$100,000/acre. The rapid US production and market growth along with our current dependence on imports raise concerns about saffron quality, which is strongly affected by processing and storage, as well as by common adulteration; yet federal regulation of product quality is lacking. In fact, due to its high retail value, saffron is one of the most commonly adulterated spices. Therefore, readily accessible sensing technology for saffron quality control is needed by an increasing number of saffron growers, traders, and consumers in the US.The overall goal of this project is to invent paper-based nanosensors to test the quality of saffron. Current analysis requires specialized laboratory equipment and expertise and is cost-prohibitive to growers and/or distributors. Current methods developed by the International Organization for Standardization (ISO) to assess saffron quality are primarily based on visual inspection and spectrophotometric analysis, which lack accuracy in determining the concentrations of key saffron compounds, such as picrocrocin, crocin, and safranal. For instance, the ISO-standard determines picrocrocin levels by measuring the specific absorption of saffron extracts at 258nm, but many other natural compounds (including proteins, DNA, carotenoids, etc.) also absorb light at this wavelength. Growers say developing reliable, cost-effective quality testing tools is critical for this emerging industry. To meet this stakeholder need, our research will draw on advanced nanotechnology concepts to design novel nanocages that detect key saffron components, and generate the first paper-based sensors to detect them. We will computationally design, synthesize, and investigate selective nanoscale environments, which will allow for unique sensitivity. When detecting a specific component in saffron, our nanocages will change color, thanks to a tunable FRET mechanism. These concentration-dependent color changes -- which can directly be observed on the paper strip under a yellow LED lamp -- will tell growers how much picrocrocin is in their saffron (analogous to a pH test strip).Ultimately, the proposed sensing technology will benefit the survival of small-to-medium family farms in the US, which make up >97% of all the farms in this country. Many of these farms are struggling economically and the key to their future success is diversification. They must exploit high-value crops like saffron suitable for their region, and maximize on niche markets to sell their products for the highest returns. We have shown that saffron is an ideal crop for diversification in much of the US. Thus, hundreds of farmers nationwide are now growing it and all of them need readily accessible, real-time testing capabilities for saffron to confirm quality and achieve good pricing for their crop. If the US saffron industry is to thrive, growers need options for testing their product to ensure they can obtain top dollar for it. Finally, this project will also lay the groundwork for future expanded research on the medicinal properties of this plant, that have been reported widely overseas, but lack confirmation in the US. Last but not least, the test systems we create for saffron could also lead to nanocage-sensor development for other food products in the future.' />
Source: UNIVERSITY OF VERMONT submitted to NRP
NANOCAGES FOR ASSESSING SAFFRON QUALITY: ADVANCED TOOLS FOR AN EMERGING HIGH-VALUE CROP
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1019021
Grant No.
2019-67022-29456
Cumulative Award Amt.
$180,830.00
Proposal No.
2018-07583
Multistate No.
(N/A)
Project Start Date
May 15, 2019
Project End Date
May 14, 2022
Grant Year
2019
Program Code
[A1511]- Agriculture Systems and Technology: Nanotechnology for Agricultural and Food Systems
Recipient Organization
UNIVERSITY OF VERMONT
(N/A)
BURLINGTON,VT 05405
Performing Department
Chemistry
Non Technical Summary
Saffron is the most expensive spice in the world ($3,000-9,000/lb). It is the dried stigmas of a fall-blooming crocus (Crocus sativus), which contain hundreds of aromatic compounds, most importantly picrocrocin (for flavor), crocins (for color), and safranal (for aroma). Saffron is commonly used as a spice in gourmet cuisine, but is also in rising demand for medical and cosmetic applications, which further increases its economic value above other spices. The US imports over 46 tons per year, with a projected annual growth rate of 5-12%. In the past three years, as a result of the University of Vermont's research, growers across the US are beginning to produce saffron to increase their revenues with this high value specialty crop. Based on recent US-based research, saffron could generate >$100,000/acre. The rapid US production and market growth along with our current dependence on imports raise concerns about saffron quality, which is strongly affected by processing and storage, as well as by common adulteration; yet federal regulation of product quality is lacking. In fact, due to its high retail value, saffron is one of the most commonly adulterated spices. Therefore, readily accessible sensing technology for saffron quality control is needed by an increasing number of saffron growers, traders, and consumers in the US.The overall goal of this project is to invent paper-based nanosensors to test the quality of saffron. Current analysis requires specialized laboratory equipment and expertise and is cost-prohibitive to growers and/or distributors. Current methods developed by the International Organization for Standardization (ISO) to assess saffron quality are primarily based on visual inspection and spectrophotometric analysis, which lack accuracy in determining the concentrations of key saffron compounds, such as picrocrocin, crocin, and safranal. For instance, the ISO-standard determines picrocrocin levels by measuring the specific absorption of saffron extracts at 258nm, but many other natural compounds (including proteins, DNA, carotenoids, etc.) also absorb light at this wavelength. Growers say developing reliable, cost-effective quality testing tools is critical for this emerging industry. To meet this stakeholder need, our research will draw on advanced nanotechnology concepts to design novel nanocages that detect key saffron components, and generate the first paper-based sensors to detect them. We will computationally design, synthesize, and investigate selective nanoscale environments, which will allow for unique sensitivity. When detecting a specific component in saffron, our nanocages will change color, thanks to a tunable FRET mechanism. These concentration-dependent color changes -- which can directly be observed on the paper strip under a yellow LED lamp -- will tell growers how much picrocrocin is in their saffron (analogous to a pH test strip).Ultimately, the proposed sensing technology will benefit the survival of small-to-medium family farms in the US, which make up >97% of all the farms in this country. Many of these farms are struggling economically and the key to their future success is diversification. They must exploit high-value crops like saffron suitable for their region, and maximize on niche markets to sell their products for the highest returns. We have shown that saffron is an ideal crop for diversification in much of the US. Thus, hundreds of farmers nationwide are now growing it and all of them need readily accessible, real-time testing capabilities for saffron to confirm quality and achieve good pricing for their crop. If the US saffron industry is to thrive, growers need options for testing their product to ensure they can obtain top dollar for it. Finally, this project will also lay the groundwork for future expanded research on the medicinal properties of this plant, that have been reported widely overseas, but lack confirmation in the US. Last but not least, the test systems we create for saffron could also lead to nanocage-sensor development for other food products in the future.
Animal Health Component
10%
Research Effort Categories
Basic
80%
Applied
10%
Developmental
10%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
5012220200050%
5022235100050%
Knowledge Area
501 - New and Improved Food Processing Technologies; 502 - New and Improved Food Products;

