any bulk destabilization is seen (e.g., oiling-off, creaming, gelation). From this experiment, we will simplify our experimental design so we can complete a more thorough characterization of the more successful samples in a second 10-week experiment where we will measure kinetic changes in particle size and shape as well as crystal morphology. In parallel we will localize the BLI within the solid emulsion droplets by EPR. We expect the rate of probe oxidation will decrease beta-tending solid>liquid>alpha-tending solid and fine>coarse particles reflecting the different availability of the probes in these systems. Task 2: Chemical Stability of BLI in Emulsion-Based Delivery Systems. We hypothesize that the location of the bioactives as determined in Task 1 will determine the availability of these compounds to oxidation/acid catalyzed hydrolysis and the rate of degradation in beta-tending solid particle>liquid nanoemulsion>alpha-tending solid particle and fine>coarse. Task 3: Application of Emulsion Based-Delivery Systems to Real Foods. We will apply optimized emulsion delivery systems by using them as ingredients to incorporate bioactive lipids into in either a carbonated beverage (e.g. if the emulsion-based delivery system contains an aroma), or salad dressing (e.g. if the emulsion based delivery system contains vitamins).
Project Methods
In most of our studies, we will use glyceryltripalmitate and hydrogenated palm oil as our simple and complex solid fat. As a liquid control oil we will use Miglyol 812. As our aqueous phase we will use polyoxyethylene sorbitan monolaurate (Tween 20) in phosphate buffer (50 mM, pH 7) containing sodium azide (0.01 wt%) to limit microbial growth. As our model BLI we will use beta-carotene and limonene. Hot liquid carrier lipid will be mixed with an aqueous emulsifier solution at the same temperature as the lipid phase using a thermostated high speed blender to a final lipid concentration of 10 wt%. The coarse emulsion will be homogenized using a microfluidizer (Microfluidics, Newton, MA) or high pressure valve homogenizer (Niro Panda, Hudson WI) thermostated at a high temperature (above the melting point of lipid phase). Finally, the droplets will be cooled in ice water to induce droplet crystallization and stored at 5oC. Physical Structure. Particle size measurements will be made by dynamic light scattering (DLS, NanoZS, Malvern Instruments, Malvern, Worcester, England or ZetaPALS, Brookhaven Instruments, Holtsville, NY) after suitable dilution in buffer. While we will get some indication of changes in shape as changes in apparent droplet size, we will confirm this by measuring the shape of the droplets by atomic force microscopy. The crystal morphology within lipid droplets is one of the key properties that influence their ability to maintain chemical stability of bioactive components and is most readily measured by X-ray diffraction. EPR Methods. PTMIO (10 uM) will be added to the lipid phase before homogenization. Samples will be loaded into micropipettes and sealed with Critoseal. EPR spectra will be recorded using a Bruker e-scan R analyzer (Bruker Biospin, Billerica, MA). Absolute radical concentration will be determined by comparing the double integration of the observed EPR spectra with a reference sample. Simulation and fitting of the EPR spectra will be performed using the PEST WinSIM program. Chemical Characterization. We will add an appropriate catalysts to induce oxidation of the BLI (i.e., 15 uM Fe(III)/ 100 uM ascorbic acid) and remove aliquots at intervals and determine the kinetics degradation. Carotenoid concentration in the lipid extract will be measured by reverse-phase HPLC. The degradation of limonene in emulsions will be monitored by measuring loss of this compound as well as formation of the degradation products, limonene oxide, p-cymene and p-methylacetophenone by gas chromatography (GC). We will measure the functionality of the carotene as a pigment using a colorimeter (Labscan II, Hunter Associates Laboratory, Reston, VA). We will measure the functionality of limonene as an aroma by measuring the headspace composition.

