Source: PURDUE UNIVERSITY submitted to NRP
FUNCTIONALIZATION OF PEA PROTEINS USING COLD EXTRUSION FOR APPLICATIONS THAT INCLUDE EMULSIFICATION AND GELATION
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
ACTIVE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1024316
Grant No.
2021-67017-33339
Cumulative Award Amt.
$476,270.00
Proposal No.
2020-03961
Multistate No.
(N/A)
Project Start Date
Jan 1, 2021
Project End Date
May 31, 2025
Grant Year
2021
Program Code
[A1364]- Novel Foods and Innovative Manufacturing Technologies
Recipient Organization
PURDUE UNIVERSITY
(N/A)
WEST LAFAYETTE,IN 47907
Performing Department
Food Science
Non Technical Summary
Pea protein is a sustainable, vegetable-based, protein source that has shown promise in the production of meat and dairy analogues. In order to improve our knowledge and understanding of pea protein, we will produce bioinformatic models of pea protein subfractions vicilin, legumin, and convicilin, and we will identify lipid binding regions therein. These structural models will be interpreted through techniques that quantify the bonding mechanisms in pea protein secondary structures. These models will then be used to understand emulsification and gelling properties of pea protein and protein subfractions over shelf life, thus creating a full link between in silico models and real-world performance. They will also be applied in order to better understand our novel processing technique, cold extrusion. By subjecting pea protein isolate to temperatures between 0 and -30°C, the hydrophobic regions will denature, while the single screw extruder subjects the isolate to high shear forces and molecular rearrangement. This will create proteins with strong emulsion stability and activity, as well as uniquely soft gels that are held together through hydrophobic bonds. Combining structure function relationships between in silico models and real world performance, introducing cold extrusion as a form of protein modification, and confirming the shelf life of pea protein gels through established rheological techniques.
Animal Health Component
35%
Research Effort Categories
Basic
50%
Applied
35%
Developmental
15%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
5015010202050%
5025010200020%
5015010100030%
Knowledge Area
501 - New and Improved Food Processing Technologies; 502 - New and Improved Food Products;

Subject Of Investigation
5010 - Food;

