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Ethanol Assay Kit

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00:04  Introduction
01:00   Principle
02:37    Reagent Preparation
04:11     Procedure
09:14    Calculations

Ethanol Assay Kit K-ETOH Scheme
Product code: K-ETOH

60 assays (manual) / 600 assays (microplate) / 600 assays (auto-analyser)

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Content: 60 assays (manual) / 600 assays (microplate) / 600 assays (auto-analyser)
Shipping Temperature: Ambient
Storage Temperature: Short term stability: 2-8oC,
Long term stability: See individual component labels
Stability: > 2 years under recommended storage conditions
Analyte: Ethanol
Assay Format: Spectrophotometer, Microplate, Auto-analyser
Detection Method: Absorbance
Wavelength (nm): 340
Signal Response: Increase
Linear Range: 0.25 to 12 µg of ethanol per assay
Limit of Detection: 0.093 mg/L
Reaction Time (min): ~ 5 min
Application examples: Wine, beer, cider, alcoholic fruit juices, spirits, liqueurs, low-alcoholic / non-alcoholic beverages, pickles, fruit and fruit juice, chocolate products, vinegar, jam, bread and bakery products, honey, soy sauce, dairy products, cosmetics, pharmaceuticals and other materials (e.g. biological cultures, samples, etc.).
Method recognition: Methods based on this principle have been accepted by AOAC (AOAC Method 2019.08, First Action), IFU, EBC Method 9.3.1, MEBAK and ASBC Method Beer 4-F

The Ethanol test kit is a simple, reliable and accurate method for the measurement and analysis of ethanol in beverages and foodstuffs.

Note for Content: The number of manual tests per kit can be doubled if all volumes are halved.  This can be readily accommodated using the MegaQuantTM  Wave Spectrophotometer (D-MQWAVE).

View our full range of alcohol assay kits.

Scheme-K-ETOH ETOH megayzme

  • Extended cofactors stability. Dissolved cofactors stable for > 1 year at 4oC.
  • Simple format – aldehyde dehydrogenase supplied as stable suspension 
  • Very competitive price (cost per test) 
  • All reagents stable for > 2 years after preparation 
  • Rapid reaction 
  • Mega-Calc™ software tool is available from our website for hassle-free raw data processing 
  • Standard included
  • Suitable for manual, microplate and auto-analyser formats
Validation of Methods
Megazyme publication

Determination of ethanol concentration in Kombucha beverages: Single-laboratory validation of an enzymatic method, First Action Method 2019.08.

Ivory, R., Delaney, E., Mangan & McCleary, B. V. (2020). Journal of AOAC International, qsaa122.

The Ethanol Assay Kit is an enzymatic test kit developed by Megazyme for the determination of ethanol in a variety of samples. The kit has been validated in a single laboratory for use with Kombucha fermented drinks, fruit juices and low-alcohol beer samples. The commercially available Ethanol Assay Kit (Megazyme catalogue no. K-ETOH) contains all components required for the analysis. Quantification is based on the oxidation of ethanol to acetaldehyde by alcohol dehydrogenase and further oxidation of acetaldehyde by acetaldehyde dehydrogenase with conversion of NAD+ to NADH. The single laboratory validation (SLV) outlined in this document was performed on a sample set of eight different commercial Kombucha products purchased in Ireland, a set of five Cerilliant aqueous ethanol solutions, two BCR low-alcohol beer reference materials, two alcohol-free beer samples and two fruit juice samples against SMPR 2016.001 (1). Parameters examined during the validation included Working range, Selectivity, Limit of Detection (LOD), Limit of Quantification (LOQ), Trueness (bias), Precision (reproducibility and repeatability), Robustness and Stability.

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Megazyme publication

Megazyme “advanced” wine test kits general characteristics and validation.

Charnock, S. J., McCleary, B. V., Daverede, C. & Gallant, P. (2006). Reveue des Oenologues, 120, 1-5.

Many of the enzymatic test kits are official methods of prestigious organisations such as the Association of Official Analytical Chemicals (AOAC) and the American Association of Cereal Chemists (AACC) in response to the interest from oenologists. Megazyme decided to use its long history of enzymatic bio-analysis to make a significant contribution to the wine industry, by the development of a range of advanced enzymatic test kits. This task has now been successfully completed through the strategic and comprehensive process of identifying limitations of existing enzymatic bio-analysis test kits where they occurred, and then using advanced techniques, such as molecular biology (photo 1), to rapidly overcome them. Novel test kits have also been developed for analytes of emerging interest to the oenologist, such as yeast available nitrogen (YAN; see pages 2-3 of issue 117 article), or where previously enzymes were simply either not available, or were too expensive to employ, such as for D-mannitol analysis.

