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                    U.S. DEPARTMENT OF AGRICULTURE
 Citrus and Other Subtropical Products Research: Fort Pierce, FL
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ARS Home Southeast Area Fort Pierce, Florida U.S. Horticultural Research Laboratory Citrus and Other Subtropical Products Research Research





- Plotto, Anne
- Bai, Jinhe
- 2023
- 2022
- 2021
- Publications
- Product Quality and New Uses (306)

Research Project: Determination of Flavor and Healthful Benefits of Florida-Grown Fruits and Vegetables and Development of Postharvest Treatments to Optimize Shelf Life an Quality for Their Fresh and Processed Products **Location:
                            Citrus and Other Subtropical Products Research** 2021 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**
                                

Objective 1. ARS researchers in Fort Pierce, Florida, obtained Citrus hybrids showing tolerance to Huanglongbing (HLB)from ARS collaborator for the fourth year. Fruit was washed, sanitized per protocol, juiced and frozen. Frozen juice from the previous year was prepared by ARS researchers in Fort Pierce, Florida, for sensory evaluation by organizing take-home panels during the Covid maximize telework. Taste panels were performed by panelists in their homes after virtual “Zoom” sessions for reviewing protocols from previous years. Frozen samples from the previous year harvest were chemically analyzed for flavor volatiles, sugars, bitter limonoids, and Candidatus Liberibacter asiaticus (CLas) content. Plans were made to blend some of the hybrids with standard Valencia or Hamlin juice in order to improve orange juice flavor when affected by HLB Strawberry selections from the University of Florida (UF) breeding program. Strawberries were harvested, juiced and frozen for later analysis of sugars, acids, and volatiles. Fresh fruit could not be evaluated by the trained taste panel because of Covid restrictions (limited number of people being present in the laboratory in one day).

Avocadoes from the ARS varietal collection were harvested monthly or bi-monthly, and allowed to ripen at room temperature (20 C). Fruit was only evaluated by ARS researchers in Fort Pierce, Florida, for ripeness, as none could be tasted due to personnel restrictions. This study establishes maturity windows for unknown cultivars or hybrids. Some hybrids seemed to show tolerance to laurel wilt and Phytophthora root rot, others withstood a period of cold weather. A collection of photos were taken by ARS researchers in Fort Pierce, Florida,for future use.
Twenty one papaya accessions from a University of Florida varietal collection were analyzed for aroma volatiles in the previous reporting period. Collaborator analyzed the data and a manuscript was submitted. 

Volatiles were sampled by ARS researchers in Fort Pierce, Florida, from microgreens of about 20 plant species in the previous reporting period, and were analyzed. Generally, volatile profiles of microgreens closely related to their plant classification. Some families, such as Brassicaceae species, all were similar in the major volatile components, but other families, such as Lamiaceae species, were greatly discriminated by the volatile profiles. A manuscript is in preparation.

Sugar apples from a varietal collection were sampled and sent home to employees for taste panels. Taste panel procedure were explained during a virtual “zoom” meeting. Fruit puree was frozen for later analysis of soluble solids, titratable acidity and volatiles.

Objective 2.
Commercial and experimental orange juice was spiked with orange seed protein extracts obtained by ARS collaborator, and frozen. Samples were tasted by a trained panel at home, after virtually (zoom) training for a new sensory technique. Four tests were conducted, with six to nine samples each. Some panelists were able to perceive differences between samples, with spiked samples being more bitter than control or having more citrus flavor, depending on panelists. However, the chosen base juice was very bitter, and the current seed protein extract was not sufficient to remove juice bitterness.

Spiking HLB-affected orange juice with the combination of volatiles and non-volatile compounds was not further explored due Covid 19 restrictions of research. 
An experiment was conducted to evaluate slow release carvacrol, a natural plant essential oil antibacterial, encapsulated in a pectin/sodium alginate matrix. Carvacrol effectively controlled postharvest decay, as well as fruit inoculated with Escherichia coli and Colletotrichum acutatum (anthracnose), without affecting fruit sensory quality.

A University of Florida collaborator sprayed grapefruit trees with commercial fungicides in an effort to control postharvest Diplodia stem-end-rot. PCR analysis of Candidatus Liberibacter asiaticus (by ARS scientist) in the fruit two and 15 days after fungicide application showed that presence of CLas in the fruit followed the same pattern as the development of the disease, hence confirming control of Diplodia by fungicides. 

