GLUTENIN FRACTIONS VISUALIZED WITH CONJUGATED QUANTUM DOTS AND CONFOCAL MICROSCOPY HELP UNDERSTAND RHEOLOGY AND MIXING BEHAVIOR OF WHEAT DOUGHS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1011918
• Grant No.: 2017-67017-26472
• Cumulative Award Amt.: $452,675.00
• Proposal No.: 2016-09060
• Project Start Date: May 1, 2017
• Project End Date: Apr 30, 2021
• Grant Year: 2017
• Program Code: [A1361]- Improving Food Quality

Recipient Organization
PURDUE UNIVERSITY
(N/A)
WEST LAFAYETTE,IN 47907

Performing Department
Food Science

Non Technical Summary
The quality of wheat products depend greatly on the kneading propertiesof wheat flour doughs which in turn depend strongly on the type and concentration of hydrated proteins and starch in the dough matrix as well as other minor ingredients. This resaerch will focus on how the kneading and strteching properties of wheat flour doughs can be exlained thorugh the characteristics of the protein component. We will monitor the protein components by tagging them with special molecules that enables us to see them with a special micrsocope. Studying the distribution of the protein components will help us understand the reason why the dough behaves the way it does. This knowledge gives the baker the pizza and cookie maker the flexibility to design both healthy and appetizing products.

Animal Health Component

25%

Research Effort Categories

Basic

60%

Applied

25%

Developmental

15%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
502	1549	2020	100%

Knowledge Area
502 - New and Improved Food Products;

Subject Of Investigation
1549 - Wheat, general/other;

Field Of Science
2020 - Engineering;

Keywords

clsm

hmw

lmw

antibody

conjugation

gliadin

glutenin

mixing

quantum dot

rheology

wheat dough

Goals / Objectives
Goals and Objectives of Research1. The objective of this research is to obtain LMW and HMW sub fractions of glutenin in softand hard flours with the goal of developing a mechanistic understanding of the distributionand behavior of these sub fractions during mixing and controlled large deformations. Wewill use our prior understanding with gliadin to supplement the new knowledge with LMWand HMW behavior to gain insights about the contribution of the three major proteinfractions.2. To this end, LMW and HMW sub fractions will be purified and amino acid sequences willbe obtained using MALDI-TOF/TOF; antibodies for each sub fraction will be designedand their specificity will be demonstrated using Western Blot experiments3. LMW and HMW specific antibodies will be conjugated with Quantum dots and CLSMwill be used to capture the behavior of each sub fraction during mixing and controlled largedeformation experiments.4. Studying the distribution of protein sub fractions LMW, HMW and gliadin (utilizing ourexisting knowledge base) will help us develop a mechanistic understanding of proteinproteinand protein-starch interactions in soft and hard wheat flours during mixing andlarge controlled deformations.

