Rejuvenated Brewer's Spent Grain: EverVita Ingredients as Game-Changers in Fibre-Enriched Bread.

Brewer's spent grain (BSG) is the main side-stream of brewing. BSG is a potential source for nutritionally enriched cereal products due to its high content of fibre and protein. Two novel ingredients originating from BSG, EverVita FIBRA (EVF) and EverVita PRO (EVP), were incorporated into bread in two addition levels to achieve a 'source of fibre' (3 g/100 g) and a 'high in fibre' (6 g/100 g) nutrition claim for the breads. The impact of those two ingredients on dough and bread quality as well as on nutritional value was investigated and compared to baker's flour (C1) and wholemeal flour (C2) breads. The addition of EVF performed outstandingly well in the bread system achieving high specific volumes (3.72-4.66 mL/g), a soft crumb texture (4.77-9.03 N) and a crumb structure comparable with C1. Furthermore, EVF barely restricted gluten network development and did not influence dough rheology. EVP increased the dough resistance (+150%) compared to C1 which led to a lower specific volume (2.17-4.38 mL/g) and a harder crumb (6.25-36.36 N). However, EVP increased the nutritional value of the breads by increasing protein content (+36%) and protein quality by elevating the amount of indispensable amino acids. Furthermore, a decrease in predicted glycaemic index by 26% was achieved and microbial shelf life was extended by up to 3 days. Although both ingredients originated from the same BSG, their impact on bread characteristics and nutritional value varied. EVF and EVP can be considered as game-changers in the development of bread fortified with BSG, increasing nutritional value, and promoting sustainability.

Particulate matter emissions during field application of poultry manure - The influence of moisture content and treatment.

Along with industry and transportation, agriculture is one of the main sources of primary particulate matter (PM) emissions worldwide. Bioaerosol formation and PM release during livestock manure field application and the associated threats to environmental and human health are rarely investigated. In the temperate climate zone, field fertilization with manure seasonally contributes to local PM air pollution regularly twice per year (spring and autumn). Measurements in a wind tunnel, in the field and computational fluid dynamics (CFD) simulations were performed to analyze PM aerosolization during poultry manure application and the influence of manure moisture content and treatment. A positive correlation between manure dry matter content (DM) and PM release was observed. Therefore, treatments strongly increasing the DM of poultry manure should be avoided. However, high manure DM led to reduced microbial abundance and, therefore, to a lower risk of environmental pathogen dispersion. Considering the findings of PM and microbial measurements, the optimal poultry manure DM range for field fertilization was identified as 50-70%. Maximum PM10 concentrations of approx. 10 mg per m3 of air were measured during the spreading of dried manure (DM 80%), a concentration that is classified as strongly harmful. The modeling of PM aerosolization processes indicated a low health risk beyond a distance of 400 m from the manure application source. The detailed knowledge about PM aerosolization during manure field application was improved with this study, enabling manure management optimization for lower PM aerosolization and pathogenic release into the environment.

Agricultural fertilization with poultry manure results in persistent environmental contamination with the pathogen Clostridioides difficile.

During a field experiment applying broiler manure for fertilization of agricultural land, we detected viable Clostridioides (also known as Clostridium) difficile in broiler faeces, manure, dust and fertilized soil. A large diversity of toxigenic C. difficile isolates was recovered, including PCR ribotypes common from human disease. Genomic relatedness of C. difficile isolates from dust and from soil, recovered more than 2 years after fertilization, traced their origins to the specific chicken farm that had delivered the manure. We present evidence of long-term contamination of agricultural soil with manure-derived C. difficile and demonstrate the potential for airborne dispersal of C. difficile through dust emissions during manure application. Clostridioides genome sequences virtually identical to those from manure had been recovered from chicken meat and from human infections in previous studies, suggesting broiler-associated C. difficile are capable of zoonotic transmission.

Low airborne tenacity and spread of ESBL-/AmpC-producing Escherichia coli from fertilized soil by wind erosion.

