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Acetic Acid Assay Kit (ACS Manual Format)

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0:05 Introduction
0:52 Principle
1:21  Reagent Preparation
2:04 Procedure
6:04 Calculation

Acetic Acid Assay Kit ACS Manual Format K-ACET Scheme
Product code: K-ACET

53 assays per kit

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Available for shipping

Content: 53 assays per kit
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: Acetic Acid
Assay Format: Spectrophotometer
Detection Method: Absorbance
Wavelength (nm): 340
Signal Response: Increase
Linear Range: 0.3 to 20 µg of acetic acid per assay
Limit of Detection: 0.14 mg/L
Reaction Time (min): ~ 14 min
Application examples: Wine, beer, fruit and fruit juices, soft drinks, vinegar, vegetables, pickles, dairy products (e.g. cheese), meat, fish, bread, bakery products (and baking agents), ketchup, soy sauce, mayonnaise, dressings, paper (and cardboard), tea, pharmaceuticals (e.g. infusion solutions), feed and other materials (e.g. biological cultures, samples, etc.).
Method recognition: Methods based on this principle have been accepted by EN, ISO,ICUMSA, IFU and MEBAK

The Acetic Acid (ACS Manual Format) test kit is a simple method for the rapid and reliable measurement and analysis of acetic acid/acetate in foods, beverages and other materials.

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).

See our full range of organic acid test kits.

Scheme-K-ACET ACET Megazyme

  • No wasted ACS solution (stable suspension supplied) 
  • PVP incorporated to prevent tannin inhibition 
  • All reagents stable for > 2 years after preparation
  • Very competitive price (cost per test) 
  • Mega-Calc™ software tool is available from our website for hassle-free raw data processing
Certificate of Analysis
Safety Data Sheet
FAQs Booklet Data Calculator Other automated assay procedures Product Performance Validation Report
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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Chemical Composition of Sour Beer Resulting from Supplementation the Fermentation Medium with Magnesium and Zinc Ions.

Ciosek, A., Fulara, K., Hrabia, O., Satora, P. & Poreda, A. (2020). Biomolecules, 10(12), 1599.

The bioavailability of minerals, such as zinc and magnesium, has a significant impact on the fermentation process. These metal ions are known to influence the growth and metabolic activity of yeast, but there are few reports on their effects on lactic acid bacteria (LAB) metabolism during sour brewing. This study aimed to evaluate the influence of magnesium and zinc ions on the metabolism of Lactobacillus brevis WLP672 during the fermentation of brewers’ wort. We carried out lactic acid fermentations using wort with different mineral compositions: without supplementation; supplemented with magnesium at 60 mg/L and 120 mg/L; and supplemented with zinc at 0.4 mg/L and 2 mg/L. The concentration of organic acids, pH of the wort and carbohydrate use was determined during fermentation, while aroma compounds, real extract and ethanol were measured after the mixed fermentation. The addition of magnesium ions resulted in the pH of the fermenting wort decreasing more quickly, an increase in the level of L-lactic acid (after 48 h of fermentation) and increased concentrations of some volatile compounds. While zinc supplementation had a negative impact on the L. brevis strain, resulting in a decrease in the L-lactic acid content and a higher pH in the beer. We conclude that zinc supplementation is not recommended in sour beer production using L. brevis WLP672.

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Identification, characterization, and immobilization of a novel YbfF esterase from Halomonas elongata.

Yoo, W., Kim, B., Jeon, S., Kim, K. K. & Kim, T. D. (2020). International Journal of Biological Macromolecules, 165, 1139-1148.

The YbfF esterase family, which has a bifurcated binding pocket for diverse ligands, could serve as excellent biocatalysts in industrial and biotechnological applications. Here, the identification, characterization, and immobilization of a novel YbfF esterase (YbfFHalomonas elongata) from Halomonas elongata DSM 2581 is reported. Biochemical characterization of YbfF was carried out using activity staining, chromatographic analysis, kinetic analysis, activity assay, acetic acid release, and pH-indicator-based hydrolysis. YbfFH.elongata displayed broad substrate specificity, including that for p-nitrophenyl esters, glucose pentaacetate, tert-butyl acetate, and β-lactam-containing compounds, with high efficiency. Based on a homology model of YbfFH.elongata, Trp237 in the substrate-binding pocket, a critical residue for catalytic activity and substrate specificity was identified and characterized. Furthermore, crosslinked enzyme aggregates and nanoflower formation were explored to enhance the chemical stability and recyclability of YbfFH.elongata. The present study is the first report of a YbfF esterase from extremophiles, and explains its protein stability, catalytic activity, substrate specificities and diversities, kinetics, functional residues, amyloid formation, and immobilization.