Field Of Science
2020 - Engineering; 1000 - Biochemistry and biophysics; 2000 - Chemistry;
Goals / Objectives
2. Specific Objectives of Research1. Develop structure function relationships between in silico bioinformatic models of legumin, vicilin, and convicilin, interpreted by FTIR, DSC, and SDS - PAGE, and real-world performance in gels and emulsions.2. Induce structural modifications to pea protein's hydrophobic regions by cold denaturation and cold extrusion in order to develop novel protein structures with good emulsification and gelling properties.3. Utilize modified proteins in order to create gels and emulsions and test these model food systems with rheological techniques that have been proven to be strong indicators of product quality and stability, particularly with model emulsion products, during shelf life, building relationships between bioinformatics and shelf life stability.4. Evaluate the success of cold extrusion modifications of pea proteins and fine tune/modify to obtain the most successful functional properties
Project Methods
a. Amino Acid SequencingAmino acid sequencing will be done in two parts based on previously established methods in our (Bhunia/Kokini) laboratories for the amino acid sequencing of kafirin and wheat glutenin and gliadin 13,15. Legumin rich and vicilin rich fractions will be isolated with a salt extraction method, dialyzed, and suspended in sample buffer (1.4 ml MiliQ water, 2.0 ml 0.5 M Tris-HCL, pH 6.8, 2.0 ml of 10% SDS, 2.0 ml glycerol, 0.4 ml of 0.05% bromophenol blue) and loaded into SDS - PAGE wells and run at 100V64,64,65,65. Staining will be done using Coomassie Brilliant Blue R-250. Standard kDa ladders will also be purchased and prepared in the same way. The pea proteins will be separated in the gel, based on their molecular weights similar to previous work2,5,41. Following SDS-PAGE separation, gel bands are subjected to Lysine-C/trypsin digestion and analyzed using a Q ExactiveTM HF Hybrid Quadrupole-Orbitrap Mass Spectrometer, available on a fee for service basis at Purdue's Bindley Bioscience center 13,15.b. Bioinformatic modeling and the development of 3D molecular models to understand lipid binding sites, and hydrogen bonding interaction sitesAfter amino acid sequences have been identified, the development of three-dimensional protein models will be carried out utilizing the I-TASSER software. Hydrophobic regions within specific protein secondary structures will be identified using Heliquest and ExPASy software, the earlier of which allows for the development of Eisenberg wheel projections giving hydrophobic moment data on hydrophobicity patterns in proteins, and the latter of which can show the degree of hydrophobic accessibility, as has been applied to kafirin in our laboratory, and other proteins as well 13-15,21.i. Comparison Method of high and low temperature modificationPea protein isolate, and protein fractions, will also be subjected to cold temperature treatment to promote cold denaturation (0°C to -30°C). Samples will be prepared as above, dissolving in a pH 9 medium and dispersed in a second solution of 200-proof ethanol, with ethanol being chosen for its low freezing point in order to prevent ice crystal formation during treatment. Suspended protein isolate, and protein isolate fractions, will then be placed inside an ice cream maker bowl and stirred continuously for 5, 10, or 20 minutes and at varying temperatures (-30°, -15°, and 0° C). Samples will be dried overnight, ground into a powder, and stored in air tight containers. The whole protein isolates will be referred to as heat treated (HTP) and cold treated proteins (CTP). The fractions will be referred to as heat treated legumin (HTL), cold treated legumin (CTL), heat treated vicilin (HTV), cold treated vicilin (CTV), heat treated convicilin (HTC) and cold treated convicilin (CTC).iii. Extrusion Based Protein ModificationExtrusion will be carried out on a 3 zone 19mm diameter Brabender single-screw extruder equipped with glycol / ammonia cooling lines and a 3 mm single bore die (Plasticorder, Model PL2000, C.W. Brabender Instruments, Inc. South Hackensack, N.J.). Three screws (1/1 compression, 3/1 compression, and dispersive) will be used for all experiments. High temperatures between 100°C and 130°C will be tested at moisture contents between 25% and 45%, and a screw speed of 100, 125, and 150 rpm. Moisture will be measured by using a rapid moisture analyzer (Mettler Toledo, Columbus OH). Pea protein will be tempered to the desired moisture using a combination of water and ethanol in order to minimize ice crystal formation in cold extrusion. Cold extrusion will be conducted at temperatures between 0°C and - 30°C in order to combine the impact of high shear and cold denaturation. Screw speed and moisture content will be tested in the same ranges as high temperature extrusion.HEP and CEP will be dried and ground for analysis in all subsequent steps of this proposal.Secondary structures of all pea protein modifications (HTP, CTP, HEP, CEP) and protein fractions (HTL, CTL, HTV, CTV, HTC, CTC) will be analyzed by Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy (Nicolet Nexus 470 FTIR, Thermo Fisher Scientific, Waltham MA), with spectra obtained between 800 and 3800cm-1 and the amide I peak (1600-1700cm-1) will be analyzed for secondary structure analysis based on previous work with zein in our lab15,54. Secondary structures are known to impact the development of tertiary and quaternary structures of proteins as well 15,53,53,54,57.i. Disulfide Bonding interactionsDisulfide bonding interactions will be probed using SDS-PAGE, by comparing results in unmodified and modified pea protein isolate. All modifications and fractions will all be run on SDS-PAGE gels using the same procedure outlined in the amino acid sequencing section of this proposal. Proteins will also be suspended in the same solution with added 5% mercaptoethanol in order to reduce disulfide bonding. SDS - PAGE results will be analyzed using J Image software (available in the public domain) which quantifies the intensity of bands at different molecular weights.ii. Hydrogen bonding interactionsHydrogen bonding interactions will be probed through differential scanning calorimetry (DSC), following established methods in our laboratory and previous work in pea protein8,36,37,69,70. Modified pea protein, and fractions, will be passed through a 0.25 mm mesh sieve prior to analysis. DSC will be performed with a Discovery Nano Series DSC Instrument, available in the Kokini Lab, (TA Instruments, Delaware, USA) calibrated with pure indium and sapphire. 4-5 mg samples will be heated from 20° to 300° C and cooled back to 20°C at a rate of 10° C/ min under continuous nitrogen purging at 50 ml/min.iii. Hydrophobic interactionsHydrophobic interactions will be studied with the aid of bioinformatic modeling described in section 3.2 of this proposal. Bioinformatic models are capable of identifying lipid binding regions based on their amino acid sequences and the alignment of hydrophobic and hydrophilic amino acid residues14,15,21. These models also indicate the degree of accessibility of these protein structures15. This leads to understanding of lipid binding, emulsification, and gelation in these proteins.i. Emulsification Stability and ActivityIn this study, model oil in water emulsions will be prepared based on previous work in our laboratories71 by dispersing modified protein, and fractions, in Mili-Q water at various concentrations72. The sample preparation will then be diluted using SDS to a concentration of 0.1% and the absorbance measured at 500 nm with a UV-VIS spectrophotometer.ii. Emulsification Shelf Life ModelingModified pea protein and isolate will be prepared into model emulsions by the same method as 3.6.i of this proposal71. Creep compliance values will be obtained using a DHR-3 Rheometer (TA Instruments, USA). Constant stress will be applied for 1200s and allowed to recover for 600s. Creep compliance will be obtained on days 1,3,7,10, and 14 in order to develop compliance over a two-week shelf life47,48,74.iii. GelationThe gels which are at the minimum gelling concentration will be observed by small amplitude oscillatory shear (SAOS) measurements through time sweeps, amplitude sweeps, and frequency sweeps utilizing a DHR-3 Rheometer (TA Instruments, USA). Gels at the minimum gelling concentration will also be tested at temperatures of 4°C, 25°, and 65°C on days 1,3,7,10,14 of shelf life.

Progress 01/01/24 to 05/31/24

Outputs
Target Audience:1. Food scientists in the US and around the world 2. Food engineers and chemical engineers and biological engineers and agricultural engineers in the US and around the world 3. Food industry professionals 4. High school teachers 5. High school students 6. Culinary professionals 7. Food product developers and others Changes/Problems:When we realized that the film properties of plasticized and baked pea protein films offer unique mechanical and rheological properties, we made a significant shift in the direction of part of the project with the hope to also write a new proposal next year with the goal of expanding the work we are doing in this proposal. The method we have used is unique and has the potential to lead to several patentable and transformational discoveries. We intend to use this coming year very judiciously to develop these new opportunities. We want to systematically change the molecular transformations in the pea protein films to develop the strongest and most extensible films that could lead to the development of a new generation of plant based highly resilient films that may be able to contribute to sustainability. What opportunities for training and professional development has the project provided?Professional interactions at professional meetings: all of my students attended multiple professional meetings where they interacted with the scientific community and had the opportunity to share their knowledge and acquire new ideas and new information from the professionals they interacted with. Through participation in the programs of the Whistler Carbohydrate Center they interacted with member company professionals and some of them were able to land internships especially the undergraduate students working on the projects. The students worked on product development teams and were able to use the learnings of the project to develop various products as part of the product development projects. How have the results been disseminated to communities of interest?The results of the research were disseminated first through refereed journal papers and book chapters and some of the publications received many citations. Then all of the work was presented at multiple professional society meeting and reached the relevant audience directly. Many one on one interactions occurred that occasionally led to consulting assignments were the science could be shared with industry professionals. Invitations by companies and universities allowed for closer dissemination and interactions as well. What do you plan to do during the next reporting period to accomplish the goals?We have begun to mimic the film mechanical properties especially the extensional properties of Saran wrap by using a unique approach to fabricate pea protein films by plasticizing and baking pea protein solutions after they were first cold extruded. Interestingly our initial findings are suggesting that this is a promising approach to the design and fabrication of protein films.

