Succinic Acid Assay Kit

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.

Fermentation is a type of metabolism pervasive in oxygen-deprived environments. Despite its importance, we know little about the range and traits of organisms that carry out this metabolism. Our study addresses this gap with a comprehensive analysis of the phenotype and genotype of fermentative prokaryotes. We assembled a dataset with phenotypic records of 8350 organisms plus 4355 genomes and 13.6 million genes. Our analysis reveals fermentation is both widespread (in ~30% of prokaryotes) and complex (forming ~300 combinations of metabolites). Furthermore, it points to previously uncharacterized proteins involved in this metabolism. Previous studies suggest that metabolic pathways for fermentation are well understood, but metabolic models built in our study show gaps in our knowledge. This study demonstrates the complexity of fermentation while showing that there is still much to learn about this metabolism. All resources in our study can be explored by the scientific community with an online, interactive tool.

Background: Increasing evidence highlights the involvement of metabolic disorder and calcium influx mediated by transient receptor potential channels in migraine; however, the relationship between these factors in the pathophysiology of migraine remains unknown. Gastrodin is the major component of the traditional Chinese medicine Tianma, which is extensively used in migraine therapy. Purpose: Our work aimed to explore the analgesic action of gastrodin and its regulatory mechanisms from a metabolic perspective. Methods/results: After being treated with gastrodin, the mice were given nitroglycerin (NTG) to induce migraine. Gastrodin treatment significantly raised the threshold of sensitivity in response to both mechanical and thermal stimulus evidenced by von Frey and hot plate tests, respectively, and decreased total contact numbers in orofacial operant behavioral assessment. We found that the expression of transient receptor potential melastatin 2 (TRPM2) channel was increased in the trigeminal ganglion (TG) of NTG-induced mice, resulting in a sustained Ca²⁺ influx to trigger migraine pain. The content of succinate, a metabolic biomarker, was elevated in blood samples of migraineurs, as well as in the serum and TG tissue from NTG-induced migraine mice. Calcium imaging assay indicated that succinate insult elevated TRPM2-mediated calcium flux signal in TG neurons. Mechanistically, accumulated succinate upregulated hypoxia inducible factor-1α (HIF-1α) expression and promoted its translocation into nucleus, where HIF-1α enhanced TRPM2 expression through transcriptional induction in TG neurons, evidenced by luciferase reporter measurement. Gastrodin treatment inhibited TRPM2 expression and TRPM2-dependent Ca2+ influx by attenuating succinate accumulation and downstream HIF-1α signaling, and thereby exhibited analgesic effect. Conclusion: This work revealed that succinate was a critical metabolic signaling molecule and the key mediator of migraine pain through triggering TRPM2-mediated calcium overload. Gastrodin alleviated NTG-induced migraine-like pain via inhibiting succinate/HIF-1α/TRPM2 signaling pathway in TG neurons. These findings uncovered the anti-migraine effect of gastrodin and its regulatory mechanisms from a metabolic perspective and provided a novel theoretical basis for the analgesic action of gastrodin.

In this study, organic acids were demonstrated as a promising carbon source for bisabolene production by the non-conventional yeast, Rhodosporidium toruloides, at microscale with a maximum titre of 1055 ± 7 mg/L. A 125-fold scale-up of the optimal process, enhanced bisabolene titres 2.5-fold to 2606 mg/L. Implementation of a pH controlled organic acid feeding strategy at this scale lead to a further threefold improvement in bisabolene titre to 7758 mg/L, the highest reported microbial titre. Finally, a proof-of-concept sequential bioreactor approach was investigated. Firstly, the cellulolytic bacterium Ruminococcus flavefaciens was employed to ferment cellulose, yielding 4.2 g/L of organic acids. R. toruloides was subsequently cultivated in the resulting supernatant, producing 318 ± 22 mg/L of bisabolene. This highlights the feasibility of a sequential bioprocess for the bioconversion of cellulose, into biojet fuel candidates. Future work will focus on enhancing organic acid yields and the use of real lignocellulosic feedstocks to further enhance bisabolene production.