ESBL-/AmpC-producing Escherichia coli from organic fertilizers were previously detected on soil surfaces of arable land and might be emitted by wind erosion. To investigate this potential environmental transmission path, we exposed ESBL-/AmpC-positive chicken litter, incorporated in agricultural soils, to different wind velocities in a wind tunnel and took air samples for microbiological analysis. No data exist concerning the airborne tenacity of ESBL-/AmpC-producing E. coli. Therefore, we explored the tenacity of two ESBL/AmpC E. coli strains and E. coli K12 in aerosol chamber experiments at different environmental conditions. In the wind tunnel, ESBL/AmpC-producing E. coli were detected in none of the air samples (n = 66). Non-resistant E. coli were qualitatively detected in 40.7% of air samples taken at wind velocities exceeding 7.3 m s-1 . Significantly increased emission of total viable bacteria with increasing wind velocity was observed. In the aerosol chamber trials, recovery rates of airborne E. coli ranged from 0.003% to 2.8%, indicating a low airborne tenacity. Concluding, an emission of ESBL/AmpC E. coli by wind erosion in relevant concentrations appears unlikely because of the low concentration in chicken litter compared with non-resistant E. coli and their low airborne tenacity, proven in the aerosol chamber trials.

Airborne bacterial emission fluxes from manure-fertilized agricultural soil.

This is the first study to quantify the dependence on wind velocity of airborne bacterial emission fluxes from soil. It demonstrates that manure bacteria get aerosolized from fertilized soil more easily than soil bacteria, and it applies bacterial genomic sequencing for the first time to trace environmental faecal contamination back to its source in the chicken barn. We report quantitative, airborne emission fluxes of bacteria during and following the fertilization of agricultural soil with manure from broiler chickens. During the fertilization process, the concentration of airborne bacteria culturable on blood agar medium increased more than 600 000-fold, and 1 m3 of air carried 2.9 × 105 viable enterococci, i.e. indicators of faecal contamination which had been undetectable in background air samples. Trajectory modelling suggested that atmospheric residence times and dispersion pathways were dependent on the time of day at which fertilization was performed. Measurements in a wind tunnel indicated that airborne bacterial emission fluxes from freshly fertilized soil under local climatic conditions on average were 100-fold higher than a previous estimate of average emissions from land. Faecal bacteria collected from soil and dust up to seven weeks after fertilization could be traced to their origins in the poultry barn by genomic sequencing. Comparative analyses of 16S rRNA gene sequences from manure, soil and dust showed that manure bacteria got aerosolized preferably, likely due to their attachment to low-density manure particles. Our data show that fertilization with manure may cause substantial increases of bacterial emissions from agricultural land. After mechanical incorporation of manure into soil, however, the associated risk of airborne infection is low.

Functional relationship of particulate matter (PM) emissions, animal species, and moisture content during manure application.

Livestock manure is recycled to agricultural land as organic fertilizer. Due to the extensive usage of antibiotics in conventional animal farming, antibiotic-resistant bacteria are highly prevalent in feces and manure. The spread of wind-driven particulate matter (PM) with potentially associated harmful bacteria through manure application may pose a threat to environmental and human health. We studied whether PM was aerosolized during the application of solid and dried livestock manure and the functional relationship between PM release, manure dry matter content (DM), treatment and animal species. In parallel, manure and resulting PM were investigated for the survival of pathogenic and antibiotic-resistant bacterial species. The results showed that from manure with a higher DM smaller particles were generated and more PM was emitted. A positive correlation between manure DM and PM aerosolization rate was observed. There was a species-dependent critical dryness level (poultry: 60% DM, pig: 80% DM) where manure began to release PM into the environment. The maximum PM emission potentials were 1 and 3 kg t-1 of applied poultry and pig manure, respectively. Dried manure and resulting PM contained strongly reduced amounts of investigated pathogenic and antibiotic-resistant microorganisms compared to fresh samples. An optimal manure DM regarding low PM emissions and reduced pathogen viability was defined from our results, which was 55-70% DM for poultry manure and 75-85% DM for pig manure. The novel findings of this study increase our detailed understanding and basic knowledge on manure PM emissions and enable optimization of manure management, aiming a manure DM that reduces PM emissions and pathogenic release into the environment.

Unanticipated Side Effects of Stratospheric Albedo Modification Proposals Due to Aerosol Composition and Phase.

