Acetic Acid Assay Kit (ACS Manual Format)

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.

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.

Chitin deacetylase (CDA) plays a pivotal role in converting chitin to chitosan, yet industrial applications remain constrained by low enzymatic activity, instability under process conditions, and insufficient understanding of metalloenzyme activation mechanisms. Addressing these challenges, we conducted a genome-driven investigation of 151 salt-tolerant Bacillus strains to identify robust CDAs tailored for industrial demands. Genomic analysis revealed 120 strains harboring CDA genes, with Bacillus pumilus B866 exhibiting the highest native activity (105.93 U/mL). Through systematic medium optimization-identifying lactose, yeast extract, and FeSO₄ as critical components—CDA production in B866 surged to 191.32 U/mL, a 2.39-fold increase over baseline. Heterologous expression of BpCDA in E. coli yielded a recombinant enzyme (123.27 U/mL) with superior thermostability (retaining > 42.9% activity after 24 h at 55°C) and broad pH adaptability (>81.4% activity at pH 7.0-9.0). Notably, BpCDA demonstrated unique Fe²⁺-dependent activation (186.4% activity enhancement at 1 mM), contrasting with Mg²⁺-dependent systems in prior studies. Comparative genomic and pan-genome analyses underscored evolutionary adaptations linked to saline–alkaline niches, while biosynthetic gene cluster profiling revealed strain-specific metabolic potentials independent of genome size. This study resolves critical limitations in CDA performance by integrating genome mining, targeted screening, and metalloenzyme engineering, establishing a scalable platform for sustainable chitin valorization. The optimized BpCDA, with its industrial-compatible stability and novel activation mechanism, represents a significant advancement toward efficient, eco-friendly chitosan production.

In the last thirty years, the factors driving the establishment and composition of the sourdough biota have been deeply studied. Nevertheless, to date, no study has ever evaluated the biochemical and microbial dynamics of sourdoughs propagated using the different traditional methods integrated into procedural back-slopping practices worldwide. A mature type I sourdough was propagated for 10 days according to four managing conditions (Milanese, In Water, Free and Piedmontese) entailing incubations in a jute sack, submerged in water, in a jar or a combination of them. Sourdoughs obtained under the different conditions (and corresponding breads) were extensively characterized. When processing parameters modified the sourdough environment, the microbial community changed. In the first days of propagation Fructilactobacillus sanfranciscensis was the main dominant species regardless of the type of propagation, remaining present in all sourdoughs, especially those maintained in a jar. Differences among the propagation methods emerged from the biochemical analysis. Sourdoughs propagated in water exhibited higher titratable acidity, mainly due to the acetic acid produced, and were characterized by a more complex aromatic profile which differentiated them from the others. Biochemical features of breads mainly reflected those of the corresponding sourdough, whereas nutritional (protein digestibility and glycemic index) and technological (texture profile, colorimetric coordinates) features were hardly affected by the propagation method. Thus, investigation on the effect of the variation of the ecological determinants within the same propagation methods and their role in the definition of sourdough potential could be the subject of further studies.

Once merely a culinary staple, vinegar has evolved into a multifaceted ingredient valued for its flavor enhancement and potential health benefits. Port wine vinegar from the Iberian Peninsula stands out for its rich nutrient profile and unique sensory characteristics. This study provides a comprehensive descriptive analysis of the chemical and sensory properties of Port wine vinegar alongside other types of wine vinegar, including white wine, red wine, and balsamic vinegar. The analysis highlights the distinctive characteristics of each vinegar type based on their chemical composition and sensory profiles. Chemical analyses encompassed pH levels, acetic acid concentrations, ethanol content, total phenolic content, antioxidant activity, colorimetric parameters, and phenolic composition. Sensory evaluations were conducted to discern distinctive flavor profiles. Port wine vinegar's reddish-brown hue and nuanced flavor are vital features described in this study, attributed to its aging process in oak barrels. Chemical analyses described the relatively low pH, moderate acetic acid concentration, and notable phenolic content of Port wine vinegar, including epigallocatechin and catechin gallate. Aging was associated with higher polyphenol concentrations across the samples. These phenolic compounds, known for their antioxidant properties, contribute to the potential health benefits of vinegar consumption.

