Location: Citrus and Other Subtropical Products Research
2023 Annual Report
Objectives
Objective 1: Establish commercially usable chemical and sensory characteristics of new citrus, strawberry, and avocado genotypes and new crops (microgreens) from subtropical and tropical climates.
Sub-Objective 1a: Develop chemical and sensory profiles of Citrus hybrids tolerant to citrus greening disease or Huanglongbing (HLB).
Sub-Objective 1b: Identify chemical and sensory characteristics for Florida-grown (avocado, strawberry, peach) and new crops (microgreens, tropical fruit) adapted to tropical/subtropical regions.
Objective 2: Enable real-time, commercial pre- and postharvest treatments to optimize shelf life of new genotypes and new crops using packaging, coatings, aqueous treatments, sanitizers, maturity markers and/or addition of flavor modulators.
Sub-Objective 2a: Develop methods to mask undesirable bitterness in orange and other citrus juice using flavor modulators.
Sub-Objective 2b: Develop novel deliveries of antimicrobial volatiles and/or plant essential oils using microencapsulated beads and/or coating technology, combined or not with modified atmosphere packaging to prevent decay in packaged fruit.
Objective 3: Isolate and test biomarkers in fruits (citrus, small fruit) with unique taste, flavor and healthful qualities for better commercial management strategies.
Sub-Objective 3a: Identify the best flavor combinations for an ideal orange or citrus juice.
Sub-Objective 3b: Identify the best flavor combinations for an ideal fresh strawberry.
Sub-Objective 3c: Identify biomarkers in citrus with unique taste, flavor and healthful qualities.
Approach
Fruits from breeding programs will be evaluated for eating quality and storability using sensory evaluations, chemical and texture analyses. For citrus, hybrids tolerant to HLB will be considered for juice quality and blending, in addition to eating quality as fresh fruit. Other fruit will include strawberries, peaches, avocadoes, tropical fruit (papaya, vanilla). A new crop, microgreens, will be included in the evaluations. For citrus and strawberries, sensory and flavor data will be statistically modelled in order to establish either ideal fruit quality markers, or criteria for non-acceptability, that can be used by breeders during the selection process. Furthermore, for citrus, identification of chemical off-flavor/off-taste targets will serve as a basis to test various flavor modulators that could mask undesirable flavors in orange juice. Potential flavor modulators include modified proteins, peptides, or amino acid, and other chemical family molecules will be tested when available. For strawberries, models will be validated by reconstitution experiments. For peaches, samples will consist of a diversity of new and old cultivars, some obsolete while others having withstood a long commercial life. The chemical fingerprint will help breeders understand the differences among genotypes for continuous effort in breeding and selection of new cultivars. For tropical fruit and microgreens, all being new crops, data will be more exploratory and descriptive than hypothesis-driven. Postharvest evaluations will be performed to test new methods of delivery of volatile antimicrobials in the form of spray-dried slow release powder placed in small fruit (strawberries, blueberries) clamshells. Control of postharvest decay as well as residual taste of volatiles will be evaluated. In citrus, Diplodia stem-end rot, due to Lasiodiplodia theobromae, has become more prevalent in citrus infected by HLB. This project will evaluate pre- and post-harvest fungicide treatments to control Diplodia stem-end rot. Finally, this project will evaluate phytochemical biomarkers in orange juice that have biological activity in mammals. Many flavonoids in citrus have been shown to have beneficial effects in human chronic diseases. Metabolites of flavonoids fed to experimental animals were previously extracted and isolated from various organs (liver, kidneys); they remain to be identified and quantified. Furthermore, their bioactivity will be evaluated in in vitro tests with emphasis on inflammation.
Progress Report
Citrus hybrids with various degrees of tolerance to HLB were harvested, washed and juiced. Hybrids had diverse genetic backgrounds, including oranges, mandarins and mandarins with Poncirus in their background (Poncirus-introgressed hybrids). Taste panels with trained panelists and instrumental analysis (soluble solids, titratable acidity, limonoids, volatiles) were performed for juice from the 2021-2022 season, completing data collection for about 60 hybrids over six seasons. Hybrids were classified in different groups of flavor characteristics including orange, mandarin, or grapefruit flavor, sweetness, sourness and bitterness. Off flavor descriptors included bitterness, typical “poncirus” off flavor (soapy, metallic, acrid), “pumpkin”, and general off flavor. Thirteen hybrids, each having a unique flavor characteristic, were chosen for further analysis of flavonoids. Volatile analysis showed that oranges and mandarin hybrids formed two distinct groups, while hybrids with poncirus in their background had profiles spanning a wide range of terpenes, alcohols and aldehydes. Volatiles (esters and some sesquiterpenes) along with flavonoids eriocitrin and quercetin 3-(3R-glucosylrutinoside) were positively correlated with orange flavor, while ß-ionone and eucalyptol were highly abundant in the mandarins. Some flavonoids were more abundant in poncirus hybrids with strong off-flavor and bitterness.