Subject Of Investigation
2220 - Medicinal crops, non-narcotic; 2235 - Herbs and spices;

Field Of Science
1000 - Biochemistry and biophysics; 2000 - Chemistry;
Goals / Objectives
The overarching goals of this project are to:Create nanocage-based methods on paper strips to assess picrocrocin content in saffron and compare the results with standard HPLC and spectrophotometric methods.Validate the test strips in the field by applying them to various saffron samples grown/harvested at the University of Vermont under different conditions.The specific objectives that will be undertaken to achieve these goals of this project are to:Synthesize nanocages and polymers with fluorescent dyes attachedInvestigate color changes when picrocrocin from saffron bindsComputationally optimize nanocages and computationally investigate picrocrocin bindingSynthesize optimal nanocagesCreate prototypical, paper-based picrocrocin sensors based on the knowledge learned from the previous objectivesCalibrate the paper-based sensorsGrow and harvest saffron under different conditionsValidate our nanosensors with saffron grown at the University of Vermont
Project Methods
This is a USDA NIFA SEED grant application, which is primarily focused on research. The anticipated research methods for the project are:Sensor synthesis and optimization. We will start by creating selective sensors for picrocrocin. We will chemically attach commercially available fluorescent dyes to the nanocages with click chemistry. Next, we will mix the dyed nanocages with glycol polymers that contain another dye. In the absence of picrocrocin, the polymers will bind to the nanocages and fluorescence resonance energy transfer (FRET) will occur between the dye on the glycol polymer (which will also be bound non-covalently to the cage). Next, we will mix the dyed nanocages and dyed polymers with saffron extracts. In doing so, the nanocages are anticipated to change color, because the picrocrocin in the saffron will displace the glycol polymers in the cage and stop the FRET mechanism from occurring. The picrocrocin concentration at which the sensors are most sensitive will be varied by changing the lengths (and with it the affinity for the cage) of the glycol polymers to cover a large concentration range accurately for analysis. Computer modeling will be used to design optimal nanocages, which bind to picrocrocin as selectively as possible to achieve maximum sensing efficiencies and strong color changes, that can readily be observed by eye. We will collect ~20 data points of the color patterns on the paper strips using standard samples (solutions of pure picrocrocin, which we will isolate from saffron with HPLC). Finally, the optimized nanosensors will be spotted onto paper strips, together with various dyed glycol-polymers.Initially, this type of analysis will be focused on picrocrocin. However, in future grants, we will also explore sensing of other components in saffron (e.g. crocin and safranal) as well as common saffron-adulterants. This generalization will lead to powerful sensors on paper strips for saffron quality, which can detect the concentrations of different saffron components in real time.Computer-aided sensor design. We will rationally design optimal nanocages with computer modeling before synthesis, which will allow us to predict their binding properties. Molecular dynamics (MD) simulations will be used to mimic the experimental conditions of binding and to model molecular interactions between the nanocages and its target analytes, providing direct structural evidence to predict the binding stability and stoichiometry. Accordingly, we can predict the potential selectivity and sensitivity of the nanocages. For the computational predictions, we will utilize cutting-edge high-performance computing technology accelerated with graphic processing units (GPUs), to enable efficient systematic design and screening of different nanocages. The most promising designs with optimal selectivity and sensitivity will be synthesized and further characterized in subsequent experiments. With the synergy of modeling and experiments, we expect to reduce the synthetic cost, accelerate our discovery, and enhance the likelihood of success for our nanosensors.Sensor validation with HPLC and spectrophotometry. Our paper-based sensors will be calibrated and validated by comparison with standard HPLC analytical methods of saffron samples. We will first calibrate the sensors with pure picrocrocin samples at prototypical concentrations. Next, we will determine the picrocrocin level for ~20 saffron samples from different sources with HPLC, spectrophotometry, and with our paper-based sensors. Results from the three methods will be compared to determine their correlation. Analysis of each sample will be replicated three times and error bars will be calculated with standard error analysis.Saffron production. Approximately 10 g/year of Vermont-grown saffron will be needed for the experiments, which will require 53 m2 of saffron growing area. Dutch-grown corms will be purchased from Roco Saffron, and planted in August in raised beds. Corms will be planted at a depth of 15 cm, and a density of 12 corms/ft2. Saffron flowers will be harvested by hand every 3 days from October to November. Stigmas and stamens will be separated immediately following harvest and dried separately.Saffron processing. The methods used to dry and store saffron after it has been harvested are critical to the quality of the final product. To further validate the sensitivity of our paper-based sensors, two different saffron drying methods and two storage methods will be tested for their effects on picrocrocin content. The drying methods are:In a food dehydrator a 35 ºC for 4-6 hoursAir-dried under ambient conditions for 48 hoursThese are the two most common drying methods used by growers. The saffron will be dried to 10% moisture (as determined by a moisture meter), and stored in glass vials with screw-top lids in the dark until processed for chemical analysis. Three storage methods will be tested:At room temperature in the darkIn a freezer at -18 ºCIn a refrigerator at 4 ºCThe results of these experiments will be analyzed as described above with HPLC, spectrophotometry, and our paper-based sensors.