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Production of a yeast-free focaccia with reduced salt content using a selected Leuconostoc citreum strain and seawater.

De Bellis, P., Montemurro, M., D'Imperio, M., Rizzello, C. G., Sisto, A. & Lavermicocca, P. (2020). LWT, 134, 109918.

A biotechnological protocol to produce a focaccia (a typical Italian flat bread) without bakers' yeast addition and with reduced salt was developed, to meet the current needs of the consumer. Based on its leavening capability, the Leuconostoc citreum strain C2.27 was selected to be used as a starter instead of the baker's yeast and inoculated in a liquid sourdough (type-II) for the production of the “yeast-free” focaccia. The addition of different NaCl concentrations and the replacement of the salt with food grade seawater were evaluated, and the capability of the selected strain to affect technological, nutritional and sensory features of the focaccia investigated. A significant improvement of the nutritional characteristics of the focaccia was observed compared to the control (leavened with bakers' yeast and added with NaCl 1.5 g/100 g) using 0.7 g/100 g of salt in the form of NaCl or seawater. Besides the reduced Na content (66% lower than the control), focaccia with seawater also showed a higher content of Ca2+ and Mg2+ (ca. 36% and 53%, respectively), and the lowest predicted glycemic index compared to the other experimental focaccia.

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Rethinking about flor yeast diversity and its dynamic in the “criaderas and soleras” biological aging system.

Ruiz-Muñoz, M., Cordero-Bueso, G., Benítez-Trujillo, F., Martínez, S., Pérez, F. & Cantoral, J. M. (2020). Food Microbiology, 92, 103553.

Fino wine is one of the most important Sherry wines and it is obtained through a complex and dynamic biological aging system. In this study, wine and veil of flor samples from fifty-two barrels with different aging levels and distributed in three different wineries from the Jerez-Xèrés-Sherry winemaking area have been analyzed during two years. Some of the wine compounds most deeply involved in flor yeast metabolism were analyzed to take into account the blending effect of this system. On the other hand, veil of flor was analyzed by molecular methods, finding five different species: S. cerevisiae, W. anomalus, P. membranaefaciens, P. kudriavzevii and P. manshurica, being the first time that the three last species have been reported in this biological aging system. Since S. cerevisiae was the vast majority of the isolates, its intraspecies variability was also analyzed by the simultaneous amplification of three microsatellite loci, obtaining nine different S. cerevisiae genotypes, also differentiated according to their physiological properties. Biodiversity analysis showed there were significant differences between the three wineries in the three aging scales, although the overall diversity was relatively low. Moreover, variations in the relative frequency of the different S. cerevisiae genotypes were found to be seasonal-dependent.

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Saccharomyces uvarum yeast isolate consumes acetic acid during fermentation of high sugar juice and juice with high starting volatile acidity.

Inglis, D., Kelly, J., van Dyk, S., Dowling, L., Pickering, G. & Kemp, B. (2020). OENO One, 54(2).

