Influence of Roasting Temperature on the Detectability of Potentially Allergenic Lupin by SDS-PAGE, ELISAs, LC-MS/MS, and Real-Time PCR.

Seeds of "sweet lupins" have been playing an increasing role in the food industry. Lupin proteins may be used for producing a variety of foods, including pasta, bread, cookies, dairy products, and coffee substitutes. In a small percentage of the population, lupin consumption may elicit allergic reactions, either due to primary sensitization to lupin or due to cross-allergy with other legumes. Thus, lupin has to be declared on commercial food products according to EU food regulations. In this study, we investigated the influence of roasting seeds of the L. angustifolius cultivar "Boregine" on the detectability of lupin by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), ELISAs, LC-MS/MS, and real-time PCR. Seeds were roasted by fluidized bed roasting, and samples were drawn at seed surface temperatures ranging from 98 °C to 242 °C. With increasing roasting temperature, the extractability of proteins and DNA decreased. In addition, roasting resulted in lower detectability of lupin proteins by ELISAs and LC-MS/MS and lower detectability of DNA by real-time PCR. Our results suggest reduced allergenicity of roasted lupin seeds used for the production of "lupin coffee"; however, this has to be confirmed in in vivo studies.

Dry Fractionation and Gluten-Free Sourdough Bread Baking from Quinoa and Sorghum.

The roller milling of sorghum and quinoa seeds into flour fractions (coarse, middle, and fine) was investigated, chemically analysed, and applied in the baking of gluten-free sourdough bread. The gap settings were adjusted to 0, 5, 8, and 10 for quinoa, and 3, 5, and 7 for sorghum. The fine fractions reached values of up to about 41% (gap 8) for quinoa and around 20% for sorghum (gap 5). SEM pictographs illustrated the clear separation of each fraction with the chemical analysis showing high contents of protein, TDF (total dietary fibre), and IDF (insoluble dietary fibre) in the coarse fraction. Up to 77% starch content was obtained in the fine fraction with significant amounts of SDF (soluble dietary fibre), which has good health benefits. Increasing the dough moisture up to 90% helped in decreasing the bread crumb firmness, while low Avrami parameters and RVA pasting behaviour indicated a slow bread-staling rate for both sourdough breads.

Does sourdough bread provide clinically relevant health benefits?

During the last decade, scientific interest in and consumer attention to sourdough fermentation in bread making has increased. On the one hand, this technology may favorably impact product quality, including flavor and shelf-life of bakery products; on the other hand, some cereal components, especially in wheat and rye, which are known to cause adverse reactions in a small subset of the population, can be partially modified or degraded. The latter potentially reduces their harmful effects, but depends strongly on the composition of sourdough microbiota, processing conditions and the resulting acidification. Tolerability, nutritional composition, potential health effects and consumer acceptance of sourdough bread are often suggested to be superior compared to yeast-leavened bread. However, the advantages of sourdough fermentation claimed in many publications rely mostly on data from chemical and in vitro analyzes, which raises questions about the actual impact on human nutrition. This review focuses on grain components, which may cause adverse effects in humans and the effect of sourdough microbiota on their structure, quantity and biological properties. Furthermore, presumed benefits of secondary metabolites and reduction of contaminants are discussed. The benefits claimed deriving from in vitro and in vivo experiments will be evaluated across a broader spectrum in terms of clinically relevant effects on human health. Accordingly, this critical review aims to contribute to a better understanding of the extent to which sourdough bread may result in measurable health benefits in humans.

Novel Gluten-Free Breakfast Cereals Produced by Extrusion Cooking from Rice and Teff: Effects on Microstructural, Physical and Nutritional Properties.

Current gluten-free products often have nutritional inadequacies. Teff is generating a growing interest for its excellent nutritional value. In this study, the effects of teff enrichment of extruded gluten-free breakfast cereals based on rice flour and two process parameters-feed moisture and temperature-were investigated based on their physical, microstructural and nutritional properties. The independent variables were modulated and examined by a Box-Behnken design. The incorporation of teff flour affected the sensory properties of extruded cereals, particularly lightness and crispness, with estimated linear effects of -6.91 and -8.49, respectively. The enrichment of breakfast cereals with teff flour also increased the total phenolic content and antioxidant capacity in both free and bound fractions, as well as the amount of insoluble dietary fibre. By varying all independent variables, the microstructural and physical properties of samples changed considerably. At the lowest feed moisture, wall thickness was small while showing the highest expansion. Samples with the highest teff flour addition exhibited an increased number of small pores which decreased crispness. These findings suggest that, by optimized extrusion cooking, the use of teff flour seems to be promising for the preparation of gluten-free breakfast cereals with superior nutritional properties and good structural characteristics.