Objective 3. 
Data obtained by ARS researchers in Fort Pierce, Florida, from in vitro human cell studies of the biological effects of citrus polymethoxylated flavone metabolites on hepatic lipid production were processed and compiled in a manuscript currently in preparation.  Some metabolites inhibited lipid production while others exhibited weak stimulation.  No overall structure/function relationships could be determined.  Yet these results contribute to a growing body of evidence evidence that not all polymethoxylated flavones exhibit the same biological effects, as also their metabolites.  
In a collaborative study, extracts of separate tissues of four major processing oranges and one grapefruit, healthy and HLB-affected fruit, were prepared by ARS researchers in Fort Pierce, Florida. HLB status was confirmed by qPCR to determine the CLas and CLam pathogens. Samples were taken for visual quality, sugar, acids, pectin, peel oil, juice volatile, flavonoids, limonoids, and carotenoids analysis. Most of chemical analysis were completed except for carotenoids. Differences were observed in the chemical compositions of individual portions of the fruit, and with HLB effect.  Studies of additional unknown non-volatile compounds by mass spectra are being continued.  Pectin isolated from HLB infected grapefruit exhibited a significantly lower yield and low degree of methylesterification (DM) than healthy fruit. These will impact the food industry using pectin as HLB continues to spread around the world. This will result in lower pectin yields, and altered pectin physiochemical properties. **Accomplishments**
                                