Impacts
What was accomplished under these goals? Exploration of creep ringing phenomena in cold denatured pea protein emulsions: Different cold denaturation pre-treatments of pea protein were used to study how cold denaturation impacts emulsions stabilized with the protein and xanthan gum. A combination of ethanol and low temperature led to significantly improved emulsion stability, and the creep ringing data provide greater insight into the particle network than traditional creep models, such as the Burger Model. Mechanistic understanding of protein-protein interactions in pulse flour bread doughs: Research on the characteristics of pulse-substituted bread doughs has been extensively conducted. Despite the improvement in nutritional values, many studies show that the low content of gluten protein in pulse flour substituted dough has significantly reduced the dough's extensibility, the final loaf volume upon baking, and the overall textural qualities upon consumption. We evaluated pulse flour dough systems with different gluten concentrations by monitoring the changes in the rheological properties as well as chemical properties such as starch crystallinity and protein secondary structure during fermentation. The baking performance was examined to access the final sensorial property of the product.

Publications

  • Type: Journal Articles Status: Published Year Published: 2024 Citation: Yazar, G., Kokini, J. L., & Smith, B. (2024). Impact of Endogenous Lipids on Mechanical Properties of Wheat Gluten Fractions, Gliadin and Glutenin, under Small, Medium, and Large Deformations. Lipidology, 1(1), 30-51.
  • Type: Journal Articles Status: Published Year Published: 2024 Citation: Liu, D., Helmick, H. D., Kokini, J. L., & Bhunia, A. K. (2024). Protocol to reveal the binding partner of secreted housekeeping enzymes in Listeria monocytogenes via in silico prediction to in vivo validation. STAR protocols, 5(1), 102839.


Progress 01/01/23 to 12/31/23

Outputs
Target Audience:1. food scientists in the US and around the world 2. Food engineers and chemical engineers and biological engineers and agricultural engineers in the US and around the world 3. Food industry professionals 4. High school teachers 5. High school students 6.culinary professionals 7. Food product developers and others Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Professional interactions at professional meetings: all of my students attended multiple professional meetings where they interacted with the scientific community and had the opportunity to share their knowledge and acquire new ideas and new information from the professionals they interacted with. Through participation in the programs of the Whistler Carbohydrate Center they interacted with member company professionals and some of them were able to land internships especially the undergraduate students working on the projects. The students worked on product development teams and were able to use the learnings of the project to develop various products as part of the product development projects. How have the results been disseminated to communities of interest?The results of the research were disseminated first through refereed journal papers and book chapters and some of the publications received many citations. Then all of the work was presented at multiple professional society meeting and reached the relevant audience directly. Many one on one interactions occurred that occasionally led to consulting assignments were the science could be shared with industry professionals. Invitations by companies and universities allowed for closer dissemination and interactions as well. What do you plan to do during the next reporting period to accomplish the goals?We have started working on structure function relationships for plant protein films using the bioinformatic guidance that we were able to discover through the project. We want to sharpen our understanding of the molecular interaction basis of mechanical properties of pea protein films.

Impacts
What was accomplished under these goals? Applications of Structural Bioinformatics in Understanding Molecular Origins of Pea Protein Gelation and Emulsification: This research aims to develop relationships between bioinformatic models of pea protein and give molecular insight on how pea protein forms gels and emulsions. In this work, a series of homology models have been generated for pea protein, as well as other pulses, from global sources in order to compare the amino acid similarity and differences based on cultivar and growing region. It has been found that the surface properties of the protein three-dimensional structure correlates well with surface hydrophobicity and zeta potential. Design of Cold Extrusion Processing and Structure-Function Relationships for Pea Protein Functionalization: This phenomenon is driven by decreasing hydrophobic interactions at lowering temperatures. In this work, we have subjected pea protein to cold temperatures, changes in tempering water pH, and shear forces through extrusion using twin and single screw extruders. Extrudates were characterized by measuring changes in secondary structure, zeta potential, surface hydrophobicity, and SDS-PAGE. We have shown that cold denaturation occurs during extrusion, and that it leads to hydrophobic amino acid exposure in pea protein with no changes in disulfide bonding. Development of the Sequence of physical processes (SPP) methodologies for the study of food systems in the LAOS and MAOS regions: This work studies and compares the nonlinear rheological behavior of three different classes of foods using the Sequence of Physical Processes method of Rogers et. al. and Fourier Transform coupled with Chebyshev Decomposition by Ewoldt and McKinley. The evolution of deltoid size with increasing strain allowed for the determination of a critical strain before the irreversible network breakdown occurred. The time component from SPP allowed for more accurate capture of the linear viscoelastic region and crossover points by the 3D amplitude sweep. Deltoid and trefoil profiles provided a detailed and distinctive intracycle behavior of each class of the food. Development of Relationships Between Protein Secondary Structure and Fundamental LAOS Parameters in Thermoset Protein Gels: This research aims to study the nonlinear rheological behavior of thermoset protein gels, with an emphasis on plant-based proteins. It is known that a variety of plant-based proteins form gels when a sufficient concentration of protein is suspended in water and heated above the protein's temperature of denaturation before cooling. In this project, it is hypothesized that the energy contained within those gels can be quantified through physicochemical analysis including secondary structure measurements, zeta potential, surface hydrophobicity, and SDS-PAGE. Furthermore, it is expected that the chemical energy will show a proportionality to the rheological responses of the material. These physicochemical measurements are currently being compared with non-linear rheological parameters derived from the SPP methodology to gain a molecular understanding of these rheological phenomenon and how they might change throughout the heating and cooling process. ?Application of machine learning algorithms in understanding the development of color and gloss in plant-based protein edible coatings: This work is aimed at replacing egg washes with plant based protein edible coatings in model baked goods and model color through machine learning. Coatings with variable pH, protein concentration, and glycerol concertation were applied to pie crusts and baked for differing amounts of time. These products are then evaluated for their glossiness, color, height, and texture. Furthermore, images taken of the pie crust and the color is estimated using a machine learning model, trained on >1200 images of different colors of paper. Model results show that it is possible to accurately estimate color, and current developments are working towards maintaining these results under variable lighting conditions, as would be present inside a bread oven, or if images are taken outdoors. This project aimed to identify suitable plant-based replacements for egg washes, and provide models that could be used as part of computer vision systems in bakeries.

