Improved hydrolase activity in barley and reduced malting time by adding phytase as an activator during malting steeping

Objectives

Exogenous phytase improved the activity of hydrolases to decrease the malting time.

Results

Treatment with phytase during barley steeping increased activity of hydrolases (α-/β-amylase, proteinase, β-glucanase and xylanase) in green malt. Maximal activity was observed for α-/β-amylase, β-glucanase and xylanase with 0.8 U phytase/g and proteinase with 1.2 U phytase/g. Phytase promoted acrospire growth of green malt and reduced malting process with 0.8 U phytase/g in 96 h, which is 24 h less than the control. No significant variation of malt quality was found between control malt and malt treated with 0.8 U/g or 1.2 U phytase/g (P > 0.05), which makes application of exogenous phytase during steeping process as a good option for reducing malting time.

Conclusion

Adding phytase during steeping process increases the activity of hydrolases, which reduces malting time without impacting on malt quality.

     

Enhancing the production of phenolic compounds during barley germination by using chitooligosaccharides to improve the antioxidant capacity of malt

Objective

To enhance the production of phenolic compounds during barley germination using chitooligosaccharide as an elicitor to improve the antioxidant capacity of malt.

Results

When used as an elicitor for barley germination, chitooligosaccharide with a molecular weight of 3 kDa, added at 10 mg/kg barley kernels during the first steeping cycle, led to the maximum production of phenolic compounds. Compared with the control with no chitooligosaccharide added to the steeping water, the total phenolic content was increased by 54.8%. Increases in the total phenolic content of the barley malt occurred when chitooligosaccharide was applied during the first or both the first and the second steeping cycles. Thus the antioxidant capacity of barley malt was increased significantly by adding chitooligosaccharide during the steeping process.

Conclusion

Applying chitooligosaccharides during the steeping process increased the content of phenolic compounds thus improving the antioxidant capacity of the barley malt.

     

The use of chitooligosaccharide in beer brewing for protection against beer-spoilage bacteria and its influence on beer performance

Objectives

To identify a biological preservative that can protect beer from microbial contamination, which often results in the production of turbidity and off-flavor.

Results

The antimicrobial activity of a chitooligosaccharide against beer-spoilage bacteria and its effect on the fermentation performance of brewer’s yeast was studied. Chitooligosaccharide with an average 2 kDa molecular weight was the best at inhibiting all tested beer-spoilage bacteria. The application of chitooligosaccharide in the brewing process did not influence the fermentation of brewer’s yeast. The change in beer performance induced by the contamination of Lactobacillus brevis could be effectively controlled by application of chitooligosaccharide in the beer brewing process.

Conclusion

The experimental data suggested that chitooligosaccharide should be an excellent preservative to inhibit beer-spoilage bacteria in the brewing process and in the end product.

     

Media optimization for extracellular amylase production by <Emphasis Type="Italic">Pseudomonas balearica</Emphasis> vitps19 using response surface methodology

Background

In this study, we optimized the process for enhancing amylase production from Pseudomonas balearica VITPS19 isolated from agricultural lands in Kolathur, India.

Methods

Process optimization for enhancing amylase production from the isolate was carried out by Response Surface Methodology (RSM) with optimized chemical and physical sources using Design expert v.7.0. A central composite design was used to evaluate the interaction between parameters. Interaction between four factors–maltose (C-source), malt extract (Nsource), pH, and CaCl₂ was studied.

Results

The factors pH and CaCl₂ concentration were found to affect amylase production. Validation of the experiment showed a nearly twofold increase in alpha amylase production.

Conclusion

Amylase production was thus optimized and increased yield was achieved.

     

Quantitative analysis of tetrahydrofolate metabolites from <Emphasis Type="Italic">clostridium autoethanogenum</Emphasis>

Introduction

Quantification of tetrahydrofolates (THFs), important metabolites in the Wood–Ljungdahl pathway (WLP) of acetogens, is challenging given their sensitivity to oxygen.