1. A nutritional foliar field spray maintained quality of orange oil from Huanglongbing affected fruit. Huanglongbing (HLB) is a devastating citrus disease that has affected citrus production world-wide. Growers have tried modifying cultural practices to keep their trees in production, including boosting nutrient uptake with foliar sprays. ARS researchers at Fort Pierce, Florida, have earlier demonstrated that the effect of some foliar spray programs on fruit quality were not consistent over the years, but the effect on peel oil composition had not been explored. HLB negatively altered peel oil of ‘Valencia’ oranges by increasing oxygenated terpenes and decreasing typical orange volatiles such as aldehydes (octanal, nonanal and decanal) and valencene in comparison with oil from healthy oranges. A nutritional spray program maintained a compositional profile of cold pressed oil similar to that of healthy oranges. Cold pressed citrus oil represents a $6-7 billion industry. 2. Different juice extractor types result in orange juice with different composition without affecting health benefits. It is often believed that a freshly squeezed orange contains more nutrients than commercially processed orange juice. A study was conducted by ARS researchers at Fort Pierce, Florida, and collaborators, comparing hand-squeezed and two types of commercial orange juice extractors, reamer extractor (RE) and shear force extractor (SFE).  The study was done with fruit harvested before the HLB pandemic (healthy fruit), and recently with fruit from HLB-affected trees. In general, juice obtained from SFE had more peel tissue fragments, pectin and associated secondary metabolites, including flavonoid glycosides and carotenoids, while juice from RE processing contained more peel oil and polymethoxylated flavones. The hand squeezed juice profile was closer to that of RE processed juice, but with less peel oil. All juice metabolites have demonstrated health benefits, although the high peel oil content in RE juice makes carotenoids and some flavonoids more bioavailable. On the other hand, excess flavonoids or peel oil in orange juice can induce bitterness, which is already perceivable in juice from HLB-affected fruit. This study demonstrates that all forms of orange juice have health benefits for consumers. Processors can select which extractor type depending on other targeted quality components. **Review Publications**
                            *Imahori, Y., Bai, J., Baldwin, E.A. 2021. Effect of storage temperature on chilling injury and activity of antioxidant enzymes in carambola "Arkin" fruit. Journal of Food Processing and Preservation. https://onlinelibrary.wiley.com/doi/epdf/10.1111/jfpp.15178.*
                                *Miranda, M., Sun, X.N., Ference, C.M., Plotto, A., Bai, J., Wood, D.R., Assis, O., Ferreira, M., Baldwin, E.A. 2021. Nano- and micro- carnauba wax emulsions versus shellac protective coatings on postharvest citrus quality. Journal of the American Society for Horticultural Science. 146(1):40-49. https://doi.org/10.21273/JASHS04972-20.*
                                *Fan, Z., Plotto, A., Bai, J., Whitaker, V. 2021. Volatiles influencing sensory attributes and Bayesian modeling of the soluble solids-sweetness relationship in strawberry. Frontiers in Plant Science. 12:640704. https://doi.org/10.3389/fpls.2021.640704.*
                                *Spricigo, P., Freitas, T., Purgatto, E., Mitsuyuki, M., Ferreira, M., Corrêa, D., Bai, J., Brecht, J. 2021. Visually imperceptible mechanical damage of harvested tomatoes changes ethylene production, color, enzyme activity, and volatile compounds profile. Postharvest Biology and Technology. https://doi.org/10.1016/j.postharvbio.2021.111503.*
                                *Carvalho, Jhonathan, Ramadan, Dania, Gonçalves,Vinicius, Brunetti, Iguatemy, Cesar, Thais  B., Manthey, J.A., Spolidorio, Luís C. 2021. Impact of citrus flavonoid supplementation on inflammation in lipopolysaccharide-induced periodontal disease in mice. Food & Function. https://doi.org/10.1039/D0FO03338C.*
                                *Nery Da Silva, M., Ferriera, P.S., Goncalves, D.R., Spolidorio, L.C., Manthey, J.A., Cesar, T.B. 2021. Tangeretin and heptamethoxyflavone decrease insulin resistance, fat accumulation and oxidative stress in a study of C57BL/6J mice fed a high-fat diet. Journal of Food Science and Nutrition. https://doi.org/10.1002/fsn3.2167.*
                                *Oh, Y., Barbey, C.R., Chandra, S., Bai, J., Fan, Z., Plotto, A., Folta, K., Whitaker, V., Lee, S. 2021. Genomic characterization of the fruity aroma gene, fafad1, reveals a gene dosage effect on y-decalactone production in strawberry (fragaria ×ananassa). Frontiers in Plant Science. https://doi.org/10.3389/fpls.2021.639345.*
                                *Loayza, F., Brecht, J., Simonne, A., Plotto, A., Baldwin, E.A., Bai, J., Lon-Kan, E. 2020. A Brief Hot-water Treatment Alleviates Chilling Injury Symptoms in Fresh Tomatoes. Journal of the Science of Food and Agriculture. 101:54-64. http://dx.doi.org/10.1002/jsfa.10821.*
                                *Loayza, F., Brecht, J., Simonne, A., Plotto, A., Baldwin, E.A., Bai, J., Lon-Kan, E. 2020. Synergy between hot water treatment and high temperature ethylene treatment in up-regulating the antioxidant system in mature-green tomatoes. Postharvest Biology and Technology. 170:111314. https://doi.org/10.1016/j.postharvbio.2020.111314.*
                                *Du, X., Sissons, J., Shanks, M., Plotto, A. 2021. Aroma and Flavor Profile of Raw and Roasted Agaricus bisporus Mushrooms Using a Panel Trained with Aroma Chemicals . LWT - Food Science and Technology. 138:110596. https://doi.org/10.1016/j.lwt.2020.110596.*
                                *Deterre, S., Mccollum, T.G., Leclair, C., Manthey, J.A., Bai, J., Baldwin, E.A., Raithore, S., Stover, E.W., Plotto, A. 2020. Effect of Poncirus trifoliata on chemical composition of fruits in pedigrees of Citrus scion hybrids. Scientia Horticulturae. 277. https://doi.org/10.1016/j.scienta.2020.109816.*
                                *Marin, A., Baldwin, E.A., Bai, J., Wood, D.R., Ference, C.M., Sun, X.N., Brecht, J., Plotto, A. 2021. Edible coatings as carriers of antibrowning compounds to maintain appealing appearance of fresh-cut mango. HortTechnology. https://doi.org/10.21273/HORTTECH04687-20.*
                                *Li, Q., Li, T., Baldwin, E., Manthey, J.A., Plotto, A., Zhang, Q., Gao, W., Bai, J., Shan, Y. 2021. Extraction method affects contents of carotenoids and flavonoids in Huanglongbing-affected ‘Valencia’ orange juice. Foods. https://doi.org/10.3390/foods10040783.*
                                *Sun, X.N., Yang, H., Zhao, W., Bourcier, E., Baldwin, E.A., Plotto, A., Irey, M., Bai, J. 2021. Huanglongbing and foliar spray programs affect the chemical profile of “valencia” orange peel oil. Frontiers in Plant Science. https://doi.org/10.3389/fpls.2021.611449.*
                                *Brewer, S., Plotto, A., Bai, J., Crane, J., Chamber, A. 2021. Evaluation of 21 papaya (Carica papaya L.) accessions in southern Florida for fruit quality, plant height, and yield components. Scientia Horticulturae. 288:110387. https://doi.org/10.1016/j.scienta.2021.110387.*
                                *Sun, X.N., Cameron, R.G., Plotto, A., Zhong, T., Ference, C.M., Bai, J. The effect of controlled-release carvacrol on safety and quality of blueberries stored in perforated packaging. Foods. 10:1487. 2021. https://doi.org/10.3390/foods10071487.*
                                *Ferreira, Paula S., Manthey, J.A., Nery, Marina S., Cesar, Thais B. 2021. Pharmacokinetics and biodistribution of eriocitrin in rats. Journal of Agricultural and Food Chemistry. 10.1021. https://doi.org/10.1021/acs.jafc.0c04553.*


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