Aim: A Saccharomyces uvarum isolate was assessed for its ability to metabolize acetic acid present in juice and during the fermentation of partially dehydrated grapes. The impact on other yeast metabolites was also compared using an S. uvarum isolate and an S. cerevisiae wine yeast. The upper limit of fruit concentration that allowed the S. uvarum isolate to ferment wines to < 5 g/L residual sugar was defined. Methods and results: Cabernet franc grapes were partially dehydrated to three different post-harvest sugar targets (24.5 °Brix, 26.0 °Brix, and 27.5 °Brix) along with non-dehydrated grapes (21.5 °Brix control). Musts from all treatments were vinified with either the S. uvarum isolate CN1, formerly identified as S. bayanus, or S. cerevisiae EC1118. All wines were successfully vinified to less than 5 g/L residual sugar. Fermentation kinetics between the two yeasts were similar for all wines other than 27.5 °Brix, where CN1 took three days longer. During fermentation with CN1, acetic acid peaked on day two, then decreased in concentration, resulting in final wine acetic acid lower than that measured on day two. Wines fermented with EC1118 showed an increase in acetic acid over the time-course of fermentation. Significantly lower wine oxidative compounds (acetic acid, acetaldehyde and ethyl acetate) and higher glycerol resulted in wine produced with CN1 in comparison to EC1118. Both yeasts produced comparable ethanol at each Brix level tested. Further studies showed that CN1 lowered acetic acid seven-fold from 0.48 g/L in juice to 0.07 g/L in wine whereas EC1118 reduced acetic acid to 0.18 g/L. Conclusions: The autochthonous S. uvarum yeast isolate successfully fermented partially dehydrated grapes to < 5 g/L sugar up to 27.5 ºBrix. The consumption rate of acetic acid was faster than its production during fermentation, resulting in low acetic acid, acetaldehyde and ethyl acetate in wine in comparison to a commercial S. cerevisiae yeast while consistently producing higher glycerol. Significance and impact of the study: The S. uvarum yeast isolate can metabolize acetic acid during fermentation to significantly lower acetic acid, ethyl acetate and acetaldehyde in wine. It can also reduce acetic acid by seven-fold from the starting juice to the finished wine, which could have potential application for managing sour rot arising in the vineyard or during the dehydration process in making appassimento-style wines.

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Prebiotic Activity of Poly-and Oligosaccharides Obtained from Plantago major L. Leaves.

Lukova, P., Nikolova, M., Petit, E., Elboutachfaiti, R., Vasileva, T., Katsarov, P., Manev, H., Gardarin, C., Pierre, G., Michaud, P., Iliev, I. & Delattre, C. (2020). Applied Sciences, 10(8), 2648.

The aim of the present study was to evaluate the prebiotic potential of Plantago major L. leaves water-extractable polysaccharide (PWPs) and its lower molecular fractions. The structure of PWPs was investigated by high pressure anion exchange chromatography (HPAEC), size exclusion chromatography coupled with multi-angle laser light scattering detector (SEC-MALLS) and Fourier-transform infrared (FTIR) spectroscopy. The chemical composition and monosaccharide analyses showed that galacturonic acid was the main monosaccharide of PWPs followed by glucose, arabinose, galactose, rhamnose and xylose. FTIR study indicated a strong characteristic absorption peak at 1550 cm−1 corresponding to the vibration of COO group of galacturonic acid. The PWPs was subjected to hydrolysis using commercial enzymes to obtain P. major low molecular fraction (PLM) which was successively separated by size exclusion chromatography on Biogel P2. PWPs and PLM were examined for in vitro prebiotic activity using various assays. Results gave evidence for changes in optical density of the bacteria cells and pH of the growth medium. A heterofermentative process with a lactate/acetate ratio ranged from 1:1 to 1:5 was observed. The ability of PLM to stimulate the production of certain probiotic bacteria glycohydrolases and to be fermented by Lactobacillus sp. strains was successfully proved.

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Discovering microbiota and volatile compounds of surströmming, the traditional Swedish sour herring.

Belleggia, L., Aquilanti, L., Ferrocino, I., Milanović, V., Garofalo, C., Clementi, F., Cocolin, L., Mozzon, M., Foligni, R., Haouet, M. N., Scuota, S., Framboas, M. & Osimani, A. (2020). Food Microbiology, 91, 103503.

