Secondary Lipid Oxidation Products as Modulators of Calpain-2 Functionality In Vitro.

The objective was to understand the impacts of secondary lipid oxidation products on calpain-2 activity and autolysis and, subsequently, to determine the quantity and localization of modification sites. 2-Hexenal and 4-hydroxynonenal incubation significantly decreased calpain-2 activity and slowed the progression of autolysis, while malondialdehyde had minimal impact on calpain-2 activity and autolysis. Specific modification sites were determined with LC-MS/MS, including distinct malondialdehyde modification sites on the calpain-2 catalytic and regulatory subunits. 2-Hexenal modification sites were observed on the calpain-2 catalytic subunit. Intact protein mass analysis with MALDI-MS revealed that a significant number of modifications on the calpain-2 catalytic and regulatory subunits are likely to exist. These observations confirm that specific lipid oxidation products modify calpain-2 and may affect the calpain-2 functionality. The results of these novel experiments have implications for healthy tissue metabolism, skeletal muscle growth, and post-mortem meat tenderness development.

Rootstock vigor dictates the canopy light environment that regulates metabolite profile and internal fruit quality development in peach.

Five rootstock cultivars of differing vigor: vigorous ('Atlas™' and 'Bright's Hybrid® 5'), standard ('Krymsk® 86' and 'Lovell') and dwarfing ('Krymsk® 1') grafted with 'Redhaven' as the scion were studied for their impact on productivity, mid-canopy photosynthetic active radiation transmission (i.e., light availability) and internal fruit quality. Αverage yield (kg per tree) and fruit count increased significantly with increasing vigor (trunk cross sectional area, TCSA). Α detailed peach fruit quality analysis on fruit of equal maturity (based on the index of absorbance difference, IAD) coming from trees with equal crop load (no. of fruit cm-2 of TCSA) characterized the direct impact of rootstock vigor on peach internal quality [dry matter content (DMC) and soluble solids concentration (SSC)]. DMC and SSC increased significantly with decreasing vigor and increasing light availability, potentially due to reduced intra-tree shading and better light distribution within the canopy. Physiologically characterized peach fruit mesocarp was further analyzed by non-targeted metabolite profiling using gas chromatography mass spectrometry (GC-MS). Metabolite distribution was associated with rootstock vigor class, mid-canopy light availability and fruit quality characteristics. Fructose, glucose, sorbose, neochlorogenic and quinic acids, catechin and sorbitol were associated with high light environments and enhanced quality traits, while sucrose, butanoic and malic acids related to low light conditions and inferior fruit quality. These outcomes show that while rootstock genotype and vigor are influencing peach tree productivity and yield, their effect on manipulating the light environment within the canopy also plays a significant role in fruit quality development.

Cover Crop Root Exudates Impact Soil Microbiome Functional Trajectories in Agricultural Soils.

Background: Cover cropping is an agricultural practice that uses secondary crops to support the growth of primary crops through various mechanisms including erosion control, weed suppression, nutrient management, and enhanced biodiversity. Cover crops may elicit some of these ecosystem services through chemical interactions with the soil microbiome via root exudation, or the release of plant metabolites from roots. Phytohormones are one metabolite type exuded by plants that activate the rhizosphere microbiome, yet managing this chemical interaction remains an untapped mechanism for optimizing plant-soil microbiome interactions. Currently, there is limited understanding on the diversity of cover crop phytohormone root exudation patterns and how these chemical messages selectively enrich specific microbial taxa and functionalities in agricultural soils. Results: Here, we link variability in cover crop root exudate composition to changes in soil microbiome functionality. Exudate chemical profiles from 4 cover crop species (Sorghum bicolor, Vicia villosa, Brassica napus, and Secale cereal) were used as the chemical inputs to decipher microbial responses. These distinct exudate profiles, along with a no exudate control, were amended to agricultural soil microcosms with microbial responses tracked over time using metabolomes and genome-resolved metatranscriptomes. Our findings illustrated microbial metabolic patterns were unique in response to cover crop exudate inputs over time, particularly by sorghum and cereal rye amended microcosms where we identify novel microbial members (at the genera and family level) who produced IAA and GA4 over time. We also identify broad changes in microbial nitrogen cycling in response chemical inputs. Conclusions: We highlight that root exudate amendments alter microbial community function and phytohormone metabolisms, particularly in response to root exudates isolated from cereal rye and sorghum plants. Additionally, we constructed a soil microbial genomic catalog of microorganisms responding to commonly used cover crops, a public resource for agriculturally-relevant microbes. Many of our exudate-stimulated microorganisms are representatives from poorly characterized or novel taxa, highlighting the yet to be discovered metabolic reservoir harbored in agricultural soils. Our findings emphasize the tractability of high-resolution multiomics approaches to investigate processes relevant for agricultural soils, opening the possibility of targeting specific soil biogeochemical outcomes through biological precision agricultural practices that use cover crops and the microbiome as levers for enhanced crop production.

