Grape and wine analysis: Oenologists to exploit advanced test kits.
Charnock, S. C. & McCleary, B. V. (2005). Revue des Enology, 117, 1-5.
It is without doubt that testing plays a pivotal role throughout the whole of the vinification process. To produce the best possible quality wine and to minimise process problems such as “stuck” fermentation or troublesome infections, it is now recognised that if possible testing should begin prior to harvesting of the grapes and continue through to bottling. Traditional methods of wine analysis are often expensive, time consuming, require either elaborate equipment or specialist expertise and frequently lack accuracy. However, enzymatic bio-analysis enables the accurate measurement of the vast majority of analytes of interest to the wine maker, using just one piece of apparatus, the spectrophotometer (see previous issue No. 116 for a detailed technical review). Grape juice and wine are amenable to enzymatic testing as being liquids they are homogenous, easy to manipulate, and can generally be analysed without any sample preparation.
Megazyme “advanced” wine test kits general characteristics and validation.
Charnock, S. J., McCleary, B. V., Daverede, C. & Gallant, P. (2006). Reveue des Oenologues, 120, 1-5.
Many of the enzymatic test kits are official methods of prestigious organisations such as the Association of Official Analytical Chemicals (AOAC) and the American Association of Cereal Chemists (AACC) in response to the interest from oenologists. Megazyme decided to use its long history of enzymatic bio-analysis to make a significant contribution to the wine industry, by the development of a range of advanced enzymatic test kits. This task has now been successfully completed through the strategic and comprehensive process of identifying limitations of existing enzymatic bio-analysis test kits where they occurred, and then using advanced techniques, such as molecular biology (photo 1), to rapidly overcome them. Novel test kits have also been developed for analytes of emerging interest to the oenologist, such as yeast available nitrogen (YAN; see pages 2-3 of issue 117 article), or where previously enzymes were simply either not available, or were too expensive to employ, such as for D-mannitol analysis.
Dimethylsulfide is an energy source for the heterotrophic marine bacterium Sagittula stellata.
Boden, R., Murrell, J. C. & Schäfer, H. (2011). FEMS Microbiology Letters, 322(2), 188-193.
Dimethylsulfide (DMS) is a volatile organosulfur compound, ubiquitous in the oceans, that has been credited with various roles in biogeochemical cycling and in climate control. Various oceanic sinks of DMS are known – both chemical and biological – although they are poorly understood. In addition to the utilization of DMS as a carbon or a sulfur source, some Bacteria are known to oxidize it to dimethylsulfoxide (DMSO). Sagittula stellata is a heterotrophic member of the Alphaproteobacteria found in marine environments. It has been shown to oxidize DMS during heterotrophic growth on sugars, but the reasons for and the mechanisms of this oxidation have not been investigated. Here, we show that the oxidation of DMS to DMSO is coupled to ATP synthesis in S. stellata and that DMS acts as an energy source during chemoorganoheterotrophic growth of the organism on fructose and on succinate. DMS dehydrogenase (which is responsible for the oxidation of DMS to DMSO in other marine Bacteria) and DMSO reductase activities were absent from cells grown in the presence of DMS, indicating an alternative route of DMS oxidation in this organism.
Artefacts in cell culture: α-Ketoglutarate can scavenge hydrogen peroxide generated by ascorbate and epigallocatechin gallate in cell culture media.
Long, L. H. & Halliwell, B. (2011). Biochemical and Biophysical Research Communications, 406(1), 20-24.
Ascorbate and several phenolic compounds readily oxidise in cell culture media to generate hydrogen peroxide. However, addition of α-ketoglutarate, which is known to be released by several cell types, decreased the levels of H2O2, and the α-ketoglutarate was depleted and converted to succinate. These observations could account for previous reports of the protective effects of α-ketoglutarate in promoting the growth of cells in culture, and may contribute to explaining some of the variability in the literature in reported rates of H2O2 production from autoxidisable compounds in cell culture systems.
Biochemical mechanism on GABA accumulation during fruit development in tomato.
Akihiro, T., Koike, S., Tani, R., Tominaga, T., Watanabe, S., Iijima, Y., Aoki, K., Shibata, D., Ashihara, H., Matsukura, C., Akama, K., Fujimura, T. & Ezura, H. (2008). Plant and Cell Physiology, 49(9), 1378-1389.
