Recipient Organization
PENNSYLVANIA STATE UNIVERSITY
408 Old Main
UNIVERSITY PARK,PA 16802-1505
Performing Department
Food Science
Non Technical Summary
Copper-based fungicides are commonly used in conventional and organic farming; however, copper (II) is known to destroy many components related to the aroma and antioxidant capacity vital for product quality. In plants like fruits, herbs, and flowers, aroma is a significant driver of consumer acceptability. Plant antioxidants have been shown to provide health benefits and, when extracted, can be added as natural preservatives in foods to prevent spoilage and oxidation. These compounds are oxidatively labile and once oxidized, no longer exhibit their aromatic or antioxidant properties. Their rate of oxidation may be accelerated in the presence of transition metals such as Cu (II). Further research is necessary to evaluate the effects of foliar applications of copper-based fungicides on the aroma and antioxidant quality of food components. I propose the use of hops (Humulus lupulus) as a model system for this investigation. Essential hop aroma compounds include terpenoids, esters, and thiols. Hops contain considerable concentrations of antioxidants including catechins, flavonoids, and organic acids. These aroma and antioxidant compounds are found in several other crops including wine grapes, herbs, citrus fruits, and berries. A field trial will be conducted to elucidate the effects of copper fungicide treatments on the quality of these components. Hop plants will be treated with three different concentrations of copper-based fungicides over the course of one growing season. Concentrations of aroma compounds will be determined, and sensory characteristics will be evaluated by a trained panel. Antioxidant capacity of hop extracts will be determined by standard methods.
Animal Health Component
80%
Research Effort Categories
Basic
20%
Applied
80%
Developmental
(N/A)
Goals / Objectives
Objective 1: Investigate the effects of copper (II)-containing fungicide treatments on the quality and stability of hop -derived aroma components. I hypothesize that the foliar application of copper-based fungicides will negatively impact the aroma quality of hops due to copper's ability to complex with and oxidize key varietal aroma components such as polyfunctional thiols. Copper's ability to alter and inhibit plant growth may also affect overall aroma quality. Field treatments of hops with copper-based fungicides will be conducted, concentrations of key aroma compounds will be determined, and sensory quality will be evaluated by trained panelists.Objective 2: Investigate the effects of copper (II) containing fungicide treatment on the antioxidant capacity of hop-derived components. I hypothesize that the treatment of hops with copper-based fungicides will decrease the antioxidant capacity of hop extracts. Copper has been shown to function as a pro-oxidant in vitro and in vivo, and previous research in olive leaves has indicated copper's potential to diminish antioxidant capacity. Extracts will be prepared from hops procured as described in Objective 1 and evaluated for their phenolic content, antioxidant capacity, and antioxidant functionality in a model food system.
Project Methods
1.1 Field Treatments: A field trial will be conducted at Penn State's Experimental Hopyard located at the Russell E. Larson Research and Education Center at Rock Springs. Eighteen established plots of Cascade hops, each containing 7 bines, will be used during the proposed research. Plots will be randomly assigned to a treatment group. Treatment groups will consist of a copper-free control, low-copper exposure, and high-copper exposure. The control fungicide treatment will consist of a rotation of Ranman-400SC (SummitAgro USA, LLC, EPA Reg. No. 71512-3-88783) and Zampro (BASF Corporation, EPA Reg. No. 769-302) to be applied to all plants at manufacturer recommended concentrations every 7 days from April to August. Experimental groups will receive treatments of the copper-based fungicide Champ WG (Nufarm Americas Inc, EPA Reg. No. 55146-1; 77% copper hydroxide) which will be tank-mixed with the control treatment. Low-copper exposure plants will be sprayed with Champ WG every 14 days while high-copper exposure plants will be sprayed with Champ WG every 7 days. Fungicide treatments will be applied to each plot using an air blast sprayer. Applications will cease 2 weeks prior to harvest to prevent harmful pesticide exposure during analysis and sensory evaluation.1.2 Soil Analysis: Elemental copper concentrations of the soil at the hop yard will be determined by inductively coupled plasma-atomic emission spectroscopy (EPA Method 6010) following acid digestion of the soil samples (EPA Method 3050B/3051). Samples will be randomly collected from each treatment mound prior to, and at the conclusion of, the fungicide applications. Analysis will provide total concentrations of elemental copper across treatment plots.1.3 Hop Cone Copper Content Determination: Concentrations of elemental copper in treated hop cones will be determined by inductively