The values for glutamate reported for pomegranate herein are greater than values reported for grape juice , indicating increased importance for consumers regarding marketing fraud in the pomegranate juice industry because often grape juice is used as the primary adulterating agent in pomegranate juices in the USA, along with apple and pear juice. Glutamine is the amino acid of the highest concentration in human blood , which may play into the folklore regarding pomegranate juice as a “blood tonic” . Ethanol is of importance in the food and beverage industries because it is an indicator of anaerobic respiration and metabolism in post harvest fruit products. Along with other alcohols, ethanol can contribute to off flavors or even enhance the flavor of fruit if concentrations are low . The freshly expressed juice had ethanol values that were far less than the levels prohibited by countries for religious and food safety reasons, which is typically required to be less than 0.5% ethanol in the USA for the beverage to be considered non-alcoholic; religious restrictions are typically stricter.This work demonstrates the high level of phenotypic diversity of pomegranate juices that exists in approximately 5% of the USDA available pomegranate germplasm collection. This study is the first of its kind in utilizing 1H NMR coupled with conventional post harvest juice quality methodologies to assess differences among pomegranate cultivars for metabolic, physicochemical and nutritional traits. The results indicated a great complexity of juice quality and nutritional differences among the cultivars analyzed, with many fitting the profile of Wonderful, but others differing greatly from this standard. As a replacement,blueberry production alternate or substitute candidate for ‘Wonderful’ in juice markets, ‘Al Sirin Nar,’ ‘Blaze,’ ‘Desertnyi,’ ‘Parfianka,’ ‘Phoenicia,’ ‘Purple Heart,’ and ‘Sakerdze,’ all meet a host of juice quality parameters and mostly fit the nutrition composition of ‘Wonderful.’

These varieties should be considered for further investigation via cultivar trials to determine phenotypical traits important to growers and taste panels to determine preferences of consumers. There were striking differences between the commercial juice and fresh-squeezed juices as well as significant differences between juice extraction methods for many parameters, including amino acid content, phenolic content and antioxidant activity. Potassium concentration varied greatly among cultivars, which can affect the flavor and nutritional composition of these fruits and their juices. This work also presents further evidence that pomegranate is not only a potentially healthy fruit in terms of phenolics and antioxidant activity, but also for nutrition as it relates to amino acids and mineral nutrition . It is important to note that these juice quality traits and nutritional factors can be significantly different among cultivars. Whether the cultivars with unique quality profiles appeal to consumers will need to be investigated in future research. This study is the first of its kind comparing fresh-pressed juice quality of the industry standard, Wonderful, with 13 other NCGR pomegranate cultivars using 1H NMR coupled with other physicochemical analytical techniques. Pomegranate is a deciduous tree crop that has been domesticated for thousands of years for its fruit, flowers, bark, and leaves , all of which have been believed to possess medicinal properties . Despite its long history of cultivation, limited horticultural information is available for growers, breeders, and the food and beverage industries about when fruit of a given cultivar is ready for harvest, processing and consumption . In the United States, Wonderful, the industry standard, is a tart, acidic, moderately hard-seeded fruit that has been reported to have astringent and bitter juice compared to other cultivars previously analyzed from the collection at the United States Department of Agriculture – Agricultural Research Service National Clonal Germplasm Repository , Davis, CA .

Despite these negative fruit quality traits, pomegranate cultivation in the United States remains predominantly a monoculture of ‘Wonderful.’ It is believed that cultivars in the national germplasm with desirable traits, such as soft seededness and low acidity may be candidates for commercial production. Studies have demonstrated a large variation in mature fruit size within commercial orchards of ‘Wonderful,’ which poses a problem for fresh market growers and packers. Wetzstein et al. reported a greater than five-fold range in mature fruit volume and weight in commercial ‘Wonderful’ pomegranate groves. Factors that influence fruit size and yield include aril number , cultivar , cultural practices , and plant maturity . Finding cultivars with better uniformity than Wonderful would be beneficial to the industry In addition to variable fruit size, dates of fruit maturity can play a major role in fruit quality. Late season harvests run the risk of fall rains, which have been associated with greater numbers of split fruit . Typical commercial harvest windows for ‘Wonderful’ range from late September to early November, but fruit in the Central Valley of California, USA, where the most pomegranate cultivation occurs, are typically ready to harvest starting in late October. Usually by November, effects of weather, especially rains, and pests will begin to damage the fruit. Therefore, harvest date can determine whether a cultivar is a good candidate for commercial production. In addition to the fresh fruit market, pomegranates are also utilized for juice. The beverage and wine industries utilize different fruit juices that have sufficient quantities of organic acids, carbohydrates , and phenolic compounds. Concentrations of total soluble solids , often expressed in ºBrix, for commercial pomegranates range from 12% to 16% at maturity. It is recommended that ‘Wonderful’ have at least 15% TSS at harvest , but above 17% is preferable . Hasnaoui et al. reported that citric acid is the determinant of sour flavor in pomegranate juice, despite sugar concentration. Sweet pomegranates typically have been reported to have citric acid concentrations less than 0.50% . Standards for fruit maturity of sweet cultivars are being investigated because growers often pick early-season cultivars too early in order to increase profits .

