Recipient Organization
UNIVERSITY OF MISSOURI
(N/A)
COLUMBIA,MO 65211
Performing Department
Food Systems & Bioengineering
Non Technical Summary
Proteins and polysaccharides will be used to develop food ingredients to improve texture and stability of food products as well as carriers to help improve the availability of bioactive food components in food matrices.
Animal Health Component
50%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
(N/A)
Goals / Objectives
Developing protein-polysaccharide based ingredients with improved functional properties.Developing delivery systems with controlled delivery of bioactive compounds.
Project Methods
The approach towards the first objective includes sequential task of 1) selecting biopolymers and environmental conditions for forming heated protein and polysaccharide complexes with varying physical and molecular properties, 2) characterizing the physical properties of the complexes, 3) determining their functional properties under different pH and salt conditions or further processing, and 4) determining critical factors leading to optimum functional properties. For objective 2, the task will include 1) formation of delivery systems with varying biopolymer types, concentration and ratio, 2) characterize the release properties of various bioactive ingredients using the developed delivery systems, and 3) determining factors playing important roles in the delivery of each bioactive compound. Statistical analysis will be used to analyze the results.Detailed methods are described as followed. Note that these methods have been well established in our laboratory.Selection of proteins and polysaccharides. We have extensive experience working with whey protein isolate (WPI) but will extend our investigation to other proteins (e.g., plant proteins).Formation of heated protein and polysaccharide complexes. Protein solutions (10% w/w and polysaccharide solutions (4%) will be prepared in deionized (DI) water (> 17 MΩ). Mixed protein-polysaccharide solutions will be prepared to contain certain biopolymer concentrations (e.g., 1 to 9% protein and 0 to 2% polysaccharide) and pH (5 to 7). Solutions will be heated at 85oC for 30 min and cooled. These conditions will produce heated protein and polysaccharide complexes with varying molecular and physical properties.Characterization of protein and polysaccharide complexes. Measurements will focus on particle size, charge, and rheological properties which are the major molecular properties that affect functional properties of the proteins. Particle size and zeta potential will be determined using Zetasizer Nano ZS (Malvern Instrument, Inc.). Rheological properties will be determined using a Kinexus rheometer (Malvern Instruments, Worcestershire, UK).Functional properties: Emulsification properties.Emulsions containing 5 to 40% oil, 0.5 to 6% protein and various concentration of polysaccharide will be obtained by emulsification of oil with aqueous solution through a two-stage process. Coarse emulsions will be prepared by blending oil and protein solution together using a laboratory homogenizer, Ultra Turrax T-25 (IKA Instruments, Germany) at 12,000 rpm for 1 min at room temperature. Final emulsion samples will be obtained by using an ultrasonic processor (Sonics VC 505, power 500 W, frequency 24 kHz) with a sonotrode (3 mm, approx. length 100 mm, titanium) for 5 min (30% amplitude of maximum power). After emulsification, the emulsions will be slowly acidified to desirable pH.Droplet size distribution. The mean hydrodynamic diameter of emulsion samples using the Mastersizer (Malvern Instruments Ltd., Malvern, Worcestershire, UK).Zeta potential of emulsion droplets. The zeta potential of the emulsion will be determined by the Zetasizer Nano ZS.Rheological behavior measurement. Rheological behavior of fresh emulsions will be measured using a Kinexus Rheometer.Interfacial properties. Interfacial properties will be determined using a Kinexus rheometer equipped with a bicone geometry.Creaming stability. Emulsion stability evolutions in tubes will be determined by measurements of height (millimeter units) of a distinctive clear or semi-transparent bottom serum phase layer on day 0, 7, 14, and 21 after emulsion preparation. The extent of creaming will be characterized by creaming index (CI %) = (HS/HT) × 100%, where HS is the height of the serum layer, and HT is the total height of the emulsion. Each creaming index of sample will be recorded in duplicate.Structure characterization by confocal laser scanning microscopy (CLSM): Confocal imaging of emulsion structures will be carried out using a Zeiss LSM 510 META confocal laser scanning microscope (Carl Zeiss, Jena, Germany). Fresh emulsions and emulsions during storage will be withdrawn and stained for structural characterization using the fluorescence dyes (Nile red for fat and Rhodamine B for protein).Stability against processing. Emulsions will be subjected to processing treatments such as heating and freezing-thawing in order to determine their stability.Functional properties: Foaming properties.Foam Generation. Protein foams will be generated using a Kitchen Aid Ultra Power Mixer with a 4.3 L stationary bowl and rotating beaters. A 200 mL of protein solutions will be whipped for 20 min at a speed setting of 8. Overrun. Foam will be gently scooped into a standard weigh boat (100 mL), leveled using a rubber spatula, and weighed. This procedure will be repeated 10 times per foam and will be completed within 20 min after whipping. The mean weight will be used for overrun and air phase fraction calculations.Drainage half life. Foam stability will be measured by recording the length of time required for half of the prefoam mass to drain. Special bowl with a 6 mm diameter hole will be used. After foam generation, the bowl will be placed in a ring stand over a scale with a weigh boat and the hole uncovered. Drainage half life is the time necessary for half the mass to drain. A longer drainage half life corresponds to a greater foam stability. Surface tension. Surface tension will be determined using a Ram' Hart goniometer.Confocal Laser Scanning Microscopy (CLSM) and Image Analysis. Foam microstructure will be visualized by a Zeiss LSM 510 META confocal laser scanning microscope. Rhodamine B, as a specific dye for protein, will be added into each sample prior to foam formation (0.2 mL of 0.1% w/w for 1 g protein). Image analysis of bubbles will be conducted using a Metamorph Imaging System Software (Molecular Devices Corporation, Pennsylvania, USA).Foam Rheological Analysis. A Kinexus Rheometer in oscillatory mode equipped with serrated plate-and-plate geometry will be used to measure foam viscoelastic properties.Application testing. Foaming properties in food applications such as baking and whipped pudding will be determined.Functional properties: Heat stabilityTurbidity. This will be done by measuring the optical density of solutions within the visible region (400 to 600 nm) before and after heating.Phase separation. To establish the degree of phase stability, samples will be centrifuged. If the sample exhibits phase separation, the protein content of each phase will be determined by the BCA protein assay.Viscosity. Characterization of the solution viscosity will provide insight into protein interactions (e.g., Newtonian flow vs. shear thinning) and expected sensory perception. Flow properties will be determined using a three-step shear rate ramp using a Kinexus rheometer. An up - down - up shear rate ramp will give an indication of time dependent and time independent shear thinning.Digestion and delivery properties.Emulsions stabilized by heated protein and polysaccharide complexes will be prepared as previously describe.β-carotene will be used as a model delivery system though additional bioactive compounds can be included.In-vitro digestion. Emulsions will be subjected to a static consensus in vitro digestion method consisting of gastric phase and small intestinal phase based on a standardized static in-vitro digestion method (Minekus and others 2014, Food Funct. 2014 Jun;5(6):1113-24. doi: 10.1039/c3fo60702j).Changes in droplet size of the emulsions during digestion will be determined using the Mastersizer and confocal microscopy (with Nile Red as dye).Determination of β-carotene release. The bioaccessibility of β-carotene was determined after each sample had been subjected to the gastric and intestinal digestions.