Recipient Organization
UNIV OF WISCONSIN
21 N PARK ST STE 6401
MADISON,WI 53715-1218
Performing Department
Food Science
Non Technical Summary
Food insecurity in US frequently corresponds with obesity and under-nourishment of low-income populations. An approach we propose in this project for correcting chronic energy imbalance that poses many health risks is to enable the fortification of beverages with highly-nutritious but under-utilized whey proteins without deteriorating sensory properties. The enhancement of the utilization of whey proteins is also a critical issue for the sustainability of the dairy industry in Wisconsin. The key challenge for utilizing whey proteins in beverage is that the protein tends to denature, aggregate, and precipitate during manufacturing involving thermal processing. Our strategy for preventing the quality loss arising from heat-induced aggregation of whey proteins is to utilize novel polysaccharide stabilizers we have identified in our previous studies. These polysaccharides are expected to bind to the protein surface and form a protective layer that prevents aggregation between proteins. Structures and functionalities of these polysaccharides as well as their correlations with the quality and stability of whey protein fortified beverages will be elucidated based on various physicochemical experiments including atomic force microscopy imaging and force spectroscopy.
Animal Health Component
25%
Research Effort Categories
Basic
50%
Applied
25%
Developmental
25%
Goals / Objectives
The goal of this project is to enable the development of good-tasting and healthy beverages fortified with milk whey proteins. Currently, the pH of acidic beverages fortified with proteins needs to be adjusted to be either lower than 4.3 or higher than 6.5 due to the absence of polysaccharide stabilizers that prevent aggregation and precipitation of proteins in the intermediate pH range, resulting in too much acidity and/or unnatural flavor and taste in certain beverage products. In this project, we attempt to develop two types of novel polysaccharide stabilizers: one is for improving sensory properties of strongly acidic (pH 3.7-4.3) beverages fortified with whey proteins and the other for materializing mildly acidic (pH 5.5-6.5) beverages fortified with whey proteins that are difficult to be found in the current market.Our hypothesis is that interactions between whey proteins and polysaccharide stabilizers are primarily electrostatic. Polysaccharides to be studied in this project carry negatively chargeable groups such as the carboxyl group. Negatively charged groups on polysaccharides are expected to bind to positively charged patches on proteins. Then, the unbound parts of the polysaccharide chain form a brush or hairy layer on the protein surface and physically interfere with the direct contact between proteins and hence aggregation between proteins. On the other hand, polysaccharide molecules remaining in the solution without binding to proteins contribute to an increase in the viscosity of the bulk solution. Such an increase in the viscosity is considered to correlate positively with the molecular size of the polysaccharide stabilizer. We have shown in our previous studies that soybean pectin has a fairly compact molecular structure (e.g., ~100 nm), making it one of the most promising candidates for preventing unfavorable increases in the viscosity of acidic beverages fortified with whey proteins.Conventional polysaccharide stabilizers are unable to bind to proteins and stabilize them in mildly acidic conditions (pH 5.5-6.5). Given that the interaction between protein and polysaccharide is primarily electrostatic, the absence of polysaccharide stabilizers that are capable of binding to proteins in this pH range indicates that both the amount and distribution of negatively charged groups on the polysaccharide chain need to be appropriate relative to those of the positively charged group on the protein surface. In our preliminary investigation, pectin extracted potato tuber was found to prevent heat-induced aggregation of proteins at pH 5.5-6.5. It is therefore critical to elucidate the underlying mechanism of protein stabilization by potato pectin in order to materialize mildly acidic beverages fortified with whey proteins.The specific objectives of the project are:1. To prepare soybean pectin using varied conditions for enabling the fortification of strongly acidic (pH 3-4) beverages using whey proteins without negatively impacting their sensory properties, and characterize molecular properties of the developmental soybean pectin samples;2. To elucidate the molecular mechanism of the interaction between whey proteins and the soybean pectin, and characterize the quality and stability of strongly acidic (pH 3-4) beverages fortified with whey proteins;3. To prepare potato pectin using varied conditions for enabling the development of mildly acidic (pH 5.5-6.5) beverages fortified with whey proteins, and characterize molecular properties of the developmental potato pectin samples; and4. To elucidate the molecular mechanism of the interaction between whey proteins and the potato pectin, and characterize the quality and stability of mildly acidic (pH 5.5-6.5) beverages fortified with whey proteins.
