Effect of controlled extracellular oxidation–reduction potential on microbial metabolism and proteolysis in buckwheat sourdough

The aim of this study was to keep constant the extracellular oxidation–reduction potential (ORP) and to observe the microbial activity changes in gluten-free (GF) sourdough fermentations with lactic acid bacteria (Weissella cibaria, Pediococcus pentosaceus and coculture of both microorganisms). ORP (E_h7) was held constant at ca. +350 and ?300 mV by gas sparging with air and N₂/H₂, respectively, to achieve oxidizing or reducing conditions during buckwheat sourdough fermentations. Microbial metabolism, free thiols, proteolysis and volatile compounds were monitored. Oxidizing conditions increased the acidification rate of W. cibaria and mixed culture (mix), which contains P. pentosaceus and W. cibaria. Reducing conditions exhibited a slow acidification rate and low microbial cell density upon fermentation. Oxidizing conditions changed lactic to acetic acid ratio of W. cibaria and mix culture from 7.9 ± 1.3 and 16.0 ± 0.6 (control conditions) to 0.5 ± 0.1 and 1.2 ± 0.2 (oxidizing conditions). A release of glucose and fructose was observed by W. cibaria and P. pentosaceus under oxidizing conditions. Free thiols content was increased by reducing conditions in fermentations with P. pentosaceus. Free amino nitrogen and free amino acid content were highly increased by reducing conditions in fermentations with W. cibaria. Free amino acid release was mainly influenced by low ORP and low pH values. Extracellular redox potential changes exhibited profile modifications of volatile compounds. Oxidizing conditions promoted a higher variety in volatile compounds as compared to the profile obtained under reducing conditions. It has been demonstrated that the extracellular ORP control has an influence on the microbial activity in buckwheat sourdoughs. Oxidizing and reducing conditions can influence the microbial activity differently and thus the final quality of GF sourdough. This is a new alternative approach for GF sourdough production and it could provide an improvement of raw materials, which can be used for GF bread production.     

ATP citrate lyase 1 (acl1) gene-based loop-mediated amplification assay for the detection of the Fusarium tricinctum species complex in pure cultures and in cereal samples

The combined data set of the acl1 and tef-1α gene sequences of 61 fungal strains assigned to Fusarium tricinctum, Fusarium avenaceum, Fusarium acuminatum, Fusarium arthrosporioides, Fusarium flocciferum and Fusarium torulosum were used to study the phylogenetic relations between taxa. F. tricinctum, F. acuminatum and F. avenaceum formed distinct clades. Members of the F. tricinctum/F. acuminatum clade fall into three well supported lineages, of which the largest includes the epitype of F. tricinctum. Loop-mediated isothermal amplification (LAMP) was used to amplify a 167bp portion of the acl1 gene in F. tricinctum (Corda) Saccardo. DNA amplification was detected in-tube by indirect calcein fluorescence under black light after 60min of incubation at 65.5°C. The assay had a detection limit of 0.95pg of purified genomic DNA of F. tricinctum CBS 410.86 per reaction, corresponding to ca. 18 genomic copies of the acl1 gene. Specificity of the assay was tested using purified DNA from 67 species and subspecies of Fusarium as well as 50 species comprising 22 genera of other filamentous fungi and yeasts. The assay detected 21 of the 23 F. tricinctum strains tested. Cross reactivity was observed with eight out of 13 strains in F. acuminatum but with none of 17 F. avenaceum strains tested. Specificity was further confirmed by conventional PCR with primers designed from the same gene. Detection of F. tricinctum from culture scrapings directly added to the reaction master mix, in DNA extracts from wheat, in single barley grains or in washings of bulk grain samples are proposed as possible applications showing the suitability of the method for food analysis. Finally it was demonstrated that the LAMP reaction can be run using simple lab equipment such as a heating block, water bath, hybridization oven or household equipment, e.g. a microwave oven.     

Simulated sunlight action spectra for inactivation of MS2 and PRD1 bacteriophages in clear water

Action spectra for simulated sunlight were measured in clear water for two viruses: PRD1, a double-stranded DNA bacteriophage, and MS2, a single-stranded RNA bacteriophage. Viruses were diluted into phosphate buffered saline (20 mM PBS, pH 7.5) and exposed for 22 h to simulated sunlight either directly or through one of six glass filters with 50% cutoff wavelengths ranging from 280 to 350 nm. Virus survival was measured using the double agar layer plaque method. Both UVA (320-400 nm) and UVB (280-320 nm) light were found to contribute to PRD1 inactivation, while only UVB inactivated MS2. A computational model was developed for interpreting these action spectra with 3-nm resolution. Using these methods, we provide detailed estimates of the sensitivity of MS2 and PRD1 to photoinactivation from 285 to 345 nm. The resulting sensitivity coefficients can be combined with solar spectra to estimate inactivation rates in clear water under different sunlight conditions. This approach will be useful for modeling the inactivation of viruses and other microorganisms in sunlit natural and engineered systems.     

