Final report for LNC15-367
Project Information
The overall purpose of our project was to advance the hazelnut industry in the Upper Midwest through a series of targeted research projects. Our objectives were to: 1) Advance our long-term effort to develop improved germplasm for growers through establishment of replicated germplasm trials evaluating the performance of our top selections across a range of environments, 2) Better understand the phenolic content of hazelnut involucres, shells, and leaves as possible co-products of post-harvest processing, 3) Develop chemical fingerprinting tools for flavor compounds as a tool to identify superior genotypes for flavor and to assess the impact of post-harvest handling practices on flavor, and 4) To optimize cracking procedures to maximize whole kernel recovery rates.
We have completed work analyzing the phenolic and antioxidant content of the leaves, involucres, and nuts of American and Hybrid hazelnuts and have published the results in the American Journal of Essential Oils and Natural Products. We have completed exploratory work identifying anti-quality flavor compounds (bitterness) in hazelnut kernels and will be completing work to track the changes in these compounds in storage outside the time period of this grant project. We completed the 2016 and 2017 harvest and data analysis, identified the top 12 plants for propagation and commercial release, and established replicated performance trials with these selections at 6 locations in the Upper Midwest (two each in IA, MN, and WI) Work is continuing to develop the field-ready propagules from these 12 plants through micropropagation, but progress has been slow and delayed our goal of planting at least 10 on-farm trials. We have completed development of EMC (Equilibrium Moisture Content) curves in order to optimize cracking and dry-down procedures. We completed work to evaluate the effect of moisture content on cracking and have finished quantifying deformation forces and cracking characteristics. The results of this cracking analysis have been submitted for publication in the journal of Transactions of the American Society of Agricultural and Biological Engineers. We continue to provide updates to our stakeholders and growers via a newsletter, field days, and conferences.
Outcome/Objective 1: On-Farm Evaluation of Select Hazelnut Genotypes
Achieving Objective 1 of this project will take all three years. The first year will finalize selection of the top genotypes and put those genotypes through micro-propagation. Years 2 and 3 will focus on establishment of the replicated performance trials.
Outcome/Objective 2: Optimizing Post-Harvest Handling for Processing
The first step in this objective is to develop EMC curves for the hazelnuts in order to identify optimal cracking moisture and dry down procedures.
Outcome/Objective 3: Optimizing Post-Harvest Handling for Flavor
The next step is to implement the dry-down and storage trials to determine optimal procedures for protecting hazelnut flavor.
Outcome/Objective 4: Screening Hazelnuts for Phenolic Content and Novel Compounds
The purpose of this objective is to screen hazelnut kernel, shell, and involucre for phenolic content for possible identification of novel phenolics with commercial potential.
Cooperators
Research
Our project includes a number of research trials aimed at advancing the hazelnut industry in the Upper Midwest.
We hypothesize that genetic gain in average per acre nut yields can be made through evaluation of hybrid and American hazelnut accessions and selection of the top performing plants.
We hypothesize that in-shell nut moisture content will affect whole kernel recovery rates and that different moisture contents will be needed for different cracking methods (impact vs compression).
We hypothesize there are genetic differences in flavor that can be identified through characterization and measurement of flavor-active compounds. We further hypothesize that dry-down rates and temperatures of in-husk nuts will affect flavor.
We hypothesize that phenolic levels in hazelnuts are high and that bio-active anti-oxidants are present at economically significant levels.
Outcome 1: On-Farm Evaluation of Select Hazelnut Genotypes
Production of Liners
We propose to contract with Knight Hollow Nursery of Middleton, WI to utilize their hazelnut micropropagation experience to generate rooted clonal liners of each of four genotypes for planting at each of fifteen farms and at two University research stations. Collaborating growers will purchase liners for either a Genotype (50 liners) or Agronomic Trial (200 liners) and contribute all labor and resources necessary to establish the trials. Five-hundred (500) liners will be produced for each of two research station plantings for agronomic trials to follow up on questions raised by current agronomic trials, including but not limited to weed management, fertilization, irrigation and pruning.