Progress 09/01/09 to 08/31/13

Target Audience: The target audience was the wider scientific community and the food manufacturing industry. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Several post-docs, graduate students, undergraduate students and visiting scholars have received training under this project. How have the results been disseminated to communities of interest? Results have been communicated through peer-reviewed publications, book chapters, conferences presentations and talks at other universities. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

What was accomplished under these goals? A major achievement of this work has been the development and use of an electron paramagnetic resonance (EPR) spectroscopy method to determine the distribution and reactivity of small molecules (BLI) in emulsions and related systems. We have applied this method to answer the questions set out in the proposal. In Task 1 we showed that a mixture of lecithin and bile salts is particularly effective in stabilizing the alpha polymorph of high-melting food lipids. However, the EPR data confirmed that the ingredients studied did not co-crystalize with the lipids regardless of the polymorphic form. In general, across a wide range of systems the solid lipid nanoparticles (SLN) simply excluded the added BLI into the aqueous phase, although in some cases the BLI would adsorb at the particle surface instead of being simply excluded into the aqueous phase. Liquid oil emulsions on the other hand absorbed large amounts of the hydrophobic BLI and, as expected, less or none of the hydrophilic BLI. Particle size had a small but significant effect with small droplets binding less BLI in the lipid core but more at the surface. The researchers at UMASS carried our research on the formation, stability, and functional performance of lipid particles with different physical states and sizes. There work showed that emulsion-based delivery systems could be formed with different sizes by optimizing system composition and homogenization conditions, such as surfactant type, surfactant concentration, oil phase viscosity, water phase viscosity, homogenization pressure and number of passes. Thus it was possible to tailor emulsion-based delivery systems for different applications. In Task 2 we investigated the stability of BLI in emulsions and related systems. In general the reactivity of the BLI depended on the amount present in the aqueous phase so SLN behaved more poorly than liquid oil emulsions as delivery systems. Other components in the food could play a role however and surfactant micelles in particular are capable of solubilizing BLI out of the emulsion droplet and increasing their overall reactivity. Modifying the surface charge of a emulsion droplets is a useful tool for controlling the reactivity of some highly hydrophobic BLI (e.g., w3 oils) but for many even moderately hydrophobic ingredients the reaction apparently takes place in the aqueous phase so droplet surface charge is not significant. In Task 3 we applied our findings to a vitamin E enriched model food and found again that SLN provided no special protection for the BLI. The researchers at UMASS also showed that beta-carotene could be encapsulated into liquid and solid state lipid nanoparticles, and examine a variety of factors that influence the physical and chemical stability of the encapsulate nutraceutical, such as storage temperature, particle size, particle composition, interfacial charge, antioxidants, and chelating agents. The knowledge gained from this work enabled the researchers to identify the most optimum conditions for forming Finally, the researchers at UMASS tested the in vitro digestion of nutraceutical nanoemulsions. They showed that the bioaccessibility of encapsulated beta-carotene depended on emulsion droplet size, emulsifier type, and droplet composition. The overall goal of this work was to answer the question of whether some of the new methods of delivery system offered any real advantages. In terms of ingredient stability it appears that SLN are significantly worse than conventional emulsions and nano-scale is marginally worse than conventional micron-scale emulsions. It may be that SLN offer advantages when release is the key feature and indeed we showed that an enzyme-triggered crystallization of an emulsion could cause aroma release. In some applications the greater physical stability and optical transparency of nanoemulsions will outweigh their marginal disadvantage in terms of ingredient stability. This information could be used by the food industry to formulate products specifically designed to increase the bioactivity of encapsulated nutraceuticals and for stabilizing nutraceutical nanoemulsions suitable for incorporating into food products.


