Research Project: Defining, Measuring, and Mitigating Attributes that Adversely Impact the Quality and Marketability of Foods

Location: Healthy Processed Foods Research

2020 Annual Report

Objectives
The long-term goal of this project is to develop novel methods, and define measure and mitigate attributes that adversely impact the quality of foods. Specifically, during the next five years we will focus on the following objectives: Objective 1: Enable novel commercial methods for prevention or removal of defects and contaminants in foods. • Subobjective 1A: Develop techniques for detection and removal of potatoes affected by Zebra Chip disorder from the processing line. • Subobjective 1B: Develop X-ray based alternatives to radioisotopes for irradiation. Objective 2: Integrate physical, chemical, sensory, and biological changes from raw and commercially processed food products, including, olives and grapes. • Subobjective 2A: Pinpoint and identify impact aroma compounds of raw materials and commercially processed products from specialty crops including grapes and olives using gas chromatography-olfactometry (GC-O) and gas chromatography-mass spectrometry (GC-MS). Identify precursors (and eventually pathways) of such impact aroma compounds and study flavor variation in different varieties. • Subobjective 2B: Isolate and characterize phytonutrients in raw materials and food products from grapes, olives, and other specialty crops. Determine the effects of processing on the levels of these constituents and also monitor changes in biological activity (i.e., antioxidant activity). Objective 3: Integrate measurable allergenic properties with methods to mitigate food allergens in nuts and dairy. • Subobjective 3A: Identify, characterize, and develop methods for the detection of food allergens in tree nut and other foods. • Subobjective 3B: Investigate and differentiate allergen cross-reactivity and multi-sensitization and study the effects of processing methodologies on allergenicity.

Approach
1A: Acquire x-ray images and NIR spectra from whole potatoes (reflection) and through slices (transmission). Analyze slices for moisture and sugar, followed by frying and color evaluation. Develop calibration equations for prediction of Zebra Chip (ZC), moisture content, sucrose, and glucose and investigate correlations between ZC and moisture and/or sugar. Isolate appropriate subsets of NIR absorbance values as input features for standard statistical classification techniques. Test appropriate optics that can measure absorption at the determined wavelengths for the ability to evaluate ZC. Observe x-ray images for differences between potato classes and develop detection algorithms. 1B: Design, build, and test x-ray based irradiation units was alternatives to traditional gamma based units using two technologies: traditional x-ray tubes and a higher power prototype x-ray emitter system. 2A: Extract aroma compounds from grapes and olives using GC. Identify precursors and study flavor variation in plant varieties. Have judges evaluate ability to detect odor from each compound’s effluent from a GC column, with compounds detected in the most dilute sample considered to be impact aroma compounds. Identify acquired capillary GC-MS spectra using established libraries to identify food constituents. Quantify food constituents by GC-MS by comparing the areas of characteristic mass fragment ions of the compounds with that of 2-undecanone (m/z 170). Calculate odor activity values by dividing the determined concentrations by their odor thresholds. For confirmation, compare synthetic blends of identified odorants with those of the original raw and processed products using sensory panels. 2B: Extract homogenates from processed and unprocessed food components and analyze using an HPLC-diode array detector. Identify constituents by comparison of retention times and UV/Vis spectra of unknown peaks with those of authentic standards and verify by HPLC-MS, NMR or IR if necessary. Determine weight and total phenolic content for each homogenate. Evaluate antioxidant activity by the DPPH and ABTS procedures. Determine effects of processing by comparison. 3A: Isolate 2S albumins from nuts and express recombinantly to assess their allergenicity using sera from allergic patients. Isolate and express profilin protein for comparative studies. Develop protocols to purify 2S albumin allergens in other nuts including almond, pine nut, coconut, macadamia nut, and optionally chestnut. Determine the peptide sequences by N-terminal peptide sequencing and mass spectrometry. Develop antibody based methods for detecting food allergen and allergic food. 3B: Isolate and clone hazelnut allergens following established protocols. Assay association with serum IgE from patients known to react to peanut and/or hazelnut. Frequency of IgE recognition of allergens will reflect cross-reactivity and multi-sensitization. Process samples by extrusion to investigate processing effects on allergenicity.