The presence and abundance of Candidatus Liberibacter asiaticus (CLas), the presumed agent causal of HLB disease, was measured in the juice of the hybrids from the 6-year study using qPCR and Li and LJ primers. It was hypothesized that CLas content could be used as an indicator of juice quality across citrus species. There was no clear correlation between CLas titer and sensory scores across genotypes, but there was a trend within the mandarin group, which will be further pursued.
Blends were made with Valencia and Hamlin juice, following industry standard practices (Valencia:Hamlin at 60:40 ratio – base juice), and selected mandarin or mandarin hybrids with Poncirus in their background, at 10% (maximum allowed as per 21 C.F.R. §§146.135, .140; .141, .146 and .151) or up to 20% ratio. Blends with 10% mandarin juice were not statistically different from the base juice, while when adding 20% mandarin juice, trained panelists could perceive increased mandarin flavor.
In the 2022-2023 season, two of the best hybrids identified in previous years and two oranges with presumed tolerance to HLB were processed at the JBT pilot plant and pasteurized, following standard industry practices. Blends were prepared with 10%, 15% or 20% of mandarin hybrids and compared with the 60:40 Valencia:Hamlin base juice. Untrained panelists staff of the U.S. Horticultural Research Laboratory (USHRL) were asked whether they could perceive differences between the base juice and the blend. In all cases, panelists could not detect differences between samples (various levels of statistical significance). Those who could perceive differences between the blends (less than 50% panelists) indicated they liked both juices, with no trend for preference, sweetness or orange flavor for either base or mandarin blend.
Strawberry selections from the University of Florida breeding program were tasted by the trained sensory panel in January, February and March, typical season in Florida. Fruit were also sampled and analyzed for titratable acidity, soluble solids content and volatiles.
Microgreens are young, tender, and flavorful greens that are harvested after sprouting from seeds, typically within 7-21 days. In our experiment, amaranth was chosen as the experimental material to assess flavor and nutritional value when grown under various LED light spectra. The findings revealed that microgreens grown under red + blue lights, with or without green light, exhibited enhanced redness, tender texture, and grassy and green top notes, while showing reduced sulfur off-flavor. Additionally, these microgreens contained higher levels of vitamin E and carotenoids, but lower levels of flavonoids and vitamins other than vitamin E. The utilization of red + blue lights with or without green lights resulted in increased sugar and acid content, enhancing sweetness and sourness. However, a decrease in most amino acids and nucleosides, which contribute to rich and complex flavors, was also observed.
Jicama tubers in the diet have the potential to enhance insulin sensitivity, making them suitable as an ingredient in low-sugar juice. In the 2022-23 season, an experiment was conducted to compare the quality of juice samples extracted from tubers of two cultivars, grown in different soils, and harvested at three maturities. The early harvested tubers had a lower yield. However, higher antioxidant capacity was found in early harvested tubers.
Vanillin is the primary compound responsible for the characteristic taste and flavor associated with vanilla. It is crucial to elucidate the metabolic processes involved in the production of vanillin to improve efficacy in breeding and selecting vanillin-rich varieties. Samples extracts from the University of Florida vanilla breeding program were analyzed by liquid chromatography coupled with dual mass spectrometry (LC-MS/MS) for vanillin, and its intermediates in the biosynthetic pathway. Data analysis is in progress.
Valencia and Hamlin orange juice from the 2021-22 season was provided to CRADA partner who mixed them in a 60:40 ratio as described above, and spiked with citrus protein isolates. A taste panel was performed with untrained panelists staff of USHRL. Panelists could clearly perceive the difference between spiked and non-spiked juice, and usually preferred unspiked juice.
Thymol, the main volatile component in thyme essential oil and with proven general antimicrobial activity, was tested in vitro and in vivo against two postharvest pathogens, Lasiodiplodia theobromae, causal agent of citrus stem-end rot (SER), and Penicillium digitatum, causal agent of green mold in citrus (GM). In vitro assays determined that 8 ppm delivered in the volatile phase achieved the completed inhibition of L. theobromae, while only 4 ppm was necessary to completely inhibit GM P. digitatum. In the in vivo assays with inoculated grapefruit, a 50-ppm thymol treatment resulted in 81% reduction in SER incidence and 90 % reduction in visible lesion size, while the same concentration of 50 ppm thymol reduced 90 and 99% of GM incidence and lesion sizes, respectively.
Thymol was also microencapsulated in a pectin-alginate matrix to be released in small doses during extended storage. This delivery system reduced decay incidence from gray mold on blueberries inoculated with Botrytis cinerea in commercial clamshell packaging. More experiments were designed to test the antimicrobial activity of microencapsulated thymol on berries packed in commercial clamshells and in modified humidity clamshells (lower venting). The combination of controlled-release thymol with low-venting packaging effectively controlled decay, reduced weight loss and fruit shrinkage of blueberries. The same treatments had the reverse effect on blackberries: package with low venting increased blackberry fruit decay likely due to higher humidity in the clamshell, and thymol induced fruit discoloration (color reversion).
