Progress 06/21/21 to 06/01/23
Outputs
Target Audience:Target Audience: Target audience for this work are the scientists working on casein functionality, dairy companies manufacturing micellar casein concentrate and consumer goods companies. Other target audience would be graduate and undergrad students. Target audience benefited: The data generated in this project was presented in national and international scientific conferences such as annual ADSA, IFT meetings and Food Structure Functionality Forums. Research finding were also be shared with local Dairy Industry through BUILD Dairy program. This strengthened BUILD Dairy program's commitments in the interest regional progress. We trained one post doctoral fellow (Fariba Zad Bagher Seighalani) and MS student (Nathan Pougher) and two undergrad students (Benjamin Evans and Nathan Pace) on advanced rheological techniques. Broader public benefited: The work generated from the project activities provided MCC producers to optimize their formulation to minimize their operational losses and maximize their profitability and improve over sustainability of dairy processing operations. Several students got training on advanced rheological techniques and on conducting independent research. Changes/Problems:N/A What opportunities for training and professional development has the project provided?One post doctoral fellow (Fariba Zad Bagher Seighalani), one MS students (Nathan Pougher), two undergrad students (Benjamin Evans, Nathan Pace) got trained in this project. Students, post-docs, and visiting scholars gained experience on advanced rheological characterization of food materials. They were trained in the following techniques: rheology, differential scanning calorimetry, SDS and UREA PAGE, particle size analysis, powder rehydration characteristics, electron microscopy. They also gained experience in the design and execution of experiments and in the analysis and interpretation of the results. Students working on this project have got opportunities to present their work in BUILD dairy annual meeting as well monthly meetings, IDF cheese symposium, ADSA conference, IFT conferences. Several students attended professional development courses organized under BUILD dairy program. Some students, attended basic and advanced cheese making course offered by western dairy center. How have the results been disseminated to communities of interest?Publications Pougher, N., Vollmer, A.H., and Sharma, P. (2024). Impact of pH and TSC content on cold gelling behavior of HC-MCC. LWT-Food Science and Technology. (under review) (Impact factor: 6.0) Zad Bagher Seighalani, B., McMahon, D. J., & Sharma, P. (2021). Determination of critical gel-sol transition point of Highly Concentrated Micellar Casein Concentrate using Multiple Waveform Rheological Technique. Food Hydrocolloids, 106886. https://doi.org/10.1016/j.foodhyd.2021.106886 (Impact factor: 10.7) Conference presentations Pougher N*, Vollmer A, and Sharma P (2023). Understanding the Mechanism of Cold Gel Formation in Highly Concentrated-Micellar Casein Concentrate (HC-MCC) Solutions: Impact of Calcium Chelation and pH Adjustment. 9th International Symposium of Food Rheology and Structure, Wageningen, The Netherlands, Switzerland, June 11-15. (Poster) Zad Bagher Seighalani F*, Evan B, McMahon DJ, Sharma P (2023). Rheological properties of Highly Concentrated- Micellar Casein Concentrate as affected by pH and temperature. 9th International Symposium of Food Rheology and Structure, Wageningen, The Netherlands, Switzerland, June 11-15. (Poster) Pougher N*, Vollmer A, and Sharma P (2023).Characterizing micellar casein and kappa-carrageenan gels: Ultrastructure, textural, and rheological analysis. American Dairy Science Association virtual annual meeting, Ottawa, Canada June 25-28, 2023. (Abstract no. 2128). Abstract published in Journal of Dairy Science, 106 (Suppl 1), 6. (Oral presentation) Pougher N* and Sharma P (2022). Effect of calcium chelating salts on cold gelation behavior of highly concentrated micellar casein concentrate. IFT FIRST Annual Event and Expo, July 10 - 13, 2022, Chicago, Illinois. (Poster presentation). Pougher N*, and Sharma P (2022). Effect of varying pH on the cold gelling behavior of highly concentrated micellar casein concentrate (HC-MCC). American Dairy Science Association virtual annual meeting, Kanas City, MO June 19-22, 2022. (Abstract no. 1200). Abstract published in Journal of Dairy Science, 105 (Suppl 1), 77. (Oral presentation). Zad Bagher Seighalani F*, McMahon DJ, and Sharma P (2021). A novel method of determining critical gel-sol transition point of micellar casein concentrate using multiple waveform rheological technique. American Dairy Science Association virtual annual meeting, USA, July 11-14, 2021. (Oral presentation, Abstract no. 310). What do you plan to do during the next reporting period to accomplish the goals?N/A
Impacts
What was accomplished under these goals?
