ABSTRACT
Carotenoids are plant pigments that function as antioxidants, hormone precursors, colourants and essential components of the photosynthetic apparatus. Carotenoids accumulate in nearly all ...types of plastids, not just the chloroplast, and are thus found in most plant organs and tissues, albeit at trace levels in some tissues. In this review we summarise the current knowledge of the carotenoid content of non‐green plastids and discuss what is known about the regulation of their biosynthesis in roots, fruits, flowers, tubers and seeds. The emphasis is on food crops as carotenoids are essential components of human diets, primarily as some are precursors of vitamin A. The low carotenoid content of many staple foods, such as cereals, can exacerbate dietary deficiencies. The World Health Organisation has estimated that more than 100 million children are vitamin A‐deficient and up to 500,000 of these children become blind each year. Many of these children die within 12 months of going blind. Thus, understanding the regulation of carotenoid accumulation in food crops, especially tubers and cereals, should facilitate improvements to nutritional value with potentially significant health benefits.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
During brewing, gluten proteins may be solubilized, modified, complexed, hydrolyzed, and/or precipitate. Gluten fragments that persist in conventional beers render them unsuitable for people with ...celiac disease (CD) or gluten intolerance. Barley-based beers crafted to remove gluten using proprietary precipitation and/or application of enzymes, e.g. prolyl endopeptidases (PEP) that degrade the proline-rich gluten molecules, are available commercially. Gluten measurement in fermented products remains controversial. The industry standard, a competitive ELISA, may indicate gluten values <20 mg/kg, which is deemed safe for people with CD. However, in this study, liquid chromatography–mass spectrometry analyses revealed gluten peptides derived from hydrolyzed fragments, many >30 kDa in size. Barley gluten (hordeins) were detected in all beers analyzed with peptides representing all hordein classes detected in conventional beers but also, alarmingly, in many gluten-reduced beers. It is evident that PEP digestion was incomplete in several commercial beers, and peptides comprising missed cleavages were identified, warranting further optimization of PEP application in an industrial setting.
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IJS, KILJ, NUK, PNG, UL, UM, UPUK
•An optimised protein extraction method was developed to extract ATIs from barley.•LC-MRM-MS analysis was used to compare extraction method efficiency.•A sequential two-step protocol consistently ...increased ATI yield (<12% variation).•The protocol was successfully applied to measure six ATIs across 12 barley cultivars.
Plant defense protein α-amylase trypsin inhibitors (ATIs) have been proposed as one of the triggers of non-coeliac gluten sensitivity, however there have been no focused studies on their optimal extraction and quantitation from cereal grains. The efficiency of extraction is of utmost interest for the downstream detection and characterisation. In the present study, three extraction buffers and two modified protocols were investigated using LC-MRM-MS in order to examine their ability to efficiently and repeatably extract ATIs from selected barley cultivars. Initially, three extraction buffers IPA/DTT, urea and Tris-HCl were used to extract ATIs from two selected barley cultivars, Commander and Hindmarsh. The results obtained from the preliminary study showed that IPA/DTT and urea-based buffer extraction could yield ∼70% and ∼45% more ATIs, respectively than a buffer based on Tris-HCl extraction, with all methods showing high repeatability (CV < 15%). A multi-step protocol, employing IPA/DTT and urea improved the extraction efficiency in comparison to the single buffer extraction protocols (p<0.0001). When solutions from parallel extractions using IPA/DTT and urea were combined, the results were comparable (p = 0.99) with a sequential multi-step IPA/DTT-urea protocol. However, the repeatability of the combined process was compromised, as discerned by greater variation (CV>30%). The optimised sequential two-step extraction protocol was successfully used to extract and quantify ATIs from 12 barley cultivars. LC–MS analysis revealed that cv Yagan and cv Scope contain the higher levels (∼143% relative to the average barley ATI content), whereas cultivars Fleet (61%), Baudin (77%) and Commander (79%) contained the lowest levels. The libraries of ATIs identified and the quantitative methods described here provide a foundation for the future application of MS-based quantitative methodologies to detect and quantify ATIs in barley varieties and in food products.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
4.
