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.
Production of L-lactic acid from a green microalga, Hydrodictyon reticulum, by Lactobacillus paracasei LA104 isolated from the traditional Korean food, makgeolli.
Nguyen, C. M., Kim, J. S., Hwang, H. J., Park, M. S., Choi, G. J., Choi, Y. H., Jang, K. S. & Kim, J. C. (2012). Bioresource Technology, 110, 552-559.
The freshwater microalga, Hydrodictyon reticulum, that contained 47.5% reducing sugars including 35% glucose was used as substrate for the production of L-lactic acid (LA) by LA-producing bacteria. Lactobacillus paracasei LA104 was selected for fermentation in a 5-l fermentor since it was able to grow at pH 3, 60 g LA/l, 200 g glucose/l, 125 g NaCl/l, and 45°C and produced over 97.3% optically pure L-lactic acid with glucose as a substrate. Simultaneous saccharification and cofermentation from H. reticulum to L-LA using LA104 was investigated in a jar fermentor. The yield reached 46 g/100 g H. reticulum dry material, with a final concentration of 37.11 g/l and a productivity of 1.03 g/l/h. This is the first report of the production of L-LA from a microalga, and H. reticulum could be a potential feedstock for large-scale production of L-LA by LA104.
A novel lactic acid bacterium for the production of high purity L-lactic acid, Lactobacillus paracasei subsp. paracasei CHB2121.
Moon, S. K., Wee, Y. J. & Choi, G. W. (2012). Journal of Bioscience and Bioengineering, 114(2), 155-159.
Fermentation-derived lactic acid has several potential industrial uses as an intermediate carbon chemical and a raw material for biodegradable polymer. We therefore undertook the identification of a novel bacterial strain that is capable of producing high concentrations of lactic acid and has potential commercial applications. A novel L(+)-lactic acid producing bacterium, Lactobacillus paracasei subsp. paracasei CHB2121 was isolated from soil obtained near an ethanol production factory and identified by 16S rRNA gene sequence analysis and characterization using an API 50 CHL kit. L. paracasei subsp. paracasei CHB2121 efficiently produced 192 g/L lactic acid from medium containing 200 g/L of glucose, with 3.99 g/(L•h) productivity, and 0.96 g/g yield. In addition, the optical purity of the produced lactic acid was estimated to be 96.6% L(+)-lactic acid. The newly identified L. paracasei subsp. paracasei CHB2121 efficiently produces high concentrations of lactic acid, and may be suitable for use in the industrial production of lactic acid.
Histone acetylation regulates intracellular pH.
McBrian, M. A., Behbahan, I. S., Ferrari, R., Su, T., Huang, T. W., Li, K., Hong, C. S., Christofk, H. R., Vogelauer, M., Seligson, D. B. & Kurdistani, S. K. (2013). Molecular Cell, 49(2), 310-321.
Differences in global levels of histone acetylation occur in normal and cancer cells, although the reason why cells regulate these levels has been unclear. Here we demonstrate a role for histone acetylation in regulating intracellular pH (pHi). As pHi decreases, histones are globally deacetylated by histone deacetylases (HDACs), and the released acetate anions are coexported with protons out of the cell by monocarboxylate transporters (MCTs), preventing further reductions in pHi. Conversely, global histone acetylation increases as pHi rises, such as when resting cells are induced to proliferate. Inhibition of HDACs or MCTs decreases acetate export and lowers pHi, particularly compromising pHi maintenance in acidic environments. Global deacetylation at low pH is reflected at a genomic level by decreased abundance and extensive redistribution of acetylation throughout the genome. Thus, acetylation of chromatin functions as a rheostat to regulate pHi with important implications for mechanism of action and therapeutic use of HDAC inhibitors.
Relationship between Fish Size and Metabolic Rate in the Oxyconforming Inanga Galaxias maculatus Reveals Size-Dependent Strategies to Withstand Hypoxia.
Urbina, M. A. & Glover, C. N. (2013). Physiological and Biochemical Zoology, 86(6), 740-749.
The relationship between metabolic rate and body size in animals is unlikely to be a constant but is instead shaped by a variety of intrinsic (i.e., physiological) and extrinsic (i.e., environmental) factors. This study examined the effect of environmental oxygen tension on oxygen consumption as a function of body mass in the galaxiid fish, inanga (Galaxias maculatus). As an oxyconformer, this fish lacks overt intrinsic regulation of oxygen consumption, eliminating this as a factor affecting the scaling relationship at different oxygen tensions. The relationship between oxygen consumption rate and body size was best described by a power function, with an exponent of 0.82, higher than the theoretical values of 0.66 or 0.75. The value of this exponent was significantly altered by environmental Po2, first increasing as Po2 decreased and then declining at the lowest Po2 tested. These data suggest that the scaling exponent is species specific and regulated by extrinsic factors. Furthermore, the external Po2 at which fish lost equilibrium was related to fish size, an effect explained by the scaling of anaerobic capacity with fish mass. Therefore, although bigger fish were forced to depress aerobic metabolism more rapidly than small fish when exposed to progressive hypoxia, they were better able to enact anaerobic metabolism, potentially extending their survival in hypoxia.