Publications

  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Helmick, H., Tonner, T., Hauersperger, D., Okos, M., & Kokini, J. L. (2023). Comparison of the specific mechanical energy, specific thermal energy, and functional properties of cold and hot extruded pea protein isolate. Food Research International, 174, 113603.
  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Le, A. N. M., Erturk, M. Y., Shim, Y. H., Rogers, S. A., & Kokini, J. (2023). A critical study of the nonlinear rheological properties in major classes of foods using the Sequence of Physical Processes (SPP) method and the Fourier Transform Coupled with Chebyshev Decomposition (FTC) method. Food Research International, 174, 113587.
  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Helmick, H., Tonner, T., Hauersperger, D., Ettestad, S., Hartanto, C., Okos, Kokini, J. L. (2023). Physicochemical characterization of changes in pea protein as the result of cold extrusion. Food Chemistry, 423, 136240.
  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Helmick, H., Ettestad, S., & Kokini, J. L. (2023). Estimation of cold denaturation temperature and its utilization in predicting protein stability as an aid in functionalizing pea protein isolate through cold denaturation. Innovative Food Science & Emerging Technologies, 89, 103479.
  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Asif, M., Khan, M. K. I., Khan, M. I., Maan, A. A., Helmick, H., & Kokini, J. L. (2023). Effects of citrus pomace on mechanical, sensory, phenolic, antioxidant, and gastrointestinal index properties of corn extrudates. Food Bioscience, 55, 103012.
  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Yazar, G., Kokini, J. L., & Smith, B. (2023). Comparison of mixing and non-linear viscoelastic properties of carob germ glutelins and wheat glutenin. Food Hydrocolloids, 143, 108922.
  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Helmick, H., Rodriguez, N., & Kokini, J. L. (2023). Utilization of creep ringing and bioinformatic modelling in study of cold denatured pea protein emulsions. Innovative Food Science & Emerging Technologies, 88, 103420.
  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Erturk, M. Y., Le, A. N. M., & Kokini, J. (2023). Advances in large amplitude oscillatory shear rheology of food materials. Frontiers in Food Science and Technology, 3, 1130165.
  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Fu, W., Li, S., Helmick, H., Hamaker, B. R., Kokini, J. L., & Reddivari, L. (2023). Complexation with polysaccharides enhances the stability of isolated anthocyanins. Foods, 12(9), 1846.
  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Helmick, H., Jain, A., Terashi, G., Liceaga, A., Bhunia, A. K., Kihara, D., & Kokini, J. L. (2023). Bioinformatic approaches for characterizing molecular structure and function of food proteins. Annual Review of Food Science and Technology, 14, 203-224.
  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Helmick, H., Hartanto, C., Ettestad, S., Liceaga, A., Bhunia, A. K., & Kokini, J. L. (2023). Quantitative structure-property relationships of thermoset pea protein gels with ethanol, shear, and sub-zero temperature pretreatments. Food Hydrocolloids, 135, 108066.
  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Le, A. M., Erturk, M. Y., & Kokini, J. (2023). Effect of fat on non-linear rheological behavior of processed cheese spreads using coupled amplitude-frequency sweeps, Fourier Transform-Chebyshev polynomials method, sequence of physical processes, and quantitative network analysis. Journal of Food Engineering, 336, 111193.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2023 Citation: Benbow, Kara, and Gabbie Surdyka. Extensibility of Biodegradable Films. Fall Undergraduate Research Expo, Purdue University. 2023.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2023 Citation: Benbow, Kara. Pea Protein Edible Coating as Replacement for Egg Washes. Spring Undergraduate Research Conference, Purdue University. 2023.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2023 Citation: Le, Anh Minh. A critical study of the nonlinear rheological properties in major classes of foods using the Sequence of Physical Processes (SPP) method and the Fourier Transform Coupled with Chebyshev Decomposition (FTC) method. Educational, Purdue University; Whistler Center for Carbohydrate Research, October 20, 2023.
  • Type: Book Chapters Status: Under Review Year Published: 2023 Citation: Helmick, H., Le, A. M., Kokini, J. L. (2023). Rheology of Foods Containing Plant-based proteins. Plant Proteins: Farm to Table.
  • Type: Theses/Dissertations Status: Other Year Published: 2023 Citation: Harrison Helmick (PhD thesis) (2023) Bioinformatic modelling and functionalization of pea protein through cold denaturation with applications in extrusion, gelation, and emulsification, Purdue University
  • Type: Theses/Dissertations Status: Other Year Published: 2023 Citation: Anh Minh Nghi Le ( MS thesis ) (2023) Analyzing ��food nonlinear rheology through: Fourier transform with Chebyshev decomposition (FTC) and Sequence of physical processes (SPP) methodologies, Purdue University