Root-associated bacterial communities and root metabolite composition are linked to nitrogen use efficiency in sorghum.

The development of cereal crops with high nitrogen use efficiency (NUE) is a priority for worldwide agriculture. In addition to conventional plant breeding and genetic engineering, the use of the plant microbiome offers another approach to improving crop NUE. To gain insight into the bacterial communities associated with sorghum lines that differ in NUE, a field experiment was designed comparing 24 diverse Sorghum bicolor lines under sufficient and deficient nitrogen (N). Amplicon sequencing and untargeted gas chromatography-mass spectrometry were used to characterize the bacterial communities and the root metabolome associated with sorghum genotypes varying in sensitivity to low N. We demonstrated that N stress and sorghum type (energy, sweet, and grain sorghum) significantly impacted the root-associated bacterial communities and root metabolite composition of sorghum. We found a positive correlation between sorghum NUE and bacterial richness and diversity in the rhizosphere. The greater alpha diversity in high NUE lines was associated with the decreased abundance of a dominant bacterial taxon, Pseudomonas. Multiple strong correlations were detected between root metabolites and rhizosphere bacterial communities in response to low N stress. This indicates that the shift in the sorghum microbiome due to low N is associated with the root metabolites of the host plant. Taken together, our findings suggest that host genetic regulation of root metabolites plays a role in defining the root-associated microbiome of sorghum genotypes differing in NUE and tolerance to low N stress.IMPORTANCEThe development of crops that are more nitrogen use-efficient (NUE) is critical for the future of the enhanced sustainability of agriculture worldwide. This objective has been pursued mainly through plant breeding and plant molecular engineering, but these approaches have had only limited success. Therefore, a different strategy that leverages soil microbes needs to be fully explored because it is known that soil microbes improve plant growth through multiple mechanisms. To design approaches that use the soil microbiome to increase NUE, it will first be essential to understand the relationship among soil microbes, root metabolites, and crop productivity. Using this approach, we demonstrated that certain key metabolites and specific microbes are associated with high and low sorghum NUE in a field study. This important information provides a new path forward for developing crop genotypes that have increased NUE through the positive contribution of soil microbes.

Composting post-anaerobic digestion for emerging contaminant biodegradation: Impacts of operating conditions.

Sustainable manure management technologies are needed, and combining anaerobic digestion (AD) for energy generation and aerobic composting (AC) to stabilize digestate and remove emerging contaminants (ECs), including veterinary pharmaceuticals and steroid hormones, is promising. This study identified post-AD, AC operating conditions that maximized degradation of study ECs, expected to be present in cattle manure digested using treated municipal wastewater as the water source. Study ECs included sulfamethoxazole (SMX), chlortetracycline (CTC), oxytetracycline (OTC), estrone (E1), and naproxen (NPX). Composting conditions were simulated in bench-scale reactors, with microorganisms from digestate produced in an AD system (25L scale), by varying temperatures, pH, and carbon source compositions (representing food waste/manure co-digestion with different residence times). Results indicate maximum SMX biodegradation occurred at 35°C, pH 7, and with high levels of easily degradable carbon (≥99%, 99%, and 98%), and maximum E1 biodegradation occurred at 35°C, and with low levels of easily degradable carbon (≥97% and 99%). Abiotic degradation was responsible for the nearly complete removal of tetracyclines under all conditions and for partial degradation of NPX (between 20% and 48%). Microorganisms originating from the AD system putatively capable of SMX and E1 biodegradation, or of contributing to biodegradation during the AC phase, were identified, including phylotypes previously shown to biodegrade SMX (Brevundimonas and Alcaligenes).