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.
Construction of a food-grade cell surface display system for Lactobacillus casei.
Qin, J., Wang, X., Kong, J., Ma, C. & Xu, P. (2014). Microbiological Research, 169(9-10), 733-740.
In this study, a food-grade cell surface display host/vector system for Lactobacillus casei was constructed. The food-grade host L. casei Q-5 was a lactose-deficient derivative of L. casei ATCC 334 obtained by plasmid elimination. The food-grade cell surface display vector was constructed based on safe DNA elements from lactic acid bacteria containing the following: pSH71 replicon from Lactococcus lactis, lactose metabolism genes from L. casei ATCC 334 as complementation markers, and surface layer protein gene from Lactobacillus acidophilus ATCC 4356 for cell surface display. The feasibility of the new host/vector system was verified by the expression of green fluorescent protein (GFP) on L. casei. Laser scanning confocal microscopy and immunofluorescence analysis using anti-GFP antibody confirmed that GFP was anchored on the surface of the recombinant cells. The stability of recombinant L. casei cells in artificial gastrointestinal conditions was verified, which is beneficial for oral vaccination applications. These results indicate that the food-grade host/vector system can be an excellent antigen delivery vehicle in oral vaccine construction.
Identification of spoilage marker metabolites in Irish chicken breast muscle using HPLC, GC–MS coupled with SPME and traditional chemical techniques.
Alexandrakis, D., Brunton, N. P., Downey, G. & Scannell, A. G. (2012). Food and Bioprocess Technology, 5(5), 1917-1923.
The aim of this investigation was to determine the metabolites of spoilage present on the surface of Irish chicken breast muscle in order to identify biomarkers of microbial spoilage and to verify the results of a previous study which suggested that the increase of free amino acids is the main spectral influence factor leading to the near and middle infrared detection of microbial spoilage. Irish-reared chicken breast muscle samples were individually packed and stored at 4°C for 8 days under aerobic conditions. Microbiological analysis revealed that Pseudomonas spp. and Brochothrix thermosphacta were the predominant organisms (total viable counts (TVC), 4.24, 6.37 and 8.6 colony forming unity (CFU) g-1 for days 0, 4 and 8, respectively, Pseudomonas 3.2, 5.1 and finally, on day 8 7.4 log CFU g-1). Glucose and L-lactate concentrations decreased but the concentration of water-soluble polypeptides and amino acids increased over storage time. HPLC analysis of free amino acids revealed an increase of the total concentration but the composition of the profiles did not change over time. Headspace analysis detected the following volatile compounds: ethanol, acetone, ethyl acetate, methyl benzoate, heptane, C15, C12, methyl ethyl ketone, carbon disulphide, dimethyl sulphide, hexanal, and toluene. Of interest is the fact that detection of sulphides and an increase of ethanol, acetone, and ethyl acetate concentrations occurred from day 4 to 8. The increase in free amino acids throughout storage and the production of volatile compounds after day 4 require further investigation but are selected as potential biomarkers of microbial spoilage as they could be analytically detected before the accepted levels of sensory spoilage detection.
Improvement of lactic acid production in Saccharomyces cerevisiae by cell sorting for high intracellular pH.
Valli, M., Sauer, M., Branduardi, P., Borth, N., Porro, D. & Mattanovich, D. (2006). Applied and Environmental Microbiology, 72(8), 5492-5499.
Yeast strains expressing heterologous L-lactate dehydrogenases can produce lactic acid. Although these microorganisms are tolerant of acidic environments, it is known that at low pH, lactic acid exerts a high level of stress on the cells. In the present study we analyzed intracellular pH (pHi) and viability by staining with cSNARF-4F and ethidium bromide, respectively, of two lactic-acid-producing strains of Saccharomyces cerevisiae, CEN.PK m850 and CEN.PK RWB876. The results showed that the strain producing more lactic acid, CEN.PK m850, has a higher pHi. During batch culture, we observed in both strains a reduction of the mean pHi and the appearance of a subpopulation of cells with low pHi. Simultaneous analysis of pHi and viability proved that the cells with low pHi were dead. Based on the observation that the better lactic-acid-producing strain had a higher pHi and that the cells with low pHi were dead, we hypothesized that we might find better lactic acid producers by screening for cells within the highest pHi range. The screening was performed on UV-mutagenized populations through three consecutive rounds of cell sorting in which only the viable cells within the highest pHi range were selected. The results showed that lactic acid production was significantly improved in the majority of the mutants obtained compared to the parental strains. The best lactic-acid producing strain was identified within the screening of CEN.PK m850 mutants.