Project Methods
Objective 1. Preparation and characterization of soybean pectin.Preparation of soybean pectinThe residual fibrous material from soy sauce manufacturing will be obtained from a local manufacturer. The material will be suspended in a large quantity of distilled water and stirred at 50°C for 1 hour for removing water soluble impurities. The insoluble residues will be recovered, suspended in water, adjusted to pH 3-5, and autoclaved for extracting pectin. Low-, medium-, and high-molecular weight soybean pectin fractions will be extracted by heating at 120°C and pH 3 for 2 hours, at 120°C and pH 4-5 for 2 hours, and at 130°C and pH 4-5 for 3 hours, respectively. The soybean pectin samples thus prepared will be desalted using electro-dialysis and then freeze-dried.Monosaccharide and amino acid compositions of soybean pectinMonosaccharide and amino acid compositions of the soybean pectin samples will be determined using gas chromatography coupled with mass spectroscopy (GC-MS).Molecular weight distribution of soybean pectinAliquots (50 μL) of sample solutions will be applied to two size exclusion chromatography (SEC) columns connected in series and eluted at room temperature. Elution profiles will be monitored using a multi-angle laser light scattering (MALLS) photometer equipped with a 690 nm helium-neon laser.Molecular structure of soybean pectinCharacterization of individual molecules of soybean pectin will be performed using a BioScope Catalyst atomic force microscope (Bruker, Santa Barbara, CA) located in PI's laboratory. Soybean pectin will be dissolved in an aqueous buffer solution containing 10 mM NH4HCO3 to give a concentration of 1 mg/mL. An aliquot (2 mL) will be drop-deposited onto freshly cleaved mica and allowed to stand in air for 15 minutes. Molecular images will be obtained using peak force tapping (PFT) mode.The effect of pH on the conformation of soybean pectin will be investigated using atomic force microscopy (AFM). Soybean pectin will be dissolved in 1 mM MgCl2 solution to give a concentration of 1 mg/mL and then poured onto freshly cleaved mica or highly oriented pyrolytic graphite (HOPG) placed in the AFM fluid cell. The sample will be left to incubate for 5 min before imaging directly in solution using the PFT mode. After imaging, the fluid cell will be perfused with a buffer solution adjusted to pre-specified pH in order to sequentially decrease pH to be 6.5, 6.0, 5.5, 5.0, 4.5, and 4.0. Molecular images will be obtained at each pH.Objective 2. Mechanism of interactions between soybean pectin and whey protein, and the quality and stability of strongly acidic protein fortified beverage.Preparation of strongly acidic beverage fortified with whey proteinWhey protein isolate having high protein and low carbohydrate levels will be obtained from Davisco Foods International (Eden Prairie, MN). The whey protein will be dissolved to give a protein concentration of 5%, mixed with a 50% w/w sucrose solution, a 3% soybean pectin solution, and distilled water, and adjusted to a pre-specified pH using lactic acid. The final concentrations of protein, sucrose, and soybean pectin will be 1-3% w/w, 5% w/w, and 0.2-0.5% w/w, respectively. The model beverage will be homogenized using a two-stage homogenizer, heat-treated at 141°C for 4 seconds, and stored at 4°C for 2 weeks.Characterization of the simulated beverageThe particle size distribution in the stored beverage will be determined using a Mastersizer 2000 particle size analyzer (Malvern Instruments Inc., Westborough, MA) available in a research laboratory of the Department of Food Science. The zeta potential and viscosity will be also determined using a Zetasizer Nano (Malvern Instruments Inc., Westborough, MA) and a DV-E viscometer (Brookfield Engineering Laboratories, Middleboro, MA), respectively, also available within the Department of Food Science.The morphology of protein particles in the simulated beverage will be studied using AFM. Interaction forces between a single pair of protein particles stabilized by soybean pectin will be investigated using AFM force spectroscopy. An AFM cantilever will be positioned above the center of one of the protein particles immobilized onto the mica surface and driven down onto the target particle until it penetrates the particle. The protein particle thus attached to the cantilever will be detached from the slide simply by retracting the cantilever and positioned over a second particle on the mica surface to determine interaction forces between the pair of protein particles as a function of the separation distance.Objective 3. Preparation and characterization of potato pectin.Preparation of potato pectinPotato tubers will be peeled, sliced, and separated into juice and fiber using a blender. The juice containing starch will be discarded. The fiber will be dried using a vacuum dryer after being washed repeatedly with distilled water to remove any remaining starch. Low-, medium-, and high-molecular weight potato pectin fractions will be extracted from aqueous suspensions of the insoluble residues by heating at 120°C and pH 3 for 2 hours, at 120°C and pH 4-5 for 2 hours, and at 130°C and pH 4-5 for 3 hours, respectively. The potato pectin samples thus obtained will be desalted using electric dialysis and then freeze-dried.A control experiment will be performed to extract potato pectin without the application of heat to clarify whether the heat treatment causes any modifications of the structure and functionality of potato pectin. The potato fiber after removal of starch will be used as the starting material. Potato pectin will be extracted without the application of heat as described under Objective 1.Characterization of potato pectinThe prepared potato pectin samples will be characterized as described under Objective 1. Monosaccharide and amino acid compositions will be determined using GC-MS. The molecular weight distribution will be determined using SEC-MALLS. Molecular morphology and pH-dependent conformational transitions will be investigated using AFM.Objective 4. Mechanism of interactions between potato pectin and whey protein, and the quality and stability of mildly acidic protein fortified beverage.Preparation of mildly acidic beverage fortified with whey proteinWhey proteins (Davisco Foods International, Eden Prairie, MN) will be dissolved to give a protein concentration of 5%, mixed with a 50% w/w sucrose solution, a 3% potato pectin solution, and distilled water, and adjusted to a pre-specified pH using lactic acid. The final concentrations of protein, sucrose, and potato pectin will be 1-3% w/w, 5% w/w, and 0.2-0.5% w/w, respectively. The model beverage will be homogenized using a two-stage homogenizer, heat-treated at 121°C for 30 min, and cool to room temperature before characterization.Characterization of the simulated beverageThe prepared beverages will be characterized as described under Objective 2. Particle size distribution, zeta potential, and viscosity of the simulated mildly acidic beverage will be determined using a Mastersizer 2000 particle size analyzer (Malvern Instruments Inc., Westborough, MA), a Zetasizer Nano (Malvern Instruments Inc., Westborough, MA), and a DV-E viscometer (Brookfield Engineering Laboratories, Middleboro, MA). The morphology of protein particles as well as interaction forces between two protein particles will be evaluated using AFM force spectroscopy.