Progress 05/15/19 to 05/14/22

Outputs
Target Audience:Growers currently cultivating saffron and those who intend to in the future are the primary target audience. These growers represent a wide array of producers, including small diversified farmers, dairy farmers, nursery and greenhouse operators and new Americans. In addition, home gardeners, and future retirees who seek to supplement their social security with income from saffron. Extension personnel and researchers will be key to dissemination of information on saffron production and pest management practices. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?One graduate student (Olav Vestrheim) has been partially supported by this award during the reporting period. Olav has been trained in computational modeling, as well as in organic synthesis and spectroscopy. Furthermore, the postdoctoral fellow working on the project (Dr. Arash Ghalehgolabbehbahani) -- who has been promoted to a Research Associate position -- was further trained in specialized methods of chemical extraction, as well as in NMR spectroscopy. Continued effort has been expended by the authors to maintain the Center's listserve, Saffronnet. It continues to serve as an excellent tool to use for disseminating our research results and information about saffron production and pest management. Current and future saffron growers were the primary audience at multiple presentations as detailed in the products section. How have the results been disseminated to communities of interest?Yes, the results have been published in the peer-reviewed literature, and also disseminated to Growers with Saffronnet and conferences organized as detailed in the products section. Furthermore, Prof. Schneebeli has presented the results at the following departmental seminars during the reporting period: 1. University of Würzburg, Department of Chemistry, Würzburg, Germany (May 2022). 2. Hong Kong University of Science and Technology (Virtual Seminar), Advanced Materials Thrust, Hong Kong/Guangzhou, P. R. China (May 2022) 3. Purdue University, Departments of Chemistry as well as Industrial and Physical Pharmacy, West Lafayette, IN (April 2022). 4. Hunter College (CUNY, Virtual Seminar), New York, NY, Department of Chemistry (Feb 2022). 5. Soh Group Seminar Series (Virtual Seminar), School of Engineering, Stanford University, CA (December 2021). 6. University of California, Riverside, CA (October 2021). What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? During this NCE period, the main objective was to publish our results on a new analysis method for saffron (and the corresponding optimal drying method for saffron) in the peer-reviewed literature. We have accomplished this goal as detailed in the products section. We have also been able to further refined the synthesis of our nanocages (and a provional patent has been filed on our greatly improved process during this reporting period, which is key to enable future sensing applications with the nanocages for agricultural products). Last but not least, we have pushed the sensor applications further along, and have studied the binding kinetics of picrocrocin binding to the nanocages. While there is still some work left to be done (mostly due to delays caused by the global pandemic), we strive to further refine andpublish the picrocrocin sensing results in the peer-reviewed literature during the next year.