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Megazyme publication

Grape and wine analysis: Oenologists to exploit advanced test kits.

Charnock, S. C. & McCleary, B. V. (2005). Revue des Enology, 117, 1-5.

It is without doubt that testing plays a pivotal role throughout the whole of the vinification process. To produce the best possible quality wine and to minimise process problems such as “stuck” fermentation or troublesome infections, it is now recognised that if possible testing should begin prior to harvesting of the grapes and continue through to bottling. Traditional methods of wine analysis are often expensive, time consuming, require either elaborate equipment or specialist expertise and frequently lack accuracy. However, enzymatic bio-analysis enables the accurate measurement of the vast majority of analytes of interest to the wine maker, using just one piece of apparatus, the spectrophotometer (see previous issue No. 116 for a detailed technical review). Grape juice and wine are amenable to enzymatic testing as being liquids they are homogenous, easy to manipulate, and can generally be analysed without any sample preparation.

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Turmeric extract (Curcuma longa L.) regulates hepatic toxicity in a single ethanol binge rat model.

Lee, H. Y., Lee, G. H., Hoang, T. H., Kim, S. W., Kang, C. G., Jo, J. H., Chung, M. J., Min, K. & Chae, H. J. (2022). Heliyon, 8(9), e10737.

Hepatic alcohol clearance is a key factor to overcome alcohol hangovers, and over the period, alcohol hangovers may lead to inflammation and oxidative stress. Natural food products with high antioxidant and anti-inflammatory effects might contribute to hepatic alcohol clearance, a hypothesis in this study. The present study aimed to evaluate the influence of turmeric (Curcuma longa L., Zingiberaceae) is an herbal product having antioxidant and anti-inflammatory activities, on alcohol metabolism using binge alcohol drinking rat model. In vivo investigations revealed that pretreatment with turmeric extract enhanced alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) activities upon binge ethanol (3 g/kg). Additionally, pretreatment with turmeric extract regulated CYP2E1 activity and levels of reactive oxygen species (ROS), Bax, Bcl-2, and inflammatory mediators like IL-1β, IL-6, and TNF-α. Moreover, turmeric extract upregulated superoxide dismutase, catalase, and glutathione peroxidase activities in liver tissues. Together, these observations shed light on the potential beneficial effects of turmeric extract against acute liver toxicity. The results offer an alternative natural functional food product, turmeric extract, to prevent the negative implications of binge drinking.

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Effect of herbal extracts and supplement mixture on alcohol metabolism in Sprague Dawley-rats.

Choe, H., Yun, I., Kim, Y., Lee, J. H., Shin, H. A., Lee, Y. K. & Kim, M. Y. (2022). Journal of Food Science and Technology, 1-9.

This study aimed to investigate the effect of mixture of herbal extracts and supplementary formula (FNP-C) on hangovers and antioxidant enzymes in alcohol-induced liver damage in rats. HepG2 cells were used as the experimental cells and divided into five groups: non-treated control (normal), alcohol-induced control (control), mixture of herbal extracts (FNP-B), FNP-C, and a commercial treatment of liver diseases (Livers®); inhibition of detoxification and alcohol-induced damage was confirmed in vivo. Blood alcohol and acetaldehyde concentration after alcohol consumption were measured in a timely manner; alcohol dehydrogenase (ADH), aldehyde dehydrogenase (ALDH), superoxide dismutase (SOD), glutathione (GSH), glutathione transferase (GST), and lactate dehydrogenase (LDH) levels were measured in the liver. FNP-C exhibited the highest effect. When FNP-C was administered to alcohol-induced animals, blood alcohol and acetaldehyde concentration decreased compared to FNP-B and Livers®. FNP-C reduced ADH levels and improved LDH, GSH, GST, and SOD levels. The FNP-C group was effective in preventing alcohol-induced hangovers and liver damage. Thus, FNP-C improves hangovers and increases antioxidant activity in an alcohol-induced model. Adding amino acids and vitamins to natural ingredients can potentially enhance the effect of improving hangovers.

