To make foods safe, cyanogenic glycosides are frequently hydrolyzed through processes including grinding, pounding, boiling, steaming or during soaking or fermentation in water.Thermal treatments have also been used to effectively decrease levels of cyanogenic glycosides in foods.Commercial elderberry juice is thermally processed to inactivate enzymes, improve microbiological stability, and to degrade the potentially toxic cyanogenic glycosides found in this berry which include amygdalin, dhurrin, linamarin, and sambunigrin . The range and content of cyanogenic glycosides in the blue elderberry have not yet been identified. However, there was an occurrence of people getting sick after consuming raw blue elderberry juice at a at a religious gathering in Monterey County, California in 1983.Therefore understanding levels of cyanogenic glycosides in this subspecies is critical if it is going to be considered for use in commercial products. In European elderberry, sambunigrin is the main CNG in all parts of the plant, with the highest levels in the leaves and the lowest levels in the ripe berries .6 Sambunigrin is a diastereoisomer of prunasin, containing L-glucose instead of D-glucose.In the American subspecies, amygdalin, dhurrin, linamarin, and sambunigrin were identified in the skin, seed, and juice of the berry as well as in the stem.Juice of the American elderberry contained the lowest levels of CNGs relative to the skin, seeds and stems, averaging about 4 µg g- 1 , while the stems had the highest concentrations in the two cultivars evaluated.A recent study demonstrated that the total phenolic content of the blue elderberry is similar to the European and American subspecies,danish trolley however this subspecies has significantly lower levels of anthocyanins.

The dominant phenolic compounds identified in the blue elderberry include the flavonols rutin and isorhamnetin glucoside, the phenolic acid chlorogenic acid, a novel phenolic compound tentatively identified as 5-hydroxypyrogallol hexoside , and the anthocyanins cyanidin 3-glucoside and cyanidin 3-sambubioside .At the same time, anthocyanidins are desired by consumers for their potential health promoting bioactivity.Unfortunately, heat processing and pasteurization can lead to the degradation of anthocyanins resulting in a loss in color or a formation of brown polymers, possibly impacting the acceptability of the final product. Elderberry juice and extracts have been evaluated for their thermal stability, which have shown that anthocyanins degrade following first-order reaction kinetics.The stability of anthocyanidins can be reinforced via intra- and inter-molecular interactions with protective structures and flavonoids through a phenomenon termed copigmentation.Notably, acylated anthocyanins, such as those found in American elderberry like cyanidin 3-coumaroylsambubioside, are sometimes more stable during thermal processing due to protective properties of the coumaroyl group folding over the flavylium ion.Like the European elderberry, the blue elderberry does not contain acylated anthocyanins.The thermal stability of anthocyanidins in blue elderberry juice has not yet been evaluated. However, as elderberry juice and extracts are frequently thermally processed to make products, such as jam, syrup, or gummies, it is important to understand the thermal degradation of anthocyanidins in the juice from blue elderberry.

The purpose of this study was to determine the stability of the cyanogenic glycosides and main phenolic compounds in blue elderberry juice cooked at 72 °C and 95°C for two hoursto elucidate the kinetics of degradation of these important compounds. HPLC-grade methanol , and LCMS grade acetonitrile, methanol, and formic acid were purchased from Fisher Scientific . Ultrapure water was obtained from a Milli-Q water system . Prunasin was also obtained from Millipore Sigma. HPLC-grade acetonitrile , rutin , isorhamnetin 3-O-glucoside, caffeic acid, chlorogenic acid, -catechin, protocatechuic acid, ammoniumformate, and amygdalin were purchased from Sigma-Aldrich . Cyanidin 3-Osambubioside chloride and cyanidin 3-O-glucoside were purchased from ExtraSynthese Ripe berries were harvested in July 2019 from a farm in Winters, CA at latitude and longitude coordinates of 38.634884, -122.007502. Fruit was selected from all sides of the plant at a variety of heights to obtain a representative sample of berries from each plant. Only fully ripe berries . About 5 kg were harvested from each of shrub in three hedgerows. The plant material was transported to University of California, Davis on ice in plastic gallon bags within two hours of harvest and stored at -20 °C until analysis. A hot water bath was prepared using an immersion circulator , set to the desired cooking temperature . The temperature was also monitored with a thermometer in the water bath. Vials of juice randomized and place into the hot water bath and the bath was covered during cooking. Duplicate vials were removed at the following times: 15, 30, 45, 60, 75, 90, 105, and 120 min. An extended processing time was used to observe degradation of more heat-stable compounds as well as to make comparisons to other studies that processed juice for multiple hours at these temperatures. Once removed from the hot water bath, vials were placed immediately into an ice bath for 15 min.