Project Methods
a. Characterization of wheat flours and their doughsThe comparison of the behavior of LMW and HMW in soft and hard wheat flour during mixing and in controlled deformation experiments will be the key focus of this study.The wheat flours will be characterized for moisture content using AACC method 44-15A ash content using AACC method 08-01, protein content using AACC method 46-30 and falling number using AACC method 56-81B. Proteins will extracted from soft wheat flour because it has less protein content. In addition the flours will be characterized for wet gluten test using a Buhler Glutomatic MLKG System following AACC method 38-12. The flours will also be characterized for their dough and gluten strength tests using a Brabender Farinograph following AACC method 54-21.b. Separation of HMW and LMW glutenin protein components from each flour100 g of wheat flour are washed with chloroform in order to remove fat, 70% ethanol is used to dissolve and extract gliadin. The alcohol insoluble fraction contains glutenins. Glutenins are then dissolved in a solution containing 50% propanol, 2% 2- Mercaptoethanol and 1% acetic acid. Samples are centrifuged in order to remove starch and only the solubilized proteins remains in solution. A 1.5 M NaCl solution is added to solubilized proteins in order to dissolve and remove the water and salt soluble proteins.The samples are centrifuged again and the precipitated pellet is washed with water and lyophilized to get purified glutenins.c. Producing glutenin sub fractions from purified gluteninGlutenins are then separated into HMW and LMW sub fractions by solubilizing them in 50% 2- propanol, and 2% (w/v) DTT in an 80 mM Tris-HCL (pH 8.0) solution. This is followed by addition of acetone to the solubilized protein at a final concentration of 40%, which precipitates the HMW sub fractions. Increasing the acetone concentration to 80% is used to precipitate the LMW sub fractions.d. Validation of protein sub fractions by SDS-PAGEA standard SDS-PAGE method from Bio-Rad using G-coomassie blue as staining dye, used previously for glutenins separation by our team will be used to validate the presence of gliadin proteins.e. Proteomic identification of extracted proteins of gliadin and glutenin subfractionsIdentification of the protein sub fractions in the electrophoresis gels bands is going to be done by using mass spectroscopy. This procedure is performed by a commercial laboratory (Applied Biomics Inc. Hayward CA). The protein gel bands are digested with trypsin, extracted from the gel, desalted, and placed on a MALDI plate to perform the spectroscopic analysis. The mass spectra of the peptides are obtained using an Applied Biosystems Proteomics Analyzer. In this approach the most abundant peptides are fragmented and are subjected to another mass spectra analysis; the data are then compared with protein databases by comparing the time of flight (TOF)of the peptides. The 10 highest scores that matched our protein with proteins at the NCBI database are reported. Complete amino acid sequence of the identified protein is available in the NCBI database.f. Design and development of antibodies for glutenin sub fractionsThe idea of identifying LMW and HMW sub fractions from glutenin requires recognizing a peptide sequence that is found in each sub fraction but is not found in the other sub fraction or gliadin, not even partially because the antibody can recognize the pattern even if few amino acids are changed. A peptide for antigen design chosen for each antibody needs to be a relative short amino acid sequence, short enough to be found many times in the protein sequence, a 20 amino acid sequence for antibody development is reported by Lathrop et al, (2014). It also has to be long enough to have a strong interaction with the antibody when binding, four to six amino acid sequences asreported before (Borrebaeck and Wingren, 2009) for antibody development.g. Antibody validation by immunoassayWestern blot method will be followed as outlined by Bio-Rad. Glutenin and gliadin are going to be dissolved at the same and known final concentration in the sample solvent. The proteins will be separated in SDS-PAGE and transferred to a hydrophobic (PVDF) membrane for 1.25 h at 100 V using a tris-glycine transfer buffer with an ice pack in the transfer chamber to prevent over heating of proteins. The membranes will be blocked in 5% skimmed milk dissolved in PBST (PBS with 0.5% tween-20) for 1 h at room temperature. Then, the membranes are going to be incubated overnight at 4°C with their respective antibody dissolved in blocking buffer.h. Conjugation of antibodies with Quantum DotsAntibodies will be conjugated with Quantum Dots following the method provide by the Quantum Dot supplier (Life Technologies, Thermo Fisher). In which an antibody carbohydrate domain is modified by adding β-galactosidase, followed by azide attachment by adding Galactose-1- phosphate uridylyltransferase to UDP-Nazidoacetylgalactosamine in order to prepare the azide modification solution, and carbohydrate modified antibodies are then added to the azide modification solution. Finishing the QDs modified by dibenzocyclooctyne which link with azide residue attached to the antibody. The last step of the Quantum Dot conjugation is crucial for the experiments, in which the antibodies that are not successfully conjugated with Quantum Dots are removed by a washing step.i. Dough developmentDough sample are going to be prepared by following the AACC Method 54-21.02.j. Dough mixing and rheological properties studiesDough samples are going to be obtained from the farinograph bowl at AT, PT, DT, and 10 min after departure time for studying the mixing behavior and the rheological properties of the dough. The rheological study will be performed using a DHR3 rheometer, coupled with parallel plate geometry.k. Collection of samples for detectionSmall pieces of dough samples (0.5 cm3 ) are going to be taken from the farinograph bowl at four at the four different mixing times and placed into a beaker containing 20 ml of heparin solution ( 500 IU/ml) for 10 min to remove dough fluorescence.l. Sample staining for detectionThe staining procedure starts by placing the slides in a slide rack, immersed in a staining dish containing PBS and incubated for 5 min. PBS is then removed from slides by tipping the slides and allowing the PBS solution to drip out. Residual PBS around the samples will also be removed by gently absorbing the solution with Kim wipes without contaminating and damaging the samples.m. Sample examinationA confocal laser scanning microscope will be used to detect the three different color quantum dotsv at the same time. Starch granules will be identified by simple polarized light. The excitation wavelengths of the QDs were 405 nm in order to avoid the auto-fluorescence of quantum dots. Digital image files will be recorded with the Zen LSM software.The confocal laser scanning microscope is used with a constant z-position and motorized xy plane. This confocal microscope is based on an Axio Imager upright microscope with an AxioCam digital microscope camera with a Diode laser .n. Digital Image Processing and AnalysisAn image processing software such as Axiovision, will be used for analyzing the resulting images from the samples. Different colors in the sample will represent the presence of each specific protein or protein subunit. The distribution of the gliadin and glutenin sub fractions among the samples at different stages of mixing and strain will be used to elaborate a model to describe the function of gliadin and glutenin subunits in the wheat dough. We will measure the color intensity generated by the concentration of conjugated QDs which will be a marker of the concentration of protein sub fractions.

