Acetic Acid Assay Kit (Acetate Kinase 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.

This work proposes a biorefinery approach for the utilization of agri-food residues, such as tomato pomace (TP), through combining chemical hydrolysis with high-pressure homogenization (HPH), aiming to achieve the isolation of cellulose with tailored morphological properties from underused lignocellulose feedstocks, along with the valorization of the value-added compounds contained in the biomass. Cellulose was isolated from TP using sequential chemical hydrolysis in combination with mechanical pretreatment through HPH. The chemical and structural features of cellulose isolated from TP pretreated by HPH were compared with cellulose isolated from untreated TP through light scattering for particle size distribution, optical and scanning electron microscopy, and Fourier-transform infrared spectroscopy (FT-IR) analysis. HPH pretreatment (80 MPa, 10 passes) not only promoted a slight increase in the yield of cellulose extraction (+9%) but contributed to directly obtaining defibrillated cellulose particles, characterized by smaller irregular domains containing elongated needle-like fibers. Moreover, the selected mild chemical process produced side streams rich in bioactive molecules, evaluated in terms of total phenols and reducing activity. The liquors recovered from acid hydrolysis of TP exhibited a higher biological activity than those obtained through a conventional extraction (80% v/v acetone, 25°C, 24 h at 180 rpm).

There is growing evidence suggesting that by improving gut integrity and function, less energy is partitioned toward immune responses related to xenobiotic infiltration, sparing energy for productive purposes. Gluconic acid and its salts have previously shown prebiotic effects in the lower gut of nonruminant animals, where they serve as a precursor for butyrate, although evidence in ruminants is limited. Butyrate and its fermentative precursors have demonstrated multiple beneficial effects to gastrointestinal ecology, morphology, and function, such as the stimulation of epithelial cell proliferation and improvement of gut barrier function and ecology. The objective of this study was to evaluate changes in milk production, milk fatty acid composition, and fecal and blood parameters in lactating dairy cattle fed a hydrogenated fat-embedded calcium gluconate (HFCG) supplement designed to target the hindgut for calcium gluconate delivery. In addition, the effects of a compound feed processing method (i.e., incorporated into a mash or an extruded pellet) were tested to evaluate the effect of extrusion on product efficacy. Forty-five lactating Holstein cows at approximately 165 d in milk were used in a 3 × 3 Latin square consisting of three 28-d periods, during which animals were offered a basal ration mixed with 3 different compound feeds: a negative control in mash form containing no HFCG, or the HFCG supplement fed at a target rate of 16 g/d, delivered in either a mash or pelleted form. Supplementation of HFCG tended to increase yields of milk fat and fat- and energy-corrected milk. Total yields and concentrations of milk fatty acids ≥18 carbons in length tended to increase in response to HFCG. Plasma nonesterified fatty acids and milk urea increased in HFCG treatments. No differences were observed in fecal pH or fecal concentrations of volatile fatty acids, with the exception of isobutyrate, which decreased in HFCG-fed cows. Changes in milk fatty acid profile suggest that increased milk fat yield was driven by increased incorporation of preformed fatty acids, supported by increased circulating nonesterified fatty acid. Future research investigating the mode of action of HFCG at the level of the hindgut epithelium is warranted, as measured fecal parameters showed no response to treatment.

The yeast Lachancea thermotolerans can produce lactic acid during alcoholic fermentation (AF) and thereby acidify wines with insufficient acidity. However, little is known about the impact of L. thermotolerans on Oenococcus oeni, the primary lactic acid bacterium used in malolactic fermentation (MLF). This study explored the impact of sequential cultures of L. thermotolerans and Saccharomyces cerevisiae on MLF performance in white and red wines. Four L. thermotolerans strains were tested in Sauvignon blanc with sequential S. cerevisiae inoculation, compared to an S. cerevisiae control and the initially un-inoculated treatments. The L. thermotolerans wines showed large differences in acidification, and progression of MLF depended on lactic acid production, even at controlled pH. The highest and lowest lactic acid producing strains were tested further in Merlot fermentations with both co-inoculated and sequentially inoculated O. oeni. The low lactic acid producing strain enabled successful MLF, even when this failed in the S. cerevisiae treatment, with dramatically quicker malic acid depletion in O. oeni co-inoculation than in sequential inoculation. In contrast, a high lactic acid producing strain inhibited MLF irrespective of the O. oeni inoculation strategy. In a follow-up experiment, increasing concentrations of exogenously added lactic acid slowed MLF and reduced O. oeni growth across different matrices, with 6 g/L of lactic acid completely inhibiting MLF. The results confirm the inhibitory effect of lactic acid on O. oeni while highlighting the potential of some L. thermotolerans strains to promote MLF and the others to inhibit it.