A large amount of γ-aminobutyric acid (GABA) was found to accumulate in tomato (Solanum lycopersicum) fruits before the breaker stage. Shortly thereafter, GABA was rapidly catabolized after the breaker stage. We screened the GABA-rich tomato cultivar ‘DG03-9’ which did not show rapid GABA catabolism after the breaker stage. Although GABA hyperaccumulation and rapid catabolism in fruits is well known, the mechanisms are not clearly understood. In order to clarify these mechanisms, we performed comparative studies of ‘Micro-Tom’ and ‘DG03-9’ fruits for the analysis of gene expression levels, protein levels and enzymatic activity levels of GABA biosynthesis- and catabolism-related enzymes. During GABA accumulation, we found positive correlations among GABA contents and expression levels of SlGAD2 and SlGAD3. Both of these genes encode glutamate decarboxylase (GAD) which is a key enzyme of GABA biosynthesis. During GABA catabolism, we found a strong correlation between GABA contents and enzyme activity of α-ketoglutarate-dependent GABA transaminase (GABA-TK). The contents of glutamate and aspartate, which are synthesized from GABA and glutamate, respectively, increased with elevation of GABA-TK enzymatic activity. GABA-TK is the major GABA transaminase form in animals and appears to be a minor form in plants. In ‘DG03-9’ fruits, GAD enzymatic activity was prolonged until the ripening stage, and GABA-TK activity was significantly low. Taken together, our results suggest that GAD and GABA-TK play crucial roles in GABA accumulation and catabolism, respectively, in tomato fruits.
Isolation and characterization of a novel heterotrophic nitrifying and aerobic denitrifying bacterium Pseudomonas stutzeri KTB for bioremediation of wastewater.
Zhou, M., Ye, H. & Zhao, X. (2014). Biotechnology and Bioprocess Engineering, 19(2), 231-238.
A novel heterotrophic nitrifying and aerobic denitrifying bacterium, KTB, was isolated from activated sludge flocci collected from a biological aerated filter according to the modified Takaya method and identified as Pseudomonas stutzeri by 16S rDNA gene sequence analysis. When shaking-cultured in the presence of 4.331 mmol/L of nitrate, 4.511 mmol/L of nitrite and 4.438 mmol/L of ammonium, the strain grew fast, with µmax being 0.42, 0.45, and 0.56/h, and displayed high nitrogen removal efficiency, with nitrogen removal rate being 0.239, 0.362, and 0.361 mmol/L/h and nitrogen removal ratio being 99.1, 100.0, and 100.0% in 18 h, respectively. The removal mainly occurred in the logarithmic phase. Nitrite accumulation did not affect denitrification performance. Nitrate concentration was below the detectable limit during the whole growth cycle when ammonium was used as sole nitrogen source. It tolerated high DO level and exhibited excellent aggregation ability. A possible pathway involved in the nitrogen removal process, which demonstrated a full nitrification and denitrification route, was speculated. The strain might be a great candidate for biological removal of nitrogen compounds from wastewater.
Identification of Organic Acids in Wine That Stimulate Mechanisms of Gastric Acid Secretion.
Liszt, K. I., Walker, J. & Somoza, V. (2012). Journal of Agricultural and Food Chemistry, 60(28), 7022-7030.
Wine may cause stomach irritation due to its stimulatory effect on gastric acid secretion, although the mechanisms by which wine or components thereof activate pathways of gastric acid secretion are poorly understood. Gastric pH was measured with a noninvasive intragastric probe, demonstrating that administration of 125 mL of white or red wine to healthy volunteers stimulated gastric acid secretion more potently than the administration of equivalent amounts of ethanol. Between both beverages, red wine showed a clear trend for being more active in stimulating gastric acid secretion than white wine (p = 0.054). Quantification of the intracellular proton concentration in human gastric tumor cells (HGT-1), a well-established indicator of proton secretion and, in turn, stomach acid formation in vivo, confirmed the stronger effect of red wine as compared to white wine. RT-qPCR experiments on cells exposed to red wine also revealed a more pronounced effect than white wine on the fold change expression of genes associated with gastric acid secretion. Of the quantitatively abundant organic acids in wine, malic acid and succinic acid most actively stimulated proton secretion in vitro. However, addition of ethanol to individual organic acids attenuated the secretory effect of tartaric acid, but not that of the other organic acids. It was concluded that malic acid for white wine and succinic acid for red wine are key organic acids that contribute to gastric acid stimulation.
Succinic acid production from orange peel and wheat straw by batch fermentations of Fibrobacter succinogenes S85.