Publications

  • Type: Journal Articles Status: Published Year Published: 2022 Citation: A. Ghalehgolabbehbahani, O. Vestrheim, M. Skinner, J. Li, S. T. Schneebeli. NMR-based Quality Assessment of Vermont Grown Saffron (Crocus Sativus L.)  Optimal Drying Conditions and Mechanistic Implications ACS Food Sci Technol. 2022, 2, 315320. Cover Art.
  • Type: Websites Status: Published Year Published: 2021 Citation: The North American Center for Saffron Research and Development at the University of Vermont http://www.uvm.edu/~saffron/ 5,750 hits over the past year.
  • Type: Other Status: Published Year Published: 2021 Citation: Factsheet: Ghalehgolabbehbahani, A., M. Skinner, B.L. Parker, & S. Schneebeli. 2021. Saffron Drying Methods. UVM North American Center for Saffron Research & Development, Burlington, VT. Factsheet 2 pp.
  • Type: Other Status: Other Year Published: 2021 Citation: Saffronnet, International email listserv: Over 800 subscribers from across the US, Europe and Asia, an increase of 22 subscribers, and over 900 emails transmitted to the list over the past year.
  • Type: Other Status: Other Year Published: 2022 Citation: Event: Skinner, M., A. Ghalehgolabbehbahani & B.L. Parker. 2022. Saffron Cultivation from Here and There. 6th Annual UVM Saffron Center Workshop. 23 March and 30 March 2022. Webinar. 150 attendees.
  • Type: Other Status: Other Year Published: 2022 Citation: Event: Skinner, M. 2022. Saffron: For Good Health and Good Food. Univ. of VT. North American Center for Saffron Research & Development. Burlington, VT. Factsheet. 2 pp.
  • Type: Other Status: Other Year Published: 2021 Citation: Event: Skinner, M., B.L. Parker & A. Ghalehgolabbehbahani. 2021. To Market, To Market, to Sell your Fine Saffron, 15 Nov. 2021. UVM North American Center for Saffron Research & Dev., Burlington, VT. Webinar. 75 attendees.
  • Type: Other Status: Other Year Published: 2021 Citation: Event: Skinner, M., A. Ghalehgolabbehbahani & B.L. Parker. 2022. Saffron: From Start to Finish, Planting to Selling. 5th Annual UVM Saffron Center Workshop. 11 March and 18 March 2021. Webinar. 120 attendees.
  • Type: Other Status: Other Year Published: 2021 Citation: Skinner, M., B.L. Parker & A. Ghalehgolabbehbahani. 2021. Saffron Harvesting and Drying: Tricks of the Trade, 7 Oct. 2021. UVM North American Center for Saffron Research & Dev., Burlington, VT. Webinar. 85 attendees. https://www.youtube.com/watch?v=C3XQB1l9s2U&list=PLBDy4IUFNEtwcG4I3ZV2yNPBfqdDqyWdG&index=1


Progress 05/15/20 to 05/14/21

Outputs
Target Audience:Growers currently cultivating saffron and those who intend to in the future are the primary target audience. These growers represent a wide array of producers, including small diversified farmers, dairy farmers, nursery and greenhouse operators and new Americans. In addition, home gardeners, and future retirees who seek to supplement their social security with income from saffron. Extension personnel and researchers will be key to dissemination of information on saffron production and pest management practices. Changes/Problems:In addition to sensing with fluorescent dyes, we have also started to investigate the possibility for creating electronic sensors coated with our nanocagesfor detecting the picrocrocin content in saffron. A key advantage of electronic sensors could be their potential reusability. What opportunities for training and professional development has the project provided?Two graduate students (Nick Hamilton and Olav Vestrheim) have been partially supported by this award during the reportingperiod. Furthermore, two undergraduate students (Finley Clark and RichardAbbott) were also able to participate in this project during the summer of 2020, supported by UVM Chemistry Summer UndergraduateResearch Fellowships.All of the studentshave been trained in computational modeling, as well as in organic synthesis andspectroscopy (Nick Hamilton, Olav Vestrheim, and Richard Abbott).Furthermore, the postdoctoral fellow working on the project (Dr. Arash Ghalehgolabbehbahani) -- who has been promoted to a Research Associate position -- wasfurther trained inspecialized methods of chemical extraction, as well as in NMR spectroscopy. Continued effort has been expended by the authors to maintain the Center's listserve, Saffronnet. This listserve has over 780 members (an increase of 130 over last year). It continues to serve as an excellent tool to use for disseminating our research results and information about saffron production and pest management. Current and future saffron growers were the primary audience at multiple presentations. We gave at least 7 presentations on saffron production and quality testing, reaching over 600 growers and agricultural specialists and extension personnel. How have the results been disseminated to communities of interest? In addition to saffronnet, the listserve, a yearly workshop specifically dealing with all aspects of saffron production, marketing and quality was held in March 2021. Attendance at this workshop totaled >150 growers from all across the U.S. and Canada. Presenters from Canada and many US states were enlisted to share their experiences on production. A grower to grower approach was successful and allowed participants to delve into problems commonly encountered. Multiple presentations and factsheets were prepared and given on the value of habitat plantings, reaching over 1,000 growers, many of whom have adopted this practice which benefits the environment by supporting beneficial arthropods Presentations were held online due to the pandemic for growers across the US to introduce growers to the potential of growing saffron. The PI also presented this research at the following Departmental seminars during the reporting period: (a)S. T. Schneebeli, Northwestern University, Department of Chemistry,Stodddart Research Group Virtual Symposium, Evanston, IL(April2021); (b) S. T. Schneebeli,Rowan University Virtual Seminar,Department of Chemistry, Glassboro, NJ(March 2021); (c) S. T. Schneebeli,Penn State Erie Virtual Seminar,Department of Chemistry, Erie, PA(December 2020). What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period, we will focus on testing the sensing capability of the new nanocages we created during the last reporting period, and compare the results with our new NMR methodology for determining picrocrocin content in saffron. Results of these trials will be reported to growers at the 6th annual saffron workshop scheduled for March 2022. We will continue to maintain Saffronnet and our saffron website, produce factsheets and promote saffron production nationally. We will also continue to maintain Saffronnet and our. saffron website,producee factsheets, and promote saffron production nationally, and publish our results in the peer-reviewed literature.