Background: Biomass recalcitrance towards pretreatment and further processing can be related to the compositional and structural features of the biomass. However, the exact role and relative importance to those structural attributes has still to be further evaluated. Herein, ten different types of biomass currently considered to be important raw materials for biorefineries were chosen to be processed by the recently developed, acid-catalyzed OrganoCat pretreatment to produce cellulose-enriched pulp, sugars, and lignin with different amounts and qualities. Using wet chemistry analysis and NMR spectroscopy, the generic factors of lignocellulose recalcitrance towards OrganoCat were determined. Results: The different materials were processed applying different conditions (e.g., type of acid catalyst and temperature), and fractions with different qualities were obtained. Raw materials and products were characterized in terms of their compositional and structural features. For the first time, generic correlation coefficients were calculated between the measured chemical and structural features and the different OrganoCat product yields and qualities. Especially lignin-related factors displayed a detrimental role for enzymatic pulp hydrolysis, as well as sugar and lignin yield exhibiting inverse correlation coefficients. Hemicellulose appeared to have less impact, not being as detrimental as lignin factors, but xylan-O-acetylation was inversely correlated with product yield and qualities. Conclusion: These results illustrate the role of generic features of lignocellulosic recalcitrance towards acidic pretreatments and fractionation, exemplified in the OrganoCat strategy. Discriminating between types of lignocellulosic biomass and highlighting important compositional variables, the improved understanding of how these parameters affect OrganoCat products will ameliorate bioeconomic concepts from agricultural production to chemical products. Herein, a methodological approach is proposed.

The cell wall is a complex and dynamic structure that determines plants' performance by constant remodeling of its compounds. Although cellulose is its major load‐bearing component, pectins are crucial to determine wall characteristics. Changes in pectin physicochemical properties, due to pectin remodeling enzymes (PRE), induce the rearrangement of cell wall compounds, thus, modifying wall architecture. In this work, we tested for the first time how cell wall dynamics affect photosynthetic properties in Arabidopsis thaliana pectin methylesterase atpme17.2 and pectin acetylesterase atpae11.1 mutants in comparison to wild‐type Col‐0. Our results showed maintained PRE activities comparing mutants with wild‐type and no significant differences in cellulose, but cell wall non‐cellulosic neutral sugars contents changed. Particularly, the amount of galacturonic acid (GalA) - which represents to some extent the pectin cell wall proportion – was reduced in the two mutants. Additionally, physiological characterization revealed that mutants presented a decreased net CO₂ assimilation (A_N) because of reductions in both stomatal (g_s) and mesophyll conductances (g_m). Thus, our results suggest that atpme17.2 and atpae11.1 cell wall modifications due to genetic alterations could play a significant role in determining photosynthesis.

Among typical Portuguese sausages, the cacholeira blood sausage undoubtedly represents one of the most popular preparations. To the authors’ knowledge, a lack of information on both the microbiota and the volatile organic compounds (VOCs) of this blood-containing sausage emerges from the available scientific literature. This study represents the first characterization of physico-chemical, microbiological and volatile traits of Portuguese cacholeira blood sausage. To this end, ready-to-eat cacholeira blood sausages were collected from two production batches manufactured in summer (batch 1) and autumn (batch 2). Viable counts showed active microbial communities mainly composed by lactic acid bacteria, coagulase negative cocci, enterococci and eumycetes. The metataxonomic approach showed a simple bacterial composition, which was dominated by Lactobacillus sakei in both the analyzed batches (1 and 2) considered. Carnobacterium, Enterococcus, Kluyvera, Lactococcus and Serratia were found as minor genera. The mycobiota varied according to the production season. Batch 1 was dominated by Starmerella apicola, Debaryomyces hansenii and Candida tropicalis, whereas batch 2 was dominated by D. hansenii. Moreover, Aspergillus spp., Kurtzmaniella zeylanoides, Saccharomyces cerevisiae, Kurtzmaniella santamariae, Brettanomyces bruxellensis and Pichia kluyveri were detected in both the batches as minority species. Seventy-two volatile compounds were identified, including esters, phenols, terpenoids, acids, alcohols, ketones, aldehydes, lactones, furans, sulphur and nitrogen compounds. Significant differences were seen in the amount of some compounds, as a feasible consequence of differences in the raw materials, artisan production and seasonality.