Innate immune response cells produce high concentrations of the free radical nitric oxide (NO) in response to pathogen infection. The antimicrobial properties of NO include nonspecific damage to essential biomolecules and specific inactivation of enzymes central to aerobic metabolism. However, the molecular targets of NO in anaerobic metabolism are less understood. Here, we demonstrate that the Escherichia coli glycyl radical enzyme pyruvate formate lyase (PFL), which catalyzes the anaerobic metabolism of pyruvate, is irreversibly inhibited by NO. Using electron paramagnetic resonance and site-directed mutagenesis we show that NO destroys the glycyl radical of PFL. The activation of PFL by its cognate radical S-adenosyl-l-methionine-dependent activating enzyme (PFL-AE) is also inhibited by NO, resulting in the conversion of the essential iron–sulfur cluster to dinitrosyl iron complexes. Whole-cell EPR and metabolic flux analyses of anaerobically growing E. coli show that PFL and PFL-AE are inhibited by physiologically relevant levels of NO in bacterial cell cultures, resulting in diminished growth and a metabolic shift to lactate fermentation. The class III ribonucleotide reductase (RNR) glycyl radical enzyme and its corresponding RNR-AE are also inhibited by NO in a mechanism analogous to those observed in PFL and PFL-AE, which likely contributes to the bacteriostatic effect of NO. Based on the similarities in reactivity of the PFL/RNR and PFL-AE/RNR-AE enzymes with NO, the mechanism of inactivation by NO appears to be general to the respective enzyme classes. The results implicate an immunological role of NO in inhibiting glycyl radical enzyme chemistry in the gut.

The Maillard reaction (MR) between sugars and amino acids, peptides, or proteins is understood to occur gradually during the production and aging of sparkling wines, where it contributes to caramel, roasted, and toasted aromas. Divalent metal ions can accelerate the MR, although this has not been previously reported in wine or wine-like conditions. In this work, the effect of calcium (Ca) and magnesium (Mg) ions on the concentration of 10 Maillard reaction-associated products (MRPs) was measured in modified sparkling base wine during accelerated aging at 50 °C for four weeks. Chardonnay base wine was modified by the addition of fructose (0.02 M) and a single amino acid (lysine, glycine, cysteine; 0.01 M) in combination with Ca²⁺ or Mg²⁺ at zero, low (10 mg/L), or high (50 mg/L) dose levels. MRPs were quantified by headspace solid-phase microextraction coupled with gas chromatography–mass spectrometry (HS-SPME-GC/MS), sugar concentration was measured by enzymatic assay, and amino acids and free metal ions were monitored by capillary electrophoresis. Fructose levels did not substantially decrease during aging despite increases in all MRPs, suggesting that trace sugars or α-dicarbonyl species present in the wine matrix likely play a greater role in MRP formation than fructose. Aging duration and amino acid content had a greater effect than metal addition on the composition of the MRPs. Treatments containing cysteine and 50 mg/L Ca²⁺ had elevated concentrations of benzaldehyde and furfural ethyl ether following 4 weeks of accelerated aging. This work identified key MRPs that increase during base wine accelerated aging and informs future research on the relationship between wine composition and aging markers.

Understanding the mechanism behind the transcriptional regulation of polysaccharide O-acetylation remains a key challenge that might be regulated through transcription factors. Our earlier work revealed the upregulation of AtGELP7 in MYB46 overexpression lines, prompting us to investigate how MYB46 transcriptionally controls AtGELP7 including other cell wall acetylation pathway genes in Arabidopsis. In MYB46 overexpression lines, we observed alteration in acetylation levels on xylan, xyloglucan and pectin in different tissue types, which suggests complex and tight regulation of acetylation homeostasis in the cell wall. Further, our transcriptomic data revealed the simultaneous upregulation of both AtGELP7 (acetyl xylan esterase) and xylan-specific TBLs (xylan O-acetyltransferases) indicating sophisticated regulation of cell wall acetylation homeostasis. Using transactivation studies in Nicotiana and pAtGELP7::GUS stable lines, we found that MYB46 enhances AtGELP7 expression probably through downstream regulators which could be either MYB103 or other MYBs. In addition, extensive cell wall analysis of MYB46 overexpression lines showed differential sugar distribution, preferably to major cell wall components such as cellulose, xyloglucan, and xylan across different tissue types of different developmental stages. Moreover, the integration of RNA-sequencing and ChIP-sequencing data uncovered previously unknown, novel probable direct gene targets of MYB46 that may be involved in polysaccharide acetylation and cell wall remodeling.