Cover Crop Cultivar, Species, and Functional Diversity is Reflected in Variable Root Exudation Composition.

Cover cropping has emerged as a sustainable alternative to traditional crop rotational practices, yet the effects of variable root exudation from cover crop species and cultivars within species remains unclear. Here, we assess the chemical heterogeneity of root exudates from 16 commonly used cover crop species as well as 3 distinct cultivars of hairy vetch. Plants were grown hydroponically and analyzed via nontargeted gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-tandem mass spectrometry (LC-MS/MS), and targeted LC-MS/MS to evaluate patterns in root exudate composition across species and functional plant type. Overall, root exudation profiles are heterogeneous across crop species and cultivars. Species within legumes stand out as a unique functional group of plants capable of producing distinct chemical environments rich with complex secondary metabolites, such as triterpenoid saponins (soyasaponins), isoflavonoids, and flavonoids.

Biology and management of hemp russet mite (Acari: Eriophyidae).

Hemp is rapidly becoming a crop of global agricultural importance, and one of the more serious pests of this crop is hemp russet mite (HRM) Aculops cannabicola (Acari: Eriophyidae). Significant knowledge gaps presently exist regarding critical aspects of pest biology, quantification of crop damage, and efficacy of pesticides. Here we assessed the role of cannabidiol (CBD) on HRM performance, efficacy of sulfur treatments in field trials, and effect of hot water immersion with and without surfactants in reducing HRM counts on hemp cuttings. We found that HRM fecundity was reduced on a high-CBD cultivar compared with a low-CBD cultivar in detached leaf assays. In contrast, HRM fecundity and survival were not impacted when reared on high-CBD diet in artificial feeding assays. This suggests that cannabinoids other than CBD may aid in reduction of mite populations on the high-CBD cultivar. Sulfur sprays reduced HRM populations by up to 98% with the greatest effects seen in plants receiving dual applications, one during the vegetative period in July and the second at the initiation of flowering in August. Yields of plants treated with sulfur increased by up to 33%, and there was a further increase in cannabinoid production by up to 45% relative to untreated plants. Hot water immersion treatments with and without surfactant solution reduced HRM on infested hemp cuttings, and no phytotoxicity was observed. This study provides novel approaches to mitigating HRM at multiple stages in hemp production.

Suitability of maize crop residue fermented by Pleurotus ostreatus as feed for edible crickets: growth performance, micronutrient content, and iron bioavailability.