Impacts
What was accomplished under these goals? Synthesize nanocages andpolymers with fluorescent dyes attached. We have synthesized several new nanocages during the reporting period. Specifically, we have now been able to synthesize alkyne-functionalized cages, cages with free phenols, as well as cages with a variety of shapes. We are currently running click reactions to attachthe dyes. Investigate color changes when picrocrocin from saffron binds. We have discovered a new mechanism of how picrocrocin can bind cooperatively to our nanocages during the reporting period. Specifically, we have discovered that the geometry of the nanocages changes very significantly (from s-trans on the hydrazone bonds to s-cis) upon binding of picrocrocin to the nanocages. This finding has important implications as it helps us design more selective, cooperative nanocages with stronger color changes upon binding. Computationally optimize nanocages and computationally investigate picrocrocin binding. We have optimized nanocages at both the molecular mechanics, and density functional theory (DFT) levels during the reporting period. These detailed calculations clearly demonstrate that the s-trans conformations of the hydrazone bonds in the cage become favored in solution (without picrocrocin), which leads to a larger hydrazone cage. Interestingly, however, with less polar solvents or in vacuum, the s-cis conformation becomes favored. Thus, we have now discovered that picrocrocin binding inside of our nanocages represents a rare example of cooperative induced-fit binding in a fully synthetic receptor system. Synthesize optimal nanocages. Optimal nanocages have been synthesized and we are now finalizing the last step (clicking on different fluorescent dyes). Createprototypical, paper-based picrocrocin sensors.We are still working on this objective, and will finalize it during the NCE period. In addition to fluorescence-based sensors, we have also started to investigate electronic sensing methodologies with our nanocages attached to sensing electrodes. Calibrate the paper-based sensors.We have created an efficient way to determine the quantity of picrocrocin (as well as saffranal and crocin) in our locally-grownsaffron samples with nuclear magnetic resonance (NMR) spectroscopy. Specifically, an innovative extraction method was developed to minimize losing the saffron compounds during NMR samples' preparation. To extract the saffron secondary metabolites (scientific name of Safranal, Crocin and Picrocrocin), 100 mg of dehydrated saffron stigmas were finely milled in a mortar. Then, 30 mg of grinded saffron were transferred to a TD-NMR glass tube (Bruker, outer diameter of 7.5 mm), followed by adding 1 ml DMSO-d6 solvent (Cambridge Isotope, 10 G). The samples were sonicated for 45 minutes at 10 °C. Then sonicated samples and NMR tubes were covered by aluminum foil to restrict the impact of lighton chemical compounds in the solution. For the statistical analysis of the results,a completely randomized design (CRD) with three treatments (drying temperature regimes: 1- 100 °C for 7-10 minutes; 2- 77 °C for 25 minutes; 3- 55 °C for 60 minutes) and three replications was used. Analysis of variance (ANOVA) was conducted in SPSS ver. 25.0 to compare the concentration of picrocrocin, crocin and safranal among the treatments. The statistical significance (α = 0.05) was evaluated using Duncan's multiple ranges (DMRT). To evaluate the relationship between concentration of chemical components in Vermont grown saffron, Pearson's correlation value was computed for safranal vs. Picrocrocin and all isomers of crocin. Scatter plots were graphed in SPSS ver. 25.0 to summarize the results of correlations.Next, hierarchical cluster analysis (HCA) was performed to classify Vermont grown saffron in homogeneous clusters based on similarities inconcentration of the major chemical components. dendrogram was graphed in SPSS ver. 25.0 to illustrate the distance between the samples produced through different dehydration methods. In this study, an agglomerative hierarchical clustering (and squared Euclidean distance as the most popular distance measure were applied (Norusis, 2010). Average linkage method was used to measure the link between two clusters (Yim and Kylee 2015). Overall, we discovered, that higher temperature for drying promotes a higher concentrations of crocins in saffron.During this reporting period, the methodology has been refined, and we plan to submit a manuscript describing our results to the Journal of Agricultural and Food Chemistry during the next few weeks. Grow and harvest saffron under different conditions.We have grown and dried saffron under a variety of conditions, and this objective is now mostly complete.Specifically, an innovated extraction method was developed to minimize losing the saffron compounds during NMR samples' preparation (see above). Our results demostrate conclusively for the first time,that it is beneficial to dry saffron at higher temperature, as this leads to an overall higher crocin content. Validate our nanosensors with saffron grown at UVM.This objective is still ongoing, and will be completed during the NCE period.