  • Type: Journal Articles Status: Published Year Published: 2013 Citation: U. Yucel, R.J. Elias, J.N. Coupland (2013) Effect of Liquid Oil on the Distribution and Reactivity of a Hydrophobic Solute in Solid Lipid Nanoparticles, Journal of the American Oil Chemists Society, 90:819824.
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: C.C. Berton-Carabin, J.N Coupland, R.J. Elias (2013) Effect of the lipophilicity of model ingredients on their location and reactivity in emulsions and solid lipid nanoparticles, Colloids and Surfaces A: Physicochemical and Engineering Aspects, 431:917.
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: C.C. Berton-Carabin, J.N. Coupland, C. Qian, D.J. McClements, R.J. Elias (2013) Reactivity of a lipophilic ingredient solubilized in anionic or cationic surfactant micelles, Colloids and Surfaces A, 412:135142.
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: C.C. Berton-Carabin, R.J. Elias, J.N. Coupland (2013) Reactivity of a model lipophilic ingredient in surfactant-stabilized emulsions: Effect of droplet surface charge and ingredient location, Colloids and Surfaces A: Physicochemical and Engineering Aspects 2013, 418:6875.
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: U. Yucel, R.J. Elias, J.N. Coupland (2013) Localization and reactivity of a hydrophobic solute in lecithin and caseinate stabilized solid lipid nanoparticles and nanoemulsions, Journal of Colloid and Interface Science, 394:205.
  • Type: Journal Articles Status: Published Year Published: 2012 Citation: M. Samtlebe, U. Yucel, J. Weiss, J.N. Coupland (2012) Stability of Solid Lipid Nanoparticles in the Presence of Liquid Oil Emulsions, Journal of the American Oil Chemists Society, 89(4):609-617.
  • Type: Journal Articles Status: Published Year Published: 2012 Citation: Qian, C., E.A. Decker, H. Xiao, and D.J. McClements, Inhibition of beta-carotene degradation in oil-in-water nanoemulsions: Influence of oil-soluble and water-soluble antioxidants. Food Chemistry, 2012. 135(3):1036-1043.
  • Type: Journal Articles Status: Published Year Published: 2012 Citation: Qian, C., E.A. Decker, H. Xiao, and D.J. McClements, Nanoemulsion delivery systems: Influence of carrier oil on beta-carotene bioaccessibility. Food Chemistry, 2012. 135(3):1440-1447.
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Qian, C., E.A. Decker, H. Xiao, and D.J. McClements, Impact of lipid nanoparticle physical state on particle aggregation and beta-carotene degradation: Potential limitations of solid lipid nanoparticles. Food Research International, 2013. 52(1):342-349.
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Salvia-Trujillo, L., C. Qian, O. Martin-Belloso, and D.J. McClements, Modulating beta-carotene bioaccessibility by controlling oil composition and concentration in edible nanoemulsions. Food Chemistry, 2013. 139(1-4):878-884.
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Salvia-Trujillo, L., C. Qian, O. Martin-Belloso, and D.J. McClements, Influence of particle size on lipid digestion and beta-carotene bioaccessibility in emulsions and nanoemulsions. Food Chemistry, 2013. 141(2):1472-1480.

Progress 09/01/11 to 08/31/12

OUTPUTS: Activities conducted in this year included the development of an electron paramagnetic resonance imaging method to measure the microenvironments of probe molecules in emulsions and solid lipid nanoparticles. We exploited this method to consider the effect of particle size, crystallinity and interfacial composition (lecithin vs. casein). We also considered the effect of probe molecular structure and the role of micelles. Lastly we compared the properties of a probe molecule with those of a real food ingredient (a-tocopherol) in emulsions and solid lipid nanoparticles. Dissemination was by peer-reviewed publication and presentations at meetings. PARTICIPANTS: The PIs on this project (Drs., Coupland, Elias, Decker, McClements, Peterson) supervised the students involved, wrote papers and gave presentations. The experimental work was conducted by Umut Yucel, Claire Berton and Cheng Qian who also assisted with data analysis and writing papers. TARGET AUDIENCES: Target audiences for this work were food science researchers in universities and in industry. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