Progress 01/01/22 to 12/31/22

Outputs
Target Audience:• The food industry, especially ingredient companies and those developing meat and dairy analogues • The academic community • Food scientists • Molecular Biologists • Biotechnologist • Data Scientists • Food Engineers • Food chemists • The extension community Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?One PhD student is currently working on the aims of this grant and making progress towards getting his degree, and is expcted to graudate within the next year. Two undergraduate students have graduated from the university and are authors on at least two papers. They are now both working in the food industry. Two additional undergraduate reserachers are also currently working the project, with one set to graduate in May of 2023, and the other in May of 2024. How have the results been disseminated to communities of interest?• Publications in refereed journals (in progress) • Conference presentations at the Institute of Food Technologists (2) and Purdue Conferences (5) • Monthly contributions to the Baking and Snack magazine, a common trade magazine for the baking industry What do you plan to do during the next reporting period to accomplish the goals?We will continue to work towards the goals and objectives of the project by following the experimental strategy that we outlined. In doing this, we will obtain the outcomes of the project.

Impacts
What was accomplished under these goals? 1. Develop structure function relationships between in silico bioinformatic models of legumin, vicilin, and convicilin, interpreted by FTIR, DSC, and SDS - PAGE, and real-world performance in gels and emulsions. These charchterizations have all occurred in at least two different pea protien samples and gelation has been studied in depth. These results have been published in Food Hydrocolloids. Emulsions are currently being studied, and the data is being collected and analyzed. 2. Induce structural modifications to pea protein's hydrophobic regions by cold denaturation and cold extrusion in order to develop novel protein structures with good emulsification and gelling properties. Gelation has been studied in depth and it was found that the properties of gelation are signifcantly different than untreated protein. The gels are stabelized by hydrophobic forces, as hypothesized. Emulsions are currently being studied, and the data is being collected and analyzed. 3. Utilize modified proteins in order to create gels and emulsions and test these model food systems with rheological techniques that have been proven to be strong indicators of product quality and stability, particularly with model emulsion products, during shelf life, building relationships between bioinformatics and shelf life stability. Gelation has been studied in depth and it was found that the properties of gelation are signifcantly different than untreated protein. The gels are stabelized by hydrophobic forces, as hypothesized.Emulsions are currently being studied, and the data is being collected and analyzed. 4. Evaluate the success of cold extrusion modifications of pea proteins and fine tune/modify to obtain the most successful functional properties. Twin screw and single screw extrusion properties of pea protein at a variety of moisture contents and pH at temperatures belows 0 were studied. It was found that the extrudate is signifcantly different than heat extruded protein, and has the ability to bind both more oil and water. These extrudates may be useful in plant-based protein meat and dairy analogues. There are two manuscripts currently under review to communicate these results to the scientific community.