Contribution of the NADH-oxidase (Nox) to the aerobic life of Lactobacillus sanfranciscensis DSM20451T

Lactobacillus sanfranciscensis is the key bacterium in traditional sourdough fermentation. The molecular background of its oxygen tolerance was investigated by comparison of wild type and NADH-oxidase (Nox) knock out mutants. The nox gene of L. sanfranciscensis DSM20451^T coding for a NADH-oxidase (Nox) was inactivated by single crossover integration to yield strain L. sanfranciscensis DSM20451Δnox. By inactivation of the native NADH-oxidase gene, it was ensured that besides fructose, O₂ can react as an electron acceptor. In aerated cultures the mutant strain was only able to grow in MRS media supplemented with fructose as electron acceptor, whereas the wild type strain showed a fructose independent growth response. The use of oxygen as an external electron acceptor enables L. sanfranciscensis to shift from acetyl-phosphate into the acetate branch and gain an additionally ATP, while the reduced cofactors were regenerated by Nox-activity. In aerated cultures the wild type strain formed a fermentation ratio of lactate to acetate of 1.09 in MRS supplemented with fructose after 24 h of fermentation, while the mutant strain formed a fermentation ratio of 3.05. Additionally, L. sanfranciscensis showed manganese-dependent growth response in aerated cultures, the final OD and growth velocity was increased in media supplemented with manganese. The expression of two predicted Mn²⁺/Fe²⁺ transporters MntH1 and MntH2 in L. sanfranciscensis DSM20451^T was verified by amplification of a 318 bp fragment of MntH1 and a 239 bp fragment of MntH2 from cDNA library obtained from aerobically, exponentially growing cells of L. sanfranciscensis DSM20451^T in MRS. Moreover, the mutant strain DSM20451Δnox was more sensitive to the superoxide generating agent paraquat and showed inhibition of growth on diamide-treated MRS-plates without fructose supplementation.     

Subminimal Inhibitory Concentrations of the Disinfectant Benzalkonium Chloride Select for a Tolerant Subpopulation of Escherichia coli with Inheritable Characteristics

Exposure of Escherichia coli to a subminimal inhibitory concentration (25% below MIC) of benzalkonium chloride (BC), an antimicrobial membrane-active agent commonly used in medical and food-processing environments, resulted in cell death and changes in cell morphology (filamentation). A small subpopulation (1-5% of the initial population) survived and regained similar morphology and growth rate as non-exposed cells. This subpopulation maintained tolerance to BC after serial transfers in medium without BC. To withstand BC during regrowth the cells up regulated a drug efflux associated gene (the acrB gene, member of the AcrAB-TolC efflux system) and changed expression of outer membrane porin genes (ompFW) and several genes involved in protecting the cell from the osmotic- and oxidative stress. Cells pre-exposed to osmotic- and oxidative stress (sodium chloride, salicylic acid and methyl viologen) showed higher tolerance to BC. A control and two selected isolates showing increased BC-tolerance after regrowth in BC was genome sequenced. No common point mutations were found in the BC- isolates but one point mutation in gene rpsA (Ribosomal protein S1) was observed in one of the isolates. The observed tolerance can therefore not solely be explained by the observed point mutation. The results indicate that there are several different mechanisms responsible for the regrowth of a tolerant subpopulation in BC, both BC-specific and general stress responses, and that sub-MIC of BC may select for phenotypic variants in a sensitive E. coli culture.     

Proteomic Analysis of Albumins and Globulins from Wheat Variety Chinese Spring and Its Fine Deletion Line 3BS-8

The relationship between chromosome deletion in wheat and protein expression were investigated using Chinese Spring and fine deletion line 3BS-8. Through 2-DE (2-D electrophoresis) analysis, no differentially expressed proteins (DEPs) were found in leaf samples; however, 47 DEPs showed at least two-fold abundance variation (p < 0.05) in matured wheat grains and 21 spots were identified by tandem MALDI-TOF/TOF-MS. Among the identified spots, four were cultivar-specific, including three (spots B15, B16, and B21) in Chinese Spring and one in 3BS-8 (spot B10). Among variety-different DEPs between Chinese Spring and 3BS-8, most spots showed a higher express profile in CS; only four spots showed up-regulated expression tendency in 3BS-8. An interesting observation was that more than half of the identified protein spots were involved in storage proteins, of which 11 spots were identified as globulins. According to these results, we can presume that the encoded genes of protein spots B15, B16, and B21 were located on the chromosome segment deleted in 3BS-8.     

Towards mechanistic models for activated sludge flocculation under different conditions based on inverse problems

Experimental data of Ca-induced activated sludge flocculation under different conditions of temperature and dissolved oxygen are investigated in order to model the influence of changing physical and chemical factors. However, current kernel structures for collision frequency and efficiency are unable to describe activated sludge flocculation data. Therefore, an earlier developed methodology based on an inverse problem is applied, yielding empirical models, to find out how flocculation is affected by these different environmental conditions. This contribution shows the useful application of inverse problems to improve the understanding of complex aggregation mechanisms.