Then from each vial, 1 mL of juice was placed in a microcentrifuge tube and centrifuged at 4 °C, 15,000 rpm for 15 min . Next, the supernatant was diluted 1:10 with 1% formic acid in water, filtered with 0.2 µm PTFE filter, and placed in an HPLC vial for analysis. Five replicate juice samples were prepared and cooked at both temperatures. Composite juice samples were prepared by combing equal aliquots of the time points 0, 15, 30, 60, and 120 minutes of thermal processing for both temperatures for each juice prepared. To extract CNGs for blue elderberry juice, 0.500 mL of juice was mixed with 2.00 mL of methanol in a 5 mL centrifuge tube, then sonicated at 30 °C for 30 min. After sonication, 1.00 mL of extract was transferred to a 1 mL microcentrifuge tube and centrifuged at 15,000 rpm at 4 °C for 15 min. The supernatant was collected and filtered through 0.22 µm PTFE into an HPLC vial and used for analysis. Five replicate extractions were made of raw elderberry juice and triplicate extractions were made for the other time/temperature juices. CNGs were analyzed via ultra-high performance liquid chromatography with electrospray ionization and triple quadrupole tandem mass spectrometry using an Agilent 1290 Infinity HPLC and 6460 mass spectrometer . The UHPLC was equipped with a binary pump with an integrated vacuum degasser , an autosampler with thermostat , and a thermostated column compartment . CNGs were separated using a Kinetex F5 column at 40.0 °C. The mobile phase consisted of a linear gradient of 1 mM ammonium formate in water and 1 mM ammonium formate in 650:50 MeOH:ACNe as follows: 5% B, 0–5 min; 100%B 5-5.50 min, 95% A 5.60-6.50 min. The flow rate was 0.400 mL/min, and the injection volume was 5.0 μL. The CNGs were analyzed using negative ESI mode. The drying gas temperature was 300 °C and the flow rate was 8.0 L min-1 . The sheath gas temperature and flow rate were 350 °C and 11.0 L min-1 , respectively. The nebulizer gas pressure and capillary voltage were 45 psi and 3.5 kV, respectively. The fragmentor voltage was 160 V for amygdalin and 100 V for sambunigrin. The dwell time was 100 ms for amygdalin and 200 ms for sambunigrin. The collision energy was set to 12 V for amygdalin, 0 V for sambunigrin. The multiple reaction monitoring mode was utilized to analyze amygdalin and sambunigrin. Quantification of CNGs was performed using external calibration curves using standard addition at levels of 500, 100, 50, and 5 ng L-1 . For amygdalin, vertical aeroponic tower garden the area of m/z 456.2 to m/z 323.1 was measured. For sambunigrin, the area of m/z 340.3 to m/z 294.2 was measured. Half-life values of phenolic compounds were calculated by plotting the natural log of C0/C ratio vs heating time t, where C0 is the initial concentration of a compound, C is the concentration of the same compound at time t in hours. The slope calculated from the figure is k . Longitudinal Analysis of variance was performed with Tukey’s post-hoc test with p value at 0.05. Excel used for average and standard deviation and R Studio was used for ANOVA and post hoc analysis . CNG data was analyzed using MassHunter Quantitative Analysis to obtain peak areas for the targeted compounds. Microsoft Excel was used to create the calibration curves and determine CNG concentrations in samples . The concentrations of cyanogenic glycosides were quantified in raw and cooked blue elderberry juice for the first time. Results indicate that neoamygdalin , sambunigrin and prunasin are the primary CNGs in blue elderberry . Concentration of neoamygdalin were significantly higher than sambunigrin and prunasin . Neoamygdalin has been measured in raw bitters almonds in concentrations lower than amygdalin.133 In studies of American and European elderberry, sambunigrin is typically major CNG identified. Levels of total CNGs in blue elderberry are lower than American and European elderberry. European elderberry CNG levels range from 0.08 ± 0.01 to 0.77 ± 0.08 µg g-1 depending on the elevation and growing location.6 CNG levels in American elderberry juice range from 0.29 to 2.36 µg mL-1 .