Absolute and relative quantitation of amylase/trypsin-inhibitors by LC-MS/MS from wheat lines obtained by CRISPR-Cas9 and RNAi.

Quantitation of wheat proteins is still a challenge, especially regarding amylase/trypsin-inhibitors (ATIs). A selection of ATIs was silenced in the common wheat cultivar Bobwhite and durum wheat cultivar Svevo by RNAi and gene editing, respectively, in order to reduce the amounts of ATIs. The controls and silenced lines were analyzed after digestion to peptides by LC-MS/MS with different approaches to evaluate changes in composition of ATIs. First, a targeted method with stable isotope dilution assay (SIDA) using labeled peptides as internal standards was applied. Additionally, four different approaches for relative quantitation were conducted, in detail, iTRAQ labeled and label free quantitation (LFQ) combined with data dependent acquisition (DDA) and data independent acquisition (DIA). Quantitation was performed manually (Skyline and MASCOT) and with different proteomics software tools (PLGS, MaxQuant, and PEAKS X Pro). To characterize the wheat proteins on protein level, complementary techniques as high-performance liquid chromatography (HPLC) and gel electrophoresis were performed. The targeted approach with SIDA was able to quantitate all ATIs, even at low levels, but an optimized extraction is necessary. The labeled iTRAQ approach revealed an indistinct performance. LFQ with low resolution equipment (IonTrap) showed similar results for major ATIs, but low abundance ATIs as CM1, were not detectable. DDA measurements with an Orbitrap system and evaluation using MaxQuant showed that the relative quantitation was dependent on the wheat species. The combination of manual curation of the MaxQuant search with Skyline revealed a very good performance. The DIA approach with analytical flow found similar results compared to absolute quantitation except for some minor ATIs, which were not detected. Comparison of applied methods revealed that peptide selection is a crucial step for protein quantitation. Wheat proteomics faces challenges due to the high genetic complexity, the close relationship to other cereals and the incomplete, redundant protein database requiring sensitive, precise and accurate LC-MS/MS methods.

Physicochemical, Functional, and In Vitro Digestibility of Protein Isolates from Thai and Peru Sacha Inchi (Plukenetia volubilis L.) Oil Press-Cakes.

Proteins from Sacha inchi (SI) have been widely known for their health-benefiting properties. This study aimed to investigate the different protein isolates obtained from oil press-cakes of Thai and Peru SI. The protein content and protein recovery of Thai and Peru SI were estimated to be 93.27, 90.67%, and 49.15, 59.32%, respectively. The protein patterns of the Thai and Peru SI samples analyzed by SDS-PAGE showed glycoprotein as a major protein, with a molecular weight of 35 kDa. Both protein isolates (PI) showed water and oil holding capacities in the range of 2.97−3.09 g/g sample and 2.75−2.88 g/g sample, respectively. The emulsifying properties of the PI from Thai SI were higher than those of Peru (p < 0.05), while the foaming properties were not analogous to the emulsion properties. The Thai SI sample showed lower digestibility up to 120 min of in vitro digestion time than that of the Peru SI sample (p < 0.05). However, simulated in vitro pepsin digestion of Thai and Peru Si samples displayed hydrolyzed protein bands compared to trypsin digestion, which showed no protein patterns in both SI samples on a 4−20% gradient gel. These results suggest that the protein isolates from Thai and Peru SI exhibit marked variations in physical and techno-functional properties and have a high potential to be employed as plant-based protein additives for future non-animal-based protein-rich foods.

Wheat amylase/trypsin inhibitors (ATIs): occurrence, function and health aspects.