The need to meet the demand for transplants entails the use of steatotic livers, more vulnerable to ischemia-reperfusion (IR) injury. Therefore, finding the optimal composition of static cold storage (SCS) preservation solutions is crucial. Given that ROS regulation is a therapeutic strategy for liver IR injury, we have added increasing concentrations of PEG35 and glutathione (GSH) to the preservation solutions (IGL-1 and IGL-2) and evaluated the possible protection against energy depletion and oxidative stress. Fatty livers from obese Zücker rats were isolated and randomly distributed in the control (Sham) preserved (24 h at 4°C) in IGL-0 (without PEG35 and 3 mmol/L GSH), IGL-1 (1 g/L PEG35, and 3 mmol/L GSH), and IGL-2 (5 g/L PEG35 and 9 mmol/L GSH). Energy metabolites (ATP and succinate) and the expression of mitochondrial oxidative phosphorylation complexes (OXPHOS) were determined. Mitochondrial carrier uncoupling protein 2 (UCP2), PTEN-induced kinase 1 (PINK1), nuclear factor-erythroid 2 related factor 2 (Nrf2), heme oxygenase-1 (HO-1), and the inflammasome (NLRP3) expressions were analyzed. As biomarkers of oxidative stress, protein oxidation (AOPP) and carbonylation (DNP derivatives), and lipid peroxidation (malondialdehyde (MDA)-thiobarbituric acid (TBA) adducts) were measured. In addition, the reduced and oxidized glutathione (GSH and GSSG) and enzymatic (Cu-Zn superoxide dismutase (SOD), CAT, GSH S-T, GSH-Px, and GSH-R) antioxidant capacities were determined. Our results showed that the cold preservation of fatty liver graft depleted ATP, accumulated succinate and increased oxidative stress. In contrast, the preservation with IGL-2 solution maintained ATP production, decreased succinate levels and increased OXPHOS complexes I and II, UCP2, and PINK-1 expression, therefore maintaining mitochondrial integrity. IGL-2 also protected against oxidative stress by increasing Nrf2 and HO-1 expression and GSH levels. Therefore, the presence of PEG35 in storage solutions may be a valuable option as an antioxidant agent for organ preservation in clinical transplantation.

Background and Aims: Torulaspora delbrueckii is being used increasingly as a starter for alcoholic fermentation (AF) because of its chemical modulation of wine. Previous studies on this yeast in a natural must have shown a different Oenococcus oeni population by the end of MLF. In this study we aim to evaluate this aspect in a defined O. oeni strain consortium in a sterile grape must during winemaking. Methods and Results: Before commencing AF with either S. cerevisiae or both T. delbrueckii and S. cerevisiae, the must was inoculated with a defined population of O. oeni strains. The use of T. delbrueckii determined the bacterial population at the end of MLF. Also, the inoculation of a selected strain after AF produced wines with different chemical composition to those fermented with the initial bacterial community. Conclusions: Different yeast inoculation strategies modulate the O. oeni population, and this has an impact on the chemical composition of the wines. Moreover, the inoculation of a small O. oeni population in must leads to a process similar to spontaneous MLF. Significance of the Study: Torulaspora delbrueckii can be used as a tool to modulate the O. oeni population and enhance the aromas related to MLF.

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.