Highly Concentrated-Micellar Casein Concentrate (HC-MCC) is a dairy ingredient comprised of 17-23% protein. Obtained via microfiltration and vacuum evaporation, it can form a gel in cold conditions without any physicochemical modifications. With consumer preferences moving away from polysaccharide-based stabilizers in dairy products, there is potential for the gelling properties of HC-MCC to be applied in industry. This study investigates the gelling properties of HC-MCC various states to better understand the mechanism behind cold gelation. The first study examined combinations of physicochemical modifications (dilution, calcium chelation, pH adjustment) to optimize gel strength. The second study examined certain treatments in further detail, i.e., examining additional physical properties. Lastly, the third study examined the cold gelling ability of diluted MCC and kappa carrageenan gels. A three-stage multiwave rheological protocol was applied to HC-MCC samples to observe gel strength and the temperature of cold gelation alongside texture analysis, particle size, and zeta potential measurements to observe additional characteristics in response to treatments. Ultrastructure analysis was conducted on selected treatments using transmission electron microscopy (TEM) to observe morphological changes in casein micellar structure. We observed that pH adjustment resulted in an exponential increase in gel strength as pH increased from 6.2 to 6.8 (R2=0.99), along with significantly higher gelation temperatures. The addition of a calcium chelating salt (trisodium citrate, or TSC) significantly increased gel strength in most combinations of treatments (P<0.05), with 25mM concentrations consistently yielding the strongest gels. Ultrastructure analysis of samples showed that alkalization to pH 6.8 and 7.0 increasingly disintegrated the micellar structure of casein and released individual fragments into the aqueous phase, which was observed alongside a significantly increased gel strength and gelation temperature. The use of TSC at 25mM partially disintegrated micelles, whereas 50mM resulted in the formation of large aggregates with a concomitant decrease in gel strength. Kappa carrageenan addition resulted in gelation temperatures similar to modified higher protein samples, but gel strength was lower. TEM micrographs depicted minimal interaction between kappa carrageenan and casein, creating a biphasic solution. Overall, the gelling properties of HC-MCC can be significantly improved in response to physicochemical modification with significant changes in protein structure taking place.
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Progress 10/01/21 to 09/30/22
Outputs
Target Audience:The work reported in this period will be of interest not only to the scientific research groups around the world but also to the local dairy industries. The data generated during this period was presented in national and international scientific conferences including annual ADSA, and IFT meetings. Research finding were also shared with local Dairy Industry through BUILD Dairy program. This strengthens our BUILD Dairy program's commitments in the interest regional progress. We hired one MS student who is properly trained to run their project independently. Changes/Problems:PI got stuck in India due to Covid 19 situation during April-May-June 2021, which caused delay in the full fledge start on this project. This may necessitate no-cost extension of this project for one more year. What opportunities for training and professional development has the project provided?Nathan Pougher, grad student working on this project got opportunity to present his data in the various national and international conferences including American Dairy Science Association, Institute of Food Technologists, BUILD Dairy annual conference and monthly meetings. He participated in the Idaho Milk Processors Association (IMPA) product develop competition and won third place. He also won third place at ADSA poster competition. How have the results been disseminated to communities of interest?Nathan presented his work at BUILD dairy annual meeting held in May 2022 in Provo, UT. Pougher N and Sharma P (2022). Effect of calcium chelating salts on cold gelation behavior of highly concentrated micellar casein concentrate. IFT FIRST Annual Event and Expo, July 10 - 13, 2022, Chicago, Illinois. (Poster) Pougher N, and Sharma P (2022). Effect of varying pH on the cold gelling behavior of highly concentrated micellar casein concentrate (HC-MCC). American Dairy Science Association virtual annual meeting, Kanas City, MO June 19-22, 2022. (Oral presentation, Abstract no. 1200). Abstract published in Journal of Dairy Science, 105 (Suppl 1), 77. What do you plan to do during the next reporting period to accomplish the goals?1. Complete the remaining experiments in the Specific aim 1. related to modification of HC-MCC proteins i.e. TSC addition, alkalization. 2. Complete full set of experiments of kappa-addition at three levels in order to reduce minimum gelling concentration of HC-MCC. All the samples will be subjected for advanced rheological, particle size, zeta potential, TEM and textural analysis.