Amylase/trypsin inhibitors (ATIs) are widely consumed in cereal-based foods and have been implicated in adverse reactions to wheat exposure, such as respiratory and food allergy, and intestinal responses associated with coeliac disease and non-coeliac wheat sensitivity. ATIs occur in multiple isoforms which differ in the amounts present in different types of wheat (including ancient and modern ones). Measuring ATIs and their isoforms is an analytical challenge as is their isolation for use in studies addressing their potential effects on the human body. ATI isoforms differ in their spectrum of bioactive effects in the human gastrointestinal (GI), which may include enzyme inhibition, inflammation and immune responses and of which much is not known. Similarly, although modifications during food processing (exposure to heat, moisture, salt, acid, fermentation) may affect their structure and activity as shown in vitro, it is important to relate these changes to effects that may present in the GI tract. Finally, much of our knowledge of their potential biological effects is based on studies in vitro and in animal models. Validation by human studies using processed foods as commonly consumed is warranted. We conclude that more detailed understanding of these factors may allow the effects of ATIs on human health to be better understood and when possible, to be ameliorated, for example by innovative food processing. We therefore review in short our current knowledge of these proteins, focusing on features which relate to their biological activity and identifying gaps in our knowledge and research priorities.

Wheat ATIs: Characteristics and Role in Human Disease.

Amylase/trypsin-inhibitors (ATIs) comprise about 2-4% of the total wheat grain proteins and may contribute to natural defense against pests and pathogens. However, they are currently among the most widely studied wheat components because of their proposed role in adverse reactions to wheat consumption in humans. ATIs have long been known to contribute to IgE-mediated allergy (notably Bakers' asthma), but interest has increased since 2012 when they were shown to be able to trigger the innate immune system, with attention focused on their role in coeliac disease which affects about 1% of the population and, more recently, in non-coeliac wheat sensitivity which may affect up to 10% of the population. This has led to studies of their structure, inhibitory properties, genetics, control of expression, behavior during processing, effects on human adverse reactions to wheat and, most recently, strategies to modify their expression in the plant using gene editing. We therefore present an integrated account of this range of research, identifying inconsistencies, and gaps in our knowledge and identifying future research needs. Note  This paper is the outcome of an invited international ATI expert meeting held in Amsterdam, February 3-5 2020.

Synthesis and accumulation of amylase-trypsin inhibitors and changes in carbohydrate profile during grain development of bread wheat (Triticum aestivum L.).

BACKGROUND: Recent studies indicate that amylase-trypsin inhibitors (ATIs) and certain carbohydrates referred to as FODMAPs (fermentable oligo-, di-, monosaccharides and polyols) play an important role in promoting wheat sensitivity. Hitherto, no study has investigated the accumulation of ATIs during the development of the wheat caryopsis. We collected caryopses of common wheat cv. 'Arnold' at eight different grain developmental stages to study compositional changes in ATI and FODMAP content. RESULTS: The harvested caryopses were analysed for their size, protein and carbohydrate concentrations. ATIs were further characterized by MALDI-TOF MS, and their trypsin inhibition was evaluated by an enzymatic assay. The results showed that ATI accumulation started about 1 week after anthesis and subsequently increased steadily until physiological maturity. However, the biological activity of ATIs in terms of enzyme inhibition was not detectable before about 4 weeks after anthesis. Carbohydrate analysis revealed the abundance of short-chain fructans in early stages of grain development, whereas non-water-soluble carbohydrates increased during later developmental stages. CONCLUSIONS: The results provide new insights into the complex metabolisms during grain filling and maturation, with particular emphasis on the ATI content as well as the inhibitory potential towards trypsin. The time lag between ATI accumulation and development of their biological activity is possibly attributed to the assembling of ATIs to dimers and tetramers, which seems to be crucial for their inhibitory potential.

Insights into the Potential of Sourdough-Related Lactic Acid Bacteria to Degrade Proteins in Wheat.