Progress 05/01/17 to 04/30/21

Outputs
Target Audience:• The Food Science community • The Cereal Science community • Agricultural Science community • Food product developers • Cereal product developers • The education community • Food / Cereal industry • Bio macromolecule community • Imaging community • Cereal science equipment manufacturers • General scientific community Changes/Problems:We have developed an in depth understanding of how to obtain networking parameters, and we have applied them to show change in the structure of dough as a function of deformation and as a function of gliadin to glutenin ratios. The planning that was developed in the propsal maintained its validity throughout the study, and there were not major changes in the direction of the project in order to obtain the goals of the grant. We validated the rheology networking parameter relationships with additional work that was conduted on yogurt. What opportunities for training and professional development has the project provided? One PhD was graduated as the direct result of the work conducted during this project. 1 undergraduate student has spent1 year in our laboratory and is in the process of completing work that will lead to a publication How have the results been disseminated to communities of interest?Yes, the results of this work has been distributed in the form of publications and speaking at professional and scientific conferences. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? 1.We successfully conducted simultaneous'in situ'fluorescent imaging of gliadin, LMW glutenin and HMW glutenin in wheat dough for the first time using antibody-QD complexes specific to each sub protein fraction with their respective auto-fluorescence and no-protein controls. This has not been done before and is a first in dough/cereal science. In order to achieve this outcome we tested several procedures and fixation media. When comparing the effectiveness of 4% PFA, acetone, and methanol as fixative agents we found that methanol is the most suitable fixation medium for dough because it maintains tissue morphology and integrity during the entire immunostaining and washing procedure, keeping the antigenic epitopes available for the antibodies to bind to the respective proteins, which is crucial when doing specific immunofluorescent detection. We also found that dough auto-fluorescence can be masked using the proper microscope/detector settings when the emission signal emitted by the fluorescent dyes is strong enough. We found that heparin, Sudan black B and TrueVIEW™ auto-fluorescence blocking agents were not effective with the wheat dough studied here. We have developed a reliable protocol for performing successful immunofluorescent imaging of gliadin, LMW and HMW glutenin sub fractions in wheat flour dough systems. We have proven that the antibody-QD complexes bind to their specific proteins in an intact dough matrix, and the imaging results are not the product of dough auto-fluorescence or non-specific binding of Antibody-QD trapped non-specifically in the dough matrix. All these findings combined were used to obtain for the first time fluorescent images capable of detecting the distribution of LMW glutenins, HMW glutenins, and gliadins in dough. In future work we will compare different dough processes and the resulting distribution of these proteins as a function of processing parameters. We are also aiming to compare differences in the quantity and distribution of wheat flour proteins between different types of wheat flour doughs with different bread-making quality. 2.This technique of antibody-conjugated QDs to detect proteins wassuitable for performing several studies on wheat flours. The distribution of glutenins can be compared between different types of wheat flour doughs with different bread-making quality; also, the behavior of glutenins during different dough processes, such as mixing dough sheeting and others, can be studied. On the basis of previously published work,(18)the antibodies are also be applicable to study glutenin and gliadin distribution in baked bread and to study their mobility during baking. 3.We have gained relevant insights on the role of gliadins, LMW glutenins, and HMW glutenins in dough mixing by using three different dough systems with three different proteins composition. The three gluten subunits (LMW glutenins, HMW glutenins, and gliadins) breakdown during weak soft wheat mixing (from 450 to 350 BU), HMW glutenins agglomerate during this network disruption. LMW glutenins and gliadins are responsible for the gluten network development during stiff hard wheat dough mixing (from 650 to 570 BU). HMW glutenins do not reorganize themselves in the gluten network at that mixing stage. We have been able to visualize and measure the unique internal mobility of each gluten subfraction in a durum semolina dough that undergoes minimal strength loss (530-500 BU) due to the associative polymer model of the glutenins. The protein subunits remain co-localized and with the no significant changes network parameters during mixing proving the role that the high amount of LMW glutenins plays in keeping the semolina dough strength stable. The magnitude and specificity of the microstructural changes in the gluten subunits achieved with the 'in situ' detection and quantitative imaging techniques cannot be achieved with oscillatory rheological measurement or other conventional microstructural techniques currently used in dough products. 4. The aims of this goal are discussed in the discussion of goals 1-3.

