Unraveling the role of germination days on the aroma variations of roasted barley malts via gas chromatography-mass spectrometry based untargeted and targeted flavoromics.

Roasting imparts malts with an increased amount of hedonic aromas. However, the relationship between the production of roasted malts and the generation of characteristic malt aromas remains unclear. In this study, roasted barley malts (RM) were prepared from three consecutive germination days (3, 4, 5D), and the aroma profiles among RM and base malt were holistically compared via HS-SPME-GC-MS/O-based flavoromics. Furthermore, the wort color, free amino acids, reducing sugars, and fatty acids compositions were determined before-and-after roasting. Results showed that roasting could flatten variations of precursors regardless of germination days. Additionally, based on quantitation of 53 aromas, a PLS-DA model was applied to differentiate all malts by 17 aromas with VIP ≥ 1. As for aroma harmony, RM with 4D-germination outstood due to a pleasant nutty note with the highest sweet-to-nutty index of 0.8. This work answers how germination days would impact the aroma of RM for the first time.

Effect of treatment of Fusarium head blight infected barley grains with hop essential oil nanoemulsion on the quality and safety of malted barley.

Fusarium mycotoxin contamination of malting barley has been a persistent food safety issue for malting companies. In this study, the effect of hop essential oil (HEO) nanoemulsion on fungal biomass and mycotoxin production during the malting process was evaluated. Furthermore, the localization of fungal hyphae on the surface and inside the tissue of barley and malts was observed. The application of HEO nanoemulsion reduced fungal biomass and deoxynivalenol (DON) contents at each stage of the malting process as compared to control. During malting process, the fungal hyphae on kernel surfaces was reduced appreciably after steeping. However, the increment of hyphae was observed between the husk and testa layer of barley after germination than raw barley grains. In addition to its antifungal activity, the antioxidant activity of HEO in the treated malts suppressed the formation of aldehydes. This study lays the foundation for the utilization of HEO in the malting industry.

Antifungal activity, mycotoxin inhibitory efficacy, and mode of action of hop essential oil nanoemulsion against Fusarium graminearum.

This work aims to investigate antifungal, mycotoxin inhibitory efficacy of the hop essential oil (HEO) nanoemulsion and their mode of action (MOA) against Fusarium graminearum isolate, a fungal pathogen causing Fusarium Head Blight (FHB) in cereal crops. The HEO, primarily consisting of terpenes and terpenoids, was encapsulated in nanoemulsion droplets. Physically stable HEO-in-water nanoemulsion was fabricated using 0.5 wt% of tween 80 and 5 wt% oil phase comprising 30 % of Ostwald ripening inhibitor and 70 % of HEO. In terms of antifungal effect, HEO nanoemulsion could not only effectively inhibit mycelial growth and spore germination of F. graminearum isolates, but also remarkably suppress the production of deoxynivalenol (DON) and its derivatives in rice culture by applying 750 µg of HEO/g rice. Our studies on the MOA showed that HEO nanoemulsion could alter the contents of total lipid and chitin in outer cell membrane as well as damaging cytoplasmic membrane.

Uncovering aroma boundary compositions of barley malts by untargeted and targeted flavoromics with HS-SPME-GC-MS/olfactometry.

In this study, HS-SPME/GC-MS based untargeted and targeted flavoromics combing with olfactometry were employed to uncover aroma boundary compositions of five types of commercial barley malts with a wide range of Lovibond (L), including kilned base malts (1.8 L and 3.5 L) and roasted caramel malts (10 L, 60 L, and 120 L). Thirty-two compounds were identified as aroma-active with modified detection frequency (MF) > 50%. 3-Methylbutanal (malty), (2E)-nonenal (fatty, cardboard-like), and 2-furfural (burnt, bready) were recognized as the most influential odorants with MF > 70% in all the malts. After untargeted flavoromics, twenty-eight aromas were retained and quantitated. Furthermore, aroma boundary compositions inside/among malt groups were explored with PLS-DA. Eight aroma markers, 3-methylbutanal, 2-isopropyl-5-methyl-2-hexenal, (2E,4E)-Decadienal, 2-furfual, maltol, 2-acetylpyrrole, phenylacetaldehyde, and ethyl hexadecanoate were shortlisted for aroma boundary compositions regarding to the Lovibond of malts.