Sourdough processing contributes to better digestible wheat-based bakery products, especially due to the proteolytic activity of lactic acid bacteria (LAB). Therefore, sourdough-related LAB were screened for their capacity to degrade immunogenic proteins like gluten and alpha-amylase-trypsin inhibitors (ATIs). Firstly, the growth of 87 isolates was evaluated on a gluten-based medium. Further, the breakdown capacity of selected isolates was determined for gluten with a focus on gliadins by measuring acidification parameters and MALDI-TOF MS protein profiles. ATI degradation after 72 h of incubation within an ATI-based medium was investigated by means of acidification, HPLC, and competitive ELISA. All isolates exhibited the potential to degrade ATIs to a high degree, whereas the gliadin degradation capacity varied more greatly among tested LAB, with Lacticaseibacillus paracasei Lpa4 exhibiting the strongest alterations of the gliadin pattern, followed by Lactiplantibacillus plantarum Lpl5. ATI degradation capacities ranged from 52.3% to 85.0% by HPLC and 22.2% to 70.2% by ELISA, with Lacticaseibacillus paracasei Lpa4 showing superior breakdown properties. Hence, a selection of specific starter cultures can be used in sourdough processing for wheat-based bakery products with reduced gluten and ATI content and, further, better tolerated products for patients suffering from non-celiac wheat sensitivity (NCWS).

Reduction of Allergenic Potential in Bread Wheat RNAi Transgenic Lines Silenced for CM3, CM16 and 0.28 ATI Genes.

Although wheat is used worldwide as a staple food, it can give rise to adverse reactions, for which the triggering factors have not been identified yet. These reactions can be caused mainly by kernel proteins, both gluten and non-gluten proteins. Among these latter proteins, α-amylase/trypsin inhibitors (ATI) are involved in baker's asthma and realistically in Non Celiac Wheat Sensitivity (NCWS). In this paper, we report characterization of three transgenic lines obtained from the bread wheat cultivar Bobwhite silenced by RNAi in the three ATI genes CM3, CM16 and 0.28. We have obtained transgenic lines showing an effective decrease in the activity of target genes that, although showing a higher trypsin inhibition as a pleiotropic effect, generate a lower reaction when tested with sera of patients allergic to wheat, accounting for the important role of the three target proteins in wheat allergies. Finally, these lines show unintended differences in high molecular weight glutenin subunits (HMW-GS) accumulation, involved in technological performances, but do not show differences in terms of yield. The development of new genotypes accumulating a lower amount of proteins potentially or effectively involved in allergies to wheat and NCWS, not only offers the possibility to use them as a basis for the production of varieties with a lower impact on adverse reaction, but also to test if these proteins are actually implicated in those pathologies for which the triggering factor has not been established yet.

Characterisation and comparison of selected wheat (Triticum aestivum L.) cultivars and their blends to develop a gluten reference material.

The reliability and comparability of gluten analytical results in gluten-free foods is hampered by the lack of reference materials (RM). This is partly caused by the yet incomplete knowledge of the effect of genetic and environmental variability of wheat proteins on immunochemical analyses, which affects the choice of gluten source to be applied for RM production. We investigated the genetic variability and the effect of harvest year on the protein composition of five previously selected wheat cultivars. We also compared the magnitude of these effects on ELISA results to get closer to the question of choosing individual cultivar or a mixture as an RM. Our results proved that the application of a blend for this purpose is advantageous. The candidates were also produced on pilot scale to investigate the feasibility of upscaling. The results of comparison studies showed that the pilot scale blended flour can also be suitable for RM.

Characterization of phenolic compounds and antioxidative potential of pot-pollen produced by stingless bees (Tetragonula biroi Friese) from the Philippines.

Eight pot-pollen samples produced by Philippine stingless bees (Tetragonula biroi Friese) were analyzed for the antioxidant composition and capacity. The methanolic extracts showed values of 7.95-24.75 mg GAE/g, 16.13-35.04 mg QE/g, and 0.19-0.74 mg CGE/g for total phenolics, flavonoids, and anthocyanins, respectively. All pot-pollens contained substantial amounts of rutin (0.471-1.547 mg/g), quercetin glucoside (1.427-3.390 mg/g), other flavonol glycosides, and hydroxycinnamic acids. Additionally, pollen samples contained 24-methylenecholesterol and Δ5-avenasterol as dominant phytosterols. Antioxidant activities based on ferric ion reducing antioxidant power (FRAP), DPPH• , and ABTS•+ scavenging activities, and lipid peroxidation (LPO) inhibition showed a high correlation with phenolic and flavonoid contents, except for LPO inhibition. Results suggested that there might be synergistic effects among antioxidants. Summing up, this study revealed that T. biroi Friese pot-pollen can be used as a food supplement with high nutraceutical potential. PRACTICAL APPLICATIONS: The recognition of Tetragonula biroi Friese pot-pollen as a nutraceutical product is strongly limited because its bioactive properties are not well-studied compared to other bee pollens. The results showed a rich occurrence of compounds with antioxidative effects like flavonoids and sterols. Antioxidant activities except lipid peroxidation inhibition revealed a high correlation with analyzed ingredients and shown interactions indicated that there might be synergistic effects among antioxidants.

