Effect of blueberries addition during beer maturation on yeast metabolism

In recent years, there has been a significant interest in beverages with increased biological value, such as beer with blueberries. In this study, blueberries were added at the beginning of maturation of lager beer with an initial extract of 12, 14 and 16ºP. The effect of blueberries addition on yeast metabolism was investigated as concentration of ethanol, higher alcohols, esters, aldehydes, and vicinal diketones in the final beer were measured and compared to control samples without blueberries. The results showed that blueberries affected positively ethanol formation only when wort with initial extract of 12°P was used and had no significant effect when wort with higher extract was used. In regard to secondary metabolites, blueberries addition led to a decrease in higher alcohols concentration and an increase in esters amounts. All the carbonyl compounds (aldehydes and vicinal diketones) were higher in beer with blueberries.

Alcohol dehydrogenases AdhE and AdhB with broad substrate ranges are important enzymes for organic acid reduction in Thermoanaerobacter sp. strain X514

Background The industrial production of various alcohols from organic carbon compounds may be performed at high rates and with a low risk of contamination using thermophilic microorganisms as whole-cell catalysts. Thermoanaerobacter species that thrive around 50–75 °C not only perform fermentation of sugars to alcohols, but some also utilize different organic acids as electron acceptors, reducing them to their corresponding alcohols. Results We purified AdhE as the major NADH- and AdhB as the major NADPH-dependent alcohol dehydrogenase (ADH) from the cell extract of the organic acid-reducing Thermoanaerobacter sp. strain X514. Both enzymes were present in high amounts during growth on glucose with and without isobutyrate, had broad substrate spectra including different aldehydes, with high affinities (< 1 mM) for acetaldehyde and for NADH (AdhE) or NADPH (AdhB). Both enzymes were highly thermostable at the physiological temperature of alcohol production. In addition to AdhE and AdhB, we identified two abundant AdhA-type ADHs based on their genes, which were recombinantly produced and biochemically characterized. The other five ADHs encoded in the genome were only expressed at low levels. Conclusions According to their biochemical and kinetic properties, AdhE and AdhB are most important for ethanol formation from sugar and reduction of organic acids to alcohols, while the role of the two AdhA-type enzymes is less clear. AdhE is the only abundant aldehyde dehydrogenase for the acetyl-CoA reduction to aldehydes, however, acid reduction may also proceed directly by aldehyde:ferredoxin oxidoreductase. The role of the latter in bio-alcohol formation from sugar and in organic acid reduction needs to be elucidated in future studies.

Mitigation of pretreatment-derived inhibitors during lignocellulosic ethanol fermentation using spent grain as a nitrogen source

Nitrogen-containing nutrient sources can be used to mitigate the negative effects of pretreatment-derived inhibitors on product formation rates during bioethanol production. Process economic limitations require these nutrients to be inexpensive. A method of hydrolyzing the protein in the spent grain fraction remaining after wheat grain saccharification, using porcine pancreas trypsin, is presented. This protein hydrolysate was shown to increase the volumetric productivity of ethanol production, measured after 24 h, during fermentation of a lignocellulosic hydrolysate from 0.24 to 0.60 g/L h. Although the effects on the productivity, on a per gram basis, were lower than with yeast extract, which increased the product formation rate to 1.64 g/L h, amino acid analysis of the soluble polypeptides in the protein hydrolysate showed that the feasibility of using spent grain as a nutrient source could be increased through optimization of the hydrolysis step. Furthermore, it was shown that pretreatment-derived inhibitors could reduce cell growth without affecting ethanol formation rates and that nutrient addition could increase ethanol formation rates without increasing cell growth. Finally, it was shown that the ability of nutrients to affect the product formation rate was limited above a certain inhibitor concentration.