Publications

  • Type: Journal Articles Status: Other Year Published: 2021 Citation: A. Ghalehgolabbehbahani, O. Vestrheim, J. Li, M. Skinner, S. T. Schneebeli. "Effect of drying temperature regimes on the quality of Vermont grown saffron". Manuscript in preparation for the Journal of Agricultural and Food Chemistry
  • Type: Websites Status: Published Year Published: 2020 Citation: The North American Center for Saffron Research and Development at the University of Vermont. http://www.uvm.edu/~saffron/ Over 6,800 hits to the Saffron website were logged in over the past year.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Skinner, M., A. Ghalehgolabbehbahani & J. Pylypiv. 2021. The Potential of Saffron for Small Farmers. Podcast #14. Wisconsin Extension System. https://soundcloud.com/cutting-edge-podcast/cutting-edge-episode-14-saffron
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Skinner, M., A. Ghalehgolabbehbahani, J. Pylypiv & P. Shorey. 2021. The Market Potential of Saffron for US Farmers. Podcast #15. Wisconsin Extension System.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Skinner, M., A. Ghalehgolabbehbahani, J. Pylypiv & P. Shorey. 2021. The Finer Points of Producing Saffron in the US. Podcast #16. Wisconsin Extension System.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Ghalehgolabbehbahani, A. and M. Skinner. 2020. Saffron: A new crop for Diversified Growers. VT Vegetable and Berry Growers Association.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Ghalehgolabbehbahani, A. & R. Gheshm. 2021. Saffron Plot Preparation & Cultivation. The 5th Annual Workshop on Saffron: Saffron: From Start to Finish, Planting to Selling, 11 March, 2021, online. North American Center for Saffron Research and Development, Burlington, VT.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Ghalehgolabbehbahani, A. 2021. Saffron Drying to Maximize Quality. The 5th Annual Workshop on Saffron: Saffron: From Start to Finish, Planting to Selling, 18 March, 2021, online. North American Center for Saffron Research and Development, Burlington, VT.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Schneebeli, S. T. 2021. Saffron Quality Testing. The 5th Annual Workshop on Saffron: Saffron: From Start to Finish, Planting to Selling, 18 March, 2021, online. North American Center for Saffron Research and Development, Burlington, VT.


Progress 05/15/19 to 05/14/20

Outputs
Target Audience:Growers currently cultivating saffron and those who intend to in the future are the primary target audience.These growers represent a wide array of producers, including small diversified farmers, dairy farmers, nursery and greenhouse operators and new Americans. In addition, home gardeners, and future retirees who seek to supplement their social security with income from saffron. Extension personnel and researchers will be key to dissemination of information on saffron production and pest management practices. Changes/Problems: On March 24, 2020 the State of Vermont Governor Scott issued a "Stay Home, Stay Safe" order and directed the closure of in-person operations for all non-essential businesses. The University of Vermontfollowing the State of Vermont "Stay at Home, Stay Safe" order transitioned where possible research and sponsored project activities to remote work, with a small number of projects continuing as essential research. Projects that could not transition to remote work and have not been approved as essential have been paused, until otherwise directed, by University and State officials. The University of Vermonthas established procedures to ensure that critical research infrastructure and assets like animals, cell lines and biological assets, reagents and chemical components remain viable, cared for, and safe. The University of Vermontparamount goal, is to protect the health and safety of our University community and the community in which we work, to continue essential research and protect research assets, to help our researchers cope with the disruptions they have faced, and to maintain our research support and administrative infrastructure. Despite the current challenges of the COVID pandemic, we continue to pursue the objectives of this project. Activities that have continued with little or no interruption include: Analysis of experimental results, working on manuscripts related to this project, as well as computational work. Sponsored project activities that have been temporarily impacted due to the COVID pandemic include: Experimental laboratory activities,travel,and field work. The PI has also signed up to present this research at theNanoscale Science and Engineering for Agriculture and Food SystemsGordonResearchConference, which has been cancelled due to Covid-19. What opportunities for training and professional development has the project provided?Two graduate students (Mona Sharafiand Xiaochuan Zhao) have been partiallysupported by this award during the reporting period. They have been trained in computational modeling (both graduate students), as well as in organic synthesis and spectroscopy (Mona Sharafi). Ms.Sharafi has -- in part thanks to theresearch she was engaged in for this award -- wonthe 2020 "Best PhD Dissertation Award" from the UVM Chemistry Department. Furthermore, the postdoctoral fellow working on the project (Dr. Arash Ghalehgolabbehbahani) has been trained in specialized methods of chemical extraction, as well as in NMR spectroscopy. Last but not least, continued effort has been expended by our teamto maintain the Center's listserve, Saffronnet.This listserve has over 650 members (62% increase from last year). It continues to serve as an excellent tool to use for disseminating our research results and information about saffron production and pest management.Current and future saffron growers were the primary audience at multiple presentations. We gave over 10 presentations on saffron production, reaching over 400 growers and agricultural specialists and extension personnel. How have the results been disseminated to communities of interest? In addition to saffronnet, the listserve, a yearly workshop specifically dealing with all aspects of saffron production, marketing and quality was held in March 2020. Attendance at this workshop totaled >150 growers from all across the U.S. and Canada.Presenters from Canada and many US states were enlisted to share their experiences on production.A grower to grower approach was successful and allowed participants to delve into problems commonly encountered. Multiple presentations and factsheets were prepared and given on the value of habitat plantings, reaching at least 1,000 growers, many of whom have adopted this practice which benefits the environment by supporting beneficial arthropods. Presentations were held in several New England states to introduce growers to the potential of growing saffron. A presentation was given in Savannah, GA,by the PIto introduce our nanocages and their potential for sensing key components in saffron to the scientific community. The PI and Co-PIs also presented this research at the following Departmental seminars during the reporting period: (a) S. T. Schneebeli,SUNY Plattsburgh, Department of Chemistry, Plattsburgh, NY (March 2020); (b) S. T. Schneebeli, University of Rhode Island, Department of Chemistry, Kingston, RI (September 2019); (c) S. T. Schneebeli,University of Würzburg, Nanoscience Institute Young Investigator Symposium, Würzburg, Germany (May 2019); (d) J. Li, Boston University, Department of Chemistry, Boston, MA (November 2019). What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period, we will focus on (i) reporting our recentresults in the peer-reviewed literature, as well as (ii) on creating and testing the paper-based nanosensor strips for saffron with the saffron samples we grew/processed under a variety of conditionsduring the firstreporting period. The results of these experimentswill be reported to growers at thee 5th annual saffron workshop scheduled for March 2021. We will also continue to maintain Saffronnet and our. saffron website, producee factsheets, and promote saffron production nationally