There is growing interest in low-temperature food processing. In the baking industry, low-temperature fermentation improves the production of natural aroma compounds, which have a positive impact on the sensory profile of the final product. The aim of this study was to develop a yeast-lactic acid bacteria starter culture that effectively ferments wheat dough at a temperature of 15°C. The microorganisms were selected based on their enzymatic activity and ability to grow at low temperature. The fermentation activity of the yeast and mixed cultures was assessed enzymatically. The biosynthesis of volatile organic compounds was quantified using the HS-GC-MS technique. Samples fermented by S. cerevisiae D3 were characterized by the highest concentration of volatile organic compounds, especially esters. The addition of lactic acid bacteria increased not only the biosynthesis of volatile organic compounds but also the productivity of carbon dioxide during dough fermentation. Based on both dough expansion and the profile of volatile organic compounds, a mixed culture of S. cerevisiae D3 and L. brevis B46 was selected as the most effective starter for low-temperature fermentation.

Safe geological disposal of radioactive waste requires a thorough understanding of geochemical conditions in the host formation. Boom Clay is a potential candidate in Belgium, where active methanogenesis has been detected in its deep subsurface, influencing the local geochemistry. However, the pathways driving this process and the characteristics of the methanogenic archaea involved remain unclear. We isolated a distinct archaeal strain from Boom Clay pore water and characterized it geno- and phenotypically. Isolate TD41E1-1 belongs to a novel species of the Methanosarcina genus, for which the name Methanosarcina hadiensis sp. nov. is proposed. TD41E1-1 cells are coccus-shaped, irregularly sized cells enveloped by extracellular polymer substances. Growth and substrate utilization experiments and genomic analysis demonstrated that the strain prefers methylated compounds or hydrogen as substrates for methane production. Although it possesses a complete acetoclastic pathway, no growth was observed in the presence of acetate in the tested conditions. Based on its phylogenetic relation to other known Methanosarcina species and on the presence of c-type cytochromes, it can be concluded that the strain likely occupies an intermediate position between type I and type II Methanosarcina species. These findings provide valuable insights for assessing Boom Clay's suitability for geological disposal of radioactive waste.

In this study a plant-based spread was developed using dextran-enriched ingredients derived from pea flours, supplemented with defatted durum wheat germ and almond flour. Optimization of fermentation with Leuconostoc pseudomesenteroides DSM 20193, both with and without enzymatic hydrolysis, aimed to enhance exopolysaccharide production and the nutritional value of pea flours. Best results were achieved through enzymatic hydrolysis with Veron PS protease followed by fermentation at 25°C, resulting in elevated dextran levels and increased peptides and total free amino acid concentration in green and yellow pea-based ingredients. The yellow pea-based ingredient was selected for the final plant-based spread formulation, blended at 35% w/w, with 45% w/w defatted durum wheat germ, and 20% w/w almond flour. The resultant spread exhibited elastic and solid-like characteristics like milk-based spreadable cheese and yogurt, boasting 'high protein' (12.49 g/100g) and 'high fiber' (11.01 g/100g) designations. It maintained chemical, biochemical, and microbiological stability over a 10-day shelf-life under refrigerated conditions. Sensory evaluation confirmed the acceptability of the plant-based spread (PBS), highlighting a well-balanced aroma and a grainy, adhesive texture. This research underscores the potential of an integrated approach utilizing food-grade enzymes and fermentation for the in-situ production of dextran to create innovative, clean label, and plant-based foods.