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Blocking mitophagy does not significantly improve fuel ethanol production in bioethanol yeast Saccharomyces cerevisiae.

Eliodório, K. P., de Gois e Cunha, G. C., White, B. A., Patel, D. H., Zhang, F., Hettema, E. H., Basso, T. O., Gombert, A. K. & Raghavendran, V. (2022). Applied and Environmental Microbiology, 88(5), e02068-21.

Ethanolic fermentation is frequently performed under conditions of low nitrogen. In Saccharomyces cerevisiae, nitrogen limitation induces macroautophagy, including the selective removal of mitochondria, also called mitophagy. Previous research showed that blocking mitophagy by deletion of the mitophagy-specific gene ATG32 increased the fermentation performance during the brewing of Ginjo sake. In this study, we tested if a similar strategy could enhance alcoholic fermentation in the context of fuel ethanol production from sugarcane in Brazilian biorefineries. Conditions that mimic the industrial fermentation process indeed induce Atg32-dependent mitophagy in cells of S. cerevisiae PE-2, a strain frequently used in the industry. However, after blocking mitophagy, no significant differences in CO2 production, final ethanol titers, or cell viability were observed after five rounds of ethanol fermentation, cell recycling, and acid treatment, which is commonly performed in sugarcane biorefineries. To test if S. cerevisiae’s strain background influenced this outcome, cultivations were carried out in a synthetic medium with strains PE-2, Ethanol Red (industrial), and BY (laboratory) with and without a functional ATG32 gene and under oxic and oxygen restricted conditions. Despite the clear differences in sugar consumption, cell viability, and ethanol titers, among the three strains, we did not observe any significant improvement in fermentation performance related to the blocking of mitophagy. We concluded, with caution, that the results obtained with Ginjo sake yeast were an exception and cannot be extrapolated to other yeast strains and that more research is needed to ascertain the role of autophagic processes during fermentation.

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Production of Bioactive Substances to Alleviates Hangover and Ethanol-Induced Liver Damage through Fermentation of Oenanthe javanica Using Lactiplantibacillus plantarum.

Gam, D. H., Park, J. H., Kim, S. H., Kang, M. H., Kim, S. B. & Kim, J. W. (2022). Molecules, 27(4), 1175.

The purpose of this study is to evaluate the effect of the bioconversion products of Oenanthe javanica extract fermented by Lactiplantibacillus plantarum (OEFL) on relieving hangovers and improving liver function. In addition, the bioactive substance of the OEFL, which alleviates hangover and ethanol-induced liver damage, was identified and its bioactive property was verified through in vivo experiments. In major substances analysis using high-performance liquid chromatography, OEFL produced 9.5-fold higher p-coumaric acid than the O. Javanica extract (OE). In addition, considering that quinic acid, which is not present in the OE, was produced in the OEFL it was confirmed that chlorogenic acid was decomposed into quinic acid by bioconversion. In the in vivo experiment using Sprague-Dawley rats, the OEFL and p-coumaric acid diets reduced blood ethanol, acetaldehyde, GPT, and ALP concentrations, increasing blood albumin concentrations compared to ethanol-administered groups, demonstrating that OEFL and p-coumaric acid, the main substance in the OEFL, improved ethanol-induced liver damage. Furthermore, the OEFL and its main bioactive substance, p-coumaric acid, alleviated liver fibrosis by downregulating TGF-β, SMAD-2, SMAD-4, α-SMA, and upregulating MMP-1. Therefore, OEFL is expected to be used as a functional food or pharmaceutical material as it has been confirmed to effectively relieve hangovers, prevent liver damage, and delay liver fibrosis in ethanol-induced liver damages.

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Synergy of Cellulase Systems between Acetivibrio thermocellus and Thermoclostridium stercorarium in Consolidated-Bioprocessing for Cellulosic Ethanol.

Wang, N., Yan, Z., Liu, N., Zhang, X. & Xu, C. (2022). Microorganisms, 10(3), 502.

