Progress 01/01/13 to 12/31/14
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest? The results from this project were presented at IFT14 and an annual meeting of the American Dairy Science Association held in July 2014. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
In order to fabricate micrometer-sized whey protein particles that morphologically mimic fat particles in foods, effects of the salt concentration and pH on the kinetics of heat-induced aggregation of whey protein were investigated as the first step. Aqueous solutions containing 0.3-2.0% w/w whey protein were adjusted to pH 3, 4, 5.5, 6, 6.5, 7, 8, 9, and 10 and to give a sodium chloride concentration of 0-100 mmol/L, and heated at 80±0.2°C for up to 2 hours. A general observation from this investigation was that the rate of heat-induced aggregation became larger as pH was shifted close to the isoelectric point (pI) (~ pH 5) of whey protein and the salt concentration was increased. Whey protein solutions containing 1 or 2% w/w whey protein and being adjusted to pH 4 or 5.5 started precipitating after heating at 80°C for less than 3 min, suggesting that large clusters of protein aggregates were formed. In contrast, whey protein solutions adjusted to pH 3, 8, 9, and 10 did not develop any visually detectable precipitates after heating at 80°C for 2 hours. The effect of pH on morphological changes occurring during heat-induced aggregation of whey protein was then investigated based on atomic force microscopy (AFM). Aqueous solutions containing up to 2.0% w/w whey protein were adjusted to pH 3, 4, 5.5, 6, and 7 and to give a sodium chloride concentration of 0-100 mmol/L, and heated at 80±0.2°C. Subsamples were taken at pre-specified time intervals and quenched in an ice water bath to terminate heat-induced protein aggregation. The sample solutions were diluted to give a protein concentration of 10-100 ppm, deposited on to freshly cleaved mica surfaces, air-dried, and imaged using AFM operated in peak-force tapping mode in air. Obtained images were processed using the software associated with the microscope and then analyzed using Image J software. At pH 3, two distinct types of morphologies were observed. A relatively small fraction of protein aggregates appeared to be fibrillar, while the majority of aggregates showed particulate structure. At other pH’s, only particulate protein aggregates were observed. All of these particulate aggregates appear to be composed of smaller elementary particles, indicating that the heat-induced aggregation of whey protein is a two-step process, consisting of the formation of primary aggregates, followed by the secondary aggregation between these primary aggregates. The development of the average size of heat-induced aggregates of whey protein was highly dependent on pH. At pH 5.5, the average size of whey protein aggregates exceeded 10 μm2 in 10 min of heating at 80°C, while at pH 3, it took 90 min to reach an average size of 0.5 μm2. At pH 7, which is also approximately 2 units away from the pI of whey protein, it took 45 min to reach an average size of 1 μm2. These results suggest that pH close to pI is more advantageous in manufacturing micrometer-sized whey protein particles in terms of the energy requirement for their production. The Feret’s diameter that represents the diameter of the smallest circle that entirely covers an individual whey protein aggregate was then plotted as a function of the area occupied by the corresponding aggregate. The Feret’s diameter appears to become more dependent of the size with increasing pH, indicating that whey protein aggregates become more extended, coarse, or anisotropic with increasing pH. More detailed analysis of the surface morphology of heat-induced whey protein aggregates has also supported the proposition that the pH close to pI of whey protein is more desirable in developing micrometer-sized whey protein particles. The surface of heat-induced aggregates of whey protein appeared to become rougher with decreasing pH in AFM images. The surface roughness evaluated based on the cross-sectional height data decreased by a factor of 2 with increasing pH from 5 to 7. These results suggest that the mechanical strength of whey protein particles decreases with increasing pH so that the particles are more flattened on the mica surface during the air-drying step. The results described in the previous sections suggest that heat-induced whey protein particles become softer as pH increases from 5 to 7. Therefore, we have conducted quantitative nano-mechanical mapping (QNM) of selected samples of heat-induced whey protein particles using AFM. During QNM, the force-distance curve is obtained at each pixel in a topographical image and analyzed to calculate mechanical properties such as the elastic modulus. A 2-factor (pH and protein concentration), 3-level (pH 5.5, 6.25, and 7; protein concentration 0.3, 1.15, and 2% w/w) design of experiment was used. At least 6 images were obtained at each combination of the protein concentration (0.3, 1.15, and 2% w/w) and pH (5.5, 6.25, and 7). More than 30 force-distance curves were extracted from each image in order to calculate the distribution of the elastic modulus using the Hertzian model. The elastic modulus calculated at each pH was found to remain fairly constant with increasing protein concentration. In contrast, pH showed a remarkable impact on the elastic modulus. The elastic modulus of heat-induced whey protein particles formed at pH 5.5 and 6.25 was around 25-30 MPa, while that that at pH 7 was around 5-12 MPa. These results provide quantitative basis for understanding the effect of pH on the property of heat-induced whey protein particles. We are currently investigating the effect of heat-induced whey protein particles on textural properties of model foods. Aqueous solutions of whey protein are dispersed in a continuous oil phase to form water-in-oil emulsions. These emulsions are then heated to induce aggregation of whey protein within dispersed aqueous phases. Whey protein particles are rinsed and collected using centrifugation and then dispersed in hydrogel matrices. Effects of the size, elastic modulus, and volume fraction of whey protein particles on textural properties of gels containing whey protein particles will be investigated. The results from this project were presented at IFT14 and an annual meeting of the American Dairy Science Association held in July 2014, will be presented at IFT15 and an annual meeting of ADSA in 2015, and will be published as 2 articles in peer-reviewed journals.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2014
Citation:
Poster presentation 'Effects of pH on Morphologies and Mechanical Properties of Heat-induced Whey Protein Aggregates' at IFT14.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2014
Citation:
Oral presentation 'Effects of pH on the Morphology and Mechanical Property of Heat-induced Whey Protein Aggregates' at an annual meeting of American Dairy Science Association.