The development and publication of our electron paramagnetic resonance method is an important change of knowledge resulting from this project. In this method, an analog of the small molecule of interest is added to the food matrix and analyze spectroscopically. We have shown how the signal can be deconvoluted into one, two or three populations each characterized by a measurement of the polarity of its environment and rotational diffusion coefficient. Then the probe can be oxidized or reduced by an aqueous reagent (ascorbate or metmyoglobin respectively) to get a measure of chemical availability. We have also considered B-carotene in similar systems as a more realistic example of the effect of food structure on the stability of an ingredient. Using this method we have shown that the formation of solid lipid nanoparticles usually results in the exclusion of the small molecule into the interface/aqueous phase where it is more reactive. There is some possibility to reduce this tendency by either using lecithin as a surfactant or by using a more aliphatic molecule. Finally we developed better understandings of the effect of formulation parameters on the properties of solid lipid nanoparticles and emulsions.


  • Yucel, U., R. J. Elias, and J. N. Coupland. 2012. Solute distribution and stability in emulsion-based delivery systems: An EPR study. Journal of Colloid and Interface Science 377:105-113.
  • Qian, C., E. A. Decker, H. Xiao, and D. J. McClements. 2012. Physical and chemical stability of beta-carotene-enriched nanoemulsions: Influence of pH, ionic strength, temperature, and emulsifier type. Food Chemistry 132:1221-1229.
  • Qian, C., E. A. Decker, H. Xiao, and D. J. McClements. 2012. Solid Lipid Nanoparticles: Effect of Carrier Oil and Emulsifier Type on Phase Behavior and Physical Stability. Journal of the American Oil Chemists Society 89:17-28.
  • Qian, C. and D. J. McClements. 2011. Formation of nanoemulsions stabilized by model food-grade emulsifiers using high-pressure homogenization: Factors affecting particle size. Food Hydrocolloids 25:(5)1000-1008.

Progress 09/01/10 to 08/31/11

OUTPUTS: Experiments were conducted at both sites. At UMass work was conducted on the formulation, properties and stability of nanoemulsins and solid lipid nanoparticles. At PSU the binding of a model small molecule to nanoemlusions and solid lipid nanoparticles was measured by a novel electron paramagnetic resonance method. PARTICIPANTS: J. N. Coupland, D. G. Peterson, R. J. Elias, E. A. Decker, D. J. McClements, U. Yucel, C. Qian. Yucel and Qian were trained as researchers in their respective PhD programs. Yucel graduated fall 2011. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Various approaches to the formulation and stabilization of nanoemulsions have been developed. Importantly the use of mixed surfactans and surfactant-cosurfactant blends were shown to improve their performance as delivery systems. The electron paramagnetic spectra of a probe molecule in a complex fluid (i.e., nanoemulsion or solid lipid nanoparticle) can be deconvoluted to provide information on its distribution within the system. Liquid droplets can encapsulate hydrophobic solutes while crystalline particles tend to exclude them to the surface. Changing the surfactant type (i.e., phospholipids vs. caseiante) allows the formation of a surface domain that can retain the probe molecule. The stability of the probe molecules is inversely related to the fraction in the aqueous phase.


  • Cheng, Q. and D. J. McClements. 2011. Formation of nanoemulsions stabilized by model food-grade emulsifiers using high-pressure homogenization: Factors affecting particle size. Food Hydrocolloids. 25(5):1000-1008.
  • Roa, J., and D. J. McClements. 2011. Formation of Flavor Oil Microemulsions, Nanoemulsions and Emulsions: Influence of Composition and Preparation Method. Journal of Agricultural and Food Chemistry 59(9):5026-5035.
  • Lee, S. J., C. Seung, Y. Li. E. A. Decker and D. J. McCements. 2011. Protein-Stabilized Nanoemulsions and Emulsions: Comparison of Physicochemical Stability, Lipid Oxidation, and Lipase Digestibility. Journal Of Agricultural And Food Chemistry 59(1):415-427.
  • McClements, D. J. and R. Jiajia. 2011. Food-Grade Nanoemulsions: Formulation, Fabrication, Properties, Performance, Biological Fate, and Potential Toxicity. Food Science and Nutrition. 51(4):285-330. PII 935269214
  • McClements, D. J. 2011. Edible nanoemulsions: fabrication, properties, and functional performance. Soft Matter. 7(6):2297-2316.
  • Qian, C., D. E. Andrew, X. Hang. et al. and D. J. McClements. 2011. Comparison of Biopolymer Emulsifier Performance in Formation and Stabilization of Orange Oil-in-Water Emulsions. Journal of the American Oil Chemists Society 88(1):47-55
  • McClements, D. J. and L. Yan. 2010. Structured emulsion-based delivery systems: Controlling the digestion and release of lipophilic food components. Advances in Colloid and Interface Science. 159(2):213-228.