Objective

To develop a simple anaerobic protocol to enable reliable THFs quantification from bioreactors.

Methods

Anaerobic cultures were mixed with anaerobic acetonitrile for extraction. Targeted LC–MS/MS was used for quantification.

Results

Tetrahydrofolates can only be quantified if sampled anaerobically. THF levels showed a strong correlation to acetyl-CoA, the end product of the WLP.

Conclusion

Our method is useful for relative quantification of THFs across different growth conditions. Absolute quantification of THFs requires the use of labelled standards.

     

Extraction of natural products from bark of <Emphasis Type="Italic">Betula pendula</Emphasis> using ionic liquids

Introduction

Aqueous–methanol mixtures have successfully been applied to extract a broad range of metabolites from plant tissue. However, a certain amount of material remains insoluble.

Objectives

To enlarge the metabolic compendium, two ionic liquids were selected to extract the methanol insoluble part of trunk from Betula pendula.

Methods

The extracted compounds were analyzed by LC/MS and GC/MS.

Results

The results show that 1-butyl-3-methylimidazolium acetate (IL-Ac) predominantly resulted in fatty acids, whereas 1-ethyl-3-methylimidazolium tosylate (IL-Tos) mostly yielded phenolic structures. Interestingly, bark yielded more ionic liquid soluble metabolites compared to interior wood.

Conclusion

From this one can conclude that the application of ionic liquids may expand the metabolic snapshot.

     

Volatiles from biofumigant plants have a direct effect on carpogenic germination of sclerotia and mycelial growth of <Emphasis Type="Italic">Sclerotinia sclerotiorum</Emphasis>

Aims

Sclerotia of Sclerotinia sclerotiorum survive in soil and germinate to produce apothecia which release airborne ascospores. Current control methods rely predominantly on the use of fungicides to kill ascospores. The aim of this research was to identify potential biofumigation treatments which suppress sclerotial germination, providing a potential alternative and long-term approach to disease management.

Methods

Microcosm and in vitro experiments were conducted using dried and milled plant material from six different biofumigant crop plants to determine effects on carpogenic germination of sclerotia and mycelial growth of S. sclerotiorum.

Results

All biofumigant plants significantly reduced germination of S. sclerotiorum sclerotia in the microcosm experiments, but were less effective against larger sclerotia. In vitro experiments showed a direct effect of biofumigant volatiles on both the mycelial growth of S. sclerotiorum, and carpogenic germination of sclerotia, where the most effective treatment was B. juncea ‘Vittasso’.

Conclusions

It was clear from this study that biofumigant crop plants have potential as part of an integrated disease management system for control of S. sclerotiorum. The microcosm experiments described here provide a straightforward and reliable screening method for evaluating different biofumigants for activity.

     

Iridoid and phenylethanoid/phenylpropanoid metabolite profiles of <Emphasis Type="Italic">Scrophularia</Emphasis> and <Emphasis Type="Italic">Verbascum</Emphasis> species used medicinally in North America

Introduction

Botanicals containing iridoid and phenylethanoid/phenylpropanoid glycosides are used worldwide for the treatment of inflammatory musculoskeletal conditions that are primary causes of human years lived with disability, such as arthritis and lower back pain.

Objectives

We report the analysis of candidate anti-inflammatory metabolites of several endemic Scrophularia species and Verbascum thapsus used medicinally by peoples of North America.

Methods

Leaves, stems, and roots were analyzed by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) and partial least squares-discriminant analysis (PLS-DA) was performed in MetaboAnalyst 3.0 after processing the datasets in Progenesis QI.

Results

Comparison of the datasets revealed significant and differential accumulation of iridoid and phenylethanoid/phenylpropanoid glycosides in the tissues of the endemic Scrophularia species and Verbascum thapsus.

Conclusions

Our investigation identified several species of pharmacological interest as good sources for harpagoside and other important anti-inflammatory metabolites.