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Progress 01/01/13 to 09/30/13
Outputs
Target Audience: Scientists and Technologists in the area of dairy foods. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals? The present results suggest that the morphologies of heat-induced whey protein aggregates are fairly similar at all examined pH’s but their mechanical properties may vary, depending on pH. Therefore, an immediate next step will be to evaluate the mechanical property of heat-induced aggregates of whey protein formed at various pH levels. To avoid flattening or collapse of mechanically weak aggregates, whey protein aggregates prepared at various pH’s will be adsorbed electrostatically onto charged surfaces of derivatized mica sheets to measure force-distance curves in an aqueous buffer using AFM. The successive step will be to gain better control over the kinetics of heat-induced aggregation of whey protein. Effects of co-existing polysaccharide on the kinetics of heat-induced aggregation of whey proteins will be investigated. Co-existence of globular proteins and polysaccharides are expected to induce phase separation. Therefore, we will be able to investigate combined effects of phase separation and nucleation-and-growth processes on the morphological development of heat-induced aggregates of whey proteins.
Impacts
What was accomplished under these goals?
In order to fabricate micrometer-sized whey protein particles that morphologically mimic fat particles in foods, effects of the salt concentration and pH on the kinetics of heat-induced aggregation of whey protein were investigated as the first step. Aqueous solutions containing 0.3-2.0% w/w whey protein were adjusted to pH 3, 4, 5.5, 6, 6.5, 7, 8, 9, and 10 and to give a sodium chloride concentration of 0-100 mmol/L, and heated at 80±0.2 °C for up to 2 hours. A general observation from this investigation was that the rate of heat-induced aggregation became larger as pH was shifted close to the isoelectric point (pI) (~ pH 5) of whey protein and the salt concentration was increased. Whey protein solutions containing 1 or 2% w/w whey protein and being adjusted to pH 4 or 5 started precipitating after heating at 80 °C for less than 3 min, suggesting that large clusters of protein aggregates were formed. In contrast, whey protein solutions adjusted to pH 3, 8, 9, and 10 did not develop any visually detectable precipitates after heating at 80 °C for 2 hours. The effect of pH on morphological changes occurring during heat-induced aggregation of whey protein was then investigated based on atomic force microscopy (AFM). Aqueous solutions containing up to 2.0% w/w whey protein were adjusted to pH 3, 4, 5.5, 6, and 7 and to give a sodium chloride concentration of 0-100 mmol/L, and heated at 80±0.2 °C. Subsamples were taken at pre-specified time intervals and quenched in a 0 °C water bath to terminate heat-induced protein aggregation. The sample solutions were diluted to give a protein concentration of 10-100 ppm, deposited on to freshly cleaved mica surfaces, air-dried, and imaged using AFM operated in peak-force tapping mode in air. Obtained images were processed using the software associated with the microscope and then analyzed using Image J software. At pH 3, two distinct types of morphologies were observed. A relatively small fraction of protein aggregates appeared to be fibrillar, while the majority of aggregates showed particulate structure. At other pH’s, only particulate protein aggregates were observed. All of these particulate aggregates appear to be composed of smaller elementary particles, indicating that the heat-induced aggregation of whey protein is a two-step process, consisting of the formation of primary aggregates, followed by the secondary aggregation between these primary aggregates. The development of the average size of heat-induced aggregates of whey protein was highly dependent on pH. At pH 5.5, the average size of whey protein aggregates exceeded 10 μm2 in 10 min of heating at 80 °C, while at pH 3, it took 90 min to reach an average size of 0.5 μm2. At pH 7, which is also approximately 2 units away from the pI of whey protein, it took 45 min to reach an average size of 1 μm2. These results suggest that pH close to pI is more advantageous in manufacturing micrometer-sized whey protein particles in terms of the energy requirement for their production. The Feret’s diameter that represents the diameter of the smallest circle that entirely covers an individual whey protein aggregate was then plotted as a function of the area occupied by the corresponding aggregate. The Feret’s diameter appears to become more dependent of the size with increasing pH, indicating that whey protein aggregates become more extended, coarse, or anisotropic with increasing pH. More detailed analysis of the surface morphology of heat-induced whey protein aggregates has also supported the proposition that the pH close to pI of whey protein is more desirable in developing micrometer-sized whey protein particles. The surface of heat-induced aggregates of whey protein appeared to become rougher with decreasing pH in AFM images. The surface roughness evaluated based on the cross-sectional height data decreased by a factor of 2 with increasing pH from 5 to 7. These results suggest that the mechanical strength of whey protein particles decreases with increasing pH so that the particles are more flattened on the mica surface during the air-drying step. These results will be summarized as a manuscript and submitted to a peer-reviewed journal shortly. The results will be also presented at IFT14, an annual meeting of the Institute of Food Technologists and an annual meeting of the American Dairy Science Association.
Publications
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