Genetic Engineering in Combination with Semi-Synthesis Leads to a New Route for Gram-Scale Production of the Immunosuppressive Natural Product Brasilicardin†A.

Brasilicardin A (1) consists of an unusual anti/syn/anti-perhydrophenanthrene skeleton with a carbohydrate side chain and an amino acid moiety. It exhibits potent immunosuppressive activity, yet its mode of action differs from standard drugs that are currently in use. Further pre-clinical evaluation of this promising, biologically active natural product is hampered by restricted access to the ready material, as its synthesis requires both a low-yielding fermentation process using a pathogenic organism and an elaborate, multi-step total synthesis. Our semi-synthetic approach included a) the heterologous expression of the brasilicardin A gene cluster in different non-pathogenic bacterial strains producing brasilicardin A aglycone (5) in excellent yield and b) the chemical transformation of the aglycone 5 into the trifluoroacetic acid salt of brasilicardin A (1 a) via a short and straightforward five-steps synthetic route. Additionally, we report the first preclinical data for brasilicardin A.

Expansion of Internal Hyphal Growth in Fusarium Head Blight-Infected Grains Contributes to the Elevated Mycotoxin Production During the Malting Process.

Fusarium head blight (FHB) and the occurrence of mycotoxins is the largest food safety threat to malting and brewing grains. Worldwide surveys of commercial beers have reported that the trichothecene mycotoxin deoxynivalenol (DON) is the most frequent contaminant in beer. Although the DON content of grain generally declines during steeping due to its solubilization, Fusarium spp. can continue to grow and produce DON from steeping through the early kilning stage of malting. DON present on malt is largely extracted into beer. The objective of the current study was to localize the growth of Fusarium spp. within FHB-infected kernels by developing an improved method and to associate fungal growth with the production of DON during malting. FHB-infected barley, wheat, rye, and triticale grains that exhibited large increases in the amount of Fusarium Tri5 DNA and trichothecene mycotoxins following malting were screened for hyphal localization. The growth of fungal hyphae associated with grain and malt was imaged by scanning electron microscopy and confocal laser-scanning microscopy assisted with WGA-Alexa Fluor 488 staining, respectively. In barley, hyphae were present on or within the husk, vascular bundle, and pericarp cavities. Following malting, vast hyphal growth was observed not only in these regions but also in the aleurone layer, endosperm, and embryo. Extensive fungal growth was also observed following malting of wheat, rye, and triticale. However, these grains already had an extensive internal presence of Fusarium hyphae in the unmalted grain, thus representing an enhanced chance of fungal expansion during the malting.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

A novel LysR-type regulator negatively affects biosynthesis of the immunosuppressant brasilicardin.

Brasilicardin A (BraA) is a promising immunosuppressive compound produced naturally by the pathogenic bacterium Nocardia terpenica IFM 0406. Heterologous host expression of brasilicardin gene cluster showed to be efficient to bypass the safety issues, low production levels and lack of genetic tools related with the use of native producer. Further improvement of production yields requires better understanding of gene expression regulation within the BraA biosynthetic gene cluster (Bra-BGC); however, the only so far known regulator of this gene cluster is Bra12. In this study, we discovered the protein LysRNt, a novel member of the LysR-type transcriptional regulator family, as a regulator of the Bra-BGC. Using in vitro approaches, we identified the gene promoters which are controlled by LysRNt within the Bra-BGC. Corresponding genes encode enzymes involved in BraA biosynthesis as well as the key Bra-BGC regulator Bra12. Importantly, we provide in vivo evidence that LysRNt negatively affects production of brasilicardin congeners in the heterologous host Amycolatopsis japonicum. Finally, we demonstrate that some of the pathway related metabolites, and their chemical analogs, can interact with LysRNt which in turn affects its DNA-binding activity.

Clove oil-in-water nanoemulsion: Mitigates growth of Fusarium graminearum and trichothecene mycotoxin production during the malting of Fusarium infected barley.