Publications

• Type: Journal Articles Status: Published Year Published: 2021 Citation: Erturk, Merve Yildirim, Jose C. Bonilla, and Jozef Kokini. Relationship of Non-Linear Rheological Properties and Quantitative Network Analysis Parameters as a Function of Increasingly Large Amplitude Deformations in Non-Fat, Low-Fat and High-Fat Yogurt Products. Food Hydrocolloids 111 (2021): 106194. https://doi.org/10.1016/j.foodhyd.2020.106194.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Helmick, H., Kokini, J., 2020. Impact of ethanol, succinic acid, and the combination thereof at levels produced during sponge fermentation on hard wheat, soft wheat, and durum wheat farinograph rheology. Journal of Cereal Science 96. https://doi.org/10.1016/j.jcs.2020.103082

Progress 05/01/17 to 02/10/21

Outputs
Target Audience:• The Food Science community • The Cereal Science community • Agricultural Science community • Food product developers • Cereal product developers • The education community • Food / Cereal industry • Bio macromolecule community • Imaging community • Cereal science equipment manufacturers • General scientific community Changes/Problems:We have developed an in depth understanding of how to obtain networking parameters, and we have applied them to show change in the structure of dough as a function of deformation and as a function of gliadin to glutenin ratios. The planning that was developed in the propsal maintained its validity throughout the study, and there were not major changes in the direction of the project in order to obtain the goals of the grant. We validated the rheology networking parameter relationships with additional work that was conduted on yogurt. What opportunities for training and professional development has the project provided? One PhD was graduated as the direct result of the work conducted during this project. 1 undergraduate student has spent1 year in our laboratory and is in the process of completing work that will lead to a publication How have the results been disseminated to communities of interest?Yes, the results of this work has been distributed in the form of publications and speaking at professional and scientific conferences. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? 1.We successfully conducted simultaneous'in situ'fluorescent imaging of gliadin, LMW glutenin and HMW glutenin in wheat dough for the first time using antibody-QD complexes specific to each sub protein fraction with their respective auto-fluorescence and no-protein controls. This has not been done before and is a first in dough/cereal science. In order to achieve this outcome we tested several procedures and fixation media. When comparing the effectiveness of 4% PFA, acetone, and methanol as fixative agents we found that methanol is the most suitable fixation medium for dough because it maintains tissue morphology and integrity during the entire immunostaining and washing procedure, keeping the antigenic epitopes available for the antibodies to bind to the respective proteins, which is crucial when doing specific immunofluorescent detection. We also found that dough auto-fluorescence can be masked using the proper microscope/detector settings when the emission signal emitted by the fluorescent dyes is strong enough. We found that heparin, Sudan black B and TrueVIEW™ auto-fluorescence blocking agents were not effective with the wheat dough studied here. We have developed a reliable protocol for performing successful immunofluorescent imaging of gliadin, LMW and HMW glutenin sub fractions in wheat flour dough systems. We have proven that the antibody-QD complexes bind to their specific proteins in an intact dough matrix, and the imaging results are not the product of dough auto-fluorescence or non-specific binding of Antibody-QD trapped non-specifically in the dough matrix. All these findings combined were used to obtain for the first time fluorescent images capable of detecting the distribution of LMW glutenins, HMW glutenins, and gliadins in dough. In future work we will compare different dough processes and the resulting distribution of these proteins as a function of processing parameters. We are also aiming to compare differences in the quantity and distribution of wheat flour proteins between different types of wheat flour doughs with different bread-making quality. 