Progress Report
This expired project has completed NP 306 Office of Scientific Quality Review and has been replaced by project 2030-41430-013-00D, “New Technologies and Methodologies for Increasing Quality, Marketability and Value of Food Products and Byproducts.” Following is a final progress report for the life of the current project. In support of Sub-objective 1A, calibration equations for prediction of Zebra Chip (ZC), moisture content, sucrose, and glucose have been developed and correlations between ZC and moisture and/or sugar have been identified. Appropriate subsets of near-infrared (NIR) absorbance values at specific wavelengths have been isolated as input features for standard statistical classification techniques based on their ability to identify ZC. Different optical configurations for measuring reflected light at these specific wavelengths have been assembled and tested. Accuracy in detecting ZC was lower for the optical measurements than that achieved using the reduced NIR spectra in combinations with chemometrics, presumably due to optical limitations in isolating specific wavelengths and the rapid signal acquisition time as compared to the spectrophotometer signal acquisition time. In support of Sub-objective 1B, x-ray dose mapping (measuring the delivered dose over a discreet volume) has been performed for several x-ray irradiation units, allowing comparison with dose distributions for gamma irradiators. This helps to evaluate x-ray as a suitable substitute for gamma irradiation. Novel irradiation configurations allowing a highly uniform dose distribution among samples as well as very precise dose determination have been developed and applied to irradiation of various sample types, especially for the sterilization of insects. In support of Sub-objective 2A, impact aroma compounds in different olive varieties were identified and quantified using gas chromatography-flame ionization detector (GC-FID) and gas chromatography-mass spectrometry (GC-MS). Odor unit values were calculated to determine the contribution of individual odor constituents to the overall flavor. These results will help growers and processors to understand differences in flavor characteristics among the different olive varieties. In further support of Sub-objective 2A, 10 volatile compounds that contribute to the aroma status (aromatic or non-aromatic) of Brazilian rice genotypes were identified. It was shown that certain genotypes (i.e. elite line aromatic rice genotypes BR4 and BR5) exhibited volatile compound profiles that indicate less off-flavors (hexanal and 1-hexanol), higher 2-acetyl-1-pyrroline content, and similar cooking time and hardness to desirable genotypes. These results provide the means for rice farmers, industries and researchers to select the most desirable genotypes of aromatic rice for production and distribution worldwide. This work may also serve as a starting point for future research on rice authenticity. Also in support of Sub-objective 2A, a previously unknown biosynthetic pathway for the production of certain terpenes has been identified. Terpenes have antibacterial, antifungal, anti-cancer, antioxidant, and antifeedant properties and are widely used in the flavor and fragrance industry due to their desirable odors. Terpenes have complex structures that make their chemical syntheses difficult. Computational chemistry, i.e., density functional theory (DFT) calculations, were used to elucidate the new biosynthetic pathway. Knowledge of terpene biosynthetic pathways provides critical insight that may be used to produce desired terpenoids on an industrial scale. In support of Sub-objective 2B, the phenolic composition of grape pomace skins from four red wine cultivars (Carinan, Merlot, Cabernet Sauvignon, and Syrah) was studied. Carignan skin had the highest concentrations of caftaric acid, rutin, quercetin 3-glucoside, kaempferol 3-glucoside and resveratrol. Grape pomace consists of pressed skins, seeds and stems, which account for about 20% of the weight of the grape. Approximately 10 million tons of grape pomace are generated annually, with adverse environmental impact due to the presence of the phenolic compounds. However, phenolic compounds have potentially positive effects on human health such as anti-inflammatory, anti-ischemic, anti-obesity, and anti-platelet aggregating effects and other potentially disease preventing cellular actions. Recovery of the phenolic compounds could therefore mitigate negative environmental impact. ARS researchers collaborated with scientists in Ethiopia and Iraq to determine physiochemical properties and antioxidant composition of four mango varieties (Tommy Atkins, Apple, Keitt, and Kent) grown in Ethiopia. Keitt had the highest weight, length, diameter, and juice content by volume. The