Fall 2015 – Eight genotypes from the replicated hybrid performance trials were selected based on the 2013 and 2014 yield and performance data from the St. Paul and Bayfield trials. Rooted shoots from each of these genotypes were dug in the fall of 2014, transplanted into pots, and grown in a greenhouse. Four of the eight genotypes will be chosen for propagation based on the 2015 yield data and the success of getting the genotypes into culture.
Winter2015/Spring 2016 – The potted stock plants will be brought out of dormancy, then plant hormones and shoot tip pruning will be used to rejuvenate them and encourage production of new shoots. The shoot tips will be harvested and cultured on agar medium with hormones and nutrients per the protocol developed by Knight Hollow Nursery.
Summer/Fall 2016 – The rooted plantlets will be harvested and transplanted into soil-less potting mix for continued growth and acclimation.
Winter 2015/2016 – The plants will be greenhouse-grown through the fall, allowed to go dormant in early winter and transplanted while still dormant in spring 2017.
Trial Plantings
The research plantings will be established at the Spooner Agricultural Research Station in Spooner, WI and the Horticultural Research Center in Chanhassen, MN. The on-farm Genotype and Agronomic Trials will be established and managed by 15 growers participating in the Hazelnut Improvement Program. Participating growers have agreed to conduct all site preparation, establishment, and management at their own expense. In addition, the growers have agreed to purchase the plants at cost. The cost shall be the expenses incurred by Knight Hollow to produce the liners in excess of the costs covered by this grant project. Growers have been chosen based on interest and their record of managing plantings, and with a goal of representing a diverse range of environments.
Summer/Fall 2016 – Jason Fischbach, Lois Braun, or the hired field technician will visit each participating grower to develop and implement a site preparation plan. Site preparation at each site will be completed by freeze-up in 2016.
Spring 2017 through Oct 2018 - The plants will be established in the spring of 2017 and maintained for optimum growth in order to quantify performance. The two research station plantings, and the on-farm Agronomic Trial plantings will be used for agronomic trials including weed management, fertilization, irrigation and pruning as determined by the interests of the grower and demands of the site. Such trials have so far been confounded by lack of genetic uniformity with the trial plants.
Outcome 2: Optimizing Post-Harvest Handling for Processing
Equilibrium Moisture Content (EMC) Determination of Nuts and Nut Clusters
Saturated salt solutions are commonly used to determine the equilibrium moisture content of materials. At UW-Madison, unique equipment for more rapidly reaching equilibrium moisture content in a saturated salt solution environment has been developed. This equipment will be used to establish EMC curves for hazelnuts and hazelnut clusters (i.e., unhusked nuts). Such curves allow for modeling expected moisture content in response to different drying methods, saving the need for expensive drying experiments. A minimum of six different salt solutions will be used at three different temperatures (40F, 80F, and 120F). Original moisture content of the nuts and nut clusters will be determined using standard ASTM procedures. Nuts and nut clusters will then be put into EMC chambers and weighed daily. Temperature and relative humidity within EMC chambers will be recorded on 10 minute intervals. Once daily weighing shows that EMC has been attained, a sample of nuts and nut clusters will be removed from each chamber for moisture content determination.
Physical Properties of Hazelnuts
Nuts conditioned to six different moisture contents will be loaded at room temperature between two parallel plates to determine the specific deformation, bio-yield force, rupture force, and rupture energy required to initiate shell and kernel rupture Tests will be carried out at deformation rates of 0.5 inches per min and 10 inches per minute. One-third of the nuts will be loaded in the X-direction (X-axis is the longitudinal axis through the hilum), one-third in the Y-direction (Y-axis is the transverse axis containing the major dimension at right angles to the longitudinal axis), and one-third in the Z-direction (Z-axis is the transverse axis containing the minimum dimension). Physical characteristics of the nut (mass, dimensions, geometric mean diameter, sphericity, moisture content (MC), the broken shell (thickness, surface area, MC) and kernel (mass, dimensions, geometric mean diameter, sphericity, MC) will be determined. Nuts taken from the same conditioned batch of nuts as those loaded to failure in this study, will also be ran through two different cracking machines to obtain relationships between commercial and laboratory cracking processes with the goal being to determine the combination of moisture and cracking method yielding the least amount of damage to the kernels.
