To 630 µL of supernatant, 57 µL deuterium oxide and 6 µL of the internal standard solution of 4,4-dimethyl-4-silapentane-1-sulfonic acid-d6were added. Deuterated acetic acid-d4or ammonium deuteroxide-d4dissolved in D2O were used for pH adjustment to 3.5 ± 0.1. After centrifugation , 620 µL of sample was introduced into a 5 mm NMR tube and the 1H NMR spectrum recorded at 25 °C using WET solvent suppression . Spectra were acquired after a 2 s relaxation delay into 32,768 data points and zero filled to 131,072 points, with 1024 scans co-added and 16 dummy scans. Apodization equivalent to 0.5 Hz line broadening was applied prior to Fourier transformation. Chemical shifts were referenced relative to the internal standard DSS at 0.00 ppm. After phasing and baseline corrections, Chenomx version suite 8.1 was used to identify the resonances of selected sugars, amino acids and organic acids, and to determine their peak areas . The latter were quantified by correcting for incomplete T1 relaxation in the proton survey experiments using the Inversion-Recovery pulse sequence. As detailed by Claridge , the T1 relaxation time of each compound was determined in aril-pressed and peel-pressed pomegranate juice samples using recovery time delays , ranging from 0.05 s to 20 s . After integrating the resonances of the selected compounds, the time delays were plotted vs peak areas using Wolfram Mathematica software , to determine the longitudinal relaxation time T1 using equation 1,large plastic garden pots where Mz corresponds to the intensity of the magnetization along the longitudinal axis, τ is the recovery delay following the inversion pulse, M0 is the value of the magnetization at thermal equilibrium, and T1 is the longitudinal relaxation time.

Among the 1H-NMR signals, only glucose and fructose were identified. Concentrations of sugars, quantified by 1H NMR, were significantly different among the pomegranate cultivars . Sucrose, as reported by Zhang et al. , was not detected in these pomegranate juices. There were significant differences for glucose, with the majority of cultivars having significantly higher glucose than Haku Botan , which is a double flowered ornamental cultivar from Japan. Averaged over all cultivars and juice types, the mean concentration of fructose was 416 ± 44 mM, and this value was similar to pomegranate juice fructose levels reported in Mphahlele et al. . There were differences among the cultivars for juice fructose, with Haku Botan having lower fructose than many cultivars, including Wonderful. There were also no differences among cultivars for glucose:fructose ratio . The juice extraction method had no significant effect on glucose, which agrees with Mphahlele et al. . The cultivars in this study had large quantities of polyphenolic compounds in the juice, regardless of juicing method . There were significant differences between juicing methods, with aril-pressed juice having significantly lower quantities of TP than peel-pressed juices . Aril-pressed juice had a mean TP of 3606 ± 356 g/L GAE and peel-pressed juice had mean TP of 4464 ± 72 mg/L GAE. There were no significant differences among cultivars for TP . Due to the relatively low concentrations of individual phenolic compounds, they were neither quantified nor identified by 1H NMR in this investigation. Juice K levels were on average determined to be 1.71 ± 0.44 g/L , which is very similar to values reported for ‘Ruby’ pomegranate in Fawole and Opara . The genotypic variation in K among pomegranate cultivars was also reported in Hepaksoyet al. , who reported similar, though higher, potassium values for pomegranate juice. Haku Botan, the most acidic cultivar, was significantly different from Wonderful and had the highest K concentration in the juice, 2.30 ± 0.24 g/L.

Wonderful had a mean K concentration of 1.85 ± 0.15 g/L, which was similar to most other cultivars of high acidity with the exception of Parfianka a soft-seeded cultivar, which had significantly less K . Many low acid cultivars also had lower K values than Wonderful . This study presents the first known data on glutamate and glutamine concentrations in the juices of different pomegranate cultivars as measured by 1H NMR. There were significant differences among the cultivars in terms of glutamate . The mean concentrations of glutamate in the juices tested ranged from 0.28 ± 0.06 mM for ‘Haku Botan’ to 0.78 ± 0.30 mM for ‘Phoenicia’ , and one sample for ‘Loffani’ had a juice glutamate concentration of 1.50 mM. Phoenicia had significantly higher glutamate concentrations than many cultivars, especially Haku Botan and Sakerdze , while Wonderful had mean glutamate concentration of 0.53 ± 0.35 mM. Juice extraction method had a significant effect on the glutamate concentration , with aril-pressed juice having more glutamate than peel-pressed juice  . The mean concentrations of glutamine ranged from 0.68 ± 0.19 mM for ‘Haku Botan’ to 3.00 ± 1.59 mM for ‘Phoenicia’ . There were significant differences among pomegranate cultivars for glutamine, with Phoenicia having significantly higher concentrations than Haku Botan. The juice extraction method also had a significant effect on glutamine , with aril-pressed juice having greater concentration of glutamine than peel-pressed juice, with mean values of 2.1 and 1.4 mM, respectively .For the concentration of ethanol in the juice, Blaze had the highest mean concentration measured compared to all other cultivars, with mean ethanol juice concentration of 1.40 ± 0.79 mM, which was significantly different than Al Sirin Nar, Desertnyi, Evertsweet, Haku Botan, Parfianka, Sakerdze .