Brettanomyces yeasts have gained popularity in many sectors of the biotechnological industry, specifically in the field of beer production, but also in wine and ethanol production. Their unique properties enable Brettanomyces to outcompete conventional brewer’s yeast in industrially relevant traits such as production of ethanol and pleasant flavors. Recent advances in next-generation sequencing (NGS) and high-throughput screening techniques have facilitated large population studies allowing the selection of appropriate yeast strains with improved traits. In order to get a better understanding of Brettanomyces species and its potential for beer production, we sequenced the whole genome of 84 strains, which we make available to the scientific community and carried out several in vitro assays for brewing-relevant properties. The collection includes isolates from different substrates and geographical origin. Additionally, we have included two of the oldest Carlsberg Research Laboratory isolates. In this study, we reveal the phylogenetic pattern of Brettanomyces species by comparing the predicted proteomes of each strain. Furthermore, we show that the Brettanomyces collection is well described using similarity in genomic organization, and that there is a direct correlation between genomic background and phenotypic characteristics. Particularly, genomic patterns affecting flavor production, maltose assimilation, beta-glucosidase activity, and phenolic off-flavor (POF) production are reported. This knowledge yields new insights into Brettanomyces population survival strategies, artificial selection pressure, and loss of carbon assimilation traits. On a species-specific level, we have identified for the first time a POF negative Brettanomyces anomalus strain, without the main spoilage character of Brettanomyces species. This strain (CRL-90) has lost DaPAD1, making it incapable of converting ferulic acid to 4-ethylguaiacol (4-EG) and 4-ethylphenol (4-EP). This loss of function makes CRL-90 a good candidate for the production of characteristic Brettanomyces flavors in beverages, without the contaminant increase in POF. Overall, this study displays the potential of exploring Brettanomyces yeast species biodiversity to find strains with relevant properties applicable to the brewing industry.

Aims: This study investigated the influence of sugars (glucose and fructose) and pH on the gene expression of cite (citrate lyase β‐subunit) and the subsequent formation of metabolites associated with citrate metabolism. Methods and Results: Different levels of glucose (2·5, 50 and 115 g l⁻¹), fructose (2·5, 50 and 115 g l⁻¹) and pH (3·0, 3·5, 4·0 and 5·0) were evaluated for their effect on cite expression in four different lactic acid bacteria strains. Two Oenococcus oeni strains and two Lactobacillus plantarum strains were used, of which one strain of each species screened positive for the cite gene. Among the factors tested, fructose had the biggest influence on the relative expression of cite in O. oeni. In addition, the citrate‐positive strains produced high concentrations of diacetyl and acetoin. Conclusions: This study gives an overview of how sugar, pH and different lactic acid bacteria strains influence cite gene expression and the formation of metabolites associated with citrate metabolism closely linked to malolactic fermentation (MLF). Significance and Impact of the Study: These results can be used to make informed decisions regarding MLF when aiming to create a wine with a buttery aroma or not.

The polysaccharides that comprise bacterial cell walls are commonly O-acetylated. This modification confers resistance to hydrolases of innate immune systems and/or controls endogenous autolytic activity. Herein, we present protocols for the compositional analysis of bacterial cell wall O-acetylation, and assays for monitoring O-acetyltransferases and O-acetylesterases. The assays are amenable for the development of high-throughput screens in search of inhibitors of the respective enzymes.

During aerobic growth on glucose, Escherichia coli excretes acetate, a mechanism called “overflow metabolism.” At high concentrations, the secreted acetate inhibits growth. Several mechanisms have been proposed for explaining this phenomenon, but a thorough analysis is hampered by the diversity of experimental conditions and strains used in these studies. Here, we describe the construction of a set of isogenic strains that remove different parts of the metabolic network involved in acetate metabolism. Analysis of these strains reveals that (i) high concentrations of acetate in the medium inhibit growth without significantly perturbing central metabolism; (ii) growth inhibition persists even when acetate assimilation is completely blocked; and (iii) regulatory interactions mediated by acetyl-phosphate play a small but significant role in growth inhibition by acetate. The major contribution to growth inhibition by acetate may originate in systemic effects like the uncoupling effect of organic acids or the perturbation of the anion composition of the cell, as previously proposed. Our data suggest, however, that under the conditions considered here, the uncoupling effect plays only a limited role.