Li, Q., Siles, J. A. & Thompson, I. P. (2010). Applied Microbiology and Biotechnology, 88(3), 671-678.
Succinic acid is a platform molecule that has recently generated considerable interests. Production of succinate from waste orange peel and wheat straw by consolidated bioprocessing that combines cellulose hydrolysis and sugar fermentation, using a cellulolytic bacterium, Fibrobacter succinogenes S85, was studied. Orange peel contains D-limonene, which is a well-known antibacterial agent. Its effects on batch cultures of F. succinogenes S85 were examined. The minimal concentrations of limonene found to inhibit succinate and acetate generation and bacterial growth were 0.01%, 0.1%, and 0.06% (v/v), respectively. Both pre-treated orange peel by steam distillation to remove D-limonene and intact wheat straw were used as feedstocks. Increasing the substrate concentrations of both feedstocks, from 5 to 60 g/L, elevated succinate concentration and productivity but lowered the yield. In addition, pre-treated orange peel generated greater succinate productivities than wheat straw but had similar resultant titres. The greatest succinate titres were 1.9 and 2.0 g/L for pre-treated orange peel and wheat straw, respectively. This work demonstrated that agricultural waste such as wheat straw and orange peel can be biotransformed to succinic acid by a one-step consolidated bioprocessing. Measures to increase fermentation efficiency are also discussed.
Gamma-amino butyric acid, glutamate dehydrogenase and glutamate decarboxylase levels in phylogenetically divergent plants.
Seher, Y., Filiz, O. & Melike, B. (2013). Plant Systematics and Evolution, 299(2), 403-412.
Gamma-amino butyric acid (GABA) is a nonprotein amino acid found in a wide range of organisms including plants. Several studies have shown that GABA plays different roles in plant metabolism including carbon–nitrogen metabolism, energy balance, signaling and development. It has been suggested that the occurrence of GABA and the enzymes related to GABA biosynthesis in prokaryotes and eukaryotes may be important in evolution and diversification. However, studies of GABA biosynthesis and GABA levels in an evolutionary context are restricted to sequenced plant genomes. In this study we aimed to compare the activities of GDH and GAD enzymes and total nitrogen, and the contents of total soluble protein, succinate, glutamate, proline and GABA in plants from different phylogenetic levels including Ulva lactuca , Pseudevernia furfuracea, Nephrolepsis exaltata, Ginkgo biloba, Pinus pinea, Magnolia grandiflora, Nymphaea alba, Urtica dioica, Portulaca oleraceae, Malva sylvestris, Rosa canina, Lavandula stoechas, Washingtonia filifera, Avena barbata and Iris kaempferi. The activities of GAD and GDH enzymes differed according to the species and were not always parallel to GABA levels. The discrepancy in the contents of succinate and GABA between higher and primitive plants was also prominent. Glutamate levels were high with a few exceptions and proline contents were at similar low values as compared to other amino acids. Our results support the hypothesis that the GABA shunt plays a key role in carbon and nitrogen partitioning via linking amino acid metabolism and the tricarboxylic acid cycle which is essential for higher plant species.
Exposure to elevated temperature and Pco2 reduces respiration rate and energy status in the periwinkle Littorina littorea.
Melatunan, S., Calosi, P., Rundle, S. D., Moody, A. J. & Widdicombe, S. (2011). Physiological and Biochemical Zoology, 84(6), 583-594.
In the future, marine organisms will face the challenge of coping with multiple environmental changes associated with increased levels of atmospheric Pco2, such as ocean warming and acidification. To predict how organisms may or may not meet these challenges, an in-depth understanding of the physiological and biochemical mechanisms underpinning organismal responses to climate change is needed. Here, we investigate the effects of elevated Pco2 and temperature on the whole-organism and cellular physiology of the periwinkle Littorina littorea. Metabolic rates (measured as respiration rates), adenylate energy nucleotide concentrations and indexes, and end-product metabolite concentrations were measured. Compared with values for control conditions, snails decreased their respiration rate by 31% in response to elevated Pco2 and by 15% in response to a combination of increased Pco2 and temperature. Decreased respiration rates were associated with metabolic reduction and an increase in end-product metabolites in acidified treatments, indicating an increased reliance on anaerobic metabolism. There was also an interactive effect of elevated Pco2 and temperature on total adenylate nucleotides, which was apparently compensated for by the maintenance of adenylate energy charge via AMP deaminase activity. Our findings suggest that marine intertidal organisms are likely to exhibit complex physiological responses to future environmental drivers, with likely negative effects on growth, population dynamics, and, ultimately, ecosystem processes.
