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.

Klebsiella pneumoniae, an emerging multidrug-resistant pathogen, exhibits hypermucoviscosity (HMV) as a critical virulence trait mediated by its capsular polysaccharide (CPS). Recent discoveries have determined acetylation as a significant modification for CPS, although its impact on HMV and virulence was previously unknown. This study elucidates the roles of two enzymes: Klebsiella pneumoniae Acetylated CPS Esterase (KpACE), an esterase that removes acetyl groups from CPS, and WcsU, an acetyltransferase that adds acetyl groups to CPS. KpACE is highly upregulated in an ompR-deficient mutant lacking HMV, and its overexpression consistently reduces HMV and diminishes virulence in a mouse model of pneumonia. The esterase domain-containing KpACE effectively deacetylates model sugar substrates and CPS-K2. Site-directed mutagenesis of the conserved catalytic histidine residue at position 370 significantly reduces its enzymatic activity. This reduction correlates with decreased HMV, affecting key virulence traits including biofilm formation and serum resistance. Similarly, a deficiency in the wcsU gene abolishes CPS acetylation, and reduces HMV and virulence. These results highlight the importance of the delicate balance between CPS acetylation by WcsU and deacetylation by KpACE in regulating the pathogenicity of K. pneumoniae. Understanding this balance provides new insights into the modulation of virulence traits and potential therapeutic targets for combating K. pneumoniae infections.

Poplar plays a vital role in carbon sequestration and provides valuable raw materials for various industrial applications. This study dissects the effects of genotype and nitrogen fertilization on recalcitrance features in poplar biomass towards OrganoCat pretreatment. Three genetically different poplar hybrids were cultivated for 13 weeks at greenhouse conditions and fertilized with different forms of inorganic nitrogen. Stem material was processed with different degrees of severity. Biochemical data of the biomass and OrganoCat products was used in a multiscale analysis to determine recalcitrance parameters. The relationship between nitrogen fertilization and genotype on the subsequent processing was investigated. The genotype had a greater influence on the recalcitrance of young poplars than nitrogen fertilization. Generally, crystalline cellulose content and enzymatic glucose conversion increased after processing with a higher severity. Moreover, the overall compositional variation decreased after OrganoCat processing. Optimization of nitrogen fertilization, transfer to field experiments and lignocellulose processing techniques are crucial for maximizing economic and environmental benefits of poplar plantations.

Phelipanche ramosa is a root parasitic plant fully dependent on host plants for nutrition and development. Upon germination, the parasitic seedling develops inside the infected roots a specific organ, the haustorium, thanks to the cell wall-degrading enzymes of haustorial intrusive cells, and induces modifications in the host’s cell walls. The model plant Arabidopsis thaliana is susceptible to P. ramosa; thus, mutants in cell wall metabolism, particularly those involved in pectin remodeling, like Atpme3-1, are of interest in studying the involvement of cell wall-degrading enzymes in the establishment of plant–plant interactions. Host–parasite co-cultures in mini-rhizotron systems revealed that parasite attachments are twice as numerous and tubercle growth is quicker on Atpme3-1 roots than on WT roots. Compared to WT, the increased susceptibility in AtPME3-1 is associated with reduced PME activity in the roots and a lower degree of pectin methylesterification at the host–parasite interface, as detected immunohistochemically in infected roots. In addition, both WT and Atpme3-1 roots responded to infestation by modulating the expression of PAE- and PME-encoding genes, as well as related global enzyme activities in the roots before and after parasite attachment. However, these modulations differed between WT and Atpme3-1, which may contribute to different pectin remodeling in the roots and contrasting susceptibility to P. ramosa. With this integrative study, we aim to define a model of cell wall response to this specific biotic stress and indicate, for the first time, the role of PME3 in this parasitic plant–plant interaction.

Viili, a Finnish ropy fermented milk, is traditionally manufactured through spontaneous fermentation, by mesophilic lactic acid bacteria and yeast-like fungi, or back-slopping. This study evaluated four natural viili starters as sources of lactic acid bacteria for dairy production. Back-slopping activation of the studied viili samples was monitored through pH and titratable acidity measurements and enumeration of mesophilic lactic acid bacteria. Sixty lactic acid bacteria isolates were collected, molecularly identified, and assayed for acidification performance, enzymatic activities, production of exopolysaccharides (EPSs), presence of the histidine decarboxylase (hdcA) gene of Gram-positive bacteria, and production of bacteriocins. A neat predominance of Lactococcus lactis emerged among the isolates, followed by Enterococcus faecalis, Enterococcus faecium, Enterococcus durans, Enterococcus lactis, and Lactococcus cremoris. Most isolates exhibited proteolytic activity, whereas only a few enterococci showed lipase activity. Five isolates identified as L. cremoris, L. lactis, and E. faecalis showed a good acidification performance. Most of the isolates tested positive for leucine arylamidase, whereas only one E. durans and two L. lactis isolates were positive for valine arylamidase. A few isolates also showed a positive reaction for beta-galactosidase and alpha- and beta-glucosidase. None of the isolates produced EPSs or bacteriocins. The hdcA gene was detected in five isolates identified as L. lactis and E. faecium. A few L. cremoris and L. lactis isolates for potential use as starter or adjunct cultures for dairy processing were finally identified.