Development of an enzymatic assay for the quantitative determination of trypsin inhibitory activity in wheat.

Wheat is one of the world's most widely consumed staple food. However, the number of people suffering from wheat-related disorders has increased drastically. Amylase-trypsin inhibitors (ATIs) have recently been identified as one of the main triggers of non-celiac wheat sensitivity (NCWS). In this study, an enzymatic assay for the determination of trypsin inhibition activity in hexaploid wheat was developed. This method was optimized with respect to several parameters, such as extraction and incubation procedures, and was validated according to international standards, concerning accuracy, precision and robustness of the method. Results revealed that linear inhibition and thus accuracy occurred only in a narrow concentration range. However, after optimization of settings the novel method was found to be satisfactory for accurate determination of trypsin inhibition in wheat. Purification of the wheat extract with immobilized trypsin beads led to the identification of CM inhibitors (chloroform/methanol soluble proteins) as main contributors of trypsin inhibition.

Applicability of Yeast Fermentation to Reduce Fructans and Other FODMAPs.

A diet low in fermentable oligosaccharides, disaccharides, monosaccharides and, polyols (FODMAPs) is recommended for people affected by irritable bowel syndrome (IBS) and non-coeliac wheat sensitivity (NCWS) in order to reduce symptoms. Therefore, the aim of this study was to evaluate the impact of 13 sourdough-related yeasts on FODMAP degradation, especially fructans. First, a model system containing a typical wheat carbohydrate profile was applied to evaluate the growth rate of each yeast strain. Additionally, changes in the sugar composition, for up to four days, were monitored by high-pressure anion-exchange chromatography (HPAEC). A more realistic approach with a wheat flour suspension was used to characterize CO2 production according to the Einhorn method. The reduction of the total fructans was analyzed using an enzymatic method. Furthermore, a fingerprint of the present fructans with different degrees of polymerization was analyzed by HPAEC. The results revealed strong differences in the examined yeast strains' ability to degrade fructans, in both the model system and wheat flour. Overall, Saccharomycescerevisiae isolated from Austrian traditional sourdough showed the highest degree of degradation of the total fructan content and the highest gas building capacity, followed by Torulasporadelbrueckii. Hence, this study provides novel knowledge about the FODMAP conversion of yeast strains.

Effects of selected lactobacilli on the functional properties and stability of gluten-free sourdough bread.

The aim of this investigation was to determine the influence of seven different Lactobacillus spp. (Lb.) strains compared with a commercial starter culture (CS) on the functional properties of gluten-free (GF) sourdough-breads. The sourdough stability of selected strains was also evaluated upon back-slopping. Results showed that the bread properties were greatly affected by the Lb. strains. Millet breads achieved lower specific volumes (1.80-2.19 cm3/g), higher crumb firmness (19.01-42.19 N) and lower relative elasticities (21.5-43.4%) than buckwheat breads. Compared with the CS, Lactobacillus pentosus and Lb. hammesii positively influenced the crumb firmness of buckwheat and millet breads, respectively, while Lb. paralimentarius enhanced this property in both breads. Only one of the two Lactobacillus sanfranciscencis strains was able to improve all functional properties in both GF breads. Back-slopping of the sourdoughs revealed stable properties in case of buckwheat, while maturity of the millet sourdough could not be reached. These observations were supported by the microbial count, metabolite production and carbohydrate consumption. Mature sourdough significantly improved the crumb firmness and porosity of the GF breads. These results highlighted the importance of selecting the appropriate lactic acid bacteria strains, to maximize the quality of GF bread.

Fractionation and antioxidant properties of rice bran protein hydrolysates stimulated by in vitro gastrointestinal digestion.