Low alcohol hulless barley wine (HW) is a popular beverage among the highland areas in China. It is known to have several health benefits due to the presence of β-glucan and antioxidant compounds. Therefore, the total β-glucan content, total phenols and flavonoids of HW samples from the highland areas of Sichuan province and Tibet were determined in this study. The results indicated that HW is abundant in both β-glucan (54-76 mg/L) and phenolic compounds (131-178 mg/L). Moreover, this study also investigated the flavor and aroma characteristics of HW samples. A total of forty six volatile aroma substances were identified by GC-MS. The HWs could be classified into three distinct groups in terms of the region of origin according to the results of PCA based on the GC-MS data. These findings provide a useful foundation for further study of the health benefits and the flavor characteristics of HW in highland areas.

Peritrophic membrane (PM) is a chitin and protein-containing extracellular matrix that lines the midgut in most insect species, functioning as a barrier to exogenous toxins and pathogens. Midgut chitin deacetylases (CDAs) are chitin-modifying enzymes known to alter the mechanical property and permeability of PM. However, biochemical properties and specific roles of these enzymes remain elusive. In this study, the midgut-expressed CDAs (BmCDA6, BmCDA7 and BmCDA8) from Bombyx mori were cloned, recombinantly expressed and purified and their enzymatic activities toward PM chitin were determined. Of the three enzymes, BmCDA7 exhibited the highest activity (0.284 μmol/min/μmol), while BmCDA8 showed lower activity of 0.061 μmol/min/μmol. BmCDA6 was inactive towards PM chitin. Gene expression patterns indicated that although all three CDA genes were specifically expressed in the anterior midgut, they differed in their temporal expression patterns. BmCDA6 was expressed almost exclusively at the mid-molt stage, the stage when the PM was thick and with multiple chitin layers. Unlike BmCDA6, high expression levels of BmCDA7 and BmCDA8 were observed only at the feeding stage, the stage when the PM is thin and with fewer chitin layers. The different gene expression patterns and biochemical characteristics provide new information about the functional specialization among BmCDA6, BmCDA7 and BmCDA8 proteins.

Lactic acid bacteria (LAB) fermentation performance is essential for aroma metabolites formation and product flavour quality. Hence, this study appraises high‐gravity malt wort fermentation (HGF) by three LAB strains to improve the fermentation performance and consumer's acceptance of lactic acid‐fermented malt‐based beverages (LAFMB). HGF at 20% (w/w) provided higher amino acid content and buffering capacity that allowed greater cell development, viable cell count and sugar utilisation. Moreover, the pH change was lesser although marked lactic acid accumulation. It is noteworthy that HGF significantly incremented the content of higher alcohols (+0 – 161%), 2‐phenylethanol (+11-147%), acetaldehyde (+27–44%) and β‐damascenone (+25 - 66%) comparing to low‐gravity malt wort at 12%. Thus, HG‐fermented beverages were significantly preferred with greater hedonic scores (4.6 ± 2.1). Our results indicate that HGF is a valuable strategy for improving LAB fermentation performance in malt wort, which in turn increases key aroma compound content resulting in enhanced acceptance of LAFMB.

The emulsification capacity of process-induced tomato and broccoli sera, containing proteins and pectin with distinct molecular structures, was investigated. Targeted pectin molecular modifications were induced by applying combinations of treatments to prepare the purées, and subsequently, the serum pectin-protein mixtures were isolated and assessed in terms of their capacity to stabilize oil-in-water (o/w) emulsions at different pH conditions (pH 3.5 or 6.0). Regardless of the processing applied, broccoli sera presented a better emulsion stabilizing capacity than tomato sera due to the higher protein content and probably the presence of more acetylated and branched pectin. In tomato sera, where a low amount of protein was detected, differences in emulsification capacity were identified depending on pectin molecular structure. Tomato sera, obtained from purées treated at low temperature for the stimulation of pectin-related enzymes which led to pectin with a high molecular weight, low degree of methyl-esterification and relatively high degree of acetylation, formed stable o/w emulsions. The vegetable sera exhibited higher capacity to form stable emulsions at pH 3.5 compared to pH 6.0, which can be attributed to a lower dissociation of the pectin carboxylic groups at low pH leading to less intramolecular repulsions resulting in a more compact conformation of the adsorbed species at the oil-water interface and better electrostatic interactions with positively-charged proteins. Thus, selective processing of vegetable purées is a promising strategy to induce molecular changes in water-soluble biopolymers with potential use as natural emulsifiers in acid/acidified foods.