There are no known imposed regulations on growers in any country, meaning they can harvest early before fruit maturity without short-term consequence. The effect of picking early on consumer perception and acceptance of pomegranate fresh fruit has been shown to be associated with astringency and a low flavor preference score . Standards have been proposed for titratable acidity and total soluble solids for ‘Wonderful’ pomegranate . Generally, citric acid is the most abundant organic acid in pomegranate juice, so TA is generally expressed in citric acid equivalents. ‘Wonderful’ pomegranate fruit is considered mature when juice is lower than 1.85% TA , so fruit are picked when fruit measure below that threshold. Maturity index is a standardized measure of maturity in many fruit crops. For pomegranate, a commonly used MI is the ratio of °Brix to TA, also known as the sugar to acid ratio. This ratio is often used to determine fruit maturity, but it has been found to not be usable for sweet cultivars. Instead, fresh aril weight is used at the indicator of fruit maturity and consumer acceptance in the sweet cultivars . The optimum MI for ‘Wonderful’ has been calculated to be greater than 8.1, at which point the fruit is ready for harvest. Other cultivars may have significantly different quantities of organic sugars and acids, so MI is logically different for different types of pomegranates . Fruit quality is not only related to sugar content, titratable acidity and spoilage, but also to phenolic compounds that contribute to the fruit’s flavors, antioxidant activity, and color . Cultivar is more influential in determining fruit juice composition than site of cultivation, year of harvest, or length of storage ,blueberry in container so it is important to study differences in traits among cultivars to identify superior cultivars in germplasm resources and make them available to growers and consumers. Determining levels of phenolics and the antioxidant activity of a cultivar’s juice is important to the beverage industry and consumers, because advertisements promote high antioxidant activity as the main selling point of juice products. If any cultivar were to demonstrate similar antioxidant activity to Wonderful, it would possibly be competitive in the pomegranate market were it to meet other consumer preferences. Having lower antioxidant activity than ‘Wonderful’ could make for an undesirable candidate for commercial production, although the public is unlikely to be able to detect differences in antioxidant activities. The objectives of this research were: 1) to evaluate fruit and juice quality traits of NCGR germplasm by comparing commercial quality parameters to the industry standard, Wonderful; and 2) to determine potential harvest windows of ten preselected pomegranate cultivars, based on seasonal trends of potential maturity indices with harvest dates. Fruit were harvested from the USDA-ARS National Clonal Germplasm Repository for Tree Fruit and Nut Crops and Grapes in Davis, CA, USA for two seasons. The trees were all over nine-years-old and in full maturity. The pomegranate cultivars analyzed included: Ambrosia, Desertnyi, Eversweet, Golden Globe, Green Globe, Haku Botan, Loffani, Parfianka, Phoenicia, and Wonderful . The cultivars in this study that have been described as soft-seeded included Desertnyi, Eversweet and Parfianka. Low acid cultivars included Ambrosia, Desertnyi, Eversweet, Golden Globe, Green Globe, and Loffani. Wonderful fresh fruit was included as a control and as the standard to compare the other cultivars in this study. All cultivars are of American origin except for Haku Botan and Desertnyi and Parfianka . Up to twelve fruit of each cultivar were hand-harvested in mid-September, midOctober, and mid-November in 2014. This was repeated in 2015.

Fruit were ground shipped, and then stored at 6 °C and 98% relative humidity for 3-4 weeks until processing. Fresh market quality fruit, as defined by being well-filled, mature, and unblemished, were chosen for juice analysis from each of the 10 cultivars. Three cultivars, Golden Globe, Green Globe and Parfianka, had fruit damage in November, so less fruit were available for these cultivars during November harvest dates. ‘Golden Globe’ and ‘Parfianka’ had no fresh market quality fruit available in November 2015 and ‘Green Globe’ had no fresh market fruit available for 2014 and 2015. At this last season harvest, the trees of these cultivars had fruit that were either cracked or infested with insect pests, especially leaf footed bug.Up to 12 fruit per cultivar were weighed using a tared digital scale. The equatorial diameter and stem- to blossom-end length of each fruit and calyx length and diameter were measured with a digital caliper. After weighing and measuring, fruit were opened by scoring the peels of the fruit longitudinally with a scalpel. Arils were then manually extracted from a subsample of five fruit. Weight of 100 arils and total aril weight was determined with a tared digital scale. Only intact, non-damaged arils were weighed for the weight of 100 arils to reduce error. Edible fruit fraction was determined by dividing weight of all arils by the total weight of fruit and converting the result to percent. For juice processing, fruits were halved and 100 undamaged arils were manually removed and placed in a polyethylene bag. These arils were pressed manually with approximately 480 Newtons of force to express the juice directly into a 15 mL test tube. Raw juice samples were transferred to a centrifuge tube via pipette and were centrifuged at 1000 g for 5 min using a Becton Dickinson DYNAC Centrifuge . Aliquots of the supernatants were used for all chemical analyses. 5.2.3 °Brix Degrees Brix, also known as total soluble solids, were quantified with a Vee Gee Scientific PDX-1 Digital Refractometer , utilizing 0.5 mL sample of expressed, centrifuged juice. Individual juice samples, each one representing a single whole fruit, were transferred to the refractometer sample reservoir with a hand pipettor. The sample reservoir was cleaned by spraying with deionized water with a wash bottle and wiped dry with Kimwipes before each subsequent sample. Results were reported in °Brix and represented the relative percent sugar content of the centrifuged pomegranate juices. One measurement was taken for each juice sample. Juice TA was measured with a Hanna HI 84532 fruit juice TA mini-titrator . Samples were prepared by mixing individual centrifuged juice aliquots with 45 mL of deionized water. Each sample represented the juice of one whole fruit and 3 to 5 samples were analyzed for TA per cultivar. Because the predominant organic acid in most pomegranates is citric acid, results are expressed in citric acid equivalents, which represent percentage citric acid in juice solution.