Anaerobes harbor some of the most efficient biological machinery for cellulose degradation, especially thermophilic bacteria, such as Acetivibrio thermocellus and Thermoclostridium stercorarium, which play a fundamental role in transferring lignocellulose into ethanol through consolidated bioprocessing (CBP). In this study, we compared activities of two cellulase systems under varying kinds of hemicellulose and cellulose. A. thermocellus was identified to contribute specifically to cellulose hydrolysis, whereas T. stercorarium contributes to hemicellulose hydrolysis. The two systems were assayed in various combinations to assess their synergistic effects using cellulose and corn stover as the substrates. Their maximum synergy degrees on cellulose and corn stover were, respectively, 1.26 and 1.87 at the ratio of 3:2. Furthermore, co-culture of these anaerobes on the mixture of cellulose and xylan increased ethanol concentration from 21.0 to 40.4 mM with a high cellulose/xylan-to-ethanol conversion rate of up to 20.7%, while the conversion rates of T. stercorarium and A. thermocellus monocultures were 19.3% and 15.2%. The reason is that A. thermocellus had the ability to rapidly degrade cellulose while T. stercorarium co-utilized both pentose and hexose, the metabolites of cellulose degradation, to produce ethanol. The synergistic effect of cellulase systems and metabolic pathways in A. thermocellus and T. stercorarium provides a novel strategy for the design, selection, and optimization of ethanol production from cellulosic biomass through CBP.

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Ethanol Production from Oil Palm Trunk: A Combined Strategy Using an Effective Pretreatment and Simultaneous Saccharification and Cofermentation.

Wardani, A. K., Sutrisno, A., Faida, T. N., Yustina, R. D. & Murdiyatmo, U. (2021). International Journal of Microbiology, 2021, In Press.

Background: Oil palm trunk (OPT) with highly cellulose content is a valuable bioresource for bioethanol production. To produce ethanol from biomass, pretreatment is an essential step in the conversion of lignocellulosic biomass to fermentable sugars such as glucose and xylose. Several pretreatment methods have been developed to overcome biomass recalcitrance. In this study, the effects of different pretreatment methods such as alkali pretreatment, microwave-alkali, and alkaline peroxide combined with autoclave on the lignocellulosic biomass structure were investigated. Moreover, ethanol production from the treated biomass was performed by simultaneous saccharification and cofermentation (SSCF) under different temperatures, fermentation times, and cell ratios of Saccharomyces cerevisiae NCYC 479 and pentose-utilizing yeast, Pichia stipitis NCYC 1541. Results: Pretreatment resulted in a significant lignin removal up to 83.26% and cellulose released up to 80.74% in treated OPT by alkaline peroxide combined with autoclave method. Enzymatic hydrolysis of treated OPT resulted in an increase in fermentable sugar up to 93.22%. Optimization of SSCF by response surface method showed that the coculture could work together to produce maximum ethanol (1.89%) and fermentation efficiency (66.14%) under the optimized condition. Conclusion: Pretreatment by alkaline peroxide combined with autoclave method and SSCF process could be expected as a promising system for ethanol production from oil palm trunk and various lignocellulosic biomass.

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Molecular brewing: The molecular structural effects of starch adjuncts on barley malt brewing performances.

Hu, S., Deng, H. T., Liu, R. H. & Yu, W. W. (2021). International Journal of Biological Macromolecules, 193, 661-671.

In this study, the effects of starch adjuncts with different fine molecular structures obtained by size-exclusion chromatography on the mashing and fermentation efficiencies of barley malts were investigated. Following fermentation, violate compounds of freshly-fermented beer samples were determined by headspace-solid-phase microextraction coupled with gas chromatography-mass spectrometry analysis (HS-SMPE-GC-MS). High performance liquid chromatography results showed that depending on their molecular structures, starch adjuncts addition significantly increased wort maltose and maltotriose content, whereas reducing the glucose content and thus both the ratios of glucose and maltotriose to that of the maltose. The whole fermentation by dry beer yeast was finished within the first 48 h and reached to equilibrium for the rest 72 h, represented by the stable soluble protein content. Results also showed that the addition of starch adjuncts resulted into increased alcohol content, which was mainly attributed to the altered glucose/maltose ratio. The HS-SPME-GC-MS results showed that whether or not with starch adjuncts addition, the composition of violate compounds were not significantly influenced, their content, on the contrary, were altered, represented by different peak heights. This study provides important information concerning the molecular effects of starch adjuncts on brewing performances of barley malts, and also provides a new pathway for choosing suitable types of adjuncts for making beer with better quality.