Publications

  • Type: Journal Articles Status: Awaiting Publication Year Published: 2023 Citation: Helmick, Harrison, Anika Jain, Genki Terashi, Andrea Liceaga, Arun K. Bhunia, Daisuke Kihara, and Jozef L. Kokini. Bioinformatic Approaches for Characterizing Molecular Structure and Function of Food Proteins. Annual Review of Food Science and Technology, March 24, 2023. https://doi.org/10.1146/annurev-food-060721-022222.
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Helmick, Harrison, Christabel Hartanto, Sarah Ettestad, Andrea Liceaga, Arun K. Bhunia, and Jozef L. Kokini. Quantitative Structure-Property Relationships of Thermoset Pea Protein Gels with Ethanol, Shear, and Sub-Zero Temperature Pretreatments. Food Hydrocolloids, August 12, 2022, 108066. https://doi.org/10.1016/j.foodhyd.2022.108066.
  • Type: Journal Articles Status: Under Review Year Published: 2023 Citation: Harrison Helmick1, Troy Tonner, Daniel Hauersperger, Sarah Ettestad, Christabel Hartanto, Martin Okos, Andrea Liceaga, Arun K. Bhunia, Jozef L. Kokini. "Physicochemical characterization of changes in pea protein as the result of cold extrusion" (under review)
  • Type: Journal Articles Status: Under Review Year Published: 2022 Citation: Harrison Helmick, Troy Tonner, Daniel Hauersperger, Sarah Ettestad, Martin Okos, Jozef L. Kokini "Utilizing the free energy folding landscape of pea protein in designing a cold extrusion process with implications in protein functionalization" (under review)
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Yazar, Gamze, Jozef L. Kokini, and Brennan Smith. Effect of Endogenous Wheat Gluten Lipids on the Non-Linear Rheological Properties of the Gluten Network. Food Chemistry 367 (January 15, 2022): 130729. https://doi.org/10.1016/j.foodchem.2021.130729.
  • Type: Book Chapters Status: Under Review Year Published: 2023 Citation: Harrison Helmick, Anh Minh Le, Jozef Kokini. "Rheological behavior of plant based proteins." Book - Plant based proteins from farm to table. (Under Review)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Helmick, Harrison, Troy Tonner, Daniel Hauersperger, Sarah Ettestad, Christabel Hartanto, Arun Bhunia, Andrea Liceaga, Jozef Kokini, and Martin Okos. Determining Protein Stability Using Random Forest Regression with Applications in Cold Extrusion. Educational presented at the ECE: Elemore Center for Electrical and Computer Engineering, Purdue University, November 3, 2022.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Helmick, Harrison, Sarah Ettestad, Christabel Hartanto, Arun Bhunia, Andrea Liceaga, and Jozef Kokini. Quantitative Structure Property Relationships of Cold Denatured Pea Protein During Gelation. Educational presented at the OIGP 2022 Spring Reception, Purdue University, May 4, 2022. https://www.purdue.edu/gradschool/oigp/events/spring-reception/index.html.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Helmick, Harrison, Troy Tonner, Daniel Hauersperger, Sarah Ettestad, Christabel Hartanto, Arun Bhunia, Andrea Liceaga, and Jozef Kokini. Optimization of a Single Screw Extruder for Low Temperature Applications. Presented at the IFT: Indiana Division Meeting, Purdue University, April 22, 2022.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Ettestad, Sarah, Harrison Helmick, Christabel Hartanto, Arun Bhunia, Andrea Liceaga, and Jozef Kokini. Understanding How Protein Structure Impacts Its Function and Effect of Cold Denaturation on Protein Gelation. Educational presented at the Purdue Undergraduate Research Symposium, Purdue University, April 14, 2022. https://engineering.purdue.edu/Engr/Research/EURO/research-symposiums.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Hartanto, Christabel, Harrison Helmick, Sarah Ettestad, ,Arun Bhunia, Andrea Liceaga, and Jozef Kokini, Understanding How Protein Structure Impacts Its Function and Effect of Cold Denaturation on Protein Gelation. Educational presented at the Purdue Undergraduate Research Symposium, Purdue University, April 14, 2022. https://engineering.purdue.edu/Engr/Research/EURO/research-symposiums.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Helmick, H., Tonner, T., Hauersperger, D., Ettestad, S., Hartanto, C., Bhunia, A., Liceaga, A., & Kokini, J. (2022, March 3). Optimization of a single screw extruder for low temperature applications [Virtual Conference]. Graduate Industrial Research Symposium, Purdue University. https://girs2022.com