Impacts
What was accomplished under these goals?
SPECIFIC AIM #1: Optimizing of MCC cold gelation conditions to form a thermoreversible gel at minimum MCC concentration levels by adjusting pH, and TSC levels. Overview: Using highly concentrated Micellar Casein Concentrate (HC-MCC) as a food ingredient could yield positive effects in the dairy industry. As more consumers become ingredient conscious, a dairy based stabilizer for dairy foods could be a promising alternative to various gums used in industry. With a protein content ranging from 19%-23%, it is capable of forming a cold gel without any modifications to the casein. However, added ingredients and changing physico-chemical parameters can significantly improve the gel strength. Purpose of this study was to study effect of varying pH levels on the cold gelling behavior of HC-MCC. Materials and Methods Gelling behavior of MCC solutions with respect to time and temperature was determined using the multiwaveform rheological technique on MCR 302 (Anton Paar, Graz, Austria) using the method developed in our lab (Zad Bagher Seighalani, McMahon and Sharma, 2021). Multiwave test was performed at a range of temperatures from 5 to 70°C with temperature ramp profile of 1°C per minute by applying multiple frequencies simultaneously. Superimposed signals (harmonics of oscillation) of 1, 3, 6,12, 24, and 48 rad/s with strains of 1, 0.18, 0.16, 0.14, 0.11, 0.09, and 0.07% were used at each frequency, respectively, with fundamental frequency of 1 rad/s. Cold gelling point (time or temperature) was determined by applying Winter-Chambon (1986) criteria, and so the corresponding temperature to first point at which tan δ from three different frequencies coincide sat a single point (i.e. tan δ becoming independent of frequency). These tests were performed in combination with time and temperature sweeps. In addition, the rheometer was set up with a temperature sweep protocol, starting at 60oC and ending at 5oC, along with time sweeps at the beginning temperature of 60oC and the ending temperature of 5oC. The calcium chelating salt used was trisodium citrate (TSC), which was added to the sample at 60oC in concentrations of 10, 25, and 50mM. Additionally, HC-MCC samples were modified by diluting to a minimum protein content where gelation still occurred. This formed the basis for observing changes in cold gel temperature at varying pH (5.8-6.6) by adding glucono-delta-lactone. Results and Discussions The minimum protein concentration which could form a cold gel was determined to be 16.8%, which was then used to observe the effects of pH modifications. Addition of the acid i.e. drop in pH increased the temperature of gel-sol transition, and increased storage modulus values for low pH samples. Additionally, acidified samples continued to behave as viscoelastic liquids at elevated temperatures and viscoelastic solid at low temperatures indicating thermoreversibility of cold gels. Addition of TSC led to the increase of storage modulus (G') and loss modulus (G") across the temperature range. This effect increased with increasing concentration of the TSC, indicating a stronger gel matrix. In addition, the temperature of gelation increased with TSC concentration. The samples prepared with a 50mM concentration of TSC highest elastic strength of the cold gel, while maintained a solid disc shape even at room temperature. Overall, this study explored the possibility of modifying HC-MCC to utilize it in the future as a dairy based thickener or stabilizer. Adding a dairy based stabilizer to another dairy product could improve consumer preferability rather than using polysaccharide-based gums for similar purposes.