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Resource recovery from the anaerobic digestion of food waste is underpinned by cross-kingdom microbial activities.

Nzeteu, C., Joyce, A., Thorn, C., McDonnell, K., Shirran, S., O'Flaherty, V. & Abram, F. (2021). Bioresource Technology Reports, 16, 100847.

As the human population grows on the planet so does the generation of waste and particularly that of food waste. In order to tackle the world sustainability crisis, efforts to recover products from waste are critical. Here, we anaerobically recovered volatile fatty acids (VFAs) from food waste and analysed the microbial populations underpinning the process. An increased contribution of fungi relative to bacteria was observed throughout the reactor operation, with both kingdoms implicated into the main three steps of anaerobic digestion occurring within our systems: hydrolysis, acidogenesis and acetogenesis. Overall, Ascomycota, Proteobacteria and Firmicutes were found to drive the anaerobic digestion of food waste, with butyrate as the most abundant VFA likely produced by Clostridium using lactate as a precursor. Taken together we demonstrate that the generation of products of added-value from food waste results from cross-kingdoms microbial activities implicating fungi and bacteria.

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Nutraceutical Chewing Candy Formulations Based on Acetic, Alcoholic, and Lactofermented Apple Juice Products.

Bartkiene, E., Zokaityte, E., Zavistanaviciute, P., Mockus, E., Cernauskas, D., Ruzauskas, M., Tolpeznikaite, E. & Guiné, R. P. (2021). Foods, 10(10), 2329.

The aim of this study was to develop nutraceutical chewing candy (NCC) formulations based on acetic, alcoholic, and lactofermented apple juice (AJ) products. In addition, different texture-forming (gelatin, pectin) and sweetening (stevia, xylitol) agents were tested. To implement the aim of this study, combinations based on AJ, prepared from fresh and frozen apples, apple cider (C) samples (No.1, No.2, No.3, and No.4), and apple vinegar (V) were used. First, the most appropriate combination was selected by evaluating overall acceptability (OA) and emotions induced for consumers (EIC). In addition, the volatile compound (VC) profile, and physicochemical and antimicrobial characteristics of the developed combinations were analyzed. For AJ fermentation, lactic acid bacteria (LAB) strains possessing antimicrobial properties (LUHS122-L. plantarum and LUHS210-L. casei) were used. AJ prepared from frozen apples had 11.1% higher OA and 45.9%, 50.4%, and 33.3% higher fructose, glucose, and saccharose concentrations, respectively. All the tested C samples inhibited Bacillus subtilis and had an average OA of 6.6 points. Very strong positive correlations were found between AJ and C OA and the emotion ‘happy’; comparing lactofermented AJ, the highest OA was obtained for AJ fermented for 48 h with LUHS122, and a moderate positive correlation was found between AJ OA and the emotion ‘happy’ (r = 0.7617). This sample also showed the highest viable LAB count (7.59 log10 CFU mL−1) and the broadest spectrum of pathogen inhibition (inhibited 6 out of 10 tested pathogens). Further, acetic, alcoholic, and lactofermented AJ product combinations were tested. For the preparation of NCC, the combination consisting of 50 mL of AJ fermented with LUHS122 for 48 h + 50 mL C-No.3 + 2 mL V was selected because it showed the highest OA, induced a high intensity of the emotion ‘happy’ for the judges, and inhibited 8 out of 10 tested pathogens. Finally, the OA of the prepared NCC was, on average, 9.03 points. The combination of acetic, alcoholic, and lactofermented AJ products leads to the formation of a specific VC profile and increases the OA and antimicrobial activity of the products which could be successfully applied in the food and nutraceutical industries.

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Multi-product biorefinery from Arthrospira platensis biomass as feedstock for bioethanol and lactic acid production.

Esquivel-Hernández, D. A., Pennacchio, A., Torres-Acosta, M. A., Parra-Saldívar, R., de Souza Vandenberghe, L. P. & Faraco, V. (2021). Scientific Reports, 11(1), 1-15.