2.This technique of antibody-conjugated QDs to detect proteins wassuitable for performing several studies on wheat flours. The distribution of glutenins can be compared between different types of wheat flour doughs with different bread-making quality; also, the behavior of glutenins during different dough processes, such as mixing dough sheeting and others, can be studied. On the basis of previously published work,(18)the antibodies are also be applicable to study glutenin and gliadin distribution in baked bread and to study their mobility during baking. 3.We have gained relevant insights on the role of gliadins, LMW glutenins, and HMW glutenins in dough mixing by using three different dough systems with three different proteins composition. The three gluten subunits (LMW glutenins, HMW glutenins, and gliadins) breakdown during weak soft wheat mixing (from 450 to 350 BU), HMW glutenins agglomerate during this network disruption. LMW glutenins and gliadins are responsible for the gluten network development during stiff hard wheat dough mixing (from 650 to 570 BU). HMW glutenins do not reorganize themselves in the gluten network at that mixing stage. We have been able to visualize and measure the unique internal mobility of each gluten subfraction in a durum semolina dough that undergoes minimal strength loss (530-500 BU) due to the associative polymer model of the glutenins. The protein subunits remain co-localized and with the no significant changes network parameters during mixing proving the role that the high amount of LMW glutenins plays in keeping the semolina dough strength stable. The magnitude and specificity of the microstructural changes in the gluten subunits achieved with the 'in situ' detection and quantitative imaging techniques cannot be achieved with oscillatory rheological measurement or other conventional microstructural techniques currently used in dough products. 4. The aims of this goal are discussed in the discussion of goals 1-3.

Publications

• Type: Journal Articles Status: Published Year Published: 2021 Citation: Erturk, Merve Yildirim, Jose C. Bonilla, and Jozef Kokini. Relationship of Non-Linear Rheological Properties and Quantitative Network Analysis Parameters as a Function of Increasingly Large Amplitude Deformations in Non-Fat, Low-Fat and High-Fat Yogurt Products. Food Hydrocolloids 111 (2021): 106194. https://doi.org/10.1016/j.foodhyd.2020.106194.

Progress 05/01/19 to 04/30/20

Outputs
Target Audience: The Food Science community The Cereal Science community Agricultural Science community Food product developers Cereal product developers The education community Food / Cereal industry Bio macromolecule community Imaging community Cereal science equipment manufacturers General scientific community Changes/Problems:We have developed an in depth understanding of how to obtain networking parameters, and we have applied them to show change in the structure of dough as a function of deformation and as a function of gliadin to glutenin ratios. What opportunities for training and professional development has the project provided?We are continuing the collaboration with Professor Brenann Smith and Dr. Gamze Yazar from the University of Idaho to extend the tools developed in this project to understand the interactions between non-starch polar lipids and gluten proteins. This interaction has led to a short collaboration where Dr. Game Yazar is spending two months in our laboratory to test the feasibility of using both our non-linear rhelogical and imaging techniques to look at lipid-protein interactions. How have the results been disseminated to communities of interest?We disseminate our reserach through publications, conferences, inivted lectures, and support / collaboration with the Brabender company that reports to us that our work is helping them interpret farinogrpah results better. What do you plan to do during the next reporting period to accomplish the goals?We anticipate that the collabration will lead to the publication 2 papers to understand the impact of lipids in LAOS rheology of wheat dough. We will write a follow up proposal to the USDA on expanding the understanding of viscoelastic properties and networking parameters.