Sourdough-leavened bread improves postprandial glucose and insulin plasma levels in subjects with impaired glucose tolerance.
Maioli, M., Pes, G. M., Sanna, M., Cherchi, S., Dettori, M., Manca, E. & Farris, G. A. (2008). Acta Diabetologica, 45(2), 91-96.
Sourdough bread has been reported to improve glucose metabolism in healthy subjects. In this study postprandial glycaemic and insulinaemic responses were evaluated in subjects with impaired glucose tolerance (IGT) who had a meal containing sourdough bread leavened with lactobacilli, in comparison to a reference meal containing bread leavened with baker yeast. Sixteen IGT subjects (age range 52–75, average BMI 29.9 ± 4.2 kg/m2) were randomly given a meal containing sourdough bread (A) and a meal containing the reference bread (B) in two separate occasions at the beginning of the study and after 7 days. Sourdough bread was leavened for 8 h using a starter containing autochthonous Saccharomyces cerevisiae and several bacilli able to produce a significant amount of D-and L-lactic acid, whereas the reference bread was leavened for 2 h with commercial baker yeast containing Saccharomyces cerevisiae. Plasma glucose and insulin levels were measured at time 0, 30, 60, 120, and 180 min. In IGT subjects sourdough bread induced a significantly lower plasma glucose response at 30 minutes (p = 0.048) and a smaller incremental area under curve (AUC) Δ 0–30 and Δ 0–60 min (p = 0.020 and 0.018 respectively) in comparison to the bread leavened with baker yeast. Plasma insulin response to this type of bread showed lower values at 30 min (p = 0.045) and a smaller AUC Δ 0–30 min (p = 0.018). This study shows that in subjects with IGT glycaemic and insulinaemic responses after the consumption of sourdough bread are lower than after the bread leavened with baker yeast. This effect is likely due to the lactic acid produced during dough leavening as well as the reduced availability of simple carbohydrates. Thus, sourdough bread may potentially be of benefit in subjects with impaired glucose metabolism.
Homo-fermentative production of D-lactic acid by Lactobacillus sp. employing casein whey permeate as a raw feed-stock.
Prasad, S., Srikanth, K., Limaye, A. M. & Sivaprakasam, S. (2014). Biotechnology Letters, 36(6), 1303-1307.
Casein whey permeate (CWP), a lactose-enriched dairy waste effluent, is a viable feed stock for the production of value-added products. Two lactic acid bacteria were cultivated in a synthetic casein whey permeate medium with or without pH control. Lactobacillus lactis ATCC 4797 produced D-lactic acid (DLA) at 12.5 g l-1 in a bioreactor. The values of Leudking–Piret model parameters suggested that lactate was a growth-associated product. Batch fermentation was also performed employing CWP (35 g lactose l-1) with casein hydrolysate as a nitrogen supplement in a bioreactor. After 40 h, L. lactis produced 24.3 g lactic acid l-1 with an optical purity >98 %. Thus CWP may be regarded as a potential feed-stock for DLA production.
Assessment of the influence of biochar on rumen and silage fermentation: A laboratory-scale experiment.
Calvelo Pereira, R., Muetzel, S., Arbestain, M. C., Bishop, P., Hina, K. & Hedley, M. (2014). Animal Feed Science and Technology, 196, 22-31.