Fusarium mycotoxin contamination in malting barley is of great concerns in malting industry. Our recent study found that clove oil nanoemulsions can act as highly efficient antifungal agents in vitro. Therefore, we explored the efficacy of clove oil nanoemulsions on Fusarium growth and mycotoxin during malting process. The impact of emulsifier types (Tween 80, BSA and quillaja saponins) on the formation of clove oil nanoemulsion, the mitigation effects on mycotoxin levels and fungal biomass, and the clove oil flavor residues on malts were measured. We observed that 1.5 mg clove oil/g nanoemulsion showed a negligible influence on germinative energy of barley, while still efficiently eliminated the DON levels and toxicogenic fungal biomass as quantified by Tri5 DNA content. Tween 80-stablized clove oil nanoemulsion displayed higher mycotoxin inhibitory activity and less flavor impact on the final malt. The results indicated the potential application of essential oil nanoemulsion during the malting process.

Effect of germination time on antioxidative activity and composition of yellow pea soluble free and polar soluble bound phenolic compounds.

This research aims to study antioxidative activities of polar solvent extractable phenolic compounds from yellow peas with different germination times against oil-in-water emulsion oxidation. After germination (0, 2, 4, and 6 days), soluble free and polar soluble bound phenolic compounds were extracted and their antioxidative activity was evaluated using stripped soybean oil (SSO)-in-water emulsions. Liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry (LC-ESI-QTOF-MS) and size-exclusion chromatography with multiangle-light-scattering and refractive-index detection (SEC-MALS-RI) were employed to analyze the phenolic composition and molar mass, respectively. Antioxidative activities of soluble free phenolic compounds increased in the SSO-in-water emulsion system, while those of polar soluble bound phenolic compounds decreased with germination. On the basis of chemometric analysis, pratensein (2), phloridzin (4), quercetin (9), sayanedine (12), hesperetin (13), glyzaglabrin (14), and pinocembrin (15) were speculated as the pivotal phenolic compounds responsible for the hydrogen donating capacity. Additionally, decreased molecular weight of soluble bound phenolic compounds was accompanied by the reduction of antioxidative activity in SSO-in-water emulsions indicating that the moieties of polar soluble bound phenolic compounds also have an important impact on the antioxidative activity of phenolic compounds.

Structural Evolution of Water on ZnO(10 1 ‾ 0): From Isolated Monomers via Anisotropic H-Bonded 2D and 3D Structures to Isotropic Multilayers.

The surface chemistry of water on zinc oxides is an important topic in catalysis and photocatalysis. Interaction of D2 O with anisotropic ZnO(10 1 ‾ 0) surfaces was studied by IR reflection absorption spectroscopy using s- and p-polarized light incident along different directions. Interpretation of the experimental data is aided using isotopologues and DFT calculations. The presence of numerous species is revealed: intact monomers, a mixed 2D D2 O/OD adlayer, an anisotropic bilayer, and H-bonded 3D structures. The isolated water monomers are identified unambiguously at low temperatures. The thermally induced diffusion of water monomers occurs at elevated temperatures, forming dimers that undergo autocatalytic dissociation via proton transfer. Polarization- and azimuth-resolved IR data provide information on the orientation and strength of H-bonds within the 2D and 3D structures. Ab initio molecular dynamics simulations reveal strong anharmonic couplings within the H-bond network.

Influence of nonionic and ionic surfactants on the antifungal and mycotoxin inhibitory efficacy of cinnamon oil nanoemulsions.

The influence of ionic surfactants (cationic surfactant lauric arginate and anionic surfactant lysolecithin) on the physical properties, antifungal and mycotoxin inhibitory efficacy of Tween 80 stabilized cinnamon oil-in-water nanoemulsions was investigated. Nanoemulsion droplets of similar particle diameter (∼100 nm), but variable electrical characteristics, were formed by mixing 0.1 wt% ionic surfactant with 0.9 wt% Tween 80 before homogenization. The nanoemulsions were physically stable over 28 days at 23 °C. The antifungal activity (against mycelial growth and spore germination) and mycotoxin inhibitory activity of cinnamon oil nanoemulsions bearing positive, neutral, and negative charge surface was then evaluated against two chemotypes of Fusarium graminearum. In general, the cinnamon oil played a decisive role in the resulting antifungal and mycotoxin inhibitory activities. The surfactant charge had a limited impact on the antifungal mycotoxin inhibitory activities of cinnamon oil in the nanoemulsions. Both ionic surfactant-based cinnamon oil nanoemulsions showed greater activity in inhibiting mycelial growth and mycotoxin production of F. graminearum than those based on Tween 80. Treatment of mycelium with cinnamon oil nanoemulsions resulted in the loss of cytoplasm from fungal hyphae, and accounted for the antifungal action. These results have important implications for the design of essential oil based nanoemulsions as effective antifungal delivery systems in foods.