Small-scale farming of edible insects could help combat public health challenges such as protein energy malnutrition and anemia, but reliable low-cost feeds for insects are needed. In resource-limited contexts, where grains such as maize are prohibitively costly for use as insect feed, the feasibility of insect farming may depend on finding alternatives. Here, we explore the potential to modify plentiful maize crop residue with edible mushroom mycelium to generate a low-cost feed adjunct for the farmed two-spotted cricket, Gryllus bimaculatus. Mushroom farming, like insect agriculture, is versatile; it can yield nutritious food while increasing system circularity by utilizing lignocellulosic residues from row crops as inputs. Pleurotus ostreatus, is an edible basidiomycete capable of being cultivated on corn stover (Zea mays). Mushroom harvest results in abundant "spent" substrate, which we investigated as a candidate feed ingredient. We created six cricket feeds containing fermented Pleurotus substrate plus an unfermented control, measuring cricket mass, mortality, and maturation weekly to evaluate cricket growth performance impacts of both fungal fermentation duration and mushroom formation. Pasteurized corn stover was inoculated with P. ostreatus mycelium and fermented for 0, 2, 3, 4, or 8 weeks. Some 4 and 8-week substrates were induced to produce mushrooms through manipulations of temperature, humidity, and light conditions. Dried fermented stover (40%) was added to a 1:1 corn/soy grain mix and fed to crickets ad libitum for 44 days. The unfermented control group showed higher survivorship compared to several fermented diets. Control group mass yield was higher for 2 out of 6 fermented diets. Little variation in cricket iron content was observed via ICP-spectrometry across feeds, averaging 2.46 mg/100 g. To determine bioavailability, we conducted in vitro Caco-2 human colon epithelial cell absorption assays, showing that iron in crickets fed fruiting-induced substrates was more bioavailable than in unfruited groups. Despite more bioavailable iron in crickets reared on post-fruiting substrates, we conclude that Pleurotus-fermented stover is an unsuitable feed ingredient for G. bimaculatus due to high mortality, variability in growth responses within treatments, and low mass yield.

Lipid Peroxidation Products Influence Calpain-1 Functionality In Vitro by Covalent Binding.

The objective of the current study was to evaluate the effects of lipid peroxidation products, malondialdehyde (MDA), hexenal, and 4-hydroxynonenal (HNE), on calpain-1 function, and liquid chromatography and tandem mass spectrometry (LC-MS/MS) identification of adducts on calpain-1. Calpain-1 activity slightly increased after incubation with 100 µM MDA but not with 500 and 1000 µM MDA. However, calpain-1 activity was lowered by hexenal and HNE at 100, 500, and 1000 µM. No difference in calpain-1 autolysis was observed between the control and 1000 µM MDA. However, 1000 µM hexenal and HNE treatments slowed the calpain-1 autolysis. Adducts of MDA were detected on glutamine, arginine, lysine, histidine, and asparagine residues via Schiff base formation, while HNE adducts were detected on histidine, lysine, glutamine, and asparagine residues via Michael addition. These results are the first to demonstrate that lipid peroxidation products can impact calpain-1 activity in a concentration-dependent manner and may impact the development of meat tenderness postmortem.

Carbon sufficiency boosts phenylpropanoid biosynthesis early in peach fruit development priming superior fruit quality.

Manipulating the crop load in peach trees determines carbon supply and optimum balance between fruit yield and quality potentials. The impact of carbon supply on peach fruit quality was assessed in three development stages (S2, S3, S4) on fruit of equal maturity from trees that were carbon (C) starved (unthinned) and sufficient (thinned). Previous studies determined that primary metabolites of peach fruit mesocarp are mainly linked with developmental processes, thus, the secondary metabolite profile was assessed using non-targeted liquid chromatography mass-spectrometry (LC-MS). Carbon sufficient (C-sufficient) fruit demonstrated superior quality attributes as compared to C-starved fruit. Early metabolic shifts in the secondary metabolome appear to prime quality at harvest. Enhanced C-availability facilitated the increased and consistent synthesis of flavonoids, like catechin, epicatechin and eriodyctiol, via the phenylpropanoid pathway, providing a link between the metabolome and fruit quality, and serving as signatures of C-sufficiency during peach fruit development.

Evaluating the ability of rapid evaporative ionization mass spectrometry to differentiate beef palatability based on consumer preference.

Rapid Evaporative Ionization Mass Spectrometry (REIMS) is a type of ambient ionization mass spectrometry, which enables real-time evaluation of several complex traits from a single measurement. The objective of this study was to evaluate the capability of REIMS analysis of raw samples coupled with chemometrics to accurately identify and predict cooked beef palatability. REIMS analysis and consumer sensory evaluation were conducted for beef arm center roasts (n = 20), top loin steaks (n = 20), top sirloin steaks (n = 20), and 20% lipid ground beef (n = 20). These data were used to train predictive models for six classification sets representing different sensory traits. The maximum prediction accuracies achieved (from high to low): beefy flavor acceptance (86.25%), juiciness acceptance (83.75%), overall acceptance (81.25%), overall flavor acceptance (81.25%), grilled flavor acceptance (78.75%), and tenderness acceptance (75%). The current study demonstrates that REIMS analysis of raw meat has the potential to predict and classify cooked beef palatability. Supplementary Information: The online version contains supplementary material available at 10.1007/s13197-022-05562-6.