Impacts
What was accomplished under these goals? The project is going as plan but in the process of conducting the research we have identified advanced and insightful quantitative techniques to quantitatively determine the co-localization of different protein fractions. This has further empowered our ability to convert imaging data in quantitative predictive data. During this project period we showed the ability of the colocalization parameters of wheat dough mixing to explain the structural origins of the viscoelastic properties of dough. We are continuing to develop this aspect of the work and in order to show the braod applicability of these concepts, we also applied them to yogurts. The networking parameters have again very successfully explained the changes in the structure of yogurt during large amplitdue deformation.

Publications

• Type: Journal Articles Status: Published Year Published: 2019 Citation: Simultaneous immunofluorescent imaging of gliadins, low molecular weight glutenins, and high molecular weight glutenins in wheat flour dough with antibody-quantum dot complexes
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Mixing dynamics and molecular interactions of HMW glutenins, LMW glutenins, and gliadins analyzed by fluorescent co-localization and protein network quantification
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Distribution and function of LMW glutenins, HMW glutenins, and gliadins in wheat doughs analyzed with in situ detection and quantitative imaging techniques
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Understanding the role of gluten subunits (LMW, HMW glutenins and gliadin) in the networking behavior of a weak soft wheat dough and a strong semolina wheat flour dough and the relationship with linear and non-linear rheology
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Effect of aging at different temperatures on LAOS properties and secondary protein structure of hard wheat flour dough

Progress 05/01/18 to 04/30/19

Outputs
Target Audience:Scientific community, industrial community, cereal scientific equipment manufacturers, imaging community, general cereal science community Changes/Problems:The project is going as plan but in the process of conducting the research we have identified advanced and insightful quantitative techniques to quantitatively determine the co-localization of different protein fractions. This has further empowered our ability to convert imaging data in quantitative predictive data. Currently we are collaborating with Professor Brenann Smith and Dr. Gamze Yazar from the University of Idaho to extend the tools developed in this project to understand the interactions between non-starch polar lipids and gluten proteins. This interaction has led to a short collaboration where Dr. Game Yazar is spending two months in our laboratory to test the feasibility of using both our non-linear rhelogical and imaging techniques to look at lipid-protein interactions. We anticipate that follow up publications and proposals will emerge from this collaboration. What opportunities for training and professional development has the project provided?Travels to conferences internationally, England, China and locally, New Orleans. Networking opportunities form other institutions around the world, in particular, the conference in China that Jose Bonilla attended was a competition among 70 universities around the world, Jose interacted with a diverse group of scientists with international perspective. He was also, the recipient for the first place award in this conference, getting him worldwide recognition and exposure, helping him meet professionals from universities, industry, and government. The research project has received interest on an international level and a research scientist from HITIT University, Professor Secil Turksoy, who received a grant from TUBITAK in Turkey for a year to come as a visiting scholar to study characterization of aging processes using the imaging tools in this project. Work is currently in progress and we expect an outcome by December of 2019. How have the results been disseminated to communities of interest?In peer-reviewed journal articles, Food Research International and the Journal of Cereal Science. Book chapters, conferences. What do you plan to do during the next reporting period to accomplish the goals?We plan to study the distribution of the different gluten subunits in three different flours (soft wheat, hard wheat, and semolina). Validating this idea by looking at degradation processes and how the methods that have been develop are able to offer insights that are not otherwise accessible by other tools.

Impacts
What was accomplished under these goals? Objective 1. We have isolated the proteins and performed proteomics analysis to deeply study them Objective 2.The antibodies for LMW andHMW glutenins have been developed. Objective 3. The antibodies have been conjugated with Quantum Dots. Objective 4. We studied the distribution of LMW glutenins, HMW glutenins, and gliadins inside wheat dough during mixing.

