In studies of the European elderflower without any additional food ingredients, linalool and linalool derivates, such as -linalool pyranoxide and cis-linalool oxide, have frequently been identified as prominent. The aroma of linalool, the main aroma compound in lavender, can be described as citrus, fruity, floral, and woody. The age of the flower when harvested as well as how the flowers are stored after harvest can greatly impact the volatile profile. As expected from the other data on inter-cultivar variation, the volatile profile is heavily influenced by the cultivar. For example, wild elderflower had twice as much rose oxide and more linalool oxide than the other 12 cultivars. While this could be a challenge for manufacturers that use elderflower in products to have a consistent aroma from batch to batch, it also allows for more selectivity to find a cultivar that matches desired organoleptic properties in the product. American elderflowers have not yet been evaluated for their volatile profile, nor have blue elderflowers.Elderberry and elderflower are becoming more common ingredients and flavoring agents in beverages and food products. However, a vast majority of the products on the market today utilize the European subspecies of this plant due its more established cultivation and a deeper understand of the composition, particularly the phenolic profile, of the fruit and flower. North American subspecies, the American elderberry S. nigra ssp. canadensis and the blue elderberry S. nigra ssp. cerulea, have some information available regarding composition, but further analyses are needed to understand how they may perform in the common applications that European elderberry and elderflower are used in today.As global warming and water scarcity issues continue to impact food systems, large plastic pots for plants fire-resilient and drought-tolerant plants will become more important for supplying nutrient-rich foods. Wildfires throughout the western United States are becoming more common and more serious as seasons are hotter and drier.

California has been experiencing unprecedented levels of wildfires, including over 1.9 million acres burned in 2018, over 4.2 million acres burned in 2020, and over 2.5 million acres in 2021. One native and fire-resilient plant is the blue elderberry , which grows wild throughout the western United States and has become a popular choice to grow in hedgerows. The blue elderberry is drought-tolerant, and the roots of the blue elderberry can survive fires to regrow the next season to continue providing valuable flowers and fruit, making it an ideal choice to plant in regions of California and American West often stricken by wildfires. While European and American elderberries have been studied for decades, there is currently little information on the subspecies native to the western region of North America, S. nigra ssp. cerulea , known as blue elderberry due to a white-colored bloom on the exterior of the berry which makes it appear blue. In California, it grows wild in riparian ecosystems near rivers and streams, but is also planted in hedgerows on farms to improve water, air, and soil quality, in addition to providing a habitat for birds, pollinators, and other beneficial insects. The plant can grow several meters tall and wide and flowers from May to August, with peak fruit ripening throughout July and August. While elderberry prefer moist soil and some hedgerows may receive some irrigation during the summer months, most are not irrigated once the hedgerow has been established, about 2-4 years. That is one of the benefits of using native and drought-tolerant plants, as they can better withstand the natural climate without excess resources. Elderberries have a long history of use by Native Americans and Europeans in foods, beverages, and herbal medicines. Research exploring links between elderberry consumption and health has increased dramatically, particularly in the past decade. Numerous in vitro and in vivo studies demonstrate that elderberries have potent antioxidant, antibacterial, and antiviral properties.

Results of two randomized, double-blind, placebo-controlled clinical trials suggest that elderberry supplements reduce the duration and severity of cold symptoms. Roscheck et al. identified two non-anthocyanin flavonoids in elderberry extract that inhibited viral ability to infect host cells when bound. While most bioactivity of elderberries is assumed to result from the phenolic compounds like anthocyanins, the high-molecular weight fraction of concentrated elderberry juice was found to contain acidic polysaccharides that had potent effects against the human influenza virus. The health-promoting properties of elderberry have led to recent increases in its use in products such as supplements, syrups, gummies, and teas, as well as wine and jams. During the COVID-19 pandemic, elderberry supplements gained wide attention because of potential anti-viral activities; however, there is no strong clinical evidence that elderberry could be beneficial in preventing or treating COVID-19. The market for elderberries is expected to continue to increase, as the sales of herbal dietary supplements was over $11 billion in 2020, a 17.3% increase from 2019. Elderberry was the topselling herbal supplement, with sales over $275 million, as consumers became more interested with supporting their immune systems. In addition to the interest in elderberry as an ingredient in functional foods, elderberry can be an excellent source of natural coloring agents for food and beverage applications due to the high content of red and purple anthocyanins 35 .Characterization of the chemical composition, functional properties, and impact of processing on the bio-active compounds in elderberry is largely limited to S. nigra ssp. nigra and, to a lesser extent, S. nigra ssp. canadensis. S. nigra ssp. nigra is commonly referred to as the European black elderberry, which has many established cultivars, such as “Haschberg” and “Samyl”, and it has an established market.