Outcome 3: Optimizing Post-Harvest Handling for Flavor
We will develop a comprehensive molecular fingerprint of hazelnut flavor quality as influenced by post-harvest handing condition to provide the know how to maintain high product acceptability and freshness. The first step will be to define the principal compounds responsible for flavor and, more importantly, compounds that impart negative flavors.
Taste compounds will be identified based on methods developed by Jiang and Peterson (2013). Ethanol/water extracts will be optimized for taste isolation. The subsequent isolate will be fractionated by ultra-filtration and further screened for taste-activity. The fractions identified as taste-active will be fractionated by off-line 2D high-pressure liquid chromatography and purified peaks will be screened for taste activity. The intensity of the taste active fractions will be determined by sensory analysis and used to select compounds that contribute the most to taste. The key taste-active compounds will be structurally elucidated using advanced analytical techniques (i.e. LC/MS-Qtof, NMR). The target taste compounds will be quantified by LC/MS/MS techniques. Taste recombination studies will be conducted for validation of the taste active compounds selected. The chemical nature of the identified off-flavor compounds will provide insight into the mechanisms of formation (enzymatic or non-enzymatic) and provide a basis to optimize post-harvest handling procedures to maintain maximum nut quality. In future would we can further extrapolate this molecular flavor map to additional samples to further investigate the influence of growing region or cultivar on flavor performance.
Effects of post-harvest drying methods.
Using the EMC curves and the optimal moisture content for cracking, three different husk-drying regimes will be evaluated to achieve the desired moisture content: 68F, 68F with forced air, 130F with forced air. In the fall of 2016, all in-husk nut clusters will be harvested from at least two plants of each of 10 separate genotypes. The clusters will be placed in 1 gallon plastic mesh onion bags with three bags per drying treatment (9 gallons/genotype). A subsample from each bag will be husked and the moisture content of the in-shell nuts will be measured. The three husk-drying treatments will be implemented until the target moisture content is reached. The nuts from each treatment will be rated for occurrence of shell fissures and then cracked to immediately remove the kernels for storage at -80C for chemical analysis. Dr. Peterson’s lab will then use comprehensive analytical sensory-guided fractionation techniques coupled with LC/MS and NMR methods for compound structural elucidation to compare the levels of anti-quality flavor components in these samples.
Effects of storage conditions and duration.
In the fall of 2015, nuts will be harvested from three genotypes, dried with 68F forced air, and husked. 200 gram sub-samples of the in-shell nuts from each of the 3 genotypes will be stored in an un-insulated shed with no temperature regulation or in a climate controlled unit at a constant temperature of 68F, 35F, or 0F. A sub-sample from each sub-sample will be removed at 30 days, 90 days, and 180 days, cracked, and the kernels will be stored at -80C for chemical analysis. Again, Dr. Peterson’s lab will then compare the levels of anti-quality flavor components in these samples.
This work will result in optimized post-harvest handling procedures and an understanding of hybrid hazelnut flavor chemistry for use in subsequent food processing.
Outcome 4: Screening Hazelnuts for Phenolic Content and Novel Compounds
The extract of phenolics and antioxidant activity will be exploratory in nature, in an effort to determine if additional research is warranted. Phenolics in the nutmeat, involucres, shells, and leaves from 8 hybrid genotypes and 5 American genotypes will be analyzed. Phenolics will be extracted using methods of Shahidi et al. (2007). Briefly, samples will be ground and fats will be removed with hexane. Extraction of phenolics will be completed using a mix of ethanol and water and then vacuum dried and stored. Samples will be rehydrated and analyzed using HPLC. Antioxidant activity will be assessed using a surrogate of hydrogen peroxide scavenging activity (Shahidi et al. 2007). Briefly, hydrogen peroxide will be added to the hazelnut extracts and a spectrophotometer will be used to model the degradation of the peroxide in the samples (caused by the extracts). Levels of phenolics and antioxidant activity will be compared to values found for C. avellana by Alasalvar et al. (2007), Contini et al. (2008) and Delgado et al. (2010). The analysis will be done at the Wisconsin Institute for Sustainable Technology.