Most mesenchymal stem cells reside in a niche of low oxygen tension. Iron-chelating agents such as CoCl₂ and deferoxamine have been utilized to mimic hypoxia and promote cell growth. The purpose of the present study was to explore whether a supplement of succinate, a natural metabolite of the tricarboxylic acid (TCA) cycle, can mimic hypoxia condition to promote human periodontal ligament cells (hPDLCs). Culturing hPDLCs in hypoxia condition promoted cell proliferation, migration, and osteogenic differentiation; moreover, hypoxia shifted cell metabolism from oxidative phosphorylation to glycolysis with accumulation of succinate in the cytosol and its release into culture supernatants. The succinate supplement enhanced hPDLC proliferation, migration, and osteogenesis with decreased succinate dehydrogenase (SDH) expression and activity, as well as increased hexokinase 2 (HK2) and 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3), suggesting metabolic reprogramming from oxidative phosphorylation to glycolysis in a normal oxygen condition. The succinate supplement in cell cultures promoted intracellular succinate accumulation while stabilizing hypoxia inducible factor-1α (HIF-1α), leading to a state of pseudohypoxia. Moreover, we demonstrate that hypoxia-induced proliferation was G-protein-coupled receptor 91- (GPR91-) dependent, while exogenous succinate-elicited proliferation involved the GPR91-dependent and GPR91-independent pathway. In conclusion, the succinate supplement altered cell metabolism in hPDLCs, induced a pseudohypoxia condition, and enhanced proliferation, migration, and osteogenesis of mesenchymal stem cells in vitro.

Background: Pediatric hypertension is recognized as an emerging global health concern. Although new guidelines are developed for facilitating clinical management, the reasons for the prevalence of hypertension in children remain unknown. Genetics and environmental factors do not fully account for the growing incidence of pediatric hypertension. Because stable bacterial flora in early life are linked with health outcomes later in life, we hypothesized that reshaping of gut microbiota in early life affects blood pressure (BP) of pediatric subjects. Methods and Results: To test this hypothesis, we administered amoxicillin, the most commonly prescribed pediatric antibiotic, to alter gut microbiota of young, genetically hypertensive rats (study 1) and dams during gestation and lactation (study 2) and recorded their BP. Reshaping of microbiota with reductions in Firmicutes/Bacteriodetes ratio were observed. Amoxicillin treated rats had lower BP compared with untreated rats. In young rats treated with amoxicillin, the lowering effect on BP persisted even after antibiotics were discontinued. Similarly, offspring from dams treated with amoxicillin showed lower systolic BP compared with control rats. Remarkably, in all cases, a decrease in BP was associated with lowering of Veillonellaceae, which are succinate‐producing bacteria. Elevated plasma succinate is reported in hypertension. Accordingly, serum succinate was measured and found lower in animals treated with amoxicillin. Conclusions: Our results demonstrate a direct correlation between succinate‐producing gut microbiota and early development of hypertension and indicate that reshaping gut microbiota, especially by depleting succinate‐producing microbiota early in life, may have long‐term benefits for hypertension‐prone individuals.

Cyanobacteria are emerging as hosts for photoautotrophic production of chemicals. Recent studies have attempted to stretch the limits of photosynthetic production, typically focusing on one product at a time, possibly to minimise the additional burden of product separation. Here, we explore the simultaneous production of two products that can be easily separated: ethylene, a gaseous product, and succinate, an organic acid that accumulates in the culture medium. This was achieved by expressing a single copy of the ethylene forming enzyme (efe) under the control of P_cpcB, the inducer-free super-strong promoter of phycocyanin β subunit. We chose the recently reported, fast-growing and robust cyanobacterium, Synechococcus elongatus PCC 11801, as the host strain. A stable recombinant strain was constructed using CRISPR-Cpf1 in a first report of markerless genome editing of this cyanobacterium. Under photoautotrophic conditions, the recombinant strain shows specific productivities of 338.26 and 1044.18 μmole/g dry cell weight/h for ethylene and succinate, respectively. These results compare favourably with the reported productivities for individual products in cyanobacteria that are highly engineered. Metabolome profiling and ¹³C labelling studies indicate carbon flux redistribution and suggest avenues for further improvement. Our results show that S. elongatus PCC 11801 is a promising candidate for metabolic engineering.

When speaking of “minerality” in wines, it is common to find descriptive terms in the vocabulary of wine tasters such as flint, match smoke, kerosene, rubber eraser, slate, granite, limestone, earthy, tar, charcoal, graphite, rock dust, wet stones, salty, metallic, steel, ferrous, etc. These are just a few of the descriptors that are commonly found in the tasting notes of wines that show this sensory profile. However, not all wines show this mineral trace at the aromatic and gustatory level. This study has used the statistical tool partial least squares regression (PLS) to mathematically model the attribute of “minerality” of wine, thereby obtaining formulas where the chemical composition and sensory attributes act jointly as the predictor variables, both for white wines and red wines, so as to help understand the term and to devise a winemaking approach able to endow wines with this attribute if desired.