Data obtained under Objective 1a were compiled for all six years (2016-17 till 2021-22). Statistical models were developed using data from years 2016-17 to 2020-21, and the best model was validated with data from year 2021-22. The model identified 26 compounds important for orange flavor, among which seven esters were determined to be key to distinguish orange flavor from mandarin flavor. Six of the seven esters appeared to be under the same genetic control. Differential gene expression analysis between ester producers and non-producers identified a novel alcohol acyltransferase gene controlling ester production in orange. Its biosynthetic activity for both straight- and branched- chain esters was validated via transient overexpression assays in citrus.
Bitterness in citrus is typically induced by flavonoids or limonoids, which constitute a large portion of citrus nonvolatile compounds. The chemical structures of citrus flavonoids are incredibly diverse and small substitutions to a common backbone structure can significantly change the biological activity and taste. Another group of compounds, limonoids, can be found in citrus in the form of their precursor as a ring lactone (tasteless), or open (bitter). In order to identify the flavonoid and limonoid composition of citrus with a wide range of bitterness, eleven varieties, including Poncirus hybrids, oranges, mandarins, pummelos, grapefruit, and a tangelo were harvested and juiced. Juice was tasted by trained panelists and rated for bitterness on a scale from 0 to 15, using a reference standard. Samples were analyzed by a newly developed, widely targeted LC-MS/MS method including 55 flavonoids and 8 limonoids. As expected, known bitter contributors, limonin, nomilin, naringin, poncirin, and neohesperidin, were positively correlated with the bitterness ratings from the panelists. Two aglycones, apigenin and tricin, were correlated with bitterness on the same level as the previously mentioned bitterness contributors. Tricin, a polymethoxylated flavone, is newly identified in citrus.
Accomplishments
1. White strawberries, a new specialty fruit. Florida’s strawberry season encompasses December through March, with demand of high value specialty products during the Christmas holidays. White fruited strawberries originated in South America have the potential to fill such a niche market, but wild type fruit of Fragaria chiloensis have low yield and produce small and soft fruit. A result of several crosses with modern cultivars, Florida Pearl®‘FL 16.78-109’ is a white fruited strawberry with red achenes and a pink blush when ripe. ARS researchers at Fort Pierce, Florida, determined in sensory taste panels, that fruit of Florida Pearl® have similar firmness, sweetness and strawberry flavor than the two most commercially grown cultivars in Florida, ‘Florida Brilliance’ and Sweet Sensation®‘Florida 127’. On the other hand, sourness and titratable acidity were consistently lower than the two commercial cultivars, giving this variety a unique low-acid flavor. Florida Pearl® has been available in retail markets as a high-end fruit, and is being grown in Florida, California and Spain thus far.
2. Deciphering orange flavor and providing a new genetic tool for citrus breeders. Huanglongbing (HLB), or Citrus greening disease, has decimated citrus production in Florida and world-wide. The orange tree (Citrus sinensis) is sensitive to the disease and cultivars grown for juice have been particularly affected, impacting Florida’s agricultural economy. Over the course of six years, researchers in Fort Pierce, Florida, have analyzed hybrids showing various degrees of tolerance to the disease. Plant material included oranges, mandarins and mandarin hybrids with Poncirus in their background, a close relative of citrus imparting tolerance to cold and diseases. This large pool of data revealed that esters were necessary to impart orange flavor to juice, distinguishing it from mandarin juice. Further analysis identified a key gene in the ester pathway, and its expression was validated in citrus. A DNA marker was developed which could be used in marker-assisted breeding. This work will greatly expand the breeders’ toolbox to develop orange-like cultivars in a time of need. As new hybrids tolerant to HLB become available, processors will have the options to choose cultivars to produce juice with more orange-like or mandarin-like flavor.
Review Publications
Cruz, M., Ferrarezi, R., Plotto, A., Bai, J., Rui, L. 2023. Effect of Huanglongbing on the volatile organic compound profile of fruit juice and peel oil in ‘Ray Ruby’ grapefruit. Foods. 12:713. https://doi.org/10.3390/foods12040713.
Bai, J., Rosskopf, E.N., Jeffries, K.A., Zhao, W., Plotto, A. 2023. Soil amendment and storage effect the quality of winter melons (benincasa hispida (thunb) cogn.) and their juice. Foods. 12(1): 209. https://doi.org/10.3390/foods12010209.
Whitaker, V., Dalid, C., Osorio, L., Peres, N., Verma, S., Lee, S., Plotto, A. 2023. Florida Pearl® ‘FL 16.78-109’ Pineberry. HortScience. 58:143-146. https://doi.org/10.21273/HORTSCI16951-22.
Jia, L., Wang, L., Xia, Q., Luo, W., Qiao, X., Zhang, X., Li, J., Baldwin, E., Jiang, L., Zhao, Y., Cao, Z., Wang, L., Bai, J. 2023. Expression patterns of volatile vompounds during ‘FL 47’ tomato ripening and their response to exogenous methyl salicylate (MeSA) fumigation. Postharvest Biology and Technology. https://doi.org/10.1016/j.postharvbio.2023.112414.