In this study, the microbiota of ready-to-eat surströmming from three Swedish producers were studied using a combined approach. The pH values of the samples ranged between 6.67 ± 0.01 and 6.98 ± 0.01, whereas their aw values were between 0.911 ± 0.001 and 0.940 ± 0.001. The acetic acid concentration was between 0.289 ± 0.009 g/100 g and 0.556 ± 0.036 g/100 g. Very low concentrations of lactic acid were measured. Viable counting revealed the presence of mesophilic aerobes, mesophilic lactobacilli and lactococci as well as halophilic lactobacilli and lactococci, coagulase-negative staphylococci, halophilic aerobes and anaerobes. Negligible counts for Enterobacteriaceae, Pseudomonadaceae and total eumycetes were observed, whereas no sulfite-reducing anaerobes were detected. Listeria monocytogenes and Salmonella spp. were absent in all samples. Multiplex real-time PCR revealed the absence of the bont/A, bont/B, bont/E, bont/F, and 4gyrB (CP) genes, which encode botulinic toxins, in all the samples analyzed. Metagenomic sequencing revealed the presence of a core microbiota dominated by Halanaerobium praevalens, Alkalibacterium gilvum, Carnobacterium spp., Tetragenococcus halophilus, Clostridiisalibacter spp. and Porphyromonadaceae. Psychrobacter celer, Ruminococcaceae, Marinilactibacillus psychrotolerans, Streptococcus infantis and Salinivibrio costicola were detected as minor OTUs. GC-MS analysis of volatile components revealed the massive presence of trimethylamine and sulphur compounds. Moreover, 1,2,4-trithiolane, phenols, ketones, aldehydes, alcohols, esters and long chain aliphatic hydrocarbons were also detected. The data obtained allowed pro-technological bacteria, which are well-adapted to saline environments, to be discovered for the first time. Further analyses are needed to better clarify the extent of the contribution of either the microbiota or autolytic enzymes of the fish flesh in the aroma definition.

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Is there any still undisclosed biodiversity in Ciauscolo salami? A new glance into the microbiota of an artisan production as revealed by high-throughput sequencing.

Belleggia, L., Milanović, V., Ferrocino, I., Cocolin, L., Haouet, M. N., Scuota, S., Maoloni, A., Garofalo, C., Cardinali, F., Aquilanti, L., Mozzon, M., Foligni, R., Pasquini, M., Trombetta, M. F., Clementi, F. & Osimani, A. (2020). Meat Science, 165, 108128.

Ciauscolo is a fermented sausage with the Protected Geographical Indication (PGI) status. To disclose the microbial ecology of a model Ciauscolo salami manufacture during its natural fermentation, viable counting, amplicon-based sequencing and real-time PCR were applied. The volatilome during fermentation was also characterized. The results allowed previously undetected species to be discovered. The core microbiota was composed by Lactobacillus algidus, Leuconostoc carnosum, Lactobacillus sakei, Debaryomyces hansenii, Glomus hyderabadensis, Tilletiopsis washingtonensis, and Kurtzmaniella zeylanoides. Salmonella spp. and Listeria monocytogenes were absent in all the samples; moreover, multiplex real-time PCR revealed the absence of the target genes bont/A, bont/B, bont/E, bont/F, and 4gyrB (CP), encoding botulinic toxins. Volatilome, deeply depending on microbiological metabolism, was characterized by spices-derived components. Limonene, sabinene, α- and β-pinene, 3-carene, and α-thujene were the most represented monoterpene hydrocarbons, whereas β- and α-copaene were the most represented sesquiterpene hydrocarbons. Allyl methyl sulphide and diallyl disulphide were the major aliphatic sulphur compounds, together with diallyl sulphide and allyl methyl disulphide.

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Biodiesel and flavor compound production using a novel promiscuous cold-adapted SGNH-type lipase (HaSGNH1) from the psychrophilic bacterium Halocynthiibacter arcticus.

Le, L. T. H. L., Yoo, W., Jeon, S., Lee, C., Kim, K. K., Lee, J. H. & Kim, T. D. (2020). Biotechnology for Biofuels, 13(1), 1-13.

Background: Biodiesel and flavor compound production using enzymatic transesterification by microbial lipases provides mild reaction conditions and low energy cost compared to the chemical process. SGNH-type lipases are very effective catalysts for enzymatic transesterification due to their high reaction rate, great stability, relatively small size for convenient genetic manipulations, and ease of immobilization. Hence, it is highly important to identify novel SGNH-type lipases with high catalytic efficiencies and good stabilities. Results: A promiscuous cold-adapted SGNH-type lipase (HaSGNH1) from Halocynthiibacter arcticus was catalytically characterized and functionally explored. HaSGNH1 displayed broad substrate specificity that included tert-butyl acetate, glucose pentaacetate, and p-nitrophenyl esters with excellent stability and high efficiency. Important amino acids (N83, M86, R87, F131, and I173F) around the substrate-binding pocket were shown to be responsible for catalytic activity, substrate specificity, and reaction kinetics. Moreover, immobilized HaSGNH1 was used to produce high yields of butyl and oleic esters. Conclusions: This work provides a molecular understanding of substrate specificities, catalytic regulation, immobilization, and industrial applications of a promiscuous cold-adapted SGNH-type lipase (HaSGNH1) from H. arcticus. This is the first analysis on biodiesel and flavor synthesis using a cold-adapted halophilic SGNH-type lipase from a Halocynthiibacter species.