Progress 09/01/09 to 08/31/10

OUTPUTS: In this period we determined the appropriate operating protocols for the microfluidizers used in this work and for the electron paramagnetic resonance spectrometer used to characterize the samples. The mean particle diameter decreased with increasing homogenization pressure and number of passes, with a linear log-log relationship between mean particle diameter and homogenization pressure. The minimum droplet diameter that could be produced after 6 passes at 14 kbar depended strongly on emulsifier type and concentration: SDS < Tween 20 < B-lactoglobulin < sodium caseinate. Small molecule surfactants formed smaller droplets than proteins, which was attributed to their ability to rapidly adsorb to the droplet surfaces during homogenization. The impact of phase viscosity was examined by using different octadecane-to-corn oil ratios in the oil phase and different glycerol-to-water ratios in the aqueous phase. The minimum droplet size achievable decreased as the ratio of disperse phase to continuous phase viscosities decreased for SDS-stabilized emulsions, but was relatively independent of viscosity ratio for B-lactoglobulin-stabilized emulsions. At low viscosity ratios, much smaller mean droplet diameters could be achieved for SDS (d ~ 60 nm) than for B-lactoglobulin (d ~ 150 nm). We successfully generated large and small particles from different lipids containing different spin probes. In preliminary work we determined that it is essential to deoxygenate samples prior to electron spin resonance measurements and developed a protocol to achieve this. In our first major series of experiments on these samples we were able to show the spin probe signal is different in solid compared to liquid samples as well as in large particles compared to small ones. The solid particles characteristically had two populations of probe: one corresponding to the droplet core and one corresponding to the droplet surface. On storage however the population in the droplet core was lost, suggesting some internal restructuring of the droplets. The spin probes were vulnerable to chemical oxidation and the kinetics of oxidation was characteristic of the physical location of the probes within the droplet. Spin probe in liquid droplets is relatively stable to oxidation while the same probe in solid droplets is at the surface so less stable. This initial work used spin probes as model bioactive ingredients but we have also investigated the oxidation of B-carotene in similar systems. In this case the rate of oxidation was similar. PARTICIPANTS: John Coupland (PI) supervised the experimental work at Penn State and provided guidance on emulsion generation and characterization. Julian McClements provided a similar role at Penn State Ryan Elias (co-PI) provided advice and support on the electron paramagnetic resonance spectroscopy and oxidation studies. Eric Decker provided support on the oxidation studies at UMass. Umut Yucel is the PhD candidate at Penn State who conducted the electron paramagnetic resonance spectroscopy.Cheng Qian is a Ph.D. student working on the project (carrying out research experiments, helping write papers and scientific presentations). Pamela Jaramillo conducted some measurement on B-carotene oxidation. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

The major changes in knowledge due to our research over this period are an improved understanding of the conditions affecting the formation of nanoemulsions and solid lipid nanoparticles in a microfluidizers. We have showed that varying the oil type, surfactant type and operating pressure can all independently affect particle size. We have also learnt that electron spin resonance can effectively be used to measure the location of spin probes in nanoemulsions and solid lipid nanoparticles. The spectral data is supported by kinetic analysis of the decay of the probe in the presence of an aqueous oxidizing agent.


  • No publications reported this period