  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Le, Anh Minh, Merve Yildirim Erturk, and Jozef Kokini. Effect of Fat on Non-Linear Rheological Behavior of Processed Cheese Spreads Using Coupled Amplitude-Frequency Sweeps, Fourier Transform-Chebyshev Polynomials Method, Sequence of Physical Processes, and Quantitative Network Analysis. Journal of Food Engineering 336 (January 1, 2023): 111193. https://doi.org/10.1016/j.jfoodeng.2022.111193.
  • Type: Other Status: Published Year Published: 2023 Citation: Helmick, Harrison, Arun Bhunia, Andrea Liceaga, and Jozef Kokini. Pro Tip: Finding Suitable Egg Replacements. Baking & Snack, January 25, 2023. https://www.bakingbusiness.com/articles/58235-pro-tip-finding-suitable-egg-replacements.
  • Type: Other Status: Published Year Published: 2022 Citation: Helmick, Harrison, Arun Bhunia, Andrea Liceaga, and Jozef Kokini. Pro Tip: Using Fava Beans for Protein Supplementation in Baked Foods. Baking & Snack, December 21, 2022.
  • Type: Other Status: Published Year Published: 2022 Citation: Helmick, Harrison, Arun Bhunia, Andrea Liceaga, and Jozef Kokini. Pro Tip: Understanding the Use of Corn in Recreating Viscoelastic Dough. Baking & Snack, November 16, 2022.
  • Type: Other Status: Published Year Published: 2022 Citation: Helmick, Harrison, Arun Bhunia, Andrea Liceaga, and Jozef Kokini. Pro Tip: Use Lentils for Emulsified Baked Food Batters. Baking & Snack, October 19, 2022. https://www.bakingbusiness.com/articles/57593-pro-tip-use-lentils-for-emulsified-baked-food-batters.
  • Type: Other Status: Published Year Published: 2022 Citation: Helmick, Harrison, Arun Bhunia, Andrea Liceaga, and Jozef Kokini. Pro Tip: Use Chickpea Protein Concentrates to Increase Gel Strength. Baking & Snack, September 23, 2022. https://www.bakingbusiness.com/articles/57381-pro-tip-use-chickpea-protein-concentrates-to-increase-gel-strength.
  • Type: Other Status: Published Year Published: 2022 Citation: Helmick, Harrison, Arun Bhunia, Andrea Liceaga, and Jozef Kokini. Pro Tip: Understanding Pea Protein Structure to Improve Baked Foods. Baking & Snack, August 17, 2022. https://www.bakingbusiness.com/articles/57007-pro-tip-understanding-pea-protein-structure-to-improve-baked-foods.
  • Type: Other Status: Published Year Published: 2022 Citation: Helmick, Harrison, Arun Bhunia, Andrea Liceaga, and Jozef Kokini. Pro Tip: Understanding Protein Structure to Determine Function. Baking & Snack, July 20, 2022. https://www.bakingbusiness.com/articles/56819-pro-tip-understanding-protein-structure-to-determine-function.
  • Type: Other Status: Published Year Published: 2022 Citation: Helmick, Harrison, Arun Bhunia, Andrea Liceaga, and Jozef Kokini. Pro Tip: Quantify the Texture of Your Products. Baking & Snack, June 15, 2022. https://www.bakingbusiness.com/articles/56566-pro-tip-quantify-the-texture-of-your-products.
  • Type: Other Status: Published Year Published: 2022 Citation: Helmick, Harrison, Arun Bhunia, Andrea Liceaga, and Jozef Kokini. Pro Tip: Use Droplet Shape to Understand Protein Emulsions. Baking & Snack, May 18, 2022. https://www.bakingbusiness.com/articles/56416-pro-tip-use-droplet-shape-to-understand-protein-emulsions.
  • Type: Other Status: Published Year Published: 2022 Citation: Helmick, Harrison, Arun Bhunia, Andrea Liceaga, and Jozef Kokini. Pro Tip: Use Calorimetery to Understand Gelation and Processing in Plant-Based Proteins. Baking & Snack, April 20, 2022. https://www.bakingbusiness.com/search?page=2&q=pro+tips.
  • Type: Other Status: Published Year Published: 2022 Citation: Helmick, Harrison, Arun Bhunia, Andrea Liceaga, and Jozef Kokini. Pro Tip: Light Can Illuminate Protein Emulsification. Baking & Snack, March 16, 2022. https://www.bakingbusiness.com/search?page=2&q=pro+tips.
  • Type: Other Status: Published Year Published: 2022 Citation: Helmick, Harrison, Arun Bhunia, Andrea Liceaga, and Jozef Kokini. Pro Tip: Electrostatic Interactions Help Understand Protein Functionality. Baking & Snack, February 2, 2022. https://www.bakingbusiness.com/search?page=2&q=pro+tips.
  • Type: Other Status: Published Year Published: 2022 Citation: Helmick, Harrison, and Jozef Kokini. Engineering Pea Protein for Meat and Dairy Substitutes. InnovatED, January 22, 2022. https://www.purdue.edu/gradschool/professional-development/innovated.php.
  • Type: Other Status: Published Year Published: 2022 Citation: Helmick, Harrison, Arun Bhunia, Andrea Liceaga, and Jozef Kokini. Pro Tip: Using Rheology to Optimize Plant-Based Proteins. Baking & Snack, January 19, 2022. https://www.bakingbusiness.com/search?page=2&q=pro+tips.