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Progress 06/21/21 to 09/30/21
Outputs
Target Audience:The data generated in this project will be presented in national and international scientific conferences such as annual ADSA, IFT meetings and Food Structure Functionality Forums. Research finding will also be shared with local Dairy Industry through BUILD Dairy program. This will further strengthen BUILD Dairy program's commitments in the interest regional progress. We expect to hire one MS student and couple undergrad students and will ensure that they properly trained to run their project independently. Changes/Problems:PI got stuck in India due to Covid 19 situation during April-May-June 2021, which caused delay in the full fledge start on this project. This may necessitate extension of this project. What opportunities for training and professional development has the project provided?Grad student Nathan Pougher participated in BUILD dairy conference organized in Boise State University during Sept 2021. He learned about multiwave rheological technique. How have the results been disseminated to communities of interest?Project just started in June, 2021. Not enough data was generated to be presented in the conference and annual scientific meetings. One methodology paper was published as below: Zad Bagher Seighalani, B., McMahon, D. J., & Sharma, P. (2021). Determination of critical gel-sol transition point of Highly Concentrated Micellar Casein Concentrate using Multiple Waveform Rheological Technique. Food Hydrocolloids, 106886. https://doi.org/10.1016/j.foodhyd.2021.106886 What do you plan to do during the next reporting period to accomplish the goals?SPECIFIC AIM #1: Optimizing of MCC cold gelation conditions to form a thermoreversible gel at minimum MCC concentration levels by adjusting pH, and TSC levels. Overview: In the first specific aim, we will be identifying minimum concentration of protein needed to form a cold gel in various conditions (pH and TSC treatment) (Fig.3). Frozen HC-MCC samples (protein ~23%) used in previous study (Zad Bagher et al., 2021) will be used in this study too according to protocol suggested by Lu at al. (2015). Un-treated MCC solutions with varying protein concentration (5-20%) will be first evaluated for cold gelling temperature using time sweeps. Gelling behavior and gel characteristics will be studied using small angle oscillatory rheological techniques and texture analysis. Once we will identify lowest possible concentration to form a cold, we will then introduce pH (6.0-6.6) and TSC (0-50 mM) treatments as a mean to increase gelling temperature and further reduce the minimum gelling concentration needed for forming a cold gel. Liquid samples treated with TSC or with different pH levels will be subjected for in-situ particle size analysis, zeta potential, and colloidal and ionic calcium levels. Ultra-structure of selected samples will be evaluated using transmission electron microscopy (TEM). Gelling behavior: Gelling behavior of MCC solutions with respect to time and temperature will be determined using the multiwaveform rheological technique on MCR 302 (Anton Paar, Graz, Austria) using the method developed in our lab (Zad Bagher Seighalani, McMahon and Sharma, 2021). Multiwave test will be performed at a range of temperatures from 5 to 70°C with temperature ramp profile of 1°C per minute by applying multiple frequencies simultaneously. Superimposed signals (harmonics of oscillation) of 1, 3, 6,12, 24, and 48 rad/s with strains of 1, 0.18, 0.16, 0.14, 0.11, 0.09, and 0.07% will be used at each frequency, respectively, with fundamental frequency of 1 rad/s. Cold gelling point (time or temperature) will be determined by applying Winter-Chambon (1986) criteria, and so the corresponding temperature to first point at which tan δ from three different frequencies coincide sat a single point (i.e. tan δ becoming independent of frequency). Multiple temperature recycling will be performed to test thermoreversibility of gels using the protocol developed by Lu et al., 2015. Particle size and Zeta Potential analysis:Changes in the particle size of casein micelles with respect to pH and TSC concentrated samples will be determined by measuringhydrodynamic diameter.The hydrodynamic diameter of caseins in the MCC dispersion will be measured in-situ at 658 nm using a Litesizer-500 (Anton Paar, Graz, Austria) with a fixed scattering angle of 175° at range of temperatures from 5-55°C. Protein samples will be diluted in skim milk ultrafiltrate (SMUF) buffer. The zeta potential of casein micelles in MCC samples will be measured using a Univette low volume cuvette in the Litesizer-500 at different temperatures and TSC concentrations. Zeta potential (mV) will be estimated based on the slipping plane's electric potential using the Smoluchowski