pH, total soluble solids (TSS), total acidity (TA), and proximate composition (moisture, ash, fat, fiber, and protein) showed significant differences across varieties. Sodium, magnesium, potassium, calcium, iron and zinc were evaluated with potassium having the highest concentration at 267 mg/100 g. Total carotenoids and vitamin C (i.e. antioxidants) concentrations varied across varieties, with Apple, Kent and Keitt varieties being rich in vitamin C. The four varieties had similar physicochemical properties and antioxidant content as mangoes grown in other countries. ARS researchers performed comparative analysis of total phenolic content of both fermented and non-fermented jaboticaba pomace. Jaboticaba, a fruit native to Brazil, is used in food or fermented beverages. Since it spoils rapidly it is often used for juices, jams, liqueurs, distillates, wine and ice cream. Fermented pomace from wine production contained higher levels of the phenolics, quercetin and myricetin, compared to unfermented pomace obtained after juice extraction. Fermented pomace may be added to food products to provide color (anthocyanins), increase antimicrobial properties, and possibly help to reduce the risk of diabetes, obesity and chronic obstructive pulmonary disease. ARS scientists have linked recurring outbreaks of acute hypoglycemic encephalopathy with litchi consumption. These outbreaks have resulted in the deaths of hundreds of children per year in India. The causative agents in soapberry fruits (of which litchi is a member) are the toxic cyclopropyl amino acids, methylenecyclopropylglycine (MCPG) and hypoglycin A (HGA). A method to simultaneously quantify both MCPG and HGA from 1 µg up to 10 mg in 1 g of dried soapberry fruit (1-10,000 ppm) has been developed. HGA has been identified and quantified for the first time in litchi arils (the fleshy edible portion of the fruit). This method can be used to identify and quantify MCPG and HGA in other soapberry fruits. This knowledge is critical for preventing illness caused by consumption of soapberry fruits. The phenolic composition of five varieties of pomegranate peel (Molla Nepes, Parfianka, Purple Heart, Wonderful and Vkunsyi) have been determined. Pomegranates are an important medicinal and nutritional product, with potential for prevention of cancer and cardiovascular diseases. Pomegranate peels constitute up to 40% of the whole fruit and are a by-product of juice production. The Vkunsyi cultivar had the highest concentrations of gallic acid, catechin and the desirable ellagitannin punicalagin. This cultivar is a potentially rich source of desirable phenolic compounds. ARS scientists have determined the phenolic composition of mesquite flour. Mesquite is the common name for leguminous desert plants of the genus Prosopis with about 44 species native to North America, South America, Africa, and South Asia. Mesquite tolerates high temperatures and low rainfall, can grow in saline soil and can fix nitrogen. Consumption has been linked to antibacterial, cardioprotective and anti-inflammatory health benefits. An analytical method using liquid chromatography-mass spectrometry (LC-MS) in conjunction with reversed phase and aqueous normal phase (ANP) chromatography was developed to separate and identify the phenolic compounds in mesquite flour, enabling study of its beneficial health effects. In support of Sub-objective 3A, ARS scientists used sera obtained from patients allergic to tree nuts to identify previously unknown tree nut allergens. Two of these allergens have been given official designations (Car i 2 for pecan and Pru du 8b for almond) by the World Health Organization and International Union of Immunological Societies (WHO/IUIS) Allergen Nomenclature Sub-committee. Pru du 8 is now recognized as a single member of a previously unknown family of food allergens. The sera have also allowed assessment of the relative importance of linear and conformational IgE epitopes in peanut allergy and in milk allergy. They also provide the means to investigate the importance of multi-sensitization and cross-reactivity in food allergies. In support of Sub-objective 3B, ARS scientists have established a new humanized rat basophil cell line using RBL-2H3 cells. Model cell lines are important tools for food allergy and allergen studies. Currently available human cells grow very slowly, and the rat cell line RBL-2H3 could not be used because the rat IgE receptor has low affinity to human IgE. While a few humanized RBL-2H3 lines exist that express a component of human IgE receptor, they suffer from competition by the endogenous rat IgE component. The new cell lines will allow effective assessment of food allergen cross-reactivity and the effects of food processing on the allergenic properties of food allergens.