Progress 01/01/21 to 12/31/21

Outputs
Target Audience:• The food industry, especially ingredient companies and those developing meat and dairy analogues • The academic community • Food scientists • Molecular Biologists • Biotechnologist • Data Scientists • Food Engineers • Food chemists • The extension community Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?• One PhD student is currently working on the aims of this grant and making progress towards getting his degree. • Two undergraduate students have been hired and will develop their reserach skills as part of this grant. They have eached published one paper and are track towards additional publications. Two additional undergraduate students will be hired in the coming year to aid in this work, since the original two will have graduated. How have the results been disseminated to communities of interest? Publications in refereed journals (in progress) Conference presentations at the American Society of Baking (1), Cereals and Grains (2), and Institute of Food Technologists (2) Monthly contributions to the Baking and Snack magazine, a common trade magazine for the baking industry Contributions to magazines including Comercial Baking and InnovatED What do you plan to do during the next reporting period to accomplish the goals?We will continue to work towards the goals and objectives of the project by following the experimental strategy that we outlined. In doing this, we will obtain the outcomes of the project.

Impacts
What was accomplished under these goals? 1. In our first submitted, we modeled pea legumin, vicilin, and convicilin using homology modeling and drawn relationships between these models and FTIR, zeta potential, and dynamics light scattering. In our second publication, we used a similiar homology modeling method to predict the zeta potential of two commercial pea protein isolates successfully. We continue with these homology modeling to end use functionalities, relating the temperature of denaturation, ethalpy of denaturation, and change in heat capacity to structures of a databse of proteins which are used to predict those attributes in pea protein. A similiar bioinformatic - to - gelation set of experiments is currently being conducted. We are finding that a parametric bioinformatic approach is sufficent to provide estimates of the stiffening rate and estimates of the magnitude of G' in a rheometer. 2. In order to induce cold denaturation in pea protein a combination of food safe ethanol, pea protein, low, temperature, and the shear force of a blender were used. We have also begun the optimization process of a single screw extruder to study the impacts of low temperature extrusion as a function of pH. Thermodynamic modeling suggests that at a pH of 3.0, the protein should unfold and produce cold denatured structures which are further changed by the shear forces in the extruder. 3. We have used the ethanol, shear force, and low temperature treatment to change the structure of protein and observed changes in the emulsification and gelation behavior of these proteins. We have found some improvements in the emulsion activity index and the proportion of protein found at the oil water interface. These properties are related to changes in surface hydrophobicity, confirmed experimentally and bioinformatically. Work regarding gelation and emulsification is currently underway, and it is expected that manusripts will be submitted within the calendar year regarding these protein modifications. 4. We have conducted experimentation with a twin screw extruder with a barrel temperature of 0 degrees C. It was found that the shear forces created too much heat to be removed under these conditions. As such, we have moved to a smaller single screw extruder with greater cooling capacity. By optimizing moisture content, we have been able to obtain product outlet temperatures as low as -1 degree celcius. At an appropriate pH value, this is within the range of cold denaturation for pea protein. This was further supplmented by the development of an artificial neural network to process video data and calculate the specific mechanical energy to within 96% accuracy. This has allowed our group to begin the charachterization of physicochemical changes that occurs as the result of this low temperature processing. This work should cluminate in a manuscript within the next year.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Helmick, Harrison, Arun Bhunia, Andrea Liceaga, and Jozef Kokini. Functionalization of Pea Protein through Cold Denaturation: From Bioinformatic Modeling to the Food Industry. Presented at the Purdue University OIGP Spring Reception, 5/6/2021. https://engineering.purdue.edu/ECE/Academics/Graduates/Announcements/20210111_OIGP_Sp21_Reception.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Helmick, Harrison, Arun Bhunia, Andrea Liceaga, and Jozef Kokini. Bioinformatic Modeling of Cold Denaturation in Pea Protein, IFT FIRST 21 Virtual Conference Poster. July 19 2021
  • Type: Other Status: Published Year Published: 2021 Citation: Helmick, Harrison, Arun Bhunia, Andrea Liceaga, and Jozef Kokini. Pro Tip: Electrostatic Interactions Help Understand Protein Functionality. Baking & Snack, February 2, 2022. https://www.bakingbusiness.com/search?page=2&q=pro+tips. Helmick, Harrison, and Jozef Kokini. Engineering Pea Protein for Meat and Dairy Substitutes. InnovatED, January 22, 2022. https://www.purdue.edu/gradschool/professional-development/innovated.php. Helmick, Harrison, Arun Bhunia, Andrea Liceaga, and Jozef Kokini. Pro Tip: Using Rheology to Optimize Plant-Based Proteins. Baking & Snack, January 19, 2022. https://www.bakingbusiness.com/search?page=2&q=pro+tips. Helmick, Harrison, Arun Bhunia, Andrea Liceaga, and Jozef Kokini. The Plant-Based Alternative Ingredient Craze  Whats behind It and Why It Isnt Going Away. Commercial Baking, November 11, 2021. https://commercialbaking.com/the-plant-based-alternative-ingredient-craze-whats-behind-it-and-why-it-isnt-going-away/. Helmick, Harrison, Arun Bhunia, Andrea Liceaga, and Jozef Kokini. Pro Tip: Use Spectroscopy to See Process Changes. Baking & Snack, November 3, 2021. https://www.bakingbusiness.com/search?page=2&q=pro+tips. Helmick, Harrison, Arun Bhunia, Andrea Liceaga, and Jozef Kokini. Pro Tip: Functionalize Protein with Extrusion. Baking & Snack, October 13, 2021. https://www.bakingbusiness.com/search?page=2&q=pro+tips. Helmick, Harrison, Arun Bhunia, Andrea Liceaga, and Jozef Kokini. Pro Tip: Use Shear Denaturation for Stronger Protein Gels. Baking & Snack, September 8, 2021. https://www.bakingbusiness.com/search?page=2&q=pro+tips. Helmick, Harrison, Arun Bhunia, Andrea Liceaga, and Jozef Kokini. Pro Tip: Pressure Has Uses beyond Food Safety. Baking & Snack, August 11, 2021. https://www.bakingbusiness.com/search?page=2&q=pro+tips. Helmick, Harrison, Arun Bhunia, Andrea Liceaga, and Jozef Kokini. Pro Tip: Cold Denaturation Reveals Novel Plant-Based Protein Functionality. Baking & Snack, July 14, 2021. https://www.bakingbusiness.com/search?page=2&q=pro+tips. Helmick, Harrison, Arun Bhunia, Andrea Liceaga, and Jozef Kokini. Pro Tip: Classify Your Data to Speed up Innovation. Baking & Snack, June 16, 2021. https://www.bakingbusiness.com/search?page=2&q=pro+tips. Helmick, Harrison, Arun Bhunia, Andrea Liceaga, and Jozef Kokini. Pro Tip: Protein Denaturation Can Be a Tool to Improved Functionality. Baking & Snack, May 19, 2021. https://www.bakingbusiness.com/search?page=2&q=pro+tips. Helmick, Harrison, Arun Bhunia, Andrea Liceaga, and Jozef Kokini. Pro Tip: Bioinformatics Enable Faster Innovation. Baking & Snack, April 20, 2021. https://www.bakingbusiness.com/search?page=2&q=pro+tips.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Helmick, Harrison. Bioinformatic Modeling of Cold Denaturation in Pea Protein. Presented at the IFT FIRST Virtual Conference, July 19, 2021.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Helmick, Harrison, and Jozef Kokini. Validation of Bioinformatic Modeling for Zeta Potential of Pea Protein. Educational, Purdue University; Whistler Center for Carbohydrate Research, October 7, 2021.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Helmick, Harrison. Understanding Cold Denaturation through Bioinformatics in Pea Protein. Presented at the Cereals & Grains 21, Virtual Conference Poster, November 18, 2021.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Helmick, Harrison, Christabel Hartanto, Arun Bhunia, Andrea Liceaga, and Jozef Kokini. Understanding Cold Denaturation through Bioinformatics in Pea Protein. Cereals & Grains 21, November 18 2021.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Helmick, H., Tonner, T., Hauersperger, D., Ettestad, S., Hartanto, C., Bhunia, A., Liceaga, A., & Kokini, J. (2022, March 3). Optimization of a single screw extruder for low temperature applications [Virtual Conference]. Graduate Industrial Research Symposium, Purdue University. https://girs2022.com
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Helmick, Harrison, Hazal Turasan, Merve Yildirim, Arun Bhunia, Andrea Liceaga, and Jozef Kokini. Cold Denaturation of Proteins: Where Bioinformatics Meets Thermodynamics to Offer a Mechanistic Understanding: Pea Protein as a Case Study. Journal of Agricultural and Food Chemistry, May 24, 2021. https://doi.org/10.1021/acs.jafc.0c06558.
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Helmick, Harrison, Christabel Hartanto, Arun Bhunia, Andrea Liceaga, and Jozef L. Kokini. Validation of Bioinformatic Modeling for the Zeta Potential of Vicilin, Legumin, and Commercial Pea Protein Isolate. Food Biophysics, August 13, 2021. https://doi.org/10.1007/s11483-021-09686-8.
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Helmick, Harrison, Sarah Ettestad, Andrea Liceaga, Gaurav Nanda, and Jozef Kokini. Applying Text Mining to Identify Relevant Literature in Food Science: Cold Denaturation as a Case Study. Journal of Food Science, 2021, 114. https://doi.org/10.1111/1750-3841.15940.