Our study aims to find gene pathways that depend on acetate assimilation under diurnal conditions in the microalga Chlamydomonas. We compare the transcriptome of two strains, one control and one mutant deficient for the glyoxylate cycle essential in acetate metabolism, cultivated under day/night cycles with acetate. We apply surprisal analysis, an information-theoretic approach, to the RNA-seq data. Carrying out the analysis on groups of dark and light phase samples separately allows identifying constraints and gene pathways that discriminate between mutant and control samples. Carbon metabolism is the most important in the light phase for the control strain while the dark phase is enriched in cell division pathways. The mutant phenotype includes genes pathways of stress response and autophagy in the two phases. Cell division pathways are found in the light phase and catabolic pathways in the dark phase, highlighting a rewiring of the mutant transcriptome in these cyclic cultivation conditions.

Microalgae are a promising platform for the production of renewable fuels and oleochemicals. Despite significant research efforts to understand the mechanisms of algal lipid accumulation, the influence of commercially relevant growth conditions on the lipid metabolism is poorly understood. To characterise the impact of differing organic carbon availabilities and photoperiod on the response of the model alga Chlamydomonas reinhardtii to nitrogen stress, the expression of key genes involved in the central carbon metabolism were monitored over a time-course of nitrogen deprivation. In addition, the growth, PSII integrity, chlorophyll content, triacylglycerol (TAG) content, starch content, and fatty acid composition were characterised. Results indicate that both organic carbon availability and photoperiod regulate the lipid accumulation response of C. reinhardtii. Under mixotrophic conditions, organic carbon uptake is favoured over photosynthesis, transcript abundance of lipid synthesis genes rapidly increase and acetate is funnelled to TAG synthesis. In contrast, autotrophic cultures lacking organic carbon experienced a slower rate of photosynthetic degradation and funnelled the majority of sequestered carbon to starch synthesis. Dark periods induced catabolism of both starch and TAG in autotrophic cultures but TAG alone in mixotrophic cultures. Furthermore, diurnal light enhanced starch synthesis under mixotrophic conditions. Finally, transcript analysis indicated that PGD1, important for the routing of oleic acid to TAG, was reliant on organic carbon availability, resulting in reduced C18:1 fatty acid accumulation in autotrophic cultures.

Many areas of the ocean are nutrient-poor yet support large microbial populations, leading to intense competition for and recycling of nutrients. Organic phosphonates are frequently found in marine waters, but require specialist enzymes for catabolism. Previous studies have shown that the genes that encode these enzymes in marine systems are under Pho regulon control and so are repressed by inorganic phosphate. This has led to the conclusion that phosphonates are recalcitrant in much of the ocean, where phosphorus is not limiting despite the degradative genes being common throughout the marine environment. Here we challenge this paradigm and show, for the first time, that bacteria isolated from marine samples have the ability to mineralise 2-aminoethylphosphonate, the most common biogenic marine aminophosphonate, via substrate-inducible gene regulation rather than via Pho-regulated metabolism. Substrate-inducible, Pho-independent 2-aminoethylphosphonate catabolism therefore represents a previously unrecognised component of the oceanic carbon, nitrogen and phosphorus cycles.

The influence of mechanical tissue disintegration techniques (i.e. blending and high pressure homogenization) and the stimulation of endogenous pectin-related enzymes (i.e. pectin methyl-esterase and polygalacturonase) on tomato purée consistency, serum composition and serum pectin structure were investigated. Serum pectin structure was characterized in terms of degree of methyl-esterification, acetylation, neutral sugar composition and molecular weight (M_w) distribution. Endogenous pectin methyl-esterase and polygalacturonase stimulation resulted in the lowest purée consistency and highest serum yield. However, when such purée was homogenized, a higher purée consistency and a low serum yield were observed. Moreover, the M_w of serum pectin was exceptionally high for the homogenized purées. The low methyl-esterified, linear and remarkably high M_w tomato serum pectin of the homogenized purées partly explains their increased consistency. This work demonstrated that high pressure homogenization can at least partially restore the consistency of tomato purée despite an initial consistency loss ascribed to enzymatic pectin degradation.