Location: Citrus and Other Subtropical Products Research
2018 Annual Report
Objectives
1. Enable, from a technological standpoint, new commercial products from pectic hydrocolloids derived from citrus processing.
2. Characterize and quantify bioactive flavonoid compounds from byproducts of citrus processing, determine their in vivo pharmacokinetics and bioavailability; and enable a new commercial delivery of bioactive flavonoids in food and feed by encapsulation with pectic hydrocolloids.
2A. Characterize and quantify bioactive flavonoid compounds from byproducts of citrus processing, determine their in vivo pharmacokinetics and bioavailability.
2B. Enable a new commercial delivery of bioactive flavonoids in food and feed by encapsulation with pectic hydrocolloids.
3. Enable a novel immunologically-based assessment of structural quality and functional properties of citrus pectin in raw and processed foods and industrial products.
Approach
Experimentation is required to determine the necessary time, temperature and concentration conditions to enable pilot-scale functionalization of the released pectic hydrocolloids from steam explosion of peel material. Response surface methodology will be used to determine these parameters using analytical methods. Consequently, the variables of temperature, time and concentration of steamed peel waste will be manipulated to determine optimal conditions for functionalizing the released pectic hydrocolloids. Functionality will be assessed by measuring resulting calcium induced viscosity using a concentric cylinder viscometer and/or oscillatory measurements using a stress controlled rheometer and related to final degree of methylation, charge distribution and molecular weight (MW) of the modified pectic hydrocolloids. Compositional analysis and structural properties will be characterized by Size Exclusion Chromatography (SEC) coupled to Multi Angle Laser Light Scattering (MALLS), Refractive Index (RI) or Conductivity Detectors; High Performance Anion Exchange Chromatography (HPAEC) coupled to an Evaporative Light Scattering Detector (ELSD) or Pulsed Amperometric Detector (PAD) and enzymatic/chemical methods. Composition of the polysaccharides present in peel wash after steam explosion will be determined by enzymatic hydrolysis and liquid chromatography with electrochemical detection. Use of polysaccharide specific enzymes (arabinase, arabinofuranosidase, etc.) will allow for determination of the contribution of individual polysaccharides. Pectin populations will be examined via interaction with antibodies that bind to specific structural epitopes on individual pectin molecules. Pectin populations will be produced by enzymatic and/or chemical methods that contain various sizes of ionically-charged or neutral, methyl-protected domains. Elucidation of the modes of anti-inflammatory actions of the health promoting compounds in citrus byproducts will be accomplished by characterizing their metabolites and pharmacokinetics, and elucidating their biochemical actions at the cellular level using in vitro assay microplate technologies. These biochemical actions subsequently will be investigated in animal trials conducted through collaborations with other research laboratories or through commercial contract research laboratories. The research will first require the isolation and chemical characterization of mammalian metabolites of the test citrus byproduct compounds, and these isolations will be achieved through established chromatographic and HPLC-MS techniques.
Progress Report
For Objective 1, the exploration of steam explosion as a means of solubilizing citrus peel pectins, sugars, flavonoids, hydroxycinnamates and limonoids and promoting their recovery using a simple water wash, hence avoiding organic solvent or harsh mineral acid extraction, was continued. ARS researchers at Ft. Pierce, Florida, were able to demonstrate the effects of temperature and time at temperature on the release and recovery of all the components described above and determine optimum conditions for recovery of the individual components. The effect of these parameters was explored using a static, batch apparatus. Various citrus biomass was used as the experimental material. They included fresh, juice extracted orange peel from two varieties of sweet oranges and a dried, stabilized “pectin” peel obtained from an industrial partner. Pectic hydrocolloids recovered from trials using the “pectin” peel were characterized for macromolecular and functional properties. These characterizations indicate that there is a range of temperature and time at temperature within which the viscosity modifying functional properties of the recovered pectin can be optimized. Results using juice processed citrus peel indicate that the optimal parameters for sugar or phenolic recovery differ from those for pectic hydrocolloids. Results from these experiments advance the achievement of Objective 1B by enabling us to optimize the operating parameters for the pilot-scale, continuous steam explosion system for the release of targeted components with improved recovery, functionality and utility.
For Objective 2, in collaboration with Hypha Discovery the modifications of citrus byproduct phenolics by bacterial and fungal fermentations were investigated. Numerous individual bacteria and fungi species were evaluated for their modifications of citrus compounds, and many unique differences were observed in the profiles of new compounds as detected by high performance liquid chromatography-mass spectrometry (HPLC-MS). The cataloging of the newly formed compounds is still in early stages, but it appears that numerous polymethoxylated flavone metabolites are included in these new compounds. Results from these experiments advance the achievement of Objective 2B by enabling us to obtain otherwise difficult to obtain polymethoxylated flavone derivatives. There is a strong demand for these compounds as chromatographic standards, and as compounds needed for further biological activity structure-function studies. Further studies have been done on the influences of chemical modifications of these citrus polymethoxylated flavones, on cells involved with cholesterol biosynthesis. These studies show that not all the compounds are equal, but rather express widely different effects on mammalian cells involved with cholesterol biosynthesis and inflammation.
Citrus pectins that have been engineered to possess different distributions of negative charges within their structure were used to encapsulate an anti-microbial essential oil (carvacrol) for potential use as a shelf life extender for packaged foods. Spray drying conditions and emulsion formulations were tested for their efficacy in producing encapsulated product. This advances Objective 2B by providing baseline information on spray drying parameters and performance of engineered pectic hydrocolloids.
Accomplishments
1. Farm to Fly Feasibility Study completed. ARS researchers at Fort Pierce, Florida, participated in the Farm to Fly Feasibility Study which explored the potential for utilizing locally produced sugar beets to create affordable, sustainable alternative jet fuel which was supported by a USDA Florida State Rural Development Office grant. The work of ARS researchers was instrumental in establishing that the existing underused citrus processing infrastructure could be utilized for the processing of sugar beets for conversion to aviation jet fuel and resulted in the processing of sugar beets at local citrus processing facilities, garnering local news attention. This work addresses the greater than 70% reduction in Florida citrus production associated with citrus greening disease and severe weather events such as hurricanes that result in lost value to growers and producers by providing options for utilizing underused processing facilities and providing growers with alternative crops for sources of income.
Review Publications
Kim, Y., Cameron, R.G., Williams, M.A., Luzio, G.A. 2018. Structural and functional effects of manipulating the degree of methylesterification in a model homogalacturonan with a pseudo-random fungal pectin methylesterase followed by a processive methylesterase. Food Hydrocolloids. 77:879-886.
Cameron, R.G., Chau, H.K., Hotchkiss, A.T., Manthey, J.A. 2017. Recovery of pectic hydrocolloids and phenolics from Huanglongbing related dropped citrus fruit. Journal of the Science of Food and Agriculture. 97:4467-4475. https://doi.org/10.1002/jsfa.8310.