Physical properties, antifungal and mycotoxin inhibitory activities of five essential oil nanoemulsions: Impact of oil compositions and processing parameters.

The influence of homogenization conditions on selected essential oil (thyme, lemongrass, cinnamon, peppermint, and clove)-in-water nanoemulsion formation and stability was investigated. Physically stable essential oil nanoemulsions could be fabricated by a microfludizer under optimized processing conditions (10,000 psi and 2 passes). The chemical compositions of EOs was characterized using GC-MS. The antifungal activity and mycotoxin inhibitory activity of essential oils in both bulk and nanoemulsion forms were determined using two isolates of Fusarium graminearum. The major chemical components of essential oil had a remarkable impact on long term physical stability, antifungal activity, and inhibition of mycotoxin production. With regard to inhibition of mycotoxin production, the mycotoxin inhibitory activity of essential oils was enhanced considerably in nanoemulsion form, which was attributed to greater solubility of the essential oils. It was also noted that the same essential oils exhibited significant differences in inhibition of mycotoxin production in the two isolates of F. graminearum.

Association and genome analyses to propose putative candidate genes for malt quality traits.

BACKGROUND: We studied the genetics of nine malt quality traits using association genetics in a panel of North Dakota, ICARDA, and Ethiopian barley lines. Grain samples harvested from Bekoji in 2011 and 2012 were used. RESULTS: The mapping panel revealed strong population structure explained by inflorescence-type, geographic origin, and breeding history. North Dakota germplasm were superior in malt quality traits and they can be donors to improve malt quality properties. We identified 106 marker-trait associations (MTAs) for the nine traits, representing 81 genomic regions across all barley chromosomes. Chromosomes 3H, 5H, and 7H contained most of the MTAs (58.5%). Nearly 18.5% of these genomic regions contained two to three malt quality traits. Within ±250 kb of 81 genomic regions, we recovered 348 barley genes, with some potential impacting malt quality. These include invertase, ß-fructofuranosidase, α-glucosidase, serine carboxypeptidase, and bidirectional sugar transporter SWEET14-like protein. Eighteen of these genes were also previously reported in the Hordeum Toolbox, and 17 of them highly expressed during the germination process. CONCLUSION: The results from this study invite further follow-up functional characterization experiments to relate the genes with individual malt quality traits with higher confidence. It also provides germplasm resources for malt barley improvement. © 2018 Society of Chemical Industry.

Malting of Fusarium Head Blight-Infected Rye (Secale cereale): Growth of Fusarium graminearum, Trichothecene Production, and the Impact on Malt Quality.

This project was initiated with the goal of investigating the malt quality of winter rye cultivars and hybrids grown in the United States in 2014 and 2015, but high levels of deoxynivalenol (DON) were subsequently found in many of the malt samples. DON levels in 75% of the investigated rye samples (n = 117) were actually below 1.0 mg/kg, as quantified by a gas chromatography combined with electron capture detector (GC-ECD). However, 83% of the samples had DON in excess of 1.0 mg/kg following malting, and the average DON level in malted rye was 10.6 mg/kg. In addition, relatively high levels of 3-acetate DON (3-ADON), 15-acetate DON (15-ADON), nivalenol (NIV), and DON-3-glucoside (D3G) were observed in some rye malts. Our results show that rye grain DON is likely a poor predicator of type B trichothecenes in malt in practice, because high levels of malt DON, 15-ADONm and D3G were produced, even when the rye samples with DON levels below 0.50 mg/kg were processed. Fusarium Tri5 DNA content in rye was highly associated with malt DON levels (r = 0.83) in a small subset of samples (n = 55). The impact of Fusarium infection on malt quality was demonstrated by the significant correlations between malt DON levels and wort viscosity, ß-glucan content, wort color, wort p-coumaric acid content, and total phenolic content. Additional correlations of rye Fusarium Tri5 DNA contents with malt diastatic power (DP), wort free amino nitrogen (FAN) content, and arabinoxylan content were observed.