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Characterization of EstZY: A new acetylesterase with 7-aminocephalosporanic acid deacetylase activity from Alicyclobacillus tengchongensis.

Ding, J., Zhou, Y., Zhu, H., Deng, M., Gao, Y., Yang, Y. & Huang, Z. (2020). International Journal of Biological Macromolecules, 148, 333-341.

Deacetyl-7-aminocephalosporanic acid (D-7-ACA) is required for producing of many semisynthetic β-lactam antibiotics; therefore, enzymes capable of converting 7-aminocephalosporanic acid (7-ACA) to D-7-ACA present a valuable resource to the pharmaceutical industry. In the present study, a putative acetylesterase, EstZY, was identified and characterized from a thermophilic bacterium Alicyclobacillus tengchongensis. Sequence alignment showed that EstZY was an acetylesterase which belonged to carbohydrate esterase family 7 (CE7), with substrate preference for short-chain acyl esters p-NPC2 to p-NPC8. Maximum enzyme activity was recorded at pH 9.0 and 50°C, where Km and Vmax were calculated as 1.9 ± 0.23 mM and 258 ± 18.5 μM min−1, respectively. The residues Ser185, Asp274, and His303 were identified as the putative catalytic triad by homology modelling, site-directed mutagenesis and molecular docking. Moreover, EstZY can remove the acetyl group at C3′ position of 7-ACA to form D-7-ACA; this is the first report of a 7-ACA deacetylase from CE7 family in A. tengchongensis and may represent a new enzyme with industrial values.

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Time-dependent self-association of spruce galactoglucomannans depends on pH and mechanical shearing.

Bhattarai, M., Valoppi, F., Hirvonen, S. P., Hietala, S., Kilpeläinen, P., Aseyev, V. & Mikkonen, K. S. (2020). Food Hydrocolloids, 102, 105607.

The demand for naturally derived, functional and cost-effective raw materials for various food applications is escalating. Spruce wood is a sustainable and abundant, but underutilized source of novel hydrocolloids-galactoglucomannans (GGM). Pressurized-hot water extracted GGM with an intermediate molar mass are hypothesized to form colloidal solutions. To design superior quality products from GGM, an understanding of their colloidal stability and their potential effect in multiphasic systems is required. The present study addresses the functionality of GGM by characterizing their properties in a bi-phasic system, and for the first time, their time-dependent colloidal stability at different extrinsic conditions- pH, ionic strength and after the application of high-intensity mechanical shearing. Amongst the conditions studied, the colloidal stability of aqueous GGM solution was highly pH dependent. The results showed that an intermediate molar mass polysaccharide like GGM formed inter-/intra molecular assemblies, which grew over time, depending on the composition and processing of the aqueous medium. The molecular dispersion of GGM and their dynamic behavior was also compared to solutions of known food hydrocolloids-gum Arabic and hydroxypropylmethyl cellulose. The observed solution properties explain the hydrocolloid functionality of GGM and contribute to design of colloidal polysaccharide systems in food application.

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Extraction of low methoxylated pectin from pea hulls via RSM.

Gutöhrlein, F., Drusch, S. & Schalow, S. (2020). Food Hydrocolloids, 102, 105609.

Nowadays, low methoxylated pectin (LMP) is generated in a multi-step process from high methoxylated pectin using fruit by-products as a raw material. In this study, we prove that LMP may be directly extracted from pea hulls. Extraction was conducted according to a central composite design (CCD) and evaluated via response surface methodology (RSM). The influence of different parameters (pH, temperature, time) on yield and composition of the extracted pectic polysaccharides (PPS) was investigated using nitric acid and citric acid as extraction media. Citric acid yielded higher amounts of PPS (3.5-9.8%) compared to nitric acid (1.4-8.0%). However, there is a conflict of aims between a high yield and the purity of the extracted PPS. Composition analysis suggests that under ‘mild’ extraction conditions (pH 2, 70°C) PPS consist of homogalacturonan, xylogalacturonan and rhamnogalacturonan with arabinose and galactose side chains (RG-I). With increasing temperature (90°), yield is maximised due to an increased solubilisation of cell wall polysaccharides. Under “harsh” conditions (pH 1, 90°C) the purity of PPS increases in terms of a relatively higher content of uronic acids, but yield decreases. This is attributed to a cleavage of non-GalA components and an ongoing depolymerisation of the pectic galacturonan. PPS extracted under these conditions is characterised by a low degree of acetylation (4%) and a relatively high protein content (7%).