Accomplishments
1. Identification of citric acid as a possible source of off-flavor in Prosopis juliflora pods. Nitrogen fixing trees and shrubs of the genus Prosopis (mesquite) provide firewood, luxury quality timber for furniture and flooring, and soil enrichment through nitrogen fixation. Prosopis species are widespread in semiarid regions of the Western Hemisphere and were a major food staple for indigenous peoples in North and South America. Prosopis spp. produce pods that typically contain about 30-40% sucrose and 10-14% protein. Mesquite pods are milled to produce flour which is sold commercially and is used in artisanal food products. ARS scientists in Albany, California, have identified high levels of citric acid (2150 mg/100 g) in P. juliflora pods which may be responsible for their sour, unpalatable flavor. Determination of the organic acid composition of mesquite pods will allow production of clones with pods that are sweet and highly palatable.

2. New technology facilitates peptide production for food allergy studies. Food allergies negatively impact the utilization of agricultural products, and peanuts and tree nuts cause most of the fatal and near-fatal food allergy cases in the United States. Linear IgE epitopes play essential roles in peanut and tree nut allergies, but the prevalent IgE epitopes of most known food allergens have not been mapped. Overlapping peptides derived from food allergens are required to identify linear epitopes, but their production has presented many challenges. To overcome this problem, ARS researchers at Albany, California, developed a method to express peptides using a truncated form of the trimeric protein as an expression tag. This technology has been used to successfully produce overlapping peptides derived from two peanut allergens. The technique will facilitate the study of processing effects on the allergenicity and cross-reactivity of food allergens.

Review Publications
Haff, R.P., Ovchinnikova, I., Liang, P., Mahoney, N.E., Gee, W.S., Gomez, J., Toyofuku, N., Jackson, E.S., Hnasko, R.M., Light, D. 2020. X-ray based sterilization of larvae of the navel orangeworm (lepidoptera:pyralaide) for sterile insect technique. Journal of Economic Entomology. 113(4):1685-1693. https://doi.org/10.1093/jee/toaa111.
Lin, L., Moran, T., Peng, B., Yang, J., Culton, D., Che, H., Jiang, S., Liu, Z., Geng, S., Zhang, Y., Diaz, L., Ye, Q. 2019. Walnut antigens can trigger autoantibody development in patients with pemphigus vulgaris through a "hit-and-run" mechanism. Journal of Allergy Clinical Immunology. 144(3):720-728. https://doi.org/10.1016/j.jaci.2019.04.020.
Chen, F., Ma, H., Li, Y., Wang, H., Samad, A., Zhou, J., Zhu, L., Zhang, Y., He, J., Fan, X., Jin, T. 2019. Screening of nanobody specific for peanut major allergen Ara h 3 by phage display. Journal of Agricultural and Food Chemistry. 67:11219-11229. https://doi.org/10.1021/acs.jafc.9b02388.
Li, Y., Zeng, W., Li, Y., Fan, W., Ma, H., Fan, X., Jiang, J., Brefo-Mensah, E., Zhang, Y., Yang, M., Dong, Z., Palmer, M., Jin, T. 2019. Structure determination of the CAMP factor of streptococcus agalactiae with the aid of an MBP tag and insights into membrane-surface attachment. Acta Crystallographica, Section D: Biological Crystallography. 75:772-781. https://doi.org/10.1107/S205979831901057X.
Hughes, A.C., Zhang, Y., Bai, X., Xiong, Y., Wang, Y., Yang, X., Xu, Q., He, X. 2019. Structural and functional characterization of Stx2k, a new subtype of Shiga toxin 2. Microorganisms. 8(1):4. https://doi.org/10.3390/microorganisms8010004.
Zeng, W., Ma, H., Fan, W., Yang, Y., Zhang, C., Kombe Kombe, J., Fan, X., Zhang, Y., Dong, Z., Shen, Z., Zhou, Y., Yang, M., Jin, T. 2019. Structure determination of CAMP factor of mobiluncus curtisii and insights into structural dynamics. International Journal of Biological Macromolecules. 150:1027–1036. https://doi.org/10.1016/j.ijbiomac.2019.10.107.
Ban, X., Li, C., Zhang, Y., Gu, Z., Cheng, L., Hong, Y., Li, Z. 2019. Importance of c-terminal extension in thermophilic 1,4-a-glucan branching enzyme from geobacillus thermoglucosidans STB02. Applied Biochemistry and Biotechnology. 190:1010–1022. https://doi.org/10.1007/s12010-019-03150-7.
Yi, J., Huang, H., Liu, Y., Lu, Y., Fan, Y., Zhang, Y. 2019. Fabrication of curcumin-loaded pea protein-pectin ternary complex for the stabilization and delivery of ß-carotene emulsions. Food Chemistry. 313:126118. https://doi.org/10.1016/j.foodchem.2019.126118.