Assessing the utility of multiplexed liquid chromatography-mass spectrometry for gluten detection in Australian breakfast food products

Coeliac disease (CD) is an autoimmune disorder triggered by the ingestion of gluten that is associated with gastrointestinal issues, including diarrhea, abdominal pain, and malabsorption. Gluten is a general name for a class of cereal storage proteins of wheat, barley, and rye that are notably resistant to gastrointestinal digestion. After ingestion, immunogenic peptides are subsequently recognized by T cells in the gastrointestinal tract. The only treatment for CD is a life-long gluten-free diet. As such, it is critical to detect gluten in diverse food types, including those where one would not expect to find gluten. The utility of liquid chromatography-mass spectrometry (LC-MS) using cereal-specific peptide markers to detect gluten in heavily processed food types was assessed. A range of breakfast products, including breakfast cereals, breakfast bars, milk-based breakfast drinks, powdered drinks, and a savory spread, were tested. No gluten was detected by LC-MS in the food products labeled gluten-free, yet enzyme-linked immunosorbent assay (ELISA) measurement revealed inconsistencies in barley-containing products. In products containing wheat, rye, barley, and oats as labeled ingredients, gluten proteins were readily detected using discovery proteomics. Panels comprising ten cereal-specific peptide markers were analyzed by targeted proteomics, providing evidence that LC-MS could detect and differentiate gluten in complex matrices, including baked goods and milk-based products

Interaction between temperature and sublethal infection with the amphibian chytrid fungus impacts a susceptible frog species

The amphibian chytrid fungus Batrachochytrium dendrobatidis is an emerging infectious pathogen present on every continent except Antarctica. It causes the disease chytridiomycosis in a subset of species but does not always result in disease or death for every host. Ambient temperature influences both amphibian metabolism and chytrid pathogenicity, however the interactive effects on host physiology is not well understood. We investigated the sublethal effect of B. dendrobatidis infection on a susceptible host, Litoria aurea to test (1) whether the infection load, metabolic activity, body fat and gonad size differed in L. aurea at either 24 degrees C or 12 degrees C ambient temperatures and (2) whether previous Bd infection caused long-term changes to body fat and gonad size. Litoria aurea in 12 degrees C treatments had higher infection loads of B. dendrobatidis and lower survivorship. Metabolic rate was higher and fat mass was lower in infected individuals and in animals in 24 degrees C treatments. Male L. aurea previously infected with B. dendrobatidis had smaller testes 5 months-post clearance of infection, an effect likely to translate to fitness costs in wild populations. These experiments demonstrate a physiological cost to sublethal B. dendrobatidis infection, which suggests a reduction in host fitness mediated by temperature in the host's environment regardless of whether infection leads to mortality

Low gluten beers contain variable gluten and immunogenic epitope content

Gluten content labels inform food choice and people practicing a gluten-free diet rely upon them to avoid illness. The regulations differ between jurisdictions, especially concerning fermented foodstuffs such as beer. Gluten abundance is typically measured using ELISAs, which have come into question when testing fermented or hydrolysed foodstuffs such as beer. Mass spectrometry can be used to directly identify gluten peptides and reveal false negatives recorded by ELISA. In this survey of gluten in control and gluten-free beers, gluten protein fragments that contain known immunogenic epitopes were detected using liquid chromatography-mass spectrometry in multiple beers that claim to be gluten-free and have sufficiently low gluten content, as measured by ELISA, to qualify as being gluten-free in some jurisdictions. In fact, several purportedly gluten-free beers showed equivalent or higher hordein content than some of the untreated, control beers. The shortcomings of ELISAs for beer gluten testing are summarised, the mismatch between ELISA and mass spectrometry results are explored, and the suitability of existing regulations as they pertain to the gluten content in fermented foods in different jurisdictions are discussed

Comparison of protein extraction protocols and allergen mapping from black soldier fly Hermetia illucens

Exploration of important insect proteins — including allergens — and proteomes can be limited by protein extraction buffer selection and the complexity of the proteome. Herein, LC-MS/MS-based proteomics experiments were used to assess the protein extraction efficiencies for a suite of extraction buffers and the effect of ingredient processing on proteome and allergen detection. Discovery proteomics revealed that SDS-based buffer yields the maximum number of protein groups from three types of BSF samples. Bioinformatic analysis revealed that buffer composition and ingredient processing could influence allergen detection. Upon applying multi-level filtering criteria, 33 putative allergens were detected by comparing the detected BSF proteins to sequences from public allergen protein databases. A targeted LC-MRM-MS assay was developed for the pan-allergen tropomyosin and used to assess the influence of buffer composition and ingredient processing using peptide abundance measurements. Significance: We demonstrated that the selection of protein extraction buffer and the processing method could influence protein yield and cross-reactive allergen detection from processed and un-processed black soldier fly (BSF) samples. In total, 33 putative allergens were detected by comparing the detected BSF proteins to sequences from public allergen protein databases. An LC-MRM-MS assay was developed for tropomyosin, indicating the importance of buffer selection and processing conditions to reduce BSF samples\u27 allergenicity

Protein extraction protocols for optimal proteome measurement and arginine kinase quantitation from cricket Acheta domesticus for foodsafety assessment