Increased signal-to-noise ratios within experimental field trials by regressing spatially distributed soil properties as principal components.

Environmental variability poses a major challenge to any field study. Researchers attempt to mitigate this challenge through replication. Thus, the ability to detect experimental signals is determined by the degree of replication and the amount of environmental variation, noise, within the experimental system. A major source of noise in field studies comes from the natural heterogeneity of soil properties which create microtreatments throughout the field. In addition, the variation within different soil properties is often nonrandomly distributed across a field. We explore this challenge through a sorghum field trial dataset with accompanying plant, microbiome, and soil property data. Diverse sorghum genotypes and two watering regimes were applied in a split-plot design. We describe a process of identifying, estimating, and controlling for the effects of spatially distributed soil properties on plant traits and microbial communities using minimal degrees of freedom. Importantly, this process provides a method with which sources of environmental variation in field data can be identified and adjusted, improving our ability to resolve effects of interest and to quantify subtle phenotypes.

Variation in Root Exudate Composition Influences Soil Microbiome Membership and Function.

Root exudation is one of the primary processes that mediate interactions between plant roots, microorganisms, and the soil matrix, yet the mechanisms by which exudation alters microbial metabolism in soils have been challenging to unravel. Here, utilizing distinct sorghum genotypes, we characterized the chemical heterogeneity between root exudates and the effects of that variability on soil microbial membership and metabolism. Distinct exudate chemical profiles were quantified and used to formulate synthetic root exudate treatments: a high-organic-acid treatment (HOT) and a high-sugar treatment (HST). To parse the response of the soil microbiome to different exudate regimens, laboratory soil reactors were amended with these root exudate treatments as well as a nonexudate control. Amplicon sequencing of the 16S rRNA gene illustrated distinct microbial diversity patterns and membership in response to HST, HOT, or control amendments. Exometabolite changes reflected these microbial community changes, and we observed enrichment of organic and amino acids, as well as possible phytohormones in the HST relative to the HOT and control. Linking the metabolic capacity of metagenome-assembled genomes in the HST to the exometabolite patterns, we identified microorganisms that could produce these phytohormones. Our findings emphasize the tractability of high-resolution multiomics tools to investigate soil microbiomes, opening the possibility of manipulating native microbial communities to improve specific soil microbial functions and enhance crop production. IMPORTANCE Decrypting the chemical interactions between plant roots and the soil microbiome is a gateway for future manipulation and management of the rhizosphere, a soil compartment critical to promoting plant fitness and yields. Our experimental results demonstrate how soil microbial community and genomic diversity is influenced by root exudates of differing chemical compositions and how changes in this microbiome result in altered production of plant-relevant metabolites. Together, these findings demonstrate the tractability of high-resolution multiomics tools to investigate soil microbiomes and provide new information on plant-soil environments useful for the development of efficient and precise microbiota management strategies in agricultural systems.

Foodomics: A Data-Driven Approach to Revolutionize Nutrition and Sustainable Diets.

Globally, we are failing to meet numerous nutritional, health, and environmental targets linked to food. Defining food composition in its full chemical and quantitative diversity is central to data-driven decision making for supporting nutrition and sustainable diets. "Foodomics"-the application of omics-technology to characterize and quantify biomolecules to improve wellbeing-has the potential to comprehensively elucidate what is in food, how this composition varies across the food system, and how diet composition as an ensemble of foods guides outcomes for nutrition, health, and sustainability. Here, we outline: (i) challenges of evaluating food composition; (ii) state-of-the-art omics technology and innovations for the analysis of food; and (iii) application of foodomics as a complementary data-driven approach to revolutionize nutrition and sustainable diets. Featuring efforts of the Periodic Table of Food Initiative, a participatory effort to create a globally shared foodomics platform, we conclude with recommendations to accelerate foodomics in ways that strengthen the capacity of scientists and benefit all people.

