Location: Citrus and Other Subtropical Products Research
2017 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
The exploration of steam explosion as a means of solubilizing citrus peel flavonoids, hydroxycinnamates and limonoids and promoting their water wash recovery, hence avoiding organic solvent extraction, was conducted. Our findings showed that steam explosion promoted excellent release of nearly all of the peel phenolic and limonoid compounds. High percent recoveries were achieved. This research advances the achievement of Objective 2A by enabling “a new commercial delivery of bioactive flavonoids in food and feed” by increasing the production efficiency of the bioactive flavonoids, and by bringing to a more complete “characterization and quantification of the bioactive flavonoid compounds from byproducts, etc.” Initial trials were made with banana waste material, but equipment failure has postponed this work. Banana tissue has diametrically different phenolic antioxidant profiles compared to citrus and significant synergy between the two sets of compounds is a topic of future research. Progress was also made in our understanding of the effects of the lemon compound, eriocitrin, in combating type-2 diabetes. This advances the achievement of Objective 2A by creating more concrete and demonstrable benefit of the use and commercialization of these byproduct materials. A mouse model showed beneficial effects of eriocitrin in lowering blood glucose levels. Similar effects were seen in our work with the main orange peel compound, hesperidin. Human trials to be conducted by a Brazilian collaborator have been planned, based on these findings. This advances Objective 2A by furthering and validating the pharmacokinetic and bioavailability and beneficial effects of the bioactive flavonoids in citrus byproducts.
Steam explosion parameters (temperature and time at temperature) that are likely to effect the release of many chemical components from their intracellular entrapment were investigated. To overcome potential uncertainty that might be associated with feedstock variability during a harvesting season, a large supply of dried, stabilized citrus peel was obtained (one metric tonne). The effect of these parameters on the release and recovery from citrus biomass of soluble and insoluble sugars and polysaccharides, and the structural and functional properties of pectic hydrocolloids was first explored using a static, batch apparatus. The composition of soluble and insoluble sugars and polysaccharides was determined for each combination of temperature and time. Pectic hydrocolloids also were collected from each sample. These pectic hydrocolloids are being characterized for several structural and functional properties. Results from these experiments will enable us to modify the running parameters for the pilot-scale, continuous steam explosion system enabling the release and recovery of these components with improved functionality and utility. Parameters associated with the functionalization of pectic hydrocolloids while still present in the crude, steam exploded biomass were explored by varying the conditions used for during the calcium chelating, alkaline demethylesterification treatment. This advances Objective 1 by enabling the optimization of the steam explosion process so that increased functionality will be associated with the recovered pectic hydrocolloids and the sugars that could be exploited as biobased platform chemicals. Additional steam explosion studies were conducted on alternative biomass feedstocks such as banana plant residues, olive leaves and sugar beets but equipment failure delayed further studies. These results suggest the continuous steam explosion process may be adaptable to biomass other than citrus.
Initial trials using pectic hydrocolloids recovered from steam exploded citrus biomass as encapsulating material for a spray dried model bioactive component were performed. Pectic hydrocolloids were recovered from steam exploded citrus biomass using a simple water wash. Following precipitation and lyophilization a portion of the freeze dried material was dialyzed to remove the hemicellulose constituent. Citrus oil and polymethoxylated flavones were encapsulated in pectic hydrocolloids with or without hemicelluloses. This advances Objective 2B by providing baseline information on spray drying parameters and performance of assorted pectic hydrocolloid fractions.
Accomplishments
1. Release and recovery of valuable co-products from flavor-degrading, culled Huanglongbing citrus fruit. Huanglongbing is a bacterial disease spread by sucking insects that is devastating United States citrus production. Trees die prematurely, yields are decreased and diseased fruits sent to juice processing plants contain off-flavored juice. To avoid mixing the off-flavored juice in this fruit with good quality juice, the most obviously affected fruits may be culled and sent to the feed mill for conversion to low value animal feed. ARS researchers in Fort Pierce, Florida, demonstrated that valuable chemical co-products could be recovered from these fruit using a pilot scale, continuous, steam-explosion process. This process could recover lost value for juice processors as well as providing very large amounts of valuable co-products from a wasted resource.
Review Publications
Owen, J., Kent, L., Ralet, M., Cameron, R.G., Williams, M. 2017. A tale of two pectins: Diverse fine structures can result from identical processive PME treatments on similar high DM subtrates. Carbohydrate Polymers. 168:365-373.
Dorado, C., Cameron, R.G., Cooper, K. 2017. Steam explosion and fermentation of sugar beets from Southern Florida and the Midwestern United States. Biocatalysis and Agricultural Biotechnology. 11:26-33.. https://doi.org/10.1016/j.bcab.2017.05.007.
Kim, Y., Williams, M., Luzio, G., Cameron, R.G. 2017. Introduction and characterization of charged functional domains into an esterified pectic homogalacturonan by a citrus pectin methylesterase and comparison of its modes of action to other pectin methylesterase isozymes. Food Hydrocolloids. 69:422-431. doi: 10.1016/j.foodhyd.2017.03.009.
Cameron, R.G., Chau, H.K., Hotchkiss, A.T., Manthey, J.A. 2017. Release and recovery of pectic hydrocolloids and phenolics from culled citrus fruits. Food Hydrocolloids. 72:52-61. doi:org/10.1016/j.foodhyd.2017.05025.