The European elderberry is the most frequently used subspecies in commercial elderberry-based products and has been extensively studied for its composition, anthocyanin stability , and health benefits in European black elderberry-based products. S. nigra ssp. canadensis is commonly referred to as the American elderberry, a subspecies native to the eastern and central regions of North America. There are several cultivars of the American elderberry, including “Johns” and “Bob Gordon”. The American elderberry, which is utilized in small-batch products, has also been evaluated for its composition and health-promoting properties. The acreage grown of this subspecies has been increasing rapidly and there is a goal to grow over 2,000 acres by 2025, according to the Midwest Elderberry Cooperative. Currently, there is no information on the chemical composition of the fruit of the blue elderberry . With the recent increase in demand for elderberry, blue elderberry grown in hedgerows may be an additional and valuable source of bio-active phenolics and natural colorants. The objective of this study was to determine the moisture content, soluble solids, pH, titratable acidity, and establish the anthocyanin and phenolic profiles of blue elderberries grown in Northern California to support the use of this robust, native crop in commercial products.Hedgerows of S. nigra ssp. cerulea were identified on five farms near Davis, California in Spring 2018 with the assistance of an experienced agronomist at The Cloverleaf Farm . Farm, hedgerow, plant pots with drainage and harvest information is presented in Table 1. Blue elderberries were determined to be ripe when the berries in a cyme were deep purple, with or without the white bloom, and had no green berries present. Ripe elderberries were harvested by hand from all four quadrants of the elderberry shrub, totaling approximately 3 kg of elderberries. The berries were placed in clear plastic bags, stored on ice, and transported to the laboratory. A subsample was separated for moisture analysis, while the rest was de-stemmed and stored at -20 °C until analyzed.Elderberries were extracted by combining 5 g frozen berries with 25 mL MeOH:formic acid in a conical tube. The contents were homogenized, placed in a shaker without water at speed 7.5 for 20 min, then centrifuged at 3,000 rpm for 7 min. The supernatant was transferred to a 15 mL plastic tube and stored at -80 °C for no more than two weeks prior to analysis. Duplicate extracts were made from each shrub. TPC was determined using the Folin-Ciocalteu method. First, elderberry phenolic extract was diluted 1:4 with water. Each extract was analyzed in duplicate and averaged. In 10 mL glass tubes, 6 mL water was combined with 100 µL sample and 500 µL Folin-Ciocalteu reagent. After mixing and incubating for 8 min at room temperature, 1.5 mL 20% aqueous sodium carbonate was added. The tubes were mixed, covered with foil to avoid light exposure, placed in a water bath at 40 °C for 40 min, then cooled at room temperature for 15 min. The samples were read by a UV visible spectrophotometer at 765 nm and quantified using an external standard curve prepared with gallic acid . TPC is expressed as mg gallic acid equivalents per 100 g FW.Five grams of frozen berries were mixed with 25 mL of in a conical tube, which was then homogenized for 1 min at 7,000 rpm . The mixture was stored at 4 °C overnight, then in the morning, centrifuged at 4,000 rpm for 7 min. The supernatant was used directly for analysis. Three pooled samples were made for each hedgerow, each consisting of even amounts of berries from three distinct shrubs. Eachpooled sample was extracted once to give 3 biological replicates, and each extract was run in duplicate . Averages concentrations for compounds were determined across the hedgerow in mg per 100 g FW. The concentration of phenolic compounds in blue elderberry followed the method by Giardello et al. with some modifications. Briefly, samples were analyzed via reversed-phase liquid chromatography on an Agilent 1200 with a diode array detector and fluorescence detector . The column used was a PLRP-S 100A 3 µm 150 x 4.6 mm at 35 °C, and the injection volume was 10.0 µl. Mobile phase A was water with 1.5 % phosphoric acid, while mobile phase B was 80%/20% acetonitrile/ mobile phase A. The gradient used was 0 min 6% B, 73 to 83 min 31% B, 90 to 105 min 6% B. The DAD was used to monitor hydroxybenzoic acids at 280 nm, hydroxycinnamic acids at 320 nm, flavonols at 360 nm, and anthocyanins at 520 nm. The FLD was used to monitor flavan-3-ols, with excitation at 230 nm and emission at 321 nm. External calibration curves were prepared using chlorogenic acid for phenolic acids, rutin for flavonols, and cyanidin-3-glucoside for anthocyanins , at the following concentrations: 200, 150, 100, 75, 50, 25, 10, 5, and 2.5 mg/L. Catechin was used to quantify flavan-3-ols and standards were run at 150, 100, 75, 50, 25, 10, 5, 2.5, and 1 mg/L. Compounds were identified based on retention time and spectral comparisons with standards. Information about the linear equations and lower limits of detection and quantitation can be found in Table S1 in the supplementary material. The LLOD was calculated as 3.3 times the standard deviation of the y-intercept of the curve divided the slope, while the LLOQ was calculated as 10 times those values.Several peaks appeared in the HPLC chromatograms that could not be identified using the above parameters. Chromatographic eluents of these peaks were collected individually and dried under vacuum. These extracts were reconstituted with mobile phase A, and 5 µL were injected into the HPLC- QTOF-MS/MS for accurate mass analysis . A Poroshell 120 EC-C18 column was used at 35 °C. Mobile phase A was 1% formic acid in distilled water, and mobile phase B was 1% formic acid in acetonitrile. The gradient used was 0 min 3% B, 30 min 50% B, 31-32 min 95% B, 33-38 min 3% B. The mass spectrometer was used in negative mode, and the mass range for MS was 100 to 1000 m/z while the range for MS/MS was 20-700 m/z. Collision energies at 10, 20, and 40 V were applied. The drying gas was set to a flow of 12 L/min at 250 °C, while the sheath gas was set to 11 L/min at 350 °C. The nebulizer was set to 40 psig, the capillary voltage was 3500 V, the nozzle was set to 500 V, and the fragmentor was set to 100 V. Data was analyzed using Agilent MassHunter Workstation Qualitative Analysis 10.0 . Tentative identification was achieved by comparing the mass to charge ratio of the precursor and fragment ions to online libraries of compounds as well as using formula generation for the peaks in the spectra.Anthocyanins are a class of phenolics that contribute red, purple, and blue hues to fruits and vegetables, act as attractants for pollinators, and are potent antioxidants.