One of the most important problems in the winemaking field is the increase of ethanol content in wine. Wines with high ethanol level negatively affect wine flavor and human health. In this study, we evaluated the use of a selected strain of Metschnikowia pulcherrima in immobilized form and under different aeration conditions, to reduce the ethanol content evaluating the volatile profile of the resulting wines. In a preliminary screening the best conditions regarding free/immobilized cells, static/aerated fermentation and inoculation level were identified. Bench-Top fermentation trials with different aeration conditions showed that the use of M. pulcherrima selected strain with aeration flow of 20 mL/L/min during the first 72 h of fermentation, led an ethanol reduction of 1.38% (v/v) in comparison with Saccharomyces cerevisiae control strain. The analytical profile of the resulting wines did not show any negative feature. Indeed, the concentration of ethyl acetate, that above its sensory threshold impacts negatively the wine sensory profile, was found at an acceptable level. On the other hand, an increase in the concentration of significant fruity and flower compounds was found.

Over the past two decades, intensive efforts have been made to construct recombinant Escherichia coli or Corynebacterium glutamicum by engineering C4 or C5 pathways to improve microbial production of 5-aminolevulinic acid (ALA), which has medical application for photodynamic cancer therapy and tumor diagnosis. In this study, we explored the feasibility of enhanced production of ALA by expressing C4 pathway enzyme, ALA synthase, in Streptomyces coelicolor, and medium optimization. The hemA from Rhodobacter sphaeroides was successfully integrated into the chromosome of Streptomyces coelicolor by conjugal transformation, and recombinant Streptomyces cells expressed well the foreign hemA. Glucose promoted ALA synthesis, and yeast extract showed a strong positive effect on ALA production. Optimization of casamino acid, peptone, malt extract, glycine, and succinic acid increased the product titer. In flask cultures, cell growth and ALA production of recombinant Streptomyces were 2.3 and 3.0-fold higher, respectively, in optimal medium than those of control. The maximum ALA, 137 mg/L, was obtained at 28 h in bioreactor culture, in which 3.1-fold higher cell mass and 2.9-fold greater volumetric productivity were achieved, compared to those in flask cultures.

Purpose: Intestinal fermentation of inulin-type fructans, including oligofructose, can modulate adiposity, improve energy regulation, and increase mineral absorption. We aimed to determine whether cereal fructans had greater effects on reducing adiposity and improving mineral absorption compared with oligofructose. Methods: Thirty-two male Sprague–Dawley rats were randomly assigned to one of four dietary treatments that contained 0% fructan (control), or 5% fructan provided by oligofructose (OF), a barley grain fraction (BGF), or a wheat stem fraction (WSF). After 1 week on the diets, mineral absorption and retention was assessed. At 4 weeks, blood samples were collected for gut hormone analysis, adipose depots were removed and weighed, and caecal digesta was analyzed for pH and short-chain fatty acids (SCFA). Results: The BGF and WSF, but not OF, had lower total visceral fat weights than the Control (p < 0.05). The fructan diets all lowered caecal pH and raised caecal digesta weight and total SCFA content, in comparison to the Control. Caecal propionate levels for OF were similar to the Control and higher for WSF (p < 0.05). Plasma peptide YY and glucagon-like peptide-1 levels were elevated for all fructan groups when compared to Control (p < 0.001) and gastric inhibitory peptide was lower for the WSF compared to the other groups (p < 0.05). The fructan diets improved calcium and magnesium retention, which was highest for WSF (p < 0.05). BGF and WSF in comparison to OF showed differential effects on fermentation, gut hormone levels, and adiposity. Conclusions: Cereal fructan sources have favorable metabolic effects that suggest greater improvements in energy regulation and mineral status to those reported for oligofructose.