coupled plasma-atomic emission spectroscopy (EPA Method 6010) following dry ashing by high-temperature oxidation.1.4 Analysis of Hop Aroma and Flavor Compounds: Concentrations of key α- and β-acids will be determined by high performance liquid chromatographic (HPLC) analysis according to established methods. Terpenoids encompass a variety of signaling, defense, and aroma compounds produced by plants that contain a terpene functionality. Key terpenoids impart aromas characteristic to hops and other plants such as tomatoes, citrus fruits, berries, and herbs. Concentrations of the terpenoids linalool, geraniol, citronellol, myrcene, caryophyllene, humulene, farnesene will be extracted from hop essential oils through stir bar-sorptive extraction (SBSE) and quantified by gas chromatography-mass spectrometry (GC-MS) using carvone as an IST. 4-MMP, 3-MH, and 3-MHA will be extracted from hop essential oils through C-18 solid phase extraction, derivatized with 4,4'-dithiodipyridine and quantified by HPLC-MS. Deuterated forms of 4-MMP, 3MH, and 3-MHA will be used as internal standards.1.5 Descriptive Analysis of Hops: Trained panelists will be used to evaluate aroma characteristics of dried hops cones. Panelists will attend a total of 10, hour-long sessions during which they will be trained to identify an array of standards related to hop aroma characteristics. Each hop sample will be placed in a 2 oz. plastic cup with a lid. Panelist will be instructed to remove the lid and take a short and long sniff through the nose and then rate the intensity of the various aroma attributes on a 15cm line scale. Hops from each of the three treatments will be evaluated in triplicate by all panelists. All protocols will be approved by Penn State's Institutional Review Board prior to carrying out this objective. Results will be analyzed by principal component analysis using the R statistical package (R Foundation for Statistical Computing, Vienna, Austria).2.1 Preparation of Hop Extracts: Acetone, water, and supercritical CO2 (SCE) hop extracts will be prepared from hop cones. Samples will be suspended in 10 volumes of either 80% acetone (v/v) or in ultra-pure water under nitrogen. Extraction vessels will be placed in a sonicating water bath for 1 hr and then centrifuged at 10,000 x g for 10 min. For the preparation of the SCE, dried hop cones will be extracted with CO2 at 40°C at 150 bar for 2.5 hrs followed by a second extraction step at 300 bar for 2.5 hrs. All extracts will be decanted and kept at -80°C under nitrogen until analysis.2.2 Quantification of Extract Phenolic Content: The total phenolic content of each extract will be determined by the Folin-Ciocalteu assay. A standard curve of gallic acid will be used to quantify assay response. Total phenolic content of the extracts will be expressed in grams gallic acid equivalent per 100g extract and normalized to dry hop weight. Hop extracts will be diluted with 10 volumes of ultrapure water and incubated with 1 volume of 5% NaNO2 (w:v) for 5 min. Ten volumes of 10% ACl3 will be added to the solution followed by 5 volumes of 1N NaOH. Absorbance at 510 nm will be measured, and response quantified expressed as rutin equivalents.2.3 Evaluation of Hop Extract Antioxidant Capacity: Antioxidant (AOX) capacity of prepared hop extracts will be evaluated by 1,1-diphenyl-2-picrylhydrazyl (DPPH), oxygen radical absorbance capacity (ORAC), and ferric reducing AOX power (FRAP) assays. The DPPH and FRAP assays measure the test compound's electron transfer AOX capacity. The concentration of AOX that is able to quench 50% of a given concentration of DPPH at constant absorbance is referred to as the inhibitory concentration (EC50). The time it takes the EC50 to reach constant absorbance is denoted as TEC50 and indicate the rate at which an AOX functions. EC50 and TEC50 values will be determined for all hop extract fractions across all treatments to determine the effects of copper treatments on electron-transfer AOX capacity and reaction rate. ORAC is a measurement of hydrogen transfer AOX capacity of a given test compound. The ORAC assay measures the signal of a fluorescence probe, fluorescein (FL), over a period of 35 min and an area under the curve (AUC) of fluorescence is calculated. FL signal decays in the presence of peroxyl radicals, generated by 2,2'-azobis(amidinopropane) dihydrochloride. When an AOX is present, the FL signal decay is slower resulting in a greater AUC. AUC values will be determined for all extracts across treatments.2.4 Antioxidant Capacity of Hop Components in Model Food Systems: Hop extracts will be evaluated for their AOX functionality in a model emulsion and the products of lipid oxidations will be measured over time. Emulsions containing 5% algae oil (~50% ω-3 fatty acids) in phosphate buffer (10 mM, pH 7) will be prepared with each hop extract added to the oil pre-emulsification. Emulsions will be stored at 7°C and allowed to oxidize in the absence of light while primary (lipid hydroperoxides) and secondary (malondialdehyde) products of lipid oxidation will be monitored over time using standard methods.