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Structural, rheological and functional properties of galactose-rich pectic polysaccharide fraction from leek.

Ognyanov, M., Remoroza, C., Schols, H. A., Georgiev, Y. N., Petkova, N. T. & Krystyjan, M. (2020). Carbohydrate Polymers, 229, 115549.

An acid-extracted polysaccharide from alchohol-insoluble solids of leek was obtained. The sugar composition indicated that galactose and galacturonic acid were the major sugars, followed by small amounts of rhamnose and arabinose. The fraction contained a relatively high methyl-esterified homogalacturonan next to rhamnogalacturonan type I decorated with galactose-rich side chains. The fraction consisted of three high Mw populations, covering the range of 10-100 kDa. Enzymatic fingerprinting was performed with HG/RG-I degrading enzymes to elucidate the structure. The oligomers were analysed using LC-HILIC-MS, HPAEC, and MALDI-TOF MS. The data revealed the presence of GalA sequences, having different patterns of methyl-esterification, RG-I composed of unbranched segments and segments heavily substituted with β-(1→4)-linked galactan chains of varying length. The rheological study showed the shear-thinning, weak thixotropic, anti-thixotropic, and non-Newtonian behavior of the polysaccharide. The pectin exhibited higher water holding capacity than oil-holding capacity and the fraction did form stable foams at high concentration.

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Redox control of yeast Sir2 activity is involved in acetic acid resistance and longevity.

Espinosa, V. L., Mir, N., Garrido, L., Vived Maza, C. & Cabiscol Català, E. (2019). Redox Biology, 24, 101129.

Yeast Sir2 is an NAD-dependent histone deacetylase related to oxidative stress and aging. In a previous study, we showed that Sir2 is regulated by S-glutathionylation of key cysteine residues located at the catalytic domain. Mutation of these residues results in strains with increased resistance to disulfide stress. In the present study, these mutant cells were highly resistant to acetic acid and had an increased chronological life span. Mutant cells had increased acetyl-CoA synthetase activity, which converts acetic acid generated by yeast metabolism to acetyl.CoA. This could explain the acetic acid resistance and lower levels of this toxic acid in the extracellular media during aging. Increased acetyl-CoA levels would raise lipid droplets, a source of energy during aging, and fuel glyoxylate-dependent gluconeogenesis. The key enzyme of this pathway, phosphoenolpyruvate carboxykinase (Pck1), showed increased activity in these Sir2 mutant cells during aging. Sir2 activity decreased when cells shifted to the diauxic phase in the mutant strains, compared to the WT strain. Since Pck1 is inactivated through Sir2-dependent deacetylation, the decline in Sir2 activity explained the rise in Pck1 activity. As a consequence, storage of sugars such as trehalose would increase. We conclude that extended longevity observed in the mutants was a combination of increased lipid droplets and trehalose, and decreased acetic acid in the extracellular media. These results offer a deeper understanding of the redox regulation of Sir2 in acetic acid resistance, which is relevant in some food and industrial biotechnology and also in the metabolism associated to calorie restriction, aging and pathologies such as diabetes.

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Molecular characterization of a novel cold-active hormone-sensitive lipase (HaHSL) from Halocynthiibacter arcticus.

Le, L. T. H. L., Yoo, W., Lee, C., Wang, Y., Jeon, S., Kim, K. K., Lee, J. H. & Kim, T. D. (2019). Biomolecules, 9(11), 704.