Perspectives on Future Protein Production Colgrave, Michelle L; Dominik, Sonja; Tobin, Aarti B ...
Journal of agricultural and food chemistry,
12/2021, Volume:
69, Issue:
50
Journal Article
Peer reviewed
Open access
An increasing world population, rising affluence, urbanization, and changing eating habits are all contributing to the diversification of protein production. Protein is a building block of life and ...is an essential part of a healthy diet, providing amino acids for growth and repair. The challenges and opportunities for production of protein-rich foods from animals (meat, dairy, and aquaculture), plant-based sources (pulses), and emerging protein sources (insects, yeast, and microalgae) are discussed against the backdrop of palatability, nutrition, and sustainability.
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IJS, KILJ, NUK, PNG, UL, UM, UPUK
The efficiency of gluten extraction is of critical importance to the results derived from any analytical method for gluten detection and quantitation, whether it employs reagent-based technology ...(antibodies) or analytical instrumentation (mass spectrometry). If the target proteins are not efficiently extracted, the end result will be an under-estimation in the gluten content posing a health risk to people affected by conditions such as celiac disease (CD) and nonceliac gluten sensitivity (NCGS). Five different extraction protocols were investigated using LC-MRM-MS for their ability to efficiently and reproducibly extract gluten. The rapid and simple “IPA/DTT” protocol and related “two-step” protocol were enriched for gluten proteins, 55/86% (trypsin/chymotrypsin) and 41/68% of all protein identifications, respectively, with both methods showing high reproducibility (CV < 15%). When using multistep protocols, it was critical to examine all fractions, as coextraction of proteins occurred across fractions, with significant levels of proteins existing in unexpected fractions and not all proteins within a particular gluten class behaving the same.
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IJS, KILJ, NUK, PNG, UL, UM, UPUK
Subjects suffering from coeliac disease, gluten allergy/intolerance must adopt a lifelong avoidance of gluten. Beer contains trace levels of hordeins (gluten) which are too high to be safely consumed ...by most coeliacs. Accurate measurement of trace hordeins by ELISA is problematic.
We have compared hordein levels in sixty beers, by sandwich ELISA, with the level determined using multiple reaction monitoring mass spectrometry (MRM-MS).
Hordein levels measured by ELISA varied by four orders of magnitude, from zero (for known gluten-free beers) to 47,000 µg/mL (ppm; for a wheat-based beer). Half the commercial gluten-free beers were free of hordein by MS and ELISA. Two gluten-free and two low-gluten beers had zero ELISA readings, but contained significant hordein levels (p<0.05), or near average (60-140%) hordein levels, by MS, respectively. Six beers gave false negatives, with zero ELISA readings but near average hordein content by MS. Approximately 20% of commercial beers had ELISA readings less than 1 ppm, but a near average hordein content by MS. Several barley beers also contained undeclared wheat proteins.
ELISA results did not correlate with the relative content of hordein peptides determined by MS, with all barley based beers containing hordein. We suggest that mass spectrometry is more reliable than ELISA, as ELISA enumerates only the concentration of particular amino-acid epitopes; this may vary between different hordeins and may not be related to the absolute hordein concentration. MS quantification is undertaken using peptides that are specific and unique, enabling the quantification of individual hordein isoforms. This outlines the problem of relying solely on ELISA determination of gluten in beverages such as beer and highlights the need for the development of new sensitive and selective quantitative assay such as MS.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
•An LC-MRM-MS method was developed using a panel of chymotryptic peptide markers.•Reproducible quantification was achieved using chymotrypsin with CVs <15%.•Chymotrypsin results in a marked increase ...in non-specific cleavage.•Trypsin is preferred for quantification of the avenins, the B-, D- and γ-hordeins.•Chymotrypsin is the enzyme of choice for the C-hordeins.