The Earth has now warmed ~1.0 °C since the period 1850-1900, due in large part to the anthropogenic addition of greenhouse gases to the atmosphere. Most strategies to address this warming have called for a reduction of emissions and, often, accompanying removal of greenhouse gases. Other proposals suggest masking the increased radiative forcing by an increase in particles and/or clouds to increase scattering of incoming solar radiation. Two related recent proposals have suggested addition of calcite particles to the stratosphere, which one model suggests may enhance ozone. Here we show that the interaction of calcite with acidic materials in the stratosphere results in a more complex aerosol than has been previously considered, including aqueous and hydrate phases that can lead to ozone loss. Our study suggests particle addition to the stratosphere could also perturb global radiative balance by affecting high altitude cloud formation and properties. Experimental and modeling results suggest particles will act as the nucleation sites for polar stratospheric cloud ice and, after sedimentation into the troposphere, impact cirrus clouds in the absence of other efficient ice nucleating particles. These results show that an overly simplistic set of assumptions regarding intentional particle emissions to the atmosphere can lead to incorrect estimates of the radiative effect and fail to identify unintended consequences.

Recurrent intussusception as initial manifestation of primary intestinal melanoma: Case report and literature review.

Enteric intussusception caused by primary intestinal malignant melanoma is a very rare cause of intestinal obstruction. We herein present a case of a 42-year-old female patient with no prior medical history of malignant melanoma, who was admitted with persistent abdominal pain, nausea, and vomiting. A computed tomography scan revealed an intestinal obstruction due to ileocolic intussusception. An emergency laparoscopy and subsequent laparotomy revealed multiple small solid tumors across the whole small bowel. An oncologic resection was not feasible due to the insufficient length of the remaining small bowel. Only a small segment of ileum, which included the largest tumors causing the intussusception, was resected. The pathologic examination revealed two intestinal malignant melanoma lesions. A systematic clinical examination, endoscopic procedures, and fluorodeoxyglucose positron emission tomography-computed tomography scan all failed to reveal any indication of cutaneous, anal, or retinal melanoma. Hence, the tumor was classified as a primary intestinal malignant melanoma with multiple intestinal metastases. Since a complete oncologic resection of tumors was not possible, in order to prevent future intestinal obstruction, a surgical resection of the largest lesions was performed with palliative intention. The epidemiology, clinical manifestations, diagnosis and management of primary intestinal malignant melanoma, and intestinal intussusception in adults are discussed along with a review of the current literature.

Functional characterization of a eukaryotic melibiose transporter.

Pathogenic fungi drastically affect plant health and cause significant losses in crop yield and quality. In spite of their impact, little is known about the carbon sources used by these fungi in planta and about the fungal transporters importing sugars from the plant-fungus interface. Here, we report on the identification and characterization of MELIBIOSE TRANSPORTER1 (MBT1) from the hemibiotrophic fungus Colletotrichum graminicola (teleomorph Glomerella graminicola), the causal agent of leaf anthracnose and stalk rot disease in maize (Zea mays). Functional characterization of the MBT1 protein in baker's yeast (Saccharomyces cerevisiae) expressing the MBT1 cDNA revealed that α-D-galactopyranosyl compounds such as melibiose, galactinol, and raffinose are substrates of MBT1, with melibiose most likely being the preferred substrate. α-D-glucopyranosyl disaccharides like trehalose, isomaltose, or maltose are also accepted by MBT1, although with lower affinities. The MBT1 gene shows low and comparable expression levels in axenically grown C. graminicola and upon infection of maize leaves both during the initial biotrophic development of the fungus and during the subsequent necrotrophic phase. Despite these low levels of MBT1 expression, the MBT1 protein allows efficient growth of C. graminicola on melibiose as sole carbon source in axenic cultures. Although Δmbt1 mutants are unable to grow on melibiose, they do not show virulence defects on maize.

Hexose transporters of a hemibiotrophic plant pathogen: functional variations and regulatory differences at different stages of infection.

Plant pathogenic fungi use a wide range of different strategies to gain access to the carbon sources of their host plants. The hemibiotrophic maize pathogen Colletotrichum graminicola (teleomorph Glomerella graminicola) colonizes its host plants, and, after a short biotrophic phase, switches to destructive, necrotrophic development. Here we present the identification of five hexose transporter genes from C. graminicola, CgHXT1 to CgHXT5, the functional characterization of the encoded proteins, and detailed expression studies for these genes during vegetative and pathogenic development. Whereas CgHXT4 is expressed under all conditions analyzed, transcript abundances of CgHXT1 and CgHXT3 are transiently up-regulated during the biotrophic phase, and CgHXT2 and CgHXT5 are expressed exclusively during necrotrophic development. Analyses of the encoded proteins characterized CgHXT5 as a low-affinity/high-capacity hexose transporter with a narrow substrate specificity for glucose and mannose. In contrast, CgHXT1 to CgHXT3 are high affinity/low capacity transporters that also accept other substrates, including fructose, galactose, or xylose. CgHXT4, the largest of the identified proteins, has only little transport activity and may function as a sugar sensor. Phylogenetic studies revealed hexose transporters closely related to the five CgHXT proteins also in other pathogenic fungi suggesting conserved functions of these proteins during fungal pathogenesis.