Dough retardation is commonly used to extend dough shelf-life, but it poses a challenge for flatbreads due to their large surface. This study explored the sourdough fermentation of oats and barley, addressing challenges in the retardation of dough for flatbread. Sourdough, using flour only or flour blended with bran (3:1), was fermented with a LIVENDO LV1 starter at 30°C for 24 h. The pH value, microbial viable cell count, total titratable acidity and organic acids concentration of the sourdough were measured. The properties of dough and flatbread, depending on the retardation time (24 h and 48 h), sourdough type (oat or barley) and sourdough level (30% or 50% dough weight), were investigated. Oat flour’s limited acidification improved with the inclusion of bran, resulting in a desirable pH, TTA, and lactic to acetic acid ratio after 15 h of fermentation, which were comparable to results achieved with barley sourdough. The sourdough addition slowed down the enzymatic browning of dough during retardation. Dough retardation at 24 h reduced the phytates content (32-38%) and crumb hardness (9-16%), depending on the sourdough type and level. In dough retardation, β-glucans were degraded by up to 9% in the case of oats and by up to 28% in the samples with barley. Overall, adding oat or barley sourdough at a 30% dough weight can be recommended to enhance flatbread’s nutritional value and prolong its shelf life.

Background: Apple cider vinegar (ACV) contains many health benefits due to its antioxidant and acetic acid content. However, the adverse effects of directly consuming the vinegar should be eliminated to make it possible to regulate its intake as a dietary supplement. The objective of the study is to optimize the spray drying operating conditions to produce ACV powder with health benefits. A central composite design (CCD) of response surface methodology (RSM) was used to optimize the spray drying process of ACV. The influences of inlet temperature (130°C–170°C), gum Arabic concentration (5%–15% w/w), and feed flow rate (1–2 kg/h) on some product and process aspects were investigated. Spray-dried powders were evaluated for their moisture content, process yield, acetic acid content, solubility, and antioxidant content. Results: The relationships between the model factors and final powder properties were established with ANOVA and multiple regression analysis. The model factors affected the investigated powder properties at different significance levels, where carrier concentration was the main influencing factor. Gum Arabic contributed to the retention of volatile and heat-sensitive compounds providing health benefits. All powders showed high solubility (>94%) and high antioxidant recovery, and most treatments achieved a drying yield of higher than 50%. Conclusion: Optimization of spray drying process was achieved with application of RSM and relationships between model factors and powder properties were established. This study highlighted the feasibility of the development of ACV-based functional powder as a dietary supplement.

The emulsion forming and stabilizing capacities of water-soluble biopolymers originating from the aqueous (serum) phase of heat-treated and high pressure homogenized purées were investigated. The serum biopolymers were characterized and then utilized as emulsifier/stabilizer in simple oil-in-water emulsions. The resulting emulsions were stored at 4°C and monitored for 2 weeks. Results revealed that carrot and tomato sera contained higher amounts of pectin and lower protein compared to broccoli. The serum pectic biopolymers exhibited distinct molecular structures, depending on the vegetable origin. Given these natural biopolymer composition and characteristics, emulsions with small droplet sizes were observed at pH 3.5. However, emulsions at pH 6.0 showed large mean droplet sizes, except for the emulsion formulated with carrot serum. Regardless of the pH, emulsions containing carrot serum biopolymers exhibited high capacity to form fine emulsions that were stable during the 2-week storage period at low temperature. This study clearly shows the capacity of natural water-soluble biopolymers isolated from the serum phase of vegetable purées to form fine emulsion droplets and maintain its stability during storage, especially in the case of carrot serum biopolymers.

Fermentation is key to developing the organoleptic characteristics of cocoa beans, as dynamic changes in metabolites have a significant impact on flavors and aromas, hence modifications of this process have been investigated. In this research, the mucilage of CCN-51 cocoa beans was replaced by a mixture of passion fruit (Passiflora edulis) and plantain (Musa paradisiaca L.) pulp, and a controlled fermentation of this mixture was carried out after its spontaneous on-farm inoculation. The physicochemical changes and correlations during the five days of fermentation were evaluated. At the end of the process, the temperature reached 47 ºC in the fermentation mass and pH 5.64 was recorded in the cotyledon. In the first 48 hours, citric acid and fructose were high but at the end of fermentation were 71% and 41.17% lower than at the start of fermentation, respectively. As glucose and fructose were consumed during fermentation, acetic acid and lactic acid levels increased from day two onward, reaching values at the end of the process of 22.48 mg/g and 16.01 mg/g, respectively. In contrast, the bromatological parameters did not show greater variability when comparing each day of fermentation. The data generated and results presented in this study will contribute to the knowledge of possible sensory improvements achieved with the inclusion of pulp fruits in the fermentation stage.

The microenvironment of solid tumors is characterized by oxygen and glucose deprivation. Acss2/HIF-2 signaling coordinates essential genetic regulators including acetate-dependent acetyl CoA synthetase 2 (Acss2), Creb binding protein (Cbp), Sirtuin 1 (Sirt1), and Hypoxia Inducible Factor 2α (HIF-2α). We previously shown in mice that exogenous acetate augments growth and metastasis of flank tumors derived from fibrosarcoma-derived HT1080 cells in an Acss2/HIF-2 dependent manner. Colonic epithelial cells are exposed to the highest acetate levels in the body. We reasoned that colon cancer cells, like fibrosarcoma cells, may respond to acetate in a pro-growth manner. In this study, we examine the role of Acss2/HIF-2 signaling in colon cancer. We find that Acss2/HIF-2 signaling is activated by oxygen or glucose deprivation in two human colon cancer-derived cell lines, HCT116 and HT29, and is crucial for colony formation, migration, and invasion in cell culture studies. Flank tumors derived from HCT116 and HT29 cells exhibit augmented growth in mice when supplemented with exogenous acetate in an Acss2/HIF-2 dependent manner. Finally, Acss2 in human colon cancer samples is most frequently localized in the nucleus, consistent with it having a signaling role. Targeted inhibition of Acss2/HIF-2 signaling may have synergistic effects for some colon cancer patients.

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.