approximation method using the instrument software. Each measurement will be conducted in triplicates. Mechanical properties of gels such as rheology, texture and structural properties will be assessed using an Anton Paar MCR 302 rheometer (Anton Paar, Graz, Austria), confocal laser scanning microscopy (CLSM) (Carl Zeiss AG, Oberkochen, Germany) using methodologies described by Sharma et al. (2015, 2016, 2017). Viscoelastic properties of food materials will be determined by conducting frequency and temperature sweeps within linear viscoelastic (LVE) limit. Fracture properties will be determined on texture analyzer (Stable Micro System Ltd., Surrey, UK) in both compression and tension deformation mode. Microstructure of the experimental samples will be studied using a protein binding (Fast Green) fluorescent dye by exciting them at 633 nm on CSLM. Statistical analysis: Data collected will be analyzed using the mixed model of SAS for repeated measures. The statistical model will include the main effects of category, aging time, and their interactions. Differences will be evaluated using a Student's t-test, with P ≤ 0.05 considered statistically significant. SPECIFIC AIM #2:Studies on cold gelling behavior of protein (caseins) and polysaccharide (kappa carrageenan) associative mixtures (different ratios). OVERVIEW Three samples selected in specific aim 1 with respect to minimum concentration of protein needed to form a cold gel from untreated MCC and samples treated with pH and TSC levels. Cold gelling temperature (CGT) and thermo reversibility of will be other criteria to short list the samples from the objective 1. These samples will then be added with an associative hydrocolloid (kappa-carageenan) at three different levels i.e. 0.01, 0.1 and 0.5% using the method described by Spagnuolo et al., (2005). MCC and kappa-carrageenan mixture will then be subjected for cold gelling and thermoreversibility of cold gels. In this specific aim we will target our strategy to further reduce the minimum casein concentration needed to form a thermoreversible. Liquid samples will be subjected to particle size and zeta potential analysis using the methods described above. Water binding ability of the new gels will be determined by applying centrifugal force 20,000 g for 5 min at 4oC. Molecular changes in the proteins (caseins) will be analyzed using Urea-PAGE technique (Lamichhane et al., 2019). From the second objective we will be able to determine whether presence of kappa-carrageenan improved gelling behavior of MCC and it is possible to further reduce the minimum concentration needed for cold gel formation. Ultrastructure analysis will help us to visualize type of structures formed due to interactions between MCC and the polysaccharide. Transmission Electron Microscopy:Ultrastructure of the MCC-kappa-carrageenan mixture and gels will be determined by using TEM method described by Lu et al., (2015). After formation of gel, samples will be fixed using 2% glutaraldehyde. Fixed gel will be cut into smaller pieces with a razor blade, followed by rising with sodium cacdylate buffer (0.1 M with 16 mM calcium chloride and 4.8% sucrose at pH 7.4) for 10 min each) and postfixing in 2% osmium tetroxide solution, further treating with saturated aqueous uranyl acetate for 1 h, then dehydration through a graded series of ethanol. At the end of fixing process and dehydration, samples will be individually embedded in a flat mold and polymerized overnight at 65°C. Sections (~70 to 100 nm) will be cut on a Leica EM UC6 ultramicrotome (Leica Microsystems Inc., Buffalo Grove, IL) using a diamond knife (Diatome, Hatfield, PA). Sections will be double-stained for 20 min with saturated aqueous uranyl acetate followed by 10 min with Reynold's lead citrate. Sections will then be analyzed using a transmission electron microscope (TEM; JEM 1400 Plus, Jeol USA Inc., Peabody, MA) operated at 120 kV, and digital images will be captured with a Gatan camera (Gatan Inc., Pleasanton, CA).
Impacts
What was accomplished under these goals?
Work on the specific Aim 1: Optimizing of MCC cold gelation conditions to form a thermoreversible gel at minimum MCC concentration levels by adjusting pH, and TSC levels just started. Initial focus is on the training graduate student on advanced rheological techniques including determining cold gelling temperature of HC-MCC using multiple wave rheological techniques. Training on following equipment was given; 1. Anton Parr MCR 302 rheometer 2. Litesizer Anton Paar for particle size in nanometers and zeta potential. 3. multiple rheological techniques. 4. preparation and harvesting for transmission electron microscopy (TEM) analysis. 5. Anton Paar's particle size analyser for particle size measurements in microns. 6. SMS texture analyzer.
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