Impacts
This project has significantly expanded efforts to develop a hazelnut industry in the Upper Midwest. We have made 12 selections from our hybrid breeding populations and are moving as quickly as we can to propagate and release to growers. We feel these 12 selections are adequate for scale-up of the industry. We have developed a better understanding of the phenolic content in hazelnut leaves, husks, shells, and nuts and have continued work to identify commercially valuable phenolics. We have developed a better understanding of key bitterness compounds in hazelnuts and now have the tools to quantify changes in those compounds caused by different post-harvest handling procedures. We also have a better understanding of the nut deformation and cracking characteristics of our midwest-grown hazelnuts that is allowing us to develop better cracking technology.
Accomplishments
Outcome/Objective 1: On-Farm Evaluation of Select Hazelnut Genotypes
We have completed the 2016 and 2017 harvest seasons and data collection and have made our final selections from the more that 150+ genotypes we've been evaluating. We are continuing to work with 20 selections, but have identified the top 12 for moving into more widespread performance trials in the Upper Midwest. Through mound-layering we were able to generate enough propagules from 8 of these selections to populate Joint Performance Trials at 6 locations (Verona, WI, Bayfield, WI, St. Paul, MN, Staples, MN, Fenton, IA, Cedar Rapids, IA) and smaller trials at 4 additional locations. These Joint Performance Trials will include the top selections from our breeding program and from Grimo Nut Nursery, Rutgers, Nebraska, and Ontario. We were hoping to establish trials at 15 on-farm locations, but micropropagation continues to be slow and we don't anticipate having enough material for the on-farm trials until fall 2019.
Outcome/Objective 2: Optimizing Post-Harvest Handling for Processing
Work has been completed on developing the EMC curves for the in-shell and in-husk nuts. We also completed our work analyzing cracking forces and have submitted a paper for publication titled: "Physical Properties of Upper Midwest USA Grown Hybrid Hazelnuts". We are now learning what we've discovered to find and/or develop cracking technology that does a better job aligning the nut along the correct orientation to maximize single-fissure cracking. We also conducted trial work to evaluate the effect of moisture content on whole kernel crack out rates and determined that orientation of deformation pressure is far more important than moisture content.
Outcome/Objective 3: Optimizing Post-Harvest Handling for Flavor
Initial sensory analysis conducted on six Midwestern hazelnuts (five hybrids of Americana variety and one native Americana variety) revealed bitterness as a main off-flavor attribute. Further sensory evaluation demonstrated differences in the perceived bitterness intensity among these samples. The main focus of this work was to identify bitter compounds in mid-western hazelnuts and to define the effect of postharvest handling techniques on bitter development (drying conditions, storage time and temperature) will be evaluated. It took longer than expected to identify the main bitterness compounds and, thus, our storage and drying condition trials were delayed. With additional funding in place we are able to continue these trials and anticipate results in 2019.
The native C. Americana hazelnut variety was selected as the reference sample to identify the bitter compounds due to sample availability and relatively high perceived bitterness as compared to other varieties. First, sample preparation was optimized, including solvent extraction, ultrafiltration and solid phase extraction. An 80% methanol extraction followed by molecular weight separation yielded a representative bitter fraction of <5kDa that the highest intensity. Subsequently sensory-guided fractionation was performed on this isolate using Liquid Chromatography coupled with Mass Spectrometry and Ultraviolet detectors (LC/MS/UV). Multiple fractions were collected and freeze-dried twice to ensure the samples were free of solvent. Fractions were reconstituted in water at the endogenous concentrations in hazelnuts for sensory evaluation by a trained panel of five judges. Bitterness intensity was rated on a designated categorical scale (not present, threshold, weak, moderate and strong). The six fractions with the highest bitter intensities were chosen for further purification by additional multidimensional HPLS fractionation. The final purified isolate was analyzed by Ultra-Performance Liquid Chromatography coupled with accurate Mass Spectrometry (UPLC/MS-QTof) to identify bitter compounds. Four compounds, cyclic diarylheptanoid asadanin (C19H20O6), kaempferol-3-rhamnoside(C21H20O10), 9,12,13-trihydroxy-10(E),15(Z)-octadecadienoic acid (C18H32O5) and 9,12,13-trihydroxy-10(E)-octadecenoic acid (C18H34O5) were positively identified via LC-MS/MS fragmentation pattern, compared with commercial available standards, and reported, for the first time, as the main bitter compounds in native American hazelnuts. Additionally egonol gentiobioside (C31H38O15) was tentatively identified and its structure will be further confirmed using 1D/2D Nuclear Magnetic Resonance (NMR) technique. Structure elucidation of the last bitter compound is currently on-going.