Identifying and assessing the impact of wine acid-related genes in yeast.
Chidi, B. S., Rossouw, D. & Bauer, F. F. (2016). Current genetics, 62(1), 149-164.
Saccharomyces cerevisiae strains used for winemaking show a wide range of fermentation phenotypes, and the genetic background of individual strains contributes significantly to the organoleptic properties of wine. This strain-dependent impact extends to the organic acid composition of the wine, an important quality parameter. However, little is known about the genes which may impact on organic acids during grape must fermentation. To generate novel insights into the genetic regulation of this metabolic network, a subset of genes was identified based on a comparative analysis of the transcriptomes and organic acid profiles of different yeast strains showing different production levels of organic acids. These genes showed significant inter-strain differences in their transcription levels at one or more stages of fermentation and were also considered likely to influence organic acid metabolism based on existing functional annotations. Genes selected in this manner were ADH3, AAD6, SER33, ICL1, GLY1, SFC1, SER1, KGD1, AGX1, OSM1 and GPD2. Yeast strains carrying deletions for these genes were used to conduct fermentations and determine organic acid levels at various stages of alcoholic fermentation in synthetic grape must. The impact of these deletions on organic acid profiles was quantified, leading to novel insights and hypothesis generation regarding the role/s of these genes in wine yeast acid metabolism under fermentative conditions. Overall, the data contribute to our understanding of the roles of selected genes in yeast metabolism in general and of organic acid metabolism in particular.
Pseudomonas putida KT2440 strain metabolizes glucose through a cycle formed by enzymes of the Entner-Doudoroff, Embden-Meyerhof-Parnas, and pentose phosphate pathways.
Nikel, P. I., Chavarría, M., Fuhrer, T., Sauer, U. & de Lorenzo, V. (2015). Journal of Biological Chemistry, 290(43), 25920-25932.
The soil bacterium Pseudomonas putida KT2440 lacks a functional Embden-Meyerhof-Parnas (EMP) pathway, and glycolysis is known to proceed almost exclusively through the Entner-Doudoroff (ED) route. To investigate the raison d'être of this metabolic arrangement, the distribution of periplasmic and cytoplasmic carbon fluxes was studied in glucose cultures of this bacterium by using 13C-labeled substrates, combined with quantitative physiology experiments, metabolite quantification, and in vitro enzymatic assays under both saturating and non-saturating, quasi in vivo conditions. Metabolic flux analysis demonstrated that 90% of the consumed sugar was converted into gluconate, entering central carbon metabolism as 6-phosphogluconate and further channeled into the ED pathway. Remarkably, about 10% of the triose phosphates were found to be recycled back to form hexose phosphates. This set of reactions merges activities belonging to the ED, the EMP (operating in a gluconeogenic fashion), and the pentose phosphate pathways to form an unforeseen metabolic architecture (EDEMP cycle). Determination of the NADPH balance revealed that the default metabolic state of P. putida KT2440 is characterized by a slight catabolic overproduction of reducing power. Cells growing on glucose thus run a biochemical cycle that favors NADPH formation. Because NADPH is required not only for anabolic functions but also for counteracting different types of environmental stress, such a cyclic operation may contribute to the physiological heftiness of this bacterium in its natural habitats.
SUCNR1-mediated chemotaxis of macrophages aggravates obesity-induced inflammation and diabetes.
van Diepen, J. A., Robben, J. H., Hooiveld, G. J., Carmone, C., Alsady, M., Boutens, L., Bekkenkamp-Grovenstein, M. B., Hijmans, A., Engelke, U. F. H.,Wevers, R. A., Netea, M. G., Tack, C. J., Stienstra, R. & Deen, P. M. T. (2017). Diabetologia, 1-10.