Differences between the subspecies may be due to genetic variation, impact of growing environment such as altitude, or methodology used to extract and analyze CNG content in the fruit and fruit juice, including how berries were handled prior to juice and juicing method. The degradation of neoamygdalin > sambunigrin > prunasin was observed during cooking and the rate of degradation was faster at 95 °C as compared to 72 °C . However, degradation in juice at 72 °C was not linear, such that sambunigrin levels in juice cooked at significantly increased during the final timepoint measured . This may be attributed to neoamygdalin breaking down resulting in sambunigrin and a glucose molecule, an equivalent pathway to amygdalin degrading to prunasin and a glucose molecule. However, some of the resulting sambunigrin from that reaction would also have to be degrading since the decrease in concentration of neoamygdalin did not cause an equivalent increase in sambunigrin. An increase in sambunigrin at the end of the processing time was not observed in the juice cooked at 95 °C. In the juice processed at 95 °C, the combined concentration of neoamygdalin and sambunigrin decreased at each measured time point and did not increase at any time points like the juice processed at 72 °C. Prunasin levels did not significantly change in the elderberry juice cooked at 72 °C but prunasin did degrade significantly when processed at 95 °C . It appears that sambunigrin is more stable than prunasin in the elderberry juice; retaining about 70% of the original concentration in the juice heated to 95 °C. Neoamygdalin levels decreased significantly in the elderberry juice at both processing temperatures, with increased degradation at 95 °C as compared with the treatments at 72 °C. As previously mentioned, sambunigrin is the expected breakdown product from neoamygladin, but sambunigrin levels did not have concomitant increase due to thermal degradation of sambunigrin as well. Thermal processing has been seen to degrade CNGs in elderberry, flaxseed, and almond in previous studies.Furthermore, studies have seen enzymatic activity contributing to the breakdown of CNGs in nuts to reduce after exposure to heat.If the β-glucosidases for the CNGs present in blue elderberry are similar, they would also be inactivated during thermal processing at 72 and 95 °C, indicating thermal degradation is the main contribution to CNG levels decreasing in the present study. Because enzymatic degradation of CNGs was not measured during the thawing and juicing steps, the impact of the enzymes before the thermal processing cannot be evaluated here. The presence of neoamygdalin instead of amygdalin is unexpected. Amygdalin can convert to neoamygdalin with heat and in alkaline conditions. However, herein the raw elderberry juice had significantly higher levels of neoamygdalin as compared to amygdalin. In a study of amygdalin content in almond varieties, amygdalin was found to convert to neoamygdalin during extraction , but the addition of acetic acid prevented the conversion.Blue elderberry naturally contain citric and malic acids, with an average titratable acidity of 0.60 ± 0.10 to 0.65 ± 0.07 g citric acid per 100 g FW.The average pH value of the juices in the present study was 3.76 ± 0.11. Therefore, there may not be enough acid in the matrix to prevent the conversion. In contrast, another study of amygdalin and derivatives in almonds found that heat of cooking caused neoamygdalin and amygdalin amide to convert to amygdalin, which was not observed in the present study.Further analysis of conversion of amygdalin to neoamygdalin in the blue elderberry could uncover why this epimer is dominant.