Impacts
What was accomplished under these goals? Overall Impact of our Results: With the COVID-19'shistoriceconomicimpact,many small-to-medium farms will or already face existencial problems. Thus, in these challenging times, it is especially important for farmers to diversify their production to secure their future. Saffron(the world's most expensive spice) offers a great opportunity in this regard, as it can be grown in the off-season. However, ensuring the highest possible saffron quality has been challenging and achieving this goal requires (i) not only advanced new protocols for growing saffron, but (ii)also better methodology for analyzing saffron.With funding support from this grant, we have -- during the reporting period -- made significant progress toward ensuring that U.S. farmers will be able to enter the growing saffron market with homegrown, high-quality product. Specifically we have (i) investigated better ways to harvest and dry saffron, (ii)invented a new way to extract and analyze saffron with a common spectroscopic technique (which wastes less valuable saffron product than currently available protocols), as well as (iii) made progress toward a nanocage-based sensor strip (which is designed to ultimately help farmers analyze their saffron product directly after the saffron is dried). Accomplishments of Specific Objectives during the Reporting Period: Synthesize nanocages and polymers with fluorescent dyes attached.We have been able to synthesize new nanocages, which are currently serving as the direct precursors for the versions functionalized with fluorescent dyes. Furthermore, we have been synthesizing a variety of polymers and studied their binding to the nanocages, both experimentally (with NMR spectroscopy) as well as computationally (with molecular dynamics simulations). These results now provide (together with our new extraction method, vide infra) the basis to create functional paper strips during the next reporting period for saffron analysis with a focus on detecting the amount of picrocrocin in saffron samples. Investigate color changes when picrocrocin from saffron binds. This is next on our to-do list, as soon as we have the final, functionalized nanocages in hand. Computationally optimize nanocages and computationally investigate picrocrocin binding. New nanocages have been designed with molecular dynamics simulations. We have been able to also utilize our computational methods to predict which other analytes might bind to our nanocages. For example, we predicted computationally that curcumin would likely also bind to our nanocages and we indeed observed significant binding to one of our nanocages experimentally very recently. A manuscript describing these results is currently in preparation. Synthesize optimal nanocages. We are in the process of doing this at the moment. Create prototypical, paper-based picrocrocin sensors based on the knowledge learned from the previous objectives. This is the key task for the second project period. Calibrate the paper-based sensors. We invented a new, and greatly improved/simplifiedprotocol for extracting and analyzing saffron quality with nuclear magnetic resonance (NMR) spectroscopy. This new protocol (which will be reported in the peer-reviewed literature during the next reporting period) provides the basis to calibrate our sensors strips during the following reporting period. Grow and harvest saffron under different conditions.In July 2019, we ordered 7,000 saffron corms.On-site soil was mixed with 2 cubic yards of compost to prepare the planting beds. Soil samples were taken from the beds and sent to the UVM soil test laboratory. Based on the soil test results, fertilizer (N-P-K) was added to the soil in April 2020.The corms were planted in mid-September at theUVM Horticulture Research Center (HRC).Saffron blooming season started at October 20, 2019 and it peaked on October 30. A total of 1,788 saffron flowers were harvested from October 22 to November 11, 2019. Separation the saffron stigmas was done at the day the flowers were picked. All saffron samples were dehydrated in an oven at the North American Center for Saffron Research and Development. Based on the trial goals, stigmas were dehydrated at three different temperatures.Three replications were used to assess the significant differences among the treatments (3 drying methods). Afterwards, we invented a new extraction and analylsis method (based on nuclear magnetic resonance, NMR spectroscopy)for saffron.Our new NMR-based analysis methodwas then used to assess the concentration of safranal, crocin and picrocrocin. Please note that our innovative, newextraction method (manuscript in preparation) was developed to (i) minimize losing the saffron compounds during NMR samples' preparation, (ii) reduce the amount of (very valuable!)saffron sample needed for analysis, and (iii) provide an accurate baseline to calibrate our paper-based sensor strips during the next reporting period. Validate our nanosensors with saffron grown at the University of Vermont. This objective will be worked on primarily during the next reporting period, with the saffron samples we produced during the firstreporting period.