     

Metabolic response of porcine colon explants to in vitro infection by <Emphasis Type="Italic">Brachyspira hyodysenteriae</Emphasis>: a leap into disease pathophysiology

Introduction

Swine dysentery caused by Brachyspira hyodysenteriae is a production limiting disease in pig farming. Currently antimicrobial therapy is the only treatment and control method available.

Objective

The aim of this study was to characterize the metabolic response of porcine colon explants to infection by B. hyodysenteriae.

Methods

Porcine colon explants exposed to B. hyodysenteriae were analyzed for histopathological, metabolic and pro-inflammatory gene expression changes.

Results

Significant epithelial necrosis, increased levels of l-citrulline and IL-1α were observed on explants infected with B. hyodysenteriae.

Conclusions

The spirochete induces necrosis in vitro likely through an inflammatory process mediated by IL-1α and NO.

     

<Emphasis Type="Italic">massPix</Emphasis>: an R package for annotation and interpretation of mass spectrometry imaging data for lipidomics

Introduction

Mass spectrometry imaging (MSI) experiments result in complex multi-dimensional datasets, which require specialist data analysis tools.

Objectives

We have developed massPix—an R package for analysing and interpreting data from MSI of lipids in tissue.

Methods

massPix produces single ion images, performs multivariate statistics and provides putative lipid annotations based on accurate mass matching against generated lipid libraries.

Results

Classification of tissue regions with high spectral similarly can be carried out by principal components analysis (PCA) or k-means clustering.

Conclusion

massPix is an open-source tool for the analysis and statistical interpretation of MSI data, and is particularly useful for lipidomics applications.

     

Biocrusts enhance soil fertility and <Emphasis Type="Italic">Bromus tectorum</Emphasis> growth,and interact with warming to influence germination

Background and aims

Biocrusts are communities of cyanobacteria, mosses, and/or lichens found in drylands worldwide. Biocrusts are proposed to enhance soil fertility and productivity, but simultaneously act as a barrier to the invasive grass, Bromus tectorum, in western North America. Both biocrusts and B. tectorum are sensitive to climate change drivers, yet how their responses might interact to affect dryland ecosystems is unclear.

Methods

Using mesocosms with bare soil versus biocrust cover, we germinated B. tectorum seeds collected from warmed, warmed + watered, and ambient temperature plots within a long-term climate change experiment on the Colorado Plateau, USA. We characterized biocrust influences on soil fertility and grass germination, morphology, and chemistry.

Results

Biocrusts increased soil fertility and B. tectorum biomass, specific leaf area (SLA), and root:shoot ratios. Germination rates were unaffected by mesocosm cover-type. Biocrusts delayed germination timing while also interacting with the warmed treatment to advance, and with the warmed + watered treatment to delay germination.

Conclusions

Biocrusts promoted B. tectorum growth, likely through positive influence on soil fertility which was elevated in biocrust mesocosms, and interacted with seed treatment-provenance to affect germination. Understanding how anticipated losses of biocrusts will affect invasion dynamics will require further investigation of how plant plasticity/adaptation to specific climate drivers interact with soil and biocrust properties.

     

Degradation of carbazole,dibenzothiophene and polyaromatic hydrocarbons by recombinant <Emphasis Type="Italic">Rhodococcus</Emphasis> sp.

Objectives

With the view of designing a single biocatalyst for biorefining, carbazole dioxygenase was cloned from Pseudomonas sp. and expressed in Rhodococcus sp.

Results

The recombinant, IGTS8, degraded both carbazole and dibenzothiophene at 400 mg/l in 24 h. Maximum carbazole degradation was in 1:1 (v/v) hexadecane/aqueous phase. Anthracene, phenanthrene, pyrene, fluoranthene and fluorine were also degraded without affecting the aliphatic component.

Conclusions

Recombinant Rhodococcus sp. IGTS8 can function as a single biocatalyst for removing major contaminants of fossil fuels viz. dibenzothiophene, carbazole and polyaromatic compounds.

     

Optimization of fecal sample preparation for untargeted LC-HRMS based metabolomics

Introduction

Collecting feces is easy. It offers direct outcome to endogenous and microbial metabolites.

