Performing Department
Soil & Crop Sciences
Non Technical Summary
Color is a primary determinant of food quality perception by consumers; for this reason, the vast majority of manufactured foods contain added colors, especially synthetic dyes. Consumer demand for clean label, natural colors, and healthy ingredients has expanded rapidly. Plant-derived pigments are widely used as water-soluble colorants in food and beverages, but tend to have poor stability to food processing and handling conditions, and are relatively expensive to process. Specific sorghums accumulate high levels of unique and highly stable color compounds, with major potential as food colorants, and bioactive food additives. The pigments are located in sorghum bran, and other waste tissue that are easy to sustainably concentrate and store. Unfortunately, the sorghum pigment compounds are difficult to extract, and also readily precipitate in water-based systems that characterize most beverage applications, for instance, limiting commercial exploitation.This application aims to develop technologies that significantly enhance extraction efficiency of sorghum pigment compounds and prevent their aggregation in water-based systems. We will, i) use microwave-solvent technology to facilitate release of sorghum pigment compounds and beneficial phenolics from plant tissue, and ii) develop technologies that use selected natural polysaccharides to protect the pigment compounds from precipitating under conditions relevant to food processing and handling. The proposed work will lead to technologies that can be applied to commercially improve food quality, and potential bioactive properties, using the uniquely functional natural sorghum pigments.
Animal Health Component
50%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
(N/A)
Goals / Objectives
Color is the most important determinant of food quality perception by consumers; for this reason, the vast majority of manufactured foods contain added colors, mostly synthetic petroleum-based FD&C dyes. Consumer demand for clean label and natural colors has expanded rapidly out of concerns over potential adverse effect of artificial additives. Legislative mandates have further pushed the pursuit of natural colorants. The US accounts for 30% of global natural food colors market, valued at about $1.9 billion in 2017, with an expected annual growth of 8.5% between 2018-2026. Plant-derived anthocyanins are the most widely used water-soluble colorants in food and beverages. Unfortunately, anthocyanins have various drawbacks that limit their food use, primarily poor stability to food processing and handling conditions, and high cost. The anthocyanins tend to fade, brown, or change hue easily under various food processing conditions; they are also mostly commercially derived from bulky (high moisture) plant tissues with high enzyme activity that are expensive to store and process.Unlike other plants, sorghum exclusively accumulates unique and highly stable anthocyanin analogs, 3-deoxyanthocyanins (3DXA) with major potential as commercial food colorants. The pigments are located in sorghum grain pericarp, and other dry tissue that are easy to concentrate and store. The 3DXA pigments also have unique bioactive properties. Interest in these unique pigments has grown among food and ingredient companies. Unfortunately two major challenges stand in the way of commercial exploitation of these valuable pigments; i) the 3DXA are relatively difficult to extract using standard solvent systems, and ii) the 3DXA have high tendency to self-associate in aqueous systems, limiting use, especially in dairy and beverages.Our goal is to develop practical strategies that can enable the food industry to improve food quality and health promoting properties. Through this project we aim to address the two major challenges facing 3DXA commercial exploitation. We hypothesize that the poor extractability of the 3DXA is due to the sorghum pericarp cell wall structure (neutral polysaccharides with high levels of phenolate cross-linkages) and relatively high partition coefficient of the 3DXA that limits their affinity for anthocyanin-based solvents. Furthermore, we hypothesize that the self-association in aqueous systems is due to hydrophobic effect induced by the naked domain between C4 (C-ring) and C5´ (B-ring) in their structure (due to unsubstituted C3). We propose to use the widely available and practical microwave technology to effectively disrupt sorghum pericarp cell wall and enable efficient extraction of the sorghum 3DXA, and use encapsulation technology to prevent aqueous self-association of the pigments. Our preliminary data demonstrate that microwave energy, coupled with appropriate solvent system, can dramatically increase extractability of 3DXA from sorghum to achieve yields of 15 - 35 g/kg bran, compared to 0.5 - 1.8 g/kg typical for anthocyanins from fruits/vegetables. Furthermore, we found that, depending on the composition of the 3DXA and solution pH, very low levels of amphiphilic ionic polysaccharides (negligible impact on viscosity) can almost completely prevent self-association of these pigments in aqueous solution without affecting solution color (hue and chroma). This suggests specific and