The AMP-forming acetyl-CoA synthetase is regulated by lysine acetylation both in bacteria and eukaryotes. However, the underlying mechanism is poorly understood. The Bacillus subtilis acetyltransferase AcuA and the AMP-forming acetyl-CoA synthetase AcsA form an AcuA•AcsA complex, dissociating upon lysine acetylation of AcsA by AcuA. Crystal structures of AcsA from Chloroflexota bacterium in the apo form and in complex with acetyl-adenosine-5'-monophosphate (acetyl-AMP) support the flexible C-terminal domain adopting different conformations. AlphaFold2 predictions suggest binding of AcuA stabilizes AcsA in an undescribed conformation. We show the AcuA•AcsA complex dissociates upon acetyl-coenzyme A (acetyl-CoA) dependent acetylation of AcsA by AcuA. We discover an intrinsic phosphotransacetylase activity enabling AcuA•AcsA generating acetyl-CoA from acetyl-phosphate (AcP) and coenzyme A (CoA) used by AcuA to acetylate and inactivate AcsA. Here, we provide mechanistic insights into the regulation of AMP-forming acetyl-CoA synthetases by lysine acetylation and discover an intrinsic phosphotransacetylase allowing modulation of its activity based on AcP and CoA levels.

A key aspect of sustainable bioeconomy is the recirculation of renewable, agricultural waste streams as substrates for microbial production of high-value compounds. One approach is the bioconversion of corn stover, an abundant maize crop byproduct, using the fungal maize pathogen Ustilago maydis. U. maydis is already used as a unicellular biocatalyst in the production of several industrially-relevant compounds using plant biomass hydrolysates. In this study, we demonstrate that U. maydis can grow using untreated corn stover as its sole carbon source. We developed a small-scale bioreactor platform to investigate U. maydis processing of corn stover, combining online monitoring of fungal growth and metabolic activity profiles with biochemical analyses of the pre- and post-fermentation residues. Our results reveal that U. maydis primarily utilizes soluble sugars i.e., glucose, sucrose and fructose present in corn stover, with only limited exploitation of the abundant lignocellulosic carbohydrates. Thus, we further explored the biotechnological potential of enhancing U. maydis´ lignocellulosic utilization. Additive performance improvements of up to 120 % were achieved when using a maize mutant with increased biomass digestibility, co-fermentation with a commercial cellulolytic enzyme cocktail, and exploiting engineered fungal strains expressing diverse lignocellulose-degrading enzymes. This work represents a key step towards scaling up the production of sustainable compounds from corn stover using U. maydis and provides a tool for the detailed monitoring of the fungal processing of plant biomass substrates.

This work was aimed to characterize functional and biochemical parameters of a bakery ingredient prepared with durum wheat by-products (micronized bran and middling) fermented by a selected microbial consortium composed of yeasts and lactic acid bacteria. The unfermented milling by-products mixture and the mixture fermented by a baker’s yeast were used as reference. The innovative ingredient showed more stable colour indexes compared to the references, a more complex profile in volatile molecules characterized by a higher presence of alcohols, ketones and acids compared to the references. A significant increase in the content of peptides, short chain fatty acids, total phenols, antioxidant activity and prebiotic activity together with a reduction in phytic acid content was observed in the samples fermented by the selected microbial consortium compared to the references. This work provides information on the impact of lactic acid bacteria and yeasts on functional and biochemical characteristics of fermented milling by-products.

Plant-based milk alternatives have gained massive popularity among consumers because of their sustainable production compared to bovine milk and because of meeting the nutritional requests of consumers affected by cow milk allergies and lactose intolerance. In this work, hemp flour, in a blend with rice flour, was used to design a novel lactose- and gluten-free yogurt-like (YL) product with suitable nutritional, functional, and sensory features. The growth and the acidification of three different lactic acid bacteria strains were monitored to better set up the biotechnological protocol for making the YL product. Hemp flour conferred the high fiber (circa 2.6 g/100 g), protein (circa 4 g/100 g), and mineral contents of the YL product, while fermentation by selected lactic acid bacteria increased the antioxidant properties (+8%) and the soluble fiber (+0.3 g/100 g), decreasing the predicted glycemic index (-10%). As demonstrated by the sensory analysis, the biotechnological process decreased the earthy flavor (typical of raw hemp flour) and increased the acidic and creamy sensory perceptions. Supplementation with natural clean-label vanilla powder and agave syrup was proposed to further decrease the astringent and bitter flavors. The evaluation of the starter survival and biochemical properties of the product under refrigerated conditions suggests an estimated shelf-life of 30 days. This work demonstrated that hemp flour might be used as a nutritional improver, while fermentation with a selected starter represents a sustainable and effective option for exploiting its potential.