Objectives

In a context of lack of consensus about fecal sample preparation, especially in animal species, we developed a robust protocol allowing untargeted LC-HRMS fingerprinting.

Methods

The conditions of extraction (quantity, preparation, solvents, dilutions) were investigated in bovine feces.

Results

A rapid and simple protocol involving feces extraction with methanol (1/3, M/V) followed by centrifugation and a step filtration (10 kDa) was developed.

Conclusion

The workflow generated repeatable and informative fingerprints for robust metabolome characterization.

     

Metabolomic analysis of tomato seed germination

Introduction

Seed germination is inherently related to seed metabolism, which changes throughout its maturation, desiccation and germination processes. The metabolite content of a seed and its ability to germinate are determined by underlying genetic architecture and environmental effects during development.

Objective

This study aimed to assess an integrative approach to explore genetics modulating seed metabolism in different developmental stages and the link between seed metabolic- and germination traits.

Methods

We have utilized gas chromatography-time-of-flight/mass spectrometry (GC-TOF/MS) metabolite profiling to characterize tomato seeds during dry and imbibed stages. We describe, for the first time in tomato, the use of a so-called generalized genetical genomics (GGG) model to study the interaction between genetics, environment and seed metabolism using 100 tomato recombinant inbred lines (RILs) derived from a cross between Solanum lycopersicum and Solanum pimpinellifolium.

Results

QTLs were found for over two-thirds of the metabolites within several QTL hotspots. The transition from dry to 6 h imbibed seeds was associated with programmed metabolic switches. Significant correlations varied among individual metabolites and the obtained clusters were significantly enriched for metabolites involved in specific biochemical pathways.

Conclusions

Extensive genetic variation in metabolite abundance was uncovered. Numerous identified genetic regions that coordinate groups of metabolites were detected and these will contain plausible candidate genes. The combined analysis of germination phenotypes and metabolite profiles provides a strong indication for the hypothesis that metabolic composition is related to germination phenotypes and thus to seed performance.

     

Characterization of ankylosing spondylitis and rheumatoid arthritis using <Superscript>1</Superscript>H NMR-based metabolomics of human fecal extracts

Introduction

The differences in fecal metabolome between ankylosing spondylitis (AS)/rheumatoid arthritis (RA) patients and healthy individuals could be the reason for an autoimmune disorder.

Objectives

The study explored the fecal metabolome difference between AS/RA patients and healthy controls to clarify human immune disturbance.

Methods

Fecal samples from 109 individuals (healthy controls 34, AS 40, and RA 35) were analyzed by ¹H NMR spectroscopy. Data were analyzed with principal component analysis (PCA) and orthogonal projection to latent structure discriminant (OPLS-DA) analysis.

Results

Significant differences in the fecal metabolic profiles could distinguish AS/RA patients from healthy controls but could not distinguish between AS and RA patients. The significantly decreased metabolites in AS/RA patients were butyrate, propionate, methionine, and hypoxanthine. Significantly increased metabolites in AS/RA patients were taurine, methanol, fumarate, and tryptophan.

Conclusion

The metabolome variations in feces indicated AS and RA were two homologous diseases that could not be distinguished by ¹H NMR metabolomics.

     

Crystal structure of <Emphasis Type="Italic">Escherichia coli</Emphasis> protein ybgI,a toroidal structure with a dinuclear metal site

Background

The protein encoded by the gene ybgI was chosen as a target for a structural genomics project emphasizing the relation of protein structure to function.

Results

The structure of the ybgI protein is a toroid composed of six polypeptide chains forming a trimer of dimers. Each polypeptide chain binds two metal ions on the inside of the toroid.

Conclusion

The toroidal structure is comparable to that of some proteins that are involved in DNA metabolism. The di-nuclear metal site could imply that the specific function of this protein is as a hydrolase-oxidase enzyme.