strong interactions of 3DXA with hydrophobic domains of the polysaccharides, perhaps stabilized via charge repulsion. Through this project, we aim to address three specific objectives:1) Establish effect of microwave-assisted extraction (MAE) conditions on the composition and yield of 3-deoxyanthocyanins and phenolic copigments from sorghum bran. In contrast to fruits and vegetables, sorghum bran cell wall structure is almost entirely composed of neutral sugars, with significant phenolic acid-ester cross linkages. These properties make the cell wall difficult to disrupt using traditional extraction systems. We expect that, when bran is tempered to the right moisture, high-energy microwave irradiation will generate instantaneous heat and steam pressure within the sorghum bran tissue cells to cause 'popcorn-type' cell wall rupture and allows the pigments to efficiently diffuse into the extracting medium. Even though MAE has been widely investigated for extracting anthocyanins (and other phenolics) from plant tissues, its overall application (and advantage) is limited by poor stability of these compounds to microwave energy and heat. The unique stability of 3DXA will likely overcome this problem, and thus lead to dramatic increase in pigment yield. A likely secondary benefit would be altered bran cell wall structure to increase soluble dietary fiber, and likely prebiotic properties of bran residue.2) Establish the effect of amphiphilic and ionic properties of polysaccharides on encapsulation efficiency of the sorghum 3-deoxyanthocyaninsm and their stability in aqueous systems. Self-aggregation in aqueous systems severely limits application of the sorghum 3DXA. We have evidence to suggest that the self-association is largely driven by hydrophobic attractions due to the naked domain between C4 and C5´ of 3DXA molecule. Interaction of the 3DXA with low levels of ionic polysaccharides with hydrophobic moieties appear to inhibit the self-aggregation. Here we propose to investigate precise the mechanisms that govern the 3DXA-polysaccharide interactions in aqueous systems to identify appropriate technologies to expand their use in food and beverages.3) Establish effect of ionic properties of solution on the stability of the 3DXA-polysaccharide complexes. We will use model beverage systems to evaluate stability and performance of the 3DXA-polysaccharide encapsulates under practically relevant processing and handling conditions, including thermal processing, presence of salts and mono/multivalent ions, reducing sugars, different pH, and different storage conditions typical to food beverages.
Project Methods
Objective 1Sorghum Material: Two types of black sorghum will be used: A non-tannin black sorghum, and a tannin-containing black sorghum. The presence of tannins can alter behavior of the pigments both during extraction, and in products. Tannins generally have a stabilizing effect on anthocyanins through copigmentation and other mechanisms.Bran preparation. The bran will be obtained from decortication of the grains at 15% decortication level. The bran will then be ground to pass through 0.5 mm screen and stored at -20oC until use.Microwave-assisted extraction (MAE). Microwave Accelerated Reaction System (MARSXpress, CEM, Matthews, NC) will be used to carry out the extraction. Energy level, extraction temperature, and solvent ratio will be preselected based on previously optimized conditions. The variables that are most likely to maximize yield of 3DXA include solvent properties (relative permittivity and partition coefficient), extraction time, and moisture content of the bran. Extraction efficiency of each treatment will be evaluated by the quantity and composition of pigments and total polyphenols extracted.1) 3-Dexyanthocyanin pigment and phenolic copigments yield: For 3DXA pigment yield, samples will be diluted in methanol at pH 1.0 and absorbance read using a Shimadzu UV-Vis spectrophotometer by scanning from 250 - 750 nm. To estimate relative extractability of phenolic copigments under MAE, phenols will be estimated using the Folin-Ciocalteu method.2) Visual color parameters: Visual color parameters will be measured using a Minolta Chroma Meter.3) Effect of MAE on 3DXA and other phenolic composition: The composition of extracted 3DXA pigments and other phenolic constituents will be determined by UPLC-MS/MS. This information is practically important because pigment and copigments composition is affects color stability and functionality in a food matrix. 4) Effect of MAE on tannin structure and MW profile: We will use normal phase HPLC-FLD to evaluate the effect of MAE on tannin MW profile as previously described. We expect that the larger MW tannins will partially depolymerize and oxidize to anthocyanidins which may modify color properties and behavior of 3DXA extracted from the tannin-containing sorghum.5) Changes to bran cell wall structure due to MAE: Bright field and Field emission scanning electron microscopy will be used to image physical changes to bran tissue under MAE.Objective 2Design. Based on our observations with alginate and gum Arabic, we will use pectin of varying degrees of O-methyl ester substitution (DM; 20 - 80%) to investigate the precise interactive effect of ionic and hydrophobic interactions in stabilizing the 3DXA in aqueous solution. Additionally, we will use different ratios of alginate-gum Arabic to determine potential effect of blending the ionic and amphiphilic polysaccharides on the 3DXA aqueous stability. This is of practical relevance because most food applications use gum blends to optimize functionality.Model system. Water suspension of pectin, sodium alginate, and gum Arabic will be prepared and blended with 3DXA extracts in buffers to final concentrations of 0-1.0 g/L polysaccharide. Testing buffer pH will be 2.0 - 7.0. 