With the aim to reach the maximum recovery of bulk and specialty bioproducts while minimizing waste generation, a multi-product biorefinery for ethanol and lactic acid production from the biomass of cyanobacterium Arthrospira platensis was investigated. Therefore, the residual biomass resulting from different pretreatments consisting of supercritical fluid extraction (SF) and microwave assisted extraction with non-polar (MN) and polar solvents (MP), previously applied on A. platensis to extract bioactive metabolites, was further valorized. In particular, it was used as a substrate for fermentation with Saccharomyces cerevisiae LPB-287 and Lactobacillus acidophilus ATCC 43121 to produce bioethanol (BE) and lactic acid (LA), respectively. The maximum concentrations achieved were 3.02 ± 0.07 g/L of BE by the MN process at 120 rpm 30°C, and 9.67 ± 0.05 g/L of LA by the SF process at 120 rpm 37°C. An economic analysis of BE and LA production was carried out to elucidate the impact of fermentation scale, fermenter costs, production titer, fermentation time and cyanobacterial biomass production cost. The results indicated that the critical variables are fermenter scale, equipment cost, and product titer; time process was analyzed but was not critical. As scale increased, costs tended to stabilize, but also more product was generated, which causes production costs per unit of product to sharply decrease. The median value of production cost was US$ 1.27 and US$ 0.39, for BE and LA, respectively, supporting the concept of cyanobacterium biomass being used for fermentation and subsequent extraction to obtain ethanol and lactic acid as end products from A. platensis.

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Rapid colorimetric detection of genome evolution in SCRaMbLEd synthetic Saccharomyces cerevisiae strains. 

Wightman, E. L., Kroukamp, H., Pretorius, I. S., Paulsen, I. T. & Nevalainen, H. K. (2020).  Microorganisms, 8(12), 1914.

Genome-scale engineering and custom synthetic genomes are reshaping the next generation of industrial yeast strains. The Cre-recombinase-mediated chromosomal rearrangement mechanism of designer synthetic Saccharomyces cerevisiae chromosomes, known as SCRaMbLE, is a powerful tool which allows rapid genome evolution upon command. This system is able to generate millions of novel genomes with potential valuable phenotypes, but the excessive loss of essential genes often results in poor growth or even the death of cells with useful phenotypes. In this study we expanded the versatility of SCRaMbLE to industrial strains, and evaluated different control measures to optimize genomic rearrangement, whilst limiting cell death. To achieve this, we have developed RED (rapid evolution detection), a simple colorimetric plate-assay procedure to rapidly quantify the degree of genomic rearrangements within a post-SCRaMbLE yeast population. RED-enabled semi-synthetic strains were mated with the haploid progeny of industrial yeast strains to produce stress-tolerant heterozygous diploid strains. Analysis of these heterozygous strains with the RED-assay, genome sequencing and custom bioinformatics scripts demonstrated a correlation between RED-assay frequencies and physical genomic rearrangements. Here we show that RED is a fast and effective method to evaluate the optimal SCRaMbLE induction times of different Cre-recombinase expression systems for the development of industrial strains.

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Dendropanax morbifera Leaf Extracts Improved Alcohol Liver Injury in Association with Changes in the Gut Microbiota of Rats.

Eom, T., Ko, G., Kim, K. C., Kim, J. S. & Unno, T. (2020). Antioxidants, 9(10), 911.

This study evaluated the protective effects of Dendropanax morbifera leaf (DML) extracts in the liver due to excessive ethanol consumption. Our results showed that the ethanol extract had better antioxidant activity than the water extract, likely due to the higher levels of total flavonoid and phenolic compounds in the former. We found that the main phenolic acid was chlorogenic acid and the major flavonoid was rutin. Results from the animal model experiment showed concentration-dependent liver protection with the distilled water extract showing better liver protection than the ethanol extract. Gut microbiota dysbiosis induced by alcohol consumption was significantly shifted by DML extracts through increasing mainly Bacteroides and Allobaculum. Moreover, predicted metabolic activities of biosynthesis of beneficial monounsaturated fatty acids such as oleate and palmitoleate were enhanced. Our results suggest that these hepatoprotective effects are likely due to the increased activities of antioxidant enzymes and partially promoted by intestinal microbiota shifts.

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Factors influencing the production of the antioxidant hydroxytyrosol during alcoholic fermentation: Yeast strain, initial tyrosine concentration and initial must.

Rebollo-Romero, I., Fernández-Cruz, E., Carrasco-Galán, F., Valero, E., Cantos-Villar, E., Cerezo, A. B., Troncosso, A. M. & Garcia-Parrilla, M. C. (2020). LWT, 130, 109631.