Measurement of the alcohol biomarker phosphatidylethanol (PEth) in dried blood spots and venous blood—importance of inhibition of post-sampling formation from ethanol

Phosphatidylethanol (PEth) is a group of phospholipids formed in cell membranes following alcohol consumption by action of the enzyme phospholipase D (PLD). PEth measurement in whole blood samples is established as a specific alcohol biomarker with clinical and forensic applications. However, in blood specimens containing ethanol, formation of PEth may continue after sampling leading to falsely elevated concentrations. This study evaluated the use of dried blood spot (DBS) and microsampling specimens to avoid post-sampling formation of PEth. Filter paper cards and three commercial devices for volumetric microsampling of finger-pricked blood were assessed, using PEth-negative and PEth-positive whole blood fortified with 2 g/L ethanol. PEth (16:0/18:1) was measured by LC–MS/MS. Post-sampling formation of PEth occurred in wet blood and in the volumetric devices, but not filter paper cards, when stored at room temperature for 48 h. Addition of an inhibitor of PLD, sodium metavanadate (NaVO3), eliminated post-sampling formation during storage and drying. In conclusion, the present study confirmed previous observations that PEth can be formed in blood samples after collection, if the specimen contains ethanol. The results further demonstrated that post-sampling formation of PEth from ethanol also occurred with commercial devices for volumetric dried blood microsampling. In order for a PEth result not to be questioned, it is recommended to use a PLD inhibitor, whether venous blood is collected in a vacutainer tube or finger-pricked blood is obtained using devices for dried blood microsampling. Graphical abstract

Production of palmitoleic acid by oleaginous yeast Scheffersomyces segobiensis DSM 27193 using systematic dissolved oxygen regulation strategy

Background: Recently, a newly isolated oleaginous yeast strain Scheffersomyces segobiensis DSM 27193 was identified with the capability of accumulating high contents of palmitoleic acid (POA) in its intracellular lipid (13.8%). However, high amounts of ethanol as the main by-product were also produced in this system. To eliminate ethanol formation and to further improve POA production, process optimization focused on dissolved oxygen (DO) regulation was conducted in this study.Results: As a result, agitation control was found to be highly beneficial for cell growth and lipid production. When keeping pO2>40% through dynamic agitation control, significant improvements toward lipid (70.12% increase) and POA (1.44 fold) production have been obtained, and the accumulation of ethanol was decreased to 2.3 g/L. While increasing the aeration rate to 2 vvm, no significant decrease has been found in ethanol accumulation. Further application of a two-stage agitation regulation (600 rpm in the first 48 h fermentation then adjusted the agitation speed to 1000 rpm) gave 10.23 g/L of lipid (50% increase) with 2.02 g/L of POA (1.15 fold) production. At last, under the best condition of 1000 rpm of agitation and 1 vvm of aeration, no ethanol was detected during the whole fermentation process, while a dry biomass concentration of 44.8 g/L with 13.43 g/L of lipid and 2.93 g/L of POA was achieved. Transcription analysis revealed that ethanol synthetic pathway was down regulated under the condition of high agitation, while the expression of the key enzymes responsible for lipid and POA accumulation were enhanced.Conclusion: This study introduces the oleaginous yeast chassis S. segobiensis DSM 27193 and an effective pO2 regulation strategy for efficient biological production of POA.

Abnormally High Blood Acetaldehyde Concentrations Suggest a Potential of Postmortem Ethanol Generation

Ethanol is one of the most commonly used and abused substances worldwide. Identifying whether the source of ethanol detected in corpses is antemortem ingestion or postmortem generation is especially important for determining the cause of death, which remains a vibrant field of research. During the synthesis of ethanol in the putrefaction process of corpses, other small molecules such as acetaldehyde and n-propanol could also be produced. According to our prospective statistical analysis based on authentic samples from forensic cases, it is rational to suspect ethanol generation after death when the concentration of acetaldehyde detected in blood exceeds 0.014 g/dL. Through in vitro simulation experiments, in addition to confirming that ethyl glucuronide and ethyl sulfate are the reliable biomarkers of antemortem ingestion of ethanol, we propose that acetaldehyde is far more sensitive than n-propanol as a potential marker in the blood of corpses for postmortem ethanol formation.