Publications

• Type: Journal Articles Status: Published Year Published: 2019 Citation: JC Bonilla, V Bernal-Crespo, JA Schaber, AK Bhunia, JL Kokini. Simultaneous immunofluorescent imaging of gliadins, low molecular weight glutenins, and high molecular weight glutenins in wheat flour dough with antibody-quantum dot complexes. Food Research International 120, 776-783
• Type: Journal Articles Status: Published Year Published: 2019 Citation: JC Bonilla, JA Schaber, AK Bhunia, JL Kokini. Mixing dynamics and molecular interactions of HMW glutenins, LMW glutenins, and gliadins analyzed by fluorescent co-localization and protein network quantification. Journal of Cereal Science, 102792
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: JOSE C. BONILLA,JAMES A SCHABER, ARUN K. BHUIA, and JOZEF L. KOKINI. Fluorescent Visualization and Image Analysis of Gliadins, LMW Glutenins, and HMW Glutenins at Different Stages of Dough Mixing in a Farinograph. IFT 2019.
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Jose Bonilla, and Jozef Kokini. Simultaneous fluorescent detection of gliadins, LMW, and HMW glutenins in wheat dough using specifically developed antibodies-quantum dots complexes. AACCI 2018
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Jose Bonilla and Jozef Kokini. In situ fluorescent detection of gliadins, LMW glutenins, and HMW glutenins in wheat dough using antibodies-quantum dots complexes. Global Food Science Student Competition, Wuxi, China, 2018
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Jose Bonilla and Jozef Kokini. Studying the Function and Interactions of Gluten subunits in wheat dough Using Antibodies-Quantum Dots Complexes. Interdisciplinary Graduate Programs Annual Meeting, Purdue University

Progress 05/01/17 to 04/30/18

Outputs
Target Audience:2017 American Association of Cereal Chemists International, oral presentation 2017 Institute of Food technologistsmeeting Changes/Problems:-compare QD conjugated techniques with flourescent dyes What opportunities for training and professional development has the project provided?- graduate student training on advanced methodologies related to the project How have the results been disseminated to communities of interest?- presentations, posters at professional society meetings and refereeed publications - converstaions with interested partiesduring annual meetings of theAACCI and IFT -Collaboration with the Brabender company who makes mixers for the crereal industry -presentations and brainstorming exercises with other graduate students presentations at the Whistler Carbohydrate Center in front of representatives/scientists from 15 industrial members companies What do you plan to do during the next reporting period to accomplish the goals?-develop methodology to detect LMW and HMW proteins in dough - make progress towards understanding the effect of mixing processing on distribution of LMW and HMW proteins in dough

Impacts
What was accomplished under these goals? Related to goals 1 and 2: Low Molecular Weight and High Molecular Weight glutenins fractions were isolated from wheat flour; we performed MALDI-TOF/TOF on them and obtained their amino acid sequences from the National Center for Biotechnology Information (NCBI) online database. We identified unique peptides within the amino acid sequences of both protein sub fractions and developed antibodies against those specific peptides. Antibodies specificity was proven though western immune blots were both antibodies reacted to glutenins in their specific molecular weight and did not show any reaction with gliadins. Related to Goals 1 and 3.We successfully conjugated the developed antibodies with dibenzocyclooctyne (DIBO)-functionalized QDs with a site-click method in which the integrity and effectiveness of the antibodies are maintained after the conjugation procedure. Related to goals 1 and 4: we have begun experiments to detect the proteins in dough.

Publications

• Type: Journal Articles Status: Published Year Published: 2018 Citation: Bonilla JC, Ryan V, Yazar G, Kokini JL, Bhunia A. 2018. Conjugation of Specifically Developed Antibodies for High- and Low-Molecular-Weight Glutenins with Fluorescent Quantum Dots as a Tool for Their Detection in Wheat Flour Dough. J. Agric. Food Chem. 66, 16, 4259-4266
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Jose Bonilla, Valerie Rayn, Arun Bhunia, and Jozef Kokini. Development of Specific Antibodies against High and Low Molecular Weight Glutenins as Research Tools by Comparative Proteomics. American Association of Cereal Chemists Annual Meeting.
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Jose Bonilla and Jozef Kokini. Studying the molecular distribution of protein, fat and starch in semolina, hard and soft wheat flour during different stages of the mixing process. Institute of Food Technologists annual meeting.