3-Deoxyanthocyanin extracts without polysaccharide will serve as control. The model systems will be stored at 24oC for at least 24 weeks to monitor color changes and encapsulate stability over time.Methods. Sample turbidity, viscosity, particle size distribution, zeta potential, and morphology will be evaluated before and after heat stability tests. Stable samples after heating will be tested for storage stability. Dispersions will be heated at 88oC/2 min or 138oC/1 min. The former is tested for acid (pH < 4.6) stability, while the latter meets the UHT requirement. Samples will be monitored for precipitation and changes in turbidity and particle size during storage.Stabilizing mechanisms of polysaccharides. Sample turbidity, viscosity, particle size distribution, zeta potential, and morphology analysis: AFM and SEM microscopy; FTIR spectroscopy, and DSC will be used as complementary techniques to distinguish the stabilization due to specific complexation between anthocyanins and polysaccharide or due to change in viscosity.Physicochemical properties of dispersions: The above dispersions before and after heating will be photographed, measured for turbidity, and characterized for other physicochemical properties.Phase solubility: the level of dissociation and 3-DXA-polyssacharide interactions in the model systems will be evaluated.Molecular forces in 3DXA-polysaccharide interactions; urea, sodium dodecyl sulfate, and dithiothreitol that disrupt hydrogen bonding, hydrophobic interactions, and disulfide bonding, respectively will be used to investigate mechanisms of interaction between polysaccharides and 3DXA. Color stability of 3-deoxyanthocyanis in polysaccharide systems: The UV-vis absorbance spectrum (200-700 nm) at day 0 will be determined as the baseline, and the spectra will be obtained for each treatment every 14 days for 4 months. The changes to the 3-DXA and phenolic copigments profile over time will be monitored by UPLC-MS.Microbial quality evaluation. Since ttreatments will be subjected to pasteurization or UHT, no food safety issues are expected. Still, a hazard analysis will be conducted and supervised. Examination will consist of collecting samples after pasteurization or UHT process. Enumeration of aerobic and anaerobic spore formers, thermoresistant and not-thermoresistant flat sour spore forming bacteria, lactic acid bacteria and yeasts/molds. These counts will be used to establish the expected levels of spore forming bacteria in UHT-processed systems and in pasteurized systems. The data will be used to establish microbiological criteria for the ingredient. Objective 3Formulation. Sugar, electrolyte (monovalent vs. multivalent), ascorbate, pH (2 - 7). The 3DXA-polysaccharide fractions will be adjusted to pH 2.0-7.0 and 0-200 mM NaCl or ferrous lactate (Fe(C3H5O3)2 with 10% w/w sucrose. To establish potential effect of the 3-DXA on fruit-based flavor sensory profile under processing and storage conditions, we will formulate the model beverages with orange and fruit punch flavors (among the most common flavors in fruit and sports drinks, that also match sensory expectation of 3-DXA colors) for sensory evaluation.Thermal processing. Pasteurization vs. sterilization the common processes used for beverages and dairy products. Dispersions will be heated at 88oC for 2 min in a water bath or 138oC for 1 min in a glycerol bath. The former is tested for acid (pH <4.6) stability, while the latter meets the UHT requirement.Storage conditions. Light (dark vs. display light), temperatures (4 oC-32 oC). Furthermore, samples of the polysaccharide-3DXA complexes will be freeze-dried and stored as powder; these will be investigated for solubility, andd stability over time.Stability; color properties, 3DXA & phenolic profile, stability of encapsulating system using methods detailed under Objectives 1 & 2 above.Microbial quality control. The model beverages will be tested over storage time to verify compliance with the microbial levels established under Objective 2 above and to determine microbial stability. Statistical analysis. For Objective 1, factorial design will be used to identify the most important variables, followed by central composite design to estimate treatment conditions that produce optimal response. Completely randomized design will be used for Objectives 2 &3 in this study. All experiments will be performed in triplicate. Treatment effects will be tested by one-way ANOVA and mean separation (p<0.05) done by Tukey test; trend analysis will be used to compare treatments over time. Pigment separation/aggregation of less than 20% compared to control after 24 weeks will be considered successful.