Insects have been consumed by people for millennia and have recently been proposed as a complementary, sustainable source of protein to feed the world’s growing population. Insects and crustaceans both belong to the arthropod family. Crustacean (shellfish) allergies are common and potentially severe; hence, the cross-reactivity of the immune system with insect proteins is a potential health concern. Herein, LC-MS/MS was used to explore the proteome of whole, roasted whole and roasted powdered cricket products. Eight protein extraction protocols were compared using the total number of protein and distinct peptide identifications. Within these data, 20 putative allergens were identified, of which three were arginine kinase (AK) proteoforms. Subsequently, a multiple reaction monitoring MS assay was developed for the AK proteoforms and applied to a subset of extracts. This targeted assay demonstrated that allergen abundance/detectability varies according to the extraction method as well as the food processing method

Protein extraction protocols for optimal proteome measurement and arginine kinase quantitation from cricket Acheta domesticus for food safety assessment

© 2021 Elsevier Ltd Insects have been consumed by people for millennia and have recently been proposed as a complementary, sustainable source of protein to feed the world\u27s growing population. Insects and crustaceans both belong to the arthropod family. Crustacean (shellfish) allergies are common and potentially severe; hence, the cross-reactivity of the immune system with insect proteins is a potential health concern. Herein, LC-MS/MS was used to explore the proteome of whole, roasted whole and roasted powdered cricket products. Eight protein extraction protocols were compared using the total number of protein and distinct peptide identifications. Within these data, 20 putative allergens were identified, of which three were arginine kinase (AK) proteoforms. Subsequently, a multiple reaction monitoring MS assay was developed for the AK proteoforms and applied to a subset of extracts. This targeted assay demonstrated that allergen abundance/detectability varies according to the extraction method as well as the food processing method

Sodium Chloride Inhibits the Growth and Infective Capacity of the Amphibian Chytrid Fungus and Increases Host Survival Rates

The amphibian chytrid fungus Batrachochytrium dendrobatidis is a recently emerged pathogen that causes the infectious disease chytridiomycosis and has been implicated as a contributing factor in the global amphibian decline. Since its discovery, research has been focused on developing various methods of mitigating the impact of chytridiomycosis on amphibian hosts but little attention has been given to the role of antifungal agents that could be added to the host's environment. Sodium chloride is a known antifungal agent used routinely in the aquaculture industry and this study investigates its potential for use as a disease management tool in amphibian conservation. The effect of 0–5 ppt NaCl on the growth, motility and survival of the chytrid fungus when grown in culture media and its effect on the growth, infection load and survivorship of infected Peron's tree frogs (Litoria peronii) in captivity, was investigated. The results reveal that these concentrations do not negatively affect the survival of the host or the pathogen. However, concentrations greater than 3 ppt significantly reduced the growth and motility of the chytrid fungus compared to 0 ppt. Concentrations of 1–4 ppt NaCl were also associated with significantly lower host infection loads while infected hosts exposed to 3 and 4 ppt NaCl were found to have significantly higher survival rates. These results support the potential for NaCl to be used as an environmentally distributed antifungal agent for the prevention of chytridiomycosis in susceptible amphibian hosts. However, further research is required to identify any negative effects of salt exposure on both target and non-target organisms prior to implementation

Face Masks and Cough Etiquette Reduce the Cough Aerosol Concentration of Pseudomonas aeruginosa in People with Cystic Fibrosis

People with cystic fibrosis (CF) generate Pseudomonas aeruginosa in droplet nuclei during coughing. The use of surgical masks has been recommended in healthcare settings to minimize pathogen transmission between patients with CF.To determine if face masks and cough etiquette reduce viable P. aeruginosa aerosolized during coughing.Twenty-five adults with CF and chronic P. aeruginosa infection were recruited. Participants performed six talking and coughing maneuvers, with or without face masks (surgical and N95) and hand covering the mouth when coughing (cough etiquette) in an aerosol-sampling device. An Andersen Cascade Impactor was used to sample the aerosol at 2 meters from each participant. Quantitative sputum and aerosol bacterial cultures were performed, and participants rated the mask comfort levels during the cough maneuvers.During uncovered coughing (reference maneuver), 19 of 25 (76%) participants produced aerosols containing P. aeruginosa, with a positive correlation found between sputum P. aeruginosa concentration (measured as cfu/ml) and aerosol P. aeruginosa colony-forming units. There was a reduction in aerosol P. aeruginosa load during coughing with a surgical mask, coughing with an N95 mask, and cough etiquette compared with uncovered coughing (P

Developing cancer warning statements for alcoholic beverages

Background: There is growing evidence of the increased cancer risk associated with alcohol consumption, but this is not well understood by the general public. This study investigated the acceptability among drinkers of cancer warning statements for alcoholic beverages. Methods: Six focus groups were conducted with Australian drinkers to develop a series of cancer-related warning statements for alcohol products. Eleven cancer warning statements and one general health warning statement were subsequently tested on 2,168 drinkers via an online survey. The statements varied by message frame (positive vs negative), cancer reference (general vs specific), and the way causality was communicated (‘increases risk of cancer’ vs ‘can cause cancer’). Results: Overall, responses to the cancer statements were neutral to favorable, indicating that they are unlikely to encounter high levels of negative reaction from the community if introduced on alcoholic beverages. Females, younger respondents, and those with higher levels of education generally found the statements to be more believable, convincing, and personally relevant. Positively framed messages, those referring to specific forms of cancer, and those using ‘increases risk of cancer’ performed better than negatively framed messages, those referring to cancer in general, and those using the term ‘can cause cancer’. Conclusion: Cancer warning statements on alcoholic beverages constitute a potential means of increasing awareness about the relationship between alcohol consumption and cancer risk