Quantitative analysis of short-chain fatty acids in human plasma and serum by GC-MS.

Short-chain fatty acids (SCFAs) are volatile fatty acids produced by gut microbial fermentation of dietary nondigestible carbohydrates. Acetate, propionate, and butyrate SCFA measures are important to clinical and nutritional studies for their established roles in promoting healthy immune and gut function. Additionally, circulating SCFAs may influence the metabolism and allied function of additional tissues and organs. The accurate quantification of SCFAs in plasma/serum is critical to understanding the biological role of SCFAs. The low concentrations of circulating SCFAs and their volatile nature present challenges for quantitative analysis. Herein, we report a sensitive method for SCFA quantification via extraction with methyl tert-butyl ether after plasma/serum acidification. The organic extract of SCFAs is injected directly with separation and detection using a polar GC column coupled to mass spectrometry. The solvent-to-sample ratio, plasma volume, and amount of HCl needed for SCFA protonation were optimized. Method validation shows good within-day and inter-day repeatability. The limit of detection was 0.3-0.6 µg/mL for acetate and 0.03-0.12 µg/mL for propionate and butyrate. Successful application of this method on clinical plasma and serum samples was demonstrated in six datasets. By simplifying the sample preparation procedure, the present method reduces the risk of contamination, lowers the cost of analysis, increases throughput, and offers the potential for automated sample preparation.

The impact of extraction protocol on the chemical profile of cannabis extracts from a single cultivar.

The last two decades have seen a dramatic shift in cannabis legislation around the world. Cannabis products are now widely available and commercial production and use of phytocannabinoid products is rapidly growing. However, this growth is outpacing the research needed to elucidate the therapeutic efficacy of the myriad of chemical compounds found primarily in the flower of the female cannabis plant. This lack of research and corresponding regulation has resulted in processing methods, products, and terminology that are variable and confusing for consumers. Importantly, the impact of processing methods on the resulting chemical profile of full spectrum cannabis extracts is not well understood. As a first step in addressing this knowledge gap we have utilized a combination of analytical approaches to characterize the broad chemical composition of a single cannabis cultivar that was processed using previously optimized and commonly used commercial extraction protocols including alcoholic solvents and super critical carbon dioxide. Significant variation in the bioactive chemical profile was observed in the extracts resulting from the different protocols demonstrating the need for further research regarding the influence of processing on therapeutic efficacy as well as the importance of labeling in the marketing of multi-component cannabis products.

Evaluation of ambient mass spectrometry tools for assessing inherent postharvest pepper quality.

Horticulturists are interested in evaluating how cultivar, environment, or production system inputs can affect postharvest quality. Ambient mass spectrometry approaches enable analysis of minimally processed samples under ambient conditions and offer an attractive high-throughput alternative for assessing quality characteristics in plant products. Here, we evaluate direct analysis in real time (DART-MS) mass spectrometry and rapid evaporative ionization-mass spectrometry (REIMS) to assess quality characteristics in various pepper (Capsicum annuum L.) cultivars. DART-MS exhibited the ability to discriminate between pod colors and pungency based on chemical fingerprints, while REIMS could distinguish pepper market class (e.g., bell, lunchbox, and popper). Furthermore, DART-MS analysis resulted in the putative detection of important bioactive compounds in human diet such as vitamin C, p-coumaric acid, and capsaicin. The results of this study demonstrate the potential for these approaches as accessible and reliable tools for high throughput screening of pepper quality.

Feature selection and causal analysis for microbiome studies in the presence of confounding using standardization.