Hydroxytyrosol is well known for its potent antioxidant activity and anticarcinogenic, antimicrobial, cardioprotective and neuroprotective properties. Main food sources are olive oil (formed from the hydrolysis of oleuropein) and wine. One possible explanation to its origin in wines is the synthesis from tyrosol, which in turn is produced from the Ehrlich pathway by yeasts. This work aims to explore the factors that could increase the content as the strain of yeast, the initial tyrosine concentrations as precursor and the effect of synthetic and sterilized natural grape musts. Alcoholic fermentations in synthetic must showed that hydroxytyrosol is produced by all the yeast strains under study. Commercial Saccharomyces cerevisiae yeasts were those which produced higher concentrations, being the Red Fruit strain the biggest producer (6.12 ng/mL). Once the strain was selected, alcoholic fermentations were performed in synthetic must, with different tyrosine concentrations. The amount of hydroxytyrosol did not increase in a proportional way as tyrosine does. On the other hand, higher concentrations of hydroxytyrosol were obtained in natural grape musts (10.46 ng/mL) than in synthetic must (4.03 ng/mL). This work confirms the capacity of winemaking yeasts to produce the bioactive hydroxytyrosol.

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Activated sludge denitrification in marine recirculating aquaculture system effluent using external and internal carbon sources.

Letelier-Gordo, C. O., Huang, X., Aalto, S. L. & Pedersen, P. B. (2020). Aquacultural Engineering, 90, 102096.

Stringent environmental legislation in Europe, especially in the Baltic Sea area, limits the discharge of nutrients to natural water bodies, limiting the aquaculture production in the region. Therefore, cost-efficient end-of-pipe treatment technologies to reduce nitrogen (N) discharge are required for the sustainable growth of marine land-based RAS. The following study examined the potential of fed batch reactors (FBR) in treating saline RAS effluents, aiming to define optimal operational conditions and evaluate the activated sludge denitrification capacity using external (acetate, propionate and ethanol) and internal carbon sources (RAS fish organic waste (FOW) and RAS fermented fish organic waste (FFOW)). The results show that between the evaluated operation cycle times (2, 4, and 6 h), the highest nitrate/nitrite removal rate was achieved at an operation cycle time of 2 h (corresponding to a hydraulic retention time of 2.5 h) when acetate was used as a carbon source. The specific denitrification rates were 98.7 ± 3.4 mg NO3-N/(h g biomass) and 93.2 ± 13.6 mg NOx-N/(h g biomass), with a resulting volumetric denitrification capacity of 1.20 kg NO3-N/(m3 reactor d). The usage of external and internal carbon sources at an operation cycle time of 4 h demonstrated that acetate had the highest nitrate removal rate (57.6 ± 6.6 mg N/(h g biomass)), followed by propionate (37.5 ± 6.3 mg NO3-N/(h g biomass)), ethanol (25.5 ± 6.0 mg NO3-N/(h g biomass)) and internal carbon sources (7.7 ± 1.6-14.1 ± 2.2 mg NO3-N/(h g biomass)). No TAN (Total Ammonia Nitrogen) or PO43- accumulation was observed in the effluent when using the external carbon sources, while 0.9 ± 0.5 mg TAN/L and 3.9 ± 1.5 mg PO43--P/L was found in the effluent when using the FOW, and 8.1±0.7 mg TAN/L and 7.3 ± 0.9 mg PO43--P/L when using FFOW. Average sulfide concentrations varied between 0.002 and 0.008 mg S2-/L when using the acetate, propionate and FOW, while using ethanol resulted in the accumulation of sulfide (0.26 ± 0.17 mg S2-/L). Altogether, it was demonstrated that FBR has a great potential for end-of-pipe denitrification in marine land-based RAS, with a reliable operation and a reduced reactor volume as compared to the other available technologies. Using acetate, the required reactor volume is less than half of what is needed for other evaluated carbon sources, due to the higher denitrification rate achieved. Additionally, combined use of both internal and external carbon sources would further reduce the operational carbon cost.

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Fermentative Microbes of Khadi, a Traditional Alcoholic Beverage of Botswana.

Motlhanka, K., Lebani, K., Boekhout, T. & Zhou, N. (2020). Fermentation, 6(2), 51.