Cultivars with detectable quantities of ethanol had mean concentrations between 0.14 and 1.40 mM. The mean values for ‘Wonderful’ was approximately an order of magnitude higher than ethanol reported in Beaulieu et al. . Values for all other cultivars were in agreement with previous studies, with ethanol values less than 1.0 mM. Ethanol was not detected in the commercial 100% PomWonderfulTM pomegranate juice. Commercial juice samples analyzed in this study had values similar to freshly pressed ‘Wonderful’ fruit juice for many parameters quantified by 1H NMR . However, there were some parameters that were significantly different between commercially-packaged and -sold ‘Wonderful’ PomWonderfulTM fruit juice and freshly pressed pomegranate juice . Both pH and ethanol were significantly different between fresh ‘Wonderful’ juice and commercial juice. This is likely due to processing of the juice,raspberry plant pot which can include freezing during storage and the addition of pomegranate-based products into the juice. Cultivars that had many similarities to both Wonderful and commercial juice and would be good candidates for juice applications were Al Sirin Nar, Blaze, Desertnyi, Parfianka, Phoenicia, Purple Heart, and Sakerdze. If K concentration is important due to labeling issues, ‘Parfianka’ would be excluded from this list because its concentration is too low relative to K labeling standards in the USA.Regression analysis indicated AA to be correlated with TP , which is logical because phenolic compounds are responsible for much of the AA in pomegranate juice . Glutamate was found to be strongly correlated with glutamine which was anticipated since they are interconverted in nitrogen metabolism. Malate was correlated with citrate , as they are both in the citric acid cycle. Both glucose and fructose were individually correlated with TSS , which is logical considering the primary disaccharide in pomegranate is sucrose, which would be reduced to its constituents, glucose and fructose when the juice is expressed from the fruit . The results of this study reveal the diversity of the juice quality of the NCGR pomegranate germplasm, indicating both stark differences and striking similarities among these cultivars, many of which have the potential to be utilized in the food and beverage industries due to their physicochemical properties. Comparing the juice quality parameters of the tested cultivars to Wonderful indicated that there were cultivars with lower acid in the juice that meet the beverage market criteria, which may appeal to a wider base of consumers. Mean values of acidity variables were similar to those found in the literature. For example, the mean pH values of cultivars in the present study were similar to those reported in Beaulieu et al. and Fawole and Opara . Results for TA are in agreement with reports on pomegranate germplasm . This high quantity of TA of ‘Haku Botan’ explains the sour flavor described in Beaulieu et al. and Preece et al. and its flavor can be described as similar to a lemon, which has a similar range of TA, so it may serve as a condiment like lemon juice. In agreement with Beaulieu et al. , citrate was the primary acid in pomegranate juices for all cultivars except Eversweet, which was higher in malate than citrate and had an extremely low concentration of citrate . This could be a factor in the fruit and juice flavor, for cultivars like Eversweet, since malic acid is less tart than citrate and is the major organic acid found in apple cultivars . The values for malate agreed with those reported by Beaulieu et al. and Mphahlele et al. . Concentrations of glucose were in agreement with Mphahlele et al. . The juice extraction method had no significant effect on glucose, which is in agreement with Mphahlele et al. .

The °Brix results agreed with Beaulieu et al. , who reported a range of 15.9-17.7 °Brix for the cultivars that they studied from the NCGR pomegranate collection. The MI values were highly variable, which agrees with values reported in Beaulieu et al., . Pomegranate cultivars that fit the criteria for Wonderful in terms of MI are Al Sirin Nar, Blaze, Desertnyi, Haku Botan, Parfianka, Phoenicia, Purple Heart, and Sakerdze . These cultivars could be good candidates for the juice industry, with the exception of Haku Botan since it has a high acidity and lack of pigment and desirable color . Because pomegranate juice AA is widely used as a marketing point, it is important to note that there were no significant differences among the cultivars tested. Tehranifar et al. reported a range of 15.6-40.7% DPPH inhibition for twenty different Iranian pomegranate cultivars, which may reflect greater genetic diversity of the Iranian germplasm as compared to the USDA pomegranate germplasm. Mean values for TP are in agreement with previous germplasm studies on pomegranate . Mean TP of aril-pressed ‘Wonderful’ juice had a 3987 ± 374 mg/L GAE which was greater than value of 3117 reported by Gil et al. for fresh arilpressed ‘Wonderful’ juice. Differences in TP between this study and Gil et al., could result from differences in methodology or differences in fruit composition, with site and year being widely known to have significant effects on phenolics in fruit crops . There are several classes of phenolic compounds in pomegranate juice. Among them, anthocyanins, ellagitannins , ellagic acid and its derivatives, and hydrolyzable tannins are most important in contributing to the antioxidant effects of pomegranate juice . Many of these compounds are found in higher concentration in the peels than in arils, explaining the effect of juice type on both AA and TP, with peel-pressed juice having significantly higher TP than arilpressed juice . Pomegranate juice is known to have high concentrations of potassium . Additionally, K is known to affect juice flavor and consumer perception of acidity due to its interactions with organic acids, which influences buffering capacity . Because K is a nutrition labeling factor, it is important for the industry to know if a cultivar used for human consumption has significantly less K than the industry standard. However, most of the sweet-tart cultivars that are possible candidates for commercial juice had similar mean K quantities compared to Wonderful. The only exception to this was ‘Parfianka,’ which had significantly lower quantities of K compared to ‘Wonderful.’ For fresh market fruit, it is important to note that sweet or cultivars have less mean K than sweet-tart and tart cultivars. The values of K reported herein demonstrate that pomegranate is a K-rich food and beverage, and can provide a large proportion of the 4.7 g of K suggested as the daily nutritional requirement for adults. Glutamic acid can be an important compound in food because it is responsible for the umami flavor in food products and can only be detected when it is in its free form in the food product. Umami has a savory flavor . Additionally, Kader reported that glutamate can also contribute sourness to fruit flavors.