Publications

  • Type: Websites Status: Published Year Published: 2019 Citation: The North American Center for Saffron Research and Development at the University of Vermont. http://www.uvm.edu/~saffron/ Over 8,000 hits to the Saffron website were logged in over the past year.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Skinner, M. A. Ghalehgolabbehbahani & B.L. Parker. 2020. Field production of saffron in Vermont. Poster. Proc. 4th Annual Saffron Workshop, 12 March 2020. Burlington, VT.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Ghalehgolabbehbahani, A., M. Skinner & B.L. Parker. 2020. Field production of saffron in Vermont. Greenhouse IPM Workshop, 9 Jan 2020. Burlington, VT.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Skinner, M. A. Ghalehgolabbehbahani & B.L. Parker. 2019. Saffron: The Next Best Thing for Crop Diversification. Mid Atlantic Vegetable & Berry Conference. 10 Dec. 2019. Manchester, NH.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Skinner, M., A. Ghalehgolabbehbahani & B.L. Parker. 2019. Saffron: A Good Fit for New England. Farmer-to-Farmer Conf., Maine Organic Farmers & Gardeners Assoc. 4 Nov. 2019. Northport, ME.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Skinner, M. & C. F. Sullivan. 2019. IPM for High Tunnel Vegetables: Practical Pathways for Organic Crop Protection. Farmer-to-Farmer Conf., Maine Organic Farmers & Gardeners Assoc. 4 Nov. Northport, ME.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Ghalehgolabbehbahani, A. 2019. UVM Saffron Tour. Shelburne/Charlotte Garden Club, 8 October 2019. University of Vermont, Burlington, VT
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Ghalehgolabbehbahani, A. 2019. UVM Saffron Tour. UVM Horticulture Research Center Farm Tour. September 2019. University of Vermont, Burlington, VT.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Skinner, M., A. Ghalehgolabbehbahani & B.L. Parker. 2019. Saffron: A Golden Opportunity for Diversified Farmers. Grantham Garden Club, 13 Sept 2019. Grantham NH.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Ghalehgolabbehbahani, A., M. Skinner, P. Reese & B.L. Parker. 2019. Saffron: A high value crop for Diversified Farmers. 28 Oct. Univ. of Vermont Osher Lifelong Learning Institute. Burlington, VT.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Ghalehgolabbehbahani, A., M. Skinner, P. Reese & B.L. Parker. 2019. Saffron Research Tour. 31 Oct. Univ. of Vermont Osher Lifelong Learning Institute. Burlington, VT.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Skinner, M., A. Ghalehgolabbehbahani & B.L. Parker. 2019. Saffron: The Next Best Thing for Crop Diversification. Penn State Univ., 13-14 August. State College, PA.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Skinner, M., A. Ghalehgolabbehbahani & B.L. Parker. 2019. Saffron: A Golden Opportunity for Diversified Farmers. UVM Hort. Research Center Open House. 6 August 2019. So. Burlington, VT.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Skinner, M., A. Ghalehgolabbehbahani & B.L. Parker. 2019. Factsheet: Saffron: A Golden Opportunity, North Amer. Center for Saffron Research & Development, Univ. of VT, Burlington VT.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Schneebeli, S. T.; Sharafi, M. 2019. Post-synthetic Catalytic Polymer Functionalization for Macromolecular Synthesis, Next Generation Smart Materials Conference, Savannah GA.
  • Type: Theses/Dissertations Status: Other Year Published: 2019 Citation: Sharafi, M. 2019. Bottom-up Synthesis of Macromolecules and their Selective Catalytic Performance at the Nanoscale, PhD Dissertation, Defended in October 2019 (currently still under embargo at ScholarWorks).