Khadi is a popular traditional alcoholic beverage in rural households in Botswana. The product is produced by fermentation of ripened sun-dried Grewia flava (Malvaceae) fruits supplemented with brown table sugar. Despite its popularity, its growing consumer acceptance, its potential nutritional value, and its contribution to the socio-economic lifestyle of Botswana, the production process remains non-standardized. Non-standardized production processes lead to discrepancies in product quality and safety as well as varying shelf life. Identification of unknown fermentative microorganisms of khadi is an important step towards standardization of its brewing process for entrance into commercial markets. The aim of this study was to isolate and identify bacteria and yeasts responsible for fermentation of khadi. Yeasts and bacteria harbored in 18 khadi samples from 18 brewers in central and northern Botswana were investigated using classic culture-dependent techniques and DNA sequencing methods. Additionally, we used the same techniques to investigate the presence of bacteria and yeasts on six batches of ripened-dried G. flava fruits used for production of the sampled brews. Our results revealed that Saccharomyces cerevisiae closely related to a commercial baker’s yeast strain sold locally was the most predominant yeast species in khadi suggesting a possible non-spontaneous brewing process. However, we also detected diverse non-Saccharomyces yeasts, which are not available commercially in retail shops in Botswana. This suggests that spontaneous fermentation is partially responsible for fermentation of khadi. This study, presenting the first microbiological characterization of a prominent traditional alcoholic beverage in Botswana, is vital for development of starter cultures for the production of a consistent product towards the commercialization of khadi.

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A microbubble-sparged yeast propagation–fermentation process for bioethanol production.

Raghavendran, V., Webb, J. P., Cartron, M. L., Springthorpe, V., Larson, T. R., Hines, M., Mohammed, H., ZimmermaN, W. B., K Poole, R., GreeN, J. & Green, J. (2020). Biotechnology for Biofuels, 13, 1-16.

Background: Industrial biotechnology will play an increasing role in creating a more sustainable global economy. For conventional aerobic bioprocesses supplying O2 can account for 15% of total production costs. Microbubbles (MBs) are micron-sized bubbles that are widely used in industry and medical imaging. Using a fluidic oscillator to generate energy-efficient MBs has the potential to decrease the costs associated with aeration. However, little is understood about the effect of MBs on microbial physiology. To address this gap, a laboratory-scale MB-based Saccharomyces cerevisiae Ethanol Red propagation-fermentation bioethanol process was developed and analysed. Results: Aeration with MBs increased O2 transfer to the propagation cultures. Titres and yields of bioethanol in subsequent anaerobic fermentations were comparable for MB-propagated and conventional, regular bubble (RB)-propagated yeast. However, transcript profiling showed significant changes in gene expression in the MB-propagated yeast compared to those propagated using RB. These changes included up-regulation of genes required for ergosterol biosynthesis. Ergosterol contributes to ethanol tolerance, and so the performance of MB-propagated yeast in fed-batch fermentations sparged with 1% O2 as either RBs or MBs were tested. The MB-sparged yeast retained higher levels of ergosteryl esters during the fermentation phase, but this did not result in enhanced viability or ethanol production compared to ungassed or RB-sparged fermentations. Conclusions: The performance of yeast propagated using energy-efficient MB technology in bioethanol fermentations is comparable to that of those propagated conventionally. This should underpin the future development of MB-based commercial yeast propagation.

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Inferring active metabolic pathways from proteomics and essentiality data.

Montero-Blay, A., Piñero-Lambea, C., Miravet-Verde, S., Lluch-Senar, M.  & Serrano, L. (2020). Cell Reports, 31(9), 107722.

Here, we propose an approach to identify active metabolic pathways by integrating gene essentiality analysis and protein abundance. We use two bacterial species (Mycoplasma pneumoniae and Mycoplasma agalactiae) that share a high gene content similarity yet show significant metabolic differences. First, we build detailed metabolic maps of their carbon metabolism, the most striking difference being the absence of two key enzymes for glucose metabolism in M. agalactiae. We then determine carbon sources that allow growth in M. agalactiae, and we introduce glucose-dependent growth to show the functionality of its remaining glycolytic enzymes. By analyzing gene essentiality and performing quantitative proteomics, we can predict the active metabolic pathways connected to carbon metabolism and show significant differences in use and direction of key pathways despite sharing the large majority of genes. Gene essentiality combined with quantitative proteomics and metabolic maps can be used to determine activity and directionality of metabolic pathways.

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