BACKGROUND: Microbiome studies have uncovered associations between microbes and human, animal, and plant health outcomes. This has led to an interest in developing microbial interventions for treatment of disease and optimization of crop yields which requires identification of microbiome features that impact the outcome in the population of interest. That task is challenging because of the high dimensionality of microbiome data and the confounding that results from the complex and dynamic interactions among host, environment, and microbiome. In the presence of such confounding, variable selection and estimation procedures may have unsatisfactory performance in identifying microbial features with an effect on the outcome. RESULTS: In this manuscript, we aim to estimate population-level effects of individual microbiome features while controlling for confounding by a categorical variable. Due to the high dimensionality and confounding-induced correlation between features, we propose feature screening, selection, and estimation conditional on each stratum of the confounder followed by a standardization approach to estimation of population-level effects of individual features. Comprehensive simulation studies demonstrate the advantages of our approach in recovering relevant features. Utilizing a potential-outcomes framework, we outline assumptions required to ascribe causal, rather than associational, interpretations to the identified microbiome effects. We conducted an agricultural study of the rhizosphere microbiome of sorghum in which nitrogen fertilizer application is a confounding variable. In this study, the proposed approach identified microbial taxa that are consistent with biological understanding of potential plant-microbe interactions. CONCLUSIONS: Standardization enables more accurate identification of individual microbiome features with an effect on the outcome of interest compared to other variable selection and estimation procedures when there is confounding by a categorical variable.

Peptidomics of an industrial gluten-free barley malt beer and its non-gluten-free counterpart: Characterisation and immunogenicity.

Recent research suggests that gluten-free beers by prolyl-endopeptidase treatment may not be safe for coeliac disease (CD) patients. Therefore, the gluten peptidome of an industrial gluten-free prolyl-endopeptidase treated malt beer (<10 ppm gluten) was compared to its untreated counterpart (58 ppm gluten) as a reference. NanoLC-HRMS analysis revealed the presence of 155 and 158 gluten peptides in the treated and reference beer, respectively. Characterisation of the peptides in treated beer showed that prolyl-endopeptidase activity was not complete with many peptides containing (multiple) internal proline-residues. Yet, prolyl-endopeptidase treatment did eliminate complete CD-immunogenic motifs, however, 18 peptides still contained partial, and potentially unsafe, motifs. In the reference beer respectively 7 and 37 gluten peptides carried (multiple) complete and/or partial CD-immunogenic motifs. Worrying is that many of these partial immunogenic gluten peptides do not contain a recognition epitope for the R5-antibody and would be overlooked in the current ELISA analysis for gluten quantification.

Microbial Community Field Surveys Reveal Abundant Pseudomonas Population in Sorghum Rhizosphere Composed of Many Closely Related Phylotypes.

While the root-associated microbiome is typically less diverse than the surrounding soil due to both plant selection and microbial competition for plant derived resources, it typically retains considerable complexity, harboring many hundreds of distinct bacterial species. Here, we report a time-dependent deviation from this trend in the rhizospheres of field grown sorghum. In this study, 16S rRNA amplicon sequencing was used to determine the impact of nitrogen fertilization on the development of the root-associated microbiomes of 10 sorghum genotypes grown in eastern Nebraska. We observed that early rhizosphere samples exhibit a significant reduction in overall diversity due to a high abundance of the bacterial genus Pseudomonas that occurred independent of host genotype in both high and low nitrogen fields and was not observed in the surrounding soil or associated root endosphere samples. When clustered at 97% identity, nearly all the Pseudomonas reads in this dataset were assigned to a single operational taxonomic unit (OTU); however, exact sequence variant (ESV)-level resolution demonstrated that this population comprised a large number of distinct Pseudomonas lineages. Furthermore, single-molecule long-read sequencing enabled high-resolution taxonomic profiling revealing further heterogeneity in the Pseudomonas lineages that was further confirmed using shotgun metagenomic sequencing. Finally, field soil enriched with specific carbon compounds recapitulated the increase in Pseudomonas, suggesting a possible connection between the enrichment of these Pseudomonas species and a plant-driven exudate profile.