     

rDolphin: a GUI R package for proficient automatic profiling of 1D <Superscript>1</Superscript>H-NMR spectra of study datasets

Introduction

Adoption of automatic profiling tools for ¹H-NMR-based metabolomic studies still lags behind other approaches in the absence of the flexibility and interactivity necessary to adapt to the properties of study data sets of complex matrices.

Objectives

To provide an open source tool that fully integrates these needs and enables the reproducibility of the profiling process.

Methods

rDolphin incorporates novel techniques to optimize exploratory analysis, metabolite identification, and validation of profiling output quality.

Results

The information and quality achieved in two public datasets of complex matrices are maximized.

Conclusion

rDolphin is an open-source R package (http://github.com/danielcanueto/rDolphin) able to provide the best balance between accuracy, reproducibility and ease of use.

     

Accumulation of Fascin<Superscript>+</Superscript> cells during experimental autoimmune neuritis

Background

Experimental autoimmune neuritis (EAN) is a well-known animal model of human demyelinating polyneuropathies and is characterized by inflammation and demyelination in the peripheral nervous system. Fascin is an evolutionarily highly conserved cytoskeletal protein of 55 kDa containing two actin binding domains that cross-link filamentous actin to hexagonal bundles.

Methods

Here we have studied by immunohistochemistry the spatiotemporal accumulation of Fascin?+?cells in sciatic nerves of EAN rats.

Results

A robust accumulation of Fascin?+?cell was observed in the peripheral nervous system of EAN which was correlated with the severity of neurological signs in EAN.

Conclusion

Our results suggest a pathological role of Fascin in EAN.

Virtual slides

The virtual slides for this article can be found here: http://www.diagnosticphatology.diagnomx.eu/vs/6734593451114811

     

Isolation and characterization of a novel <Emphasis Type="SmallCaps">l</Emphasis>-glutamate oxidase with strict substrate specificity from <Emphasis Type="Italic">Streptomyces diastatochromogenes</Emphasis>

Objectives

To find an l-glutamate oxidase (LGox), to be used for the quantitative analysis of l-glutamic acid, an lgox gene encoding LGox from Streptomyces diastatochromogenes was isolated, cloned and characterized.

Results

The gene had an ORF of 1974 bp encoding a protein of 657 amino acid residues. In comparison to the LGox precursor, the proteinase K-treated enzyme exhibited improved affinity to substrate and with a K _m of 0.15 mM and V _max of 62 μmol min^?1 mg^?1. The 50% thermal inactivation temperature of the proteinase K treated enzyme was increased from 50 to 70 °C. The enzyme exhibited strict specificity for l-glutamate.

Conclusions

LGox treated by proteinase K exhibited strict specificity for l-glutamate, good thermostability and high substrate affinity.

     

A seed-recruited microbiome protects developing seedlings from disease by altering homing responses of <Emphasis Type="Italic">Pythium aphanidermatum</Emphasis> zoospores

Aims

We investigated potential mechanisms by which a seed microbiome recruited from vermicomposted dairy manure alters Pythium aphanidermatum zoospore mediated pathogenesis in cucumber.

Methods

Bioassays were conducted to measure arrival of zoospores at the seed surface via qPCR and subsequent seedling disease incidence. Seed exudates were collected at relevant time points for use in zoospore microscopy assays. Metabolomic analysis was used to characterize seed exudates.

Results

Microbes recruited by the germinating seed from a disease suppressive substrate within 8 hours of sowing prevented zoospore arrival at the seed surface, modified seed exudates and reduced disease incidence. In vitro exposure to microbially modified seed exudates altered zoospore homing responses and reduced both encystment and germination compared to control exudates. Combining modified and control exudates failed to restore zoospore attraction to levels observed with control exudates. Observed zoosporolytic activity of the modified exudates was unique to the ethyl acetate fraction and metabolomic analysis revealed several putative zoosporolytic compounds present at higher relative abundance when compared to control exudates.

Conclusions

The observed disease suppression was likely due to the production of a specific zoosporolytic compound or set of compounds in the spermosphere by one or more members of the seed-recruited vermicompost microbiome.