Aims/hypothesis: Obesity induces macrophages to drive inflammation in adipose tissue, a crucial step towards the development of type 2 diabetes. The tricarboxylic acid (TCA) cycle intermediate succinate is released from cells under metabolic stress and has recently emerged as a metabolic signal induced by proinflammatory stimuli. We therefore investigated whether succinate receptor 1 (SUCNR1) could play a role in the development of adipose tissue inflammation and type 2 diabetes. Methods: Succinate levels were determined in human plasma samples from individuals with type 2 diabetes and non-diabetic participants. Succinate release from adipose tissue explants was studied. Sucnr1-/- and wild-type (WT) littermate mice were fed a high-fat diet (HFD) or low-fat diet (LFD) for 16 weeks. Serum metabolic variables, adipose tissue inflammation, macrophage migration and glucose tolerance were determined. Results: We show that hypoxia and hyperglycaemia independently drive the release of succinate from mouse adipose tissue (17-fold and up to 18-fold, respectively) and that plasma levels of succinate were higher in participants with type 2 diabetes compared with non-diabetic individuals (+53%; p < 0.01). Sucnr1-/- mice had significantly reduced numbers of macrophages (0.56 ± 0.07 vs 0.92 ± 0.15 F4/80 cells/adipocytes, p < 0.05) and crown-like structures (0.06 ± 0.02 vs 0.14 ± 0.02, CLS/adipocytes p < 0.01) in adipose tissue and significantly improved glucose tolerance (p < 0.001) compared with WT mice fed an HFD, despite similarly increased body weights. Consistently, macrophages from Sucnr1-/- mice showed reduced chemotaxis towards medium collected from apoptotic and hypoxic adipocytes (−59%; p < 0.05). Conclusions/interpretation: Our results reveal that activation of SUCNR1 in macrophages is important for both infiltration and inflammation of adipose tissue in obesity, and suggest that SUCNR1 is a promising therapeutic target in obesity-induced type 2 diabetes.
Bistability and Nonmonotonic Induction of the lac Operon in the Natural Lactose Uptake System.
Zander, D., Samaga, D., Straube, R. & Bettenbrock, K. (2017). Biophysical Journal, 112(9), 1984-1996.
The Escherichia coli lac operon is regulated by a positive feedback loop whose potential to generate an all-or-none response in single cells has been a paradigm for bistable gene expression. However, so far bistable lac induction has only been observed using gratuitous inducers, raising the question about the biological relevance of bistable lac induction in the natural setting with lactose as the inducer. In fact, the existing experimental evidence points to a graded rather than an all-or-none response in the natural lactose uptake system. In contrast, predictions based on computational models of the lactose uptake pathway remain controversial. Although some argue in favor of bistability, others argue against it. Here, we reinvestigate lac operon expression in single cells using a combined experimental/modeling approach. To this end, we parameterize a well-supported mathematical model using transient measurements of LacZ activity upon induction with different amounts of lactose. The resulting model predicts a monostable induction curve for the wild-type system, but indicates that overexpression of the LacI repressor would drive the system into the bistable regime. Both predictions were confirmed experimentally supporting the view that the wild-type lac induction circuit generates a graded response rather than bistability. More interestingly, we find that the lac induction curve exhibits a pronounced maximum at intermediate lactose concentrations. Supported by our data, a model-based analysis suggests that the nonmonotonic response results from saturation of the LacI repressor at low inducer concentrations and dilution of Lac enzymes due to an increased growth rate beyond the saturation point. We speculate that the observed maximum in the lac expression level helps to save cellular resources by limiting Lac enzyme expression at high inducer concentrations.
Partial characterization of phylogeny, ecology and function of the fibrolytic bacterium Ruminococcus flavefaciens OS14, newly isolated from the rumen of swamp buffalo.
Boonsaen, P., Kinjo, M., Sawanon, S., Suzuki, Y., Koike, S. & Kobayashi, Y. (2017). Animal Science Journal, In Press.
The fibrolytic rumen bacterium Ruminococcus flavefaciens OS14 was isolated from swamp buffalo and its phylogenetic, ecological and digestive properties were partially characterized. Isolates from rumen contents of four swamp buffalo were screened for fibrolytic bacteria; one of the 40 isolates showed a distinctive feature of solubilizing cellulose powder in liquid culture and was identified as R. flavefaciens based on its 16S ribosomal DNA sequence. This isolate, OS14, was employed for detection and digestion studies, for which a quantitative PCR assay was developed and defined cultures were tested with representative forages in Thailand. OS14 was phylogenetically distant from other isolated and uncultured R. flavefaciens and showed limited distribution among Thai ruminants but was absent in Japanese cattle. OS14 digested rice straw and other tropical forage to a greater extent than the type strain C94 of R. flavefaciens. OS14 produced more lactate than C94, and digested para grass to produce propionate more extensively in co-culture with lactate-utilizing Selenomonas ruminantium S137 than a co-culture of C94 with S137. These results indicate that phylogenetically distinct OS14 could digest Thai local forage more efficiently than the type strain, possibly forming a symbiotic cross-feeding relationship with lactate-utilizing bacteria. This strain might be useful for future animal and other industrial applications.