Engineering metabolic pathways in Amycolatopsis japonicum for the optimization of the precursor supply for heterologous brasilicardin congeners production.

The isoprenoid brasilicardin A is a promising immunosuppressant compound with a unique mode of action, high potency and reduced toxicity compared to today's standard drugs. However, production of brasilicardin has been hampered since the producer strain Nocardia terpenica IFM0406 synthesizes brasilicardin in only low amounts and is a biosafety level 2 organism. Previously, we were able to heterologously express the brasilicardin gene cluster in the nocardioform actinomycete Amycolatopsis japonicum. Four brasilicardin congeners, intermediates of the BraA biosynthesis, were produced. Since chemical synthesis of the brasilicardin core structure has remained elusive we intended to produce high amounts of the brasilicardin backbone for semi synthesis and derivatization. Therefore, we used a metabolic engineering approach to increase heterologous production of brasilicardin in A. japonicum. Simultaneous heterologous expression of genes encoding the MVA pathway and expression of diterpenoid specific prenyltransferases were used to increase the provision of the isoprenoid precursor isopentenyl diphosphate (IPP) and to channel the precursor into the direction of diterpenoid biosynthesis. Both approaches contributed to an elevated heterologous production of the brasilicardin backbone, which can now be used as a starting point for semi synthesis of new brasilicardin congeners with better properties.

Improvement of the Antioxidative Activity of Soluble Phenolic Compounds in Chickpea by Germination.

Our recent study found that antioxidative activity of phenolic compounds extracted from germinated chickpea was boosted in both in vitro assays and oil-in-water emulsions [ Xu et al. Food Chem. 2018 , 250 , 140 ]. The purpose of this study was to elucidate the mechanism by which germination enhances the antioxidative activity of the phenolic compounds extracted from chickpea. Liquid chromatography coupled with electrospray-ionization quadrupole time-of-flight mass spectrometry (LC-ESI-QTOF-MS) and size-exclusion chromatography with multiangle-light-scattering and refractive-index detection (SEC-MALS-RI) were employed to evaluate the phenolic composition of soluble phenolic compounds (free and bound) and molar masses of soluble bound phenolic compounds, respectively, over 6 days of germination. According to principal-component analysis of the interrelationship between germination time and phenolic composition, it is revealed that protocatechuic acid 4- O-glucoside and 6-hydroxydaidzein played a pivotal role in the soluble free phenolic compounds, whereas gentisic acid and 7,3',4'-trihydroxyflavone were important in the soluble bound phenolic compounds. Molar masses of soluble bound phenolic compounds were increased after 6 days of germination. Protective and dual antioxidative effects were proposed to explicate how the antioxidative activity of soluble bound phenolic compounds in oil-in-water emulsions was improved with germination.

Influence of oil phase composition on the antifungal and mycotoxin inhibitory activity of clove oil nanoemulsions.

The influence of oil composition on the physical properties and antifungal and mycotoxin inhibitory activity of clove oil-in-water nanoemulsions was investigated. Physically stable clove oil-in-water nanoemulsions could be fabricated by incorporating either ≥75 wt% of corn oil or ≥50 wt% of medium chain triacylglycerol (MCT) into clove oil before homogenization to prevent Ostwald ripening. The clove oil-in-water nanoemulsions with mean diameters of <150 nm showed high physical stability over 30 days storage. The antifungal activity of physically stable clove oil nanoemulsions was further evaluated using effective concentration (EC) and inhibitory activity towards mycotoxin production in two chemotypes of Fusarium graminearum isolates. The composition of the oil phase, i.e., ripening inhibitor type and concentration, in clove oil-in-water nanoemulsions had a remarkable impact on antifungal activity as well as inhibition of mycotoxin production. In general, under the same clove oil concentration in oil phase, the addition of MCT decreased the antifungal and mycotoxin inhibitory activity of clove oil more than corn oil. Compared with the bulk clove oil, this study also indicated that the mycotoxin inhibitory activity of clove was significantly enhanced when encapsulated in nanoemulsions. These results have important implications for the design of essential oil based nanoemulsions as effective antifungal and detoxification delivery systems in the food or other industries.