Gluten describes a complex mixture of proteins found in wheat, rye, barley and oats that pose a health risk to people affected by conditions such as coeliac disease and non-coeliac gluten sensitivity. Complete digestion of gluten proteins is of critical importance during quantitative analysis. To this end, chymotrypsin was investigated for its ability to efficiently and reproducibly digest specific classes of gluten in barley. Using proteomics a chymotryptic peptide marker panel was elucidated and subjected to relative quantification using LC-MRM-MS. Thorough investigation of peptide markers revealed robust and reproducible quantification with CVs <15% was possible, however a greater proportion of non-specific cleavage variants were observed relative to trypsin. The selected peptide markers were assessed to ensure their efficient liberation from their parent proteins. While trypsin remains the preferred enzyme for quantification of the avenin-like A proteins, the B-, D- and γ-hordeins, chymotrypsin was the enzyme of choice for the C-hordeins.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
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.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
α-Amylase/trypsin inhibitors (ATIs) may have a role in nonceliac wheat sensitivity (NCWS) and celiac disease (CD), but the ATI content and diversity across a range of wheat cultivars are not well ...characterized. Discovery proteomics was used to detect ATIs across two wheat cultivars: Chara and Magenta. Comprehensive mapping of detected ATIs with the ATIs from the recently published wheat genome RefSeq v1.0 shows the presence of three major subclasses: monomeric (9%), dimeric (61%), and chloroform–methanol (CM) type (30%). Subsequently, the level of 18 ATI isoforms (63 peptides) grouped into four subtypes was monitored across 15 commercial wheat cultivars and the eight parental lines from a multiparent advanced-generation intercross (MAGIC) population using liquid chromatography–multiple reaction monitoring-mass spectrometry (LC–MRM-MS). The ATI content of wheat cultivars Janz, Sunvale, Diamond Bird, and Longreach Scout was significantly lower than that of other wheat cultivars. The MAGIC parental cultivars Baxter and Xiaoyan 54 contain higher levels (∼115% relative to the average wheat ATI content), whereas cultivar Pastor contained the lowest levels (∼87%). Comprehensive sequence analysis, annotation, chromosomal locations, and epitope mapping enabled us to build an LC–MRM-MS method to monitor and quantify the immunostimulatory ATI proteins potentially related to NCWS, autoimmune diseases, and metabolic disorders. This provides an opportunity to select wheat cultivars with significantly lower levels of ATIs.
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IJS, KILJ, NUK, PNG, UL, UM
Consumers, especially those with allergies and/or intolerances, should have confidence in two critical areas of food safety: foods should be correctly labelled and free from contamination. To this ...end, global proteomic analysis employing LC-MS/MS of gluten-enriched extracts derived from 12 barley cultivars was undertaken, providing a foundation for the development of MS-based quantitative methodologies that would enable the detection of barley contamination in foods. Subsequently, a number of candidate barley-specific peptide markers were evaluated by multiple-reaction monitoring MS. From an initial panel of 26, 9 peptide markers were unique to barley, yet present in a wide range of barley varieties. The analytical method was then used to examine a range of breakfast cereals and was able to detect barley in a barley-based breakfast cereal and a muesli, but additionally allowed detection of contamination of cereals that were comprised of ancient grains and in commercially-sourced flours, including amaranth, chia, buckwheat, millet, rice, corn, oats, rye, spelt and green wheat (0.01–0.08%). LC-MS/MS provides an alternative to ELISA approaches to monitor food safety and the identification of robust and sensitive cereal-specific peptide markers is the first step toward the adoption of this technology.
Coeliac disease is a serious health issue affecting up to 70million people globally for which there is no cure. The only treatment is a life-long gluten-free diet. Contamination of foods can occur at many stages of food production from farm to fork. As such, accurate quantification and identification of the source (i.e. cereal) and type (e.g. gluten) of contamination is critical to the health and well-being of a subset of the population, including those affected by coeliac disease and non-coeliac gluten sensitivity.
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•12 economically important barley cultivars were characterised using proteomics.•9 sensitive barley-specific peptide markers were identified.•Low level barley contamination was detected in 10/15 commercially-sourced flours.•The LC-MS approach was applicable to processed food products (breakfast cereals).•Low level barley contamination found in 3/5 cereals supposedly “barley-free”.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
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