Bacterial hormone-sensitive lipases (bHSLs), which are homologous to the catalytic domains of human HSLs, have received great interest due to their uses in the preparation of highly valuable biochemicals, such as drug intermediates or chiral building blocks. Here, a novel cold-active HSL from Halocynthiibacter arcticus (HaHSL) was examined and its enzymatic properties were investigated using several biochemical and biophysical methods. Interestingly, HaHSL acted on a large variety of substrates including tertiary alcohol esters and fish oils. Additionally, this enzyme was highly tolerant to high concentrations of salt, detergents, and glycerol. Furthermore, immobilized HaHSL retained its activity for up to six cycles of use. Homology modeling suggested that aromatic amino acids (Trp23, Tyr74, Phe78, Trp83, and Phe245) in close proximity to the substrate-binding pocket were important for enzyme activity. Mutational analysis revealed that Tyr74 played an important role in substrate specificity, thermostability, and enantioselectivity. In summary, the current study provides an invaluable insight into the novel cold-active HaHSL from H. arcticus, which can be efficiently and sustainably used in a wide range of biotechnological applications.

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Structure-function relationships underlying the dual N-acetylmuramic and N-acetylglucosamine specificities of the bacterial peptidoglycan deacetylase PdaC.

Grifoll-Romero, L., Sainz-Polo, M. A., Albesa-Jové, D., Guerin, M. E., Biarnés, X. & Planas, A. (2019). Journal of Biological Chemistry, 294(50), 19066-19080.

Bacillus subtilis PdaC (BsPdaC) is a membrane-bound, multidomain peptidoglycan N-deacetylase acting on N-acetylmuramic acid (MurNAc) residues and conferring lysozyme resistance to modified cell wall peptidoglycans. BsPdaC contains a C-terminal family 4 carbohydrate esterase (CE4) catalytic domain, but unlike other MurNAc deacetylases, BsPdaC also has GlcNAc deacetylase activity on chitooligosaccharides (COSs), characteristic of chitin deacetylases. To uncover the molecular basis of this dual activity, here we determined the X-ray structure of the BsPdaC CE4 domain at 1.54 Å resolution and analyzed its mode of action on COS substrates. We found that the minimal substrate is GlcNAc3 and that activity increases with the degree of glycan polymerization. COS deacetylation kinetics revealed that BsPdaC operates by a multiple-chain mechanism starting at the internal GlcNAc units and leading to deacetylation of all but the reducing-end GlcNAc residues. Interestingly, BsPdaC shares higher sequence similarity with the peptidoglycan GlcNAc deacetylase SpPgdaA than with other MurNAc deacetylases. Therefore, we used ligand-docking simulations to analyze the dual GlcNAc- and MurNAc-binding specificities of BsPdaC and compared them with those of SpPgdA and BsPdaA, representing peptidoglycan deacetylases highly specific for GlcNAc or MurNAc residues, respectively. BsPdaC retains the conserved Asp-His-His metal-binding triad characteristic of CE4 enzymes acting on GlcNAc residues, differing from MurNAc deacetylases that lack the metal-coordinating Asp residue. BsPdaC contains short loops similar to those in SpPgdA, resulting in an open binding cleft that can accommodate polymeric substrates. We propose that PdaC is the first member of a new subclass of peptidoglycan MurNAc deacetylases.

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The Use of CRISPR-Cas9 Genome Editing to Determine the Importance of Glycerol Uptake in Wine Yeast During Icewine Fermentation.

Muysson, J., Miller, L., Allie, R. & Inglis, D. L. (2019). Fermentation, 5(4), 93.

The high concentration of sugars in Icewine juice causes formidable stress for the fermenting Saccharomyces cerevisiae, causing cells to lose water and shrink in size. Yeast can combat this stress by increasing the internal concentration of glycerol by activating the high osmolarity glycerol response to synthesize glycerol and by actively transporting glycerol into the cell from the environment. The H+/glycerol symporter, Stl1p, has been previously characterized as being glucose repressed and inactivated, despite osmotic stress induction. To further investigate the role of Stl1p in Icewine fermentations, we developed a rapid single plasmid CRISPR-Cas9-based genome editing method to construct a strain of the common Icewine yeast, S. cerevisiae K1-V1116, that lacks STL1. In an Icewine fermentation, the ∆STL1 strain had reduced fermentation performance, and elevated glycerol and acetic acid production compared to the parent. These results demonstrate that glycerol uptake by Stl1p has a significant role during osmotically challenging Icewine fermentations in K1-V1116 despite potential glucose downregulation.

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