In parallel, the shelf-life study has been initiated in December 2018. Seven varieties of in-shell American hazelnuts are stored in glass jars at -10℃, 4℃ and 35℃ for 30, 60 and 120 days. At each time point, the moisture content of each sample is measured and samples are moved to -80℃ storage. After 120 days all of the identified bitter compounds will be quantified (LC/MS) to determine the impact of storage and variety on bitter compound formation or degradation. This part of study will provide a better understanding of bitterness development during postharvest handling.
Future work will include the quantification of the bitter compounds in the shelf-life study samples as well as in a large range of American hybrid hazelnuts. Their release during consumption and impact on sensory properties will also be evaluated by quantification in expectorated saliva after mastication.
Outcome/Objective 4: Screening Hazelnuts for Phenolic Content and Novel Compounds
This work has been completed and the results published:
Demchik. M.C., Fischbach, J., and J. Hall. 2016. Phenolics and antioxidant Activity of American and hybrid hazelnuts. American Journal of Essential Oils and Natural Products. 4(1):50-52
Hazelnut leaves, involucres, shells and nutmeats all have quite high levels of phenolics and antioxidant activity. The level of phenolics was highest in the leaves and lowest in the nuts. This would be expected, as high phenolic levels are often associated with bitter flavors and bitter nuts would not be a preferred food. When corrected to the same levels, phenolics from the leaves and involucres provided the strongest antioxidant activity. Overall, the potential of hazelnut co-products, like involucres and leaves, to provide another product is exciting and deserving of additional research.
Education
As part of our outreach program we have hosted field days in both 2016 and 2017. Our 2017 Wisconsin Hazelnut Field Day was held in Spooner, WI with a focus on germplasm improvement, post-harvest processing, and establishment. The annual Upper Midwest Hazelnut Growers Conference was held in Dubuque, IA in March of 2017 and in Owatonna, MN in March of 2018. At both conferences our team provided research updates on the germplasm improvement and propagation work. We have published the results of the phenolics work and have submitted a publication reporting on our cracking experimentation. We have also published extension reports about our top 12 selections.
Project Activities
Educational & Outreach Activities
Participation Summary:
The main goal of our UMHDI project is development of research-based information for delivery to hazelnut producers, existing and prospective. Our main informational portal is www.midwesthazelnuts.org. There we post research bulletins, videos, and other information. We have published a "Choosing Hazelnut Plants" and "UMHDI 1st Generation Selections" document describing our top 12 selections. We have published one journal article about hazelnut phenolics and have submitted another article describing the physical properties of hazelnuts. We hosted field days in 2017 at two locations and our annual conference in 2017 and 2018. We presented information about our work at multiple events in MN and WI including presentations to USDA NRCS RC&D groups, Northland College students, Extension workshops, and stakeholder meetings. Jason Fischbach conducted a webinar with the Savanna Institute that is available for viewing at: http://www.savannainstitute.org/nutshells.html. Outreach education for this project continues outside the time period including our just completed 2019 hazelnut growers conference in Eau Claire. Proceedings from that conference can be found at: https://www.midwesthazelnuts.org/conference-proceedings.html
Learning Outcomes
- Key variables in hazelnut cracking, particularly as affected by moisture content and cracking method.
- Characteristics and attributes for hazelnut germplasm
- Phenolic content of hazelnut leaves, involucres, kernels, and shells.
- Challenges of hazelnut micropropagation.
Project Outcomes
Increased knowledge of hazelnut breeding and germplasm improvement to better evaluate the performance of their own plant material.
Continued work is needed in developing field-ready planting stock for the hedgerow production system. It is a balance between plant cost and rapid fill of the hedgerow with fruiting wood.
There is also a need for research on big bud mite and other arthropod pests.