The Immunosuppressant Brasilicardin: Determination of the Biosynthetic Gene Cluster in the Heterologous Host Amycolatopsis japonicum.

Nocardia terpenica IFM 0406 is the producer of the immunosuppressants brasilicardins A-D. Brasilicardin is a promising compound because of its unique mode of action and its higher potency and reduced toxicity compared to today's standard drugs. However, production of brasilicardin is so far hampered as Nocardia terpenica IFM 0406 synthesizes brasilicardin in only low amounts and represents a human pathogen (biosafety level 2 BSL2). In order to achieve a safe and high yield production of brasilicardin A (BraA), the authors heterologously express the brasilicardin gene cluster in the nocardioform actinomycete Amycolatopsis japonicum (A. japonicum::bcaAB01), which is fast growing, genetically accessible and closely related to N. terpenica IFM 0406. In A. japonicum::bcaAB01, four brasilicardin congeners, intermediates of the BraA biosynthesis, are produced. Investigation of the genes flanking the previously defined brasilicardin biosynthetic gene cluster revealed two novel genes (bra0, bra12), which are involved in brasilicardin biosynthesis: bra12 encodes a transcriptional activator of the brasilicardin gene cluster. bra0 codes for a dioxygenase involved in methoxylation of brasilicardin. Based on this finding the authors are able to revise the proposed brasilicardin biosynthesis.

Distal femoral replacement with the MML system: a single center experience with an average follow-up of 86 months.

BACKGROUND: The aim of this study was to compare the functional outcomes and complication rates after distal femoral replacement (DFR) performed with the modular Munich-Luebeck (MML) modular prosthesis (ESKA/Orthodynamics, Luebeck, Germany) in patients being treated for malignant disease or failed total knee arthroplasty. METHODS: A retrospective review of patient charts and a functional investigation (involving Musculoskeletal Tumor Society Score [MSTS], American Knee Society Score [AKSS], Oxford Knee Score [OKS], Western Ontario and McMaster Universities Osteoarthritis Index [WOMAC], Toronto Extremity Salvage Score [TESS], the 12-Item Short-Form [SF-12] Health Survey, and a failure classification system developed by Henderson et al.) of DFR cases from 2002 to 2015 were conducted. The indications for DFR were malignant tumor resection in the femur (n = 20, group A) or failure of revision total knee arthroplasty without a history of malignant disease (n = 16, group B). RESULTS: One-hundred and twenty-nine patients were treated during the study period. Of these, 82 were analyzed for complications and implant-survival. Further, 36 patients were available for functional assessment after a mean follow-up of 86 months (range: 24-154). There were 75 complications in total. The overall failure rate for DFR was 64.6% (53/82 patients). The most common failure mechanisms were type III (mechanical failure), followed by type I (soft tissue) and type II (aseptic loosening). The mean MSTS score (out of 30) was 17 for group A and 12 for group B. All the clinical outcome scores revealed an age-dependent deterioration of function. CONCLUSION: DFR is an established procedure to restore distal femoral integrity. However, complication rates are high. Post-procedure functionality depends mainly on the patient's age at initial reconstruction.

High-Quality Draft Genome Sequence of the Actinobacterium Nocardia terpenica IFM 0406, Producer of the Immunosuppressant Brasilicardins, Using Illumina and PacBio Technologies.

The bacterium Nocardia terpenica IFM 0406 is known as the producer of the immunosuppressant brasilicardin A. Here, we report the completely sequenced genome of strain IFM 0406, which facilitates the heterologous expression of the brasilicardin biosynthetic gene cluster but also unveils the intriguing biosynthetic capacity of the strain to produce secondary metabolites.