Identification of virulence genes in the corn pathogen Colletotrichum graminicola by Agrobacterium tumefaciens-mediated transformation.

A previously developed Agrobacterium tumefaciens-mediated transformation (ATMT) protocol for the plant pathogenic fungus Colletotrichum graminicola led to high rates of tandem integration of the whole Ti-plasmid, and was therefore considered to be unsuitable for the identification of pathogenicity and virulence genes by insertional mutagenesis in this pathogen. We used a modified ATMT protocol with acetosyringone present only during the co-cultivation of C. graminicola and A. tumefaciens. Analysis of 105 single-spore isolates randomly chosen from a collection of approximately 2000 transformants, indicated that almost 70% of the transformants had single T-DNA integrations. Of 500 independent transformants tested, 10 exhibited attenuated virulence in infection assays on whole plants. Microscopic analyses primarily revealed defects at different pre-penetration stages of infection-related morphogenesis. Three transformants were characterized in detail. The identification of the T-DNA integration sites was performed by amplification of genomic DNA ends after endonuclease digestion and polynucleotide tailing. In one transformant, the T-DNA had integrated into the 5'-flank of a gene with similarity to allantoicase genes of other Ascomycota. In the second and third transformants, the T-DNA had integrated into an open reading frame (ORF) and into the 5'-flank of an ORF. In both cases, the ORFs have unknown function.

Common Motifs in the Response of Cereal Primary Metabolism to Fungal Pathogens are not Based on Similar Transcriptional Reprogramming.

During compatible interactions with their host plants, biotrophic plant-pathogens subvert host metabolism to ensure the sustained provision of nutrient assimilates by the colonized host cells. To investigate, whether common motifs can be revealed in the response of primary carbon and nitrogen metabolism toward colonization with biotrophic fungi in cereal leaves, we have conducted a combined metabolome and transcriptome study of three quite divergent pathosystems, the barley powdery mildew fungus (Blumeria graminis f.sp. hordei), the corn smut fungus Ustilago maydis, and the maize anthracnose fungus Colletotrichum graminicola, the latter being a hemibiotroph that only exhibits an initial biotrophic phase during its establishment. Based on the analysis of 42 water-soluble metabolites, we were able to separate early biotrophic from late biotrophic interactions by hierarchical cluster analysis and principal component analysis, irrespective of the plant host. Interestingly, the corresponding transcriptome dataset could not discriminate between these stages of biotrophy, irrespective, of whether transcript data for genes of central metabolism or the entire transcriptome dataset was used. Strong differences in the transcriptional regulation of photosynthesis, glycolysis, the TCA cycle, lipid biosynthesis, and cell wall metabolism were observed between the pathosystems. However, increased contents of Gln, Asn, and glucose as well as diminished contents of PEP and 3-PGA were common to early post-penetration stages of all interactions. On the transcriptional level, genes of the TCA cycle, nucleotide energy metabolism and amino acid biosynthesis exhibited consistent trends among the compared biotrophic interactions, identifying the requirement for metabolic energy and the rearrangement of amino acid pools as common transcriptional motifs during early biotrophy. Both metabolome and transcript data were employed to generate models of leaf primary metabolism during early biotrophy for the three investigated interactions.

The hemibiotrophic lifestyle of Colletotrichum species.

Colletotrichum species infect several economically important crop plants. To establish a compatible parasitic interaction, a specialized infection cell, the melanized appressorium, is differentiated on the cuticle of the host. After penetration, an infection vesicle and primary hyphae are formed. These structures do not kill the host cell and show some similarities with haustoria formed by powdery mildews and rust fungi. Therefore, this stage of infection is called biotrophic. Later in the infection process, necrotrophic secondary hyphae spread within and kill the host tissue. The lifestyle of Colletotrichum species is called hemibiotrophic, as biotrophic and necrotrophic developmental stages are sequentially established. As most Colletotrichum species are accessible to molecular techniques, genes can be identified and functionally characterized. Here we demonstrate that Agrobacterium tumefaciens-mediated transformation is a well-suited method for tagging of genes mediating compatibility in the Colletotrichum graminicola-maize interaction.