Rice bran was used as a starting material to prepare protein concentrate through enzyme-assisted extraction. The hydrolysis of protein concentrate under in vitro gastrointestinal digestion (pepsin-trypsin system) greatly improved the antioxidant properties. Rice bran protein hydrolysate was further fractionated by membrane ultrafiltration (UF, F1: molecular weight (MW) <3kDa, F2: MW 3-5kDa, and F3: MW 5-10kDa). Peptides with smaller MW possessed higher antioxidant activities (P<0.05). UF showed a great efficacy to selectively separate the metal-chelating peptides. Tyrosine and phenylalanine had positive correlations with their DPPH & ABTS radicals scavenging activities and ferric reducing antioxidant power (r>0.831). A major peptide fragment was detected at m/z 1088 by a MALDI-TOF mass spectrometry. There is high potential that antioxidative peptides from rice bran might also be produced in the gastrointestinal tract of the human body.

Optimization of Arabinoxylan Isolation from Rye Bran by Adapting Extraction Solvent and Use of Enzymes.

Physicochemical and functional properties of arabinoxylans (AXs) can be significantly influenced by their isolation method. Finding balanced process conditions that allow optimal extraction yields while preserving AXs functionality is a challenge. The aim of this study was to determine the effect of different chemical solvents with neutral and alkaline pH on the intrinsic properties and extraction yield of AXs isolated from rye bran. Additionally, the application of xylanases and other cell wall degrading enzymes (Pentopan Mono BG, Deltazym XL-VR, Viscoflow BG) to solubilize bound AXs was investigated. Results show that the use of Ca(OH)2 for isolation was superior to water and Na2 CO3 , as it selectively solubilized AXs and delivered isolates with a purity of up to 43.92% AX and a moderate ferulic acid (FA) content (209.35 ± 16.79 mg FA/100 g AX). Application of xylanases was further able to duplicate these achieved AX yields (7.50 to 9.85g AX/100 g bran). Additionally, isolates displayed highest ferulic acid contents (445.18 to 616.71 mg FA/100 g AX) and lowest impurities in comparison to chemical extracted AXs. Rheological characterization of the isolates showed a pronounced shear thinning behavior which fitted well to the power-law model (R2 > 0.989). Differences in pseudoplasticity of the isolates suggested that structural and chemical properties might have been responsible for this behavior.

Growth Potential of Listeria Monocytogenes in Sliced Turkey Bresaola Packed in Modified Atmosphere.

According to EC Regulation No 2073/2005, for food business operators that produce ready-to-eat (RTE) product, it is crucial to be able to demonstrate if the product supports the growth of Listeria monocytogenes. The objective of the study was therefore to evaluate the behaviour of L. monocytogenes in sliced RTE turkey bresaola (made by cured turkey breast 4.5% NaCl, 1% sodium lactate, sodium nitrite 150 ppm and flavouring) during the shelf life of the product, simulating a contamination during the slicing operation. Considering a shelf life of 90 days, as defined by manufacturer, the packages of sliced bresaola were stored at 5°C for 7 days and at 8°C for the remaining storage time (83 days). L. monocytogenes count decreased during storage test from 1.43/1.98 log cfu/g in the three batches tested to 1.03 log cfu/g in one batch and to undetectable levels in the other two batches. The results show that the investigated product is unable to support the growth of L. monocytogenes.

Behaviour of Escherichia Coli O157:H7 During the Manufacture and Ripening of an Italian Traditional Raw Goat Milk Cheese.

Formagelle di capra is a raw goat cheese produced from whole chilled goat milk; traditional technology involving unpasteurised milk and indigenous lactic starter cultures is employed for its production in Italy. The purpose of this study was to assess the behaviour of Escherichia coli O157:H7 during the manufacturing and ripening of this raw goat milk cheese. Raw milk was experimentally inoculated with E. coli O157:H7 in a laboratory scale plant and the count was monitored during production and 30 days of ripening required for this cheese. Results showed that E. coli O157:H7 count increased to more than 1.5 Log cfu g-1 during cheese production and remained constant until the end of ripening. The evidence that E. coli O157:H7 is able to survive during the manufacturing and ripening process suggests that the 30-day ripening period alone is insufficient to eliminate levels of viable E. coli O157:H7 in Formaggelle di capra cheese and that the presence of low numbers of E. coli O157:H7 in milk destined for the production of raw goat milk cheeses could represent a potential source of infection for humans and a threat for consumers.