The addition of biochar – charcoal produced from pyrolysis of carbonaceous materials – to soil presents several challenges, mainly associated with its low bulk density, dustiness and the risk of loss when applied to hill pastures. Livestock could be an adequate vehicle for biochar delivery to New Zealand pastoral soils via dung pats; however, the potential effects of biochar on rumen metabolism need to be investigated. The objective of this study was to investigate the effect of biochar addition to grass before ensiling on the fermentation process and to test whether the addition of grass silage prepared with biochar or biochar directly to hay affected the in vitro rumen fermentation. The study included the use of different types of starting material (corn stover and pine wood chips), two pyrolysis temperatures (350 and 550°C), post-treatment (addition of different types of bio-oil at a ratio of 0.050 mL/g), and different doses of biochar. The use of biochar from either corn stover or pine pyrolysed at 550°C as silage ingredients at doses from 21 to 186 g biochar/kg dry matter had no negative effect on the final properties of the silage, and particularly on pH, NH4+-N/total N, and acetic, N-butyric and L-lactic acid concentrations. The same silage mixtures with 84 and 186 g biochar/kg dry matter were in vitro incubated with buffered rumen fluid. There was a build-up in total volatile fatty acids (VFA) production (P<0.05) in the presence of biochar – increasing at high doses – irrespective of the type of starting material considered. This increase in VFA was also observed when biochar were added to hay before in vitro incubation, and was enhanced with low-temperature biochar. None of the mixtures of biochar and hay had any significant effect on methane emissions and ammonia released. There was no effect of starting material type or post-treatment on the in vitro incubations. The results obtained in this research demonstrate the lack of negative effect of biochar mixed with grass silage, or hay, on rumen chemistry during in vitro incubations. If large-scale studies including in vivo feeding of cattle with biochar confirm these findings, the use of cattle as a delivery system could become a novel solution to safely apply biochar to New Zealand pastoral soils.
Behavioural, physiological and biochemical responses to aquatic hypoxia in the freshwater crayfish, Paranephrops zealandicus.
Broughton, R. J., Marsden, I. D., Hill, J. V. & Glover, C. N. (2017). Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 212, 72-80.
Hypoxia resulting from aquatic eutrophication threatens the population health of the New Zealand freshwater crayfish (koura), Paranephrops zealandicus. An integrated study, combining behavioural, physiological and biochemical approaches, was therefore conducted to characterise the tolerance of this species to hypoxia. When provided with a choice between water flows of high or low dissolved oxygen in short-term laboratory assays, crayfish did not preferentially inhabit waters of higher PO2. However, when an aerial refuge was provided and dissolved oxygen was progressively decreased, crayfish emersed at a PO2 of 0.56 ± 0.03 kPa, suggesting a relatively high tolerance to hypoxia. Closed-box respirometry delineated a Pcrit, the point at which crayfish transition from oxyregulating to oxyconforming, of 6.0 kPa. Simultaneous measurement of heart rate showed no changes across the PO2 range. In response to 6-h exposures to fixed dissolved oxygen levels (normoxia, 19.3 kPa; moderate hypoxia, 3.5 kPa; and severe hypoxia, 1.7 kPa), P. zealandicus showed a haemolymph PO2 that declined with the magnitude of hypoxia, and while plasma pH declined in severe hypoxia, there were no changes in plasma PO2. Plasma glucose concentrations fell, and plasma lactate increased in both hypoxic groups. There were no changes in tissue glucose or lactate concentrations. These data indicate that P. zealandicus is relatively tolerant of hypoxia, and possesses biochemical and physiological mechanisms that facilitate survival during short-term exposures to acute hypoxia. If hypoxia is severe and/or prolonged, then this species is capable of escaping to aerial refugia.
Quantitative proteomics analysis of an ethanol- and a lactate-producing mutant strain of Synechocystis sp. PCC6803.
Borirak, O., Koning, L. J., Woude, A. D., Hoefsloot, H. C., Dekker, H. L., Roseboom, W., deKoster, C. G. & Hellingwerf, K. J. (2015). Biotechnology for Biofuels, 8(1), 111.
Background: This study aimed at exploring the molecular physiological consequences of a major redirection of carbon flow in so-called cyanobacterial cell factories: quantitative whole-cell proteomics analyses were carried out on two 14N-labelled Synechocystis mutant strains, relative to their 15N-labelled wild-type counterpart. Each mutant strain overproduced one specific commodity product, i.e. ethanol or lactic acid, to such an extent that the majority of the incoming CO2 in the organism was directly converted into the product. Results: In total, 267 proteins have been identified with a significantly up- or down-regulated expression level. In the ethanol-producing mutant, which had the highest relative direct flux of carbon-to-product (>65%), significant up-regulation of several components involved in the initial stages of CO2 fixation for cellular metabolism was detected. Also a general decrease in abundance of the protein synthesizing machinery of the cells and a specific induction of an oxidative stress response were observed in this mutant. In the lactic acid overproducing mutant, that expresses part of the heterologous L-lactate dehydrogenase from a self-replicating plasmid, specific activation of two CRISPR associated proteins, encoded on the endogenous pSYSA plasmid, was observed. RT-qPCR was used to measure, of nine of the genes identified in the proteomics studies, also the adjustment of the corresponding mRNA level. Conclusion: The most striking adjustments detected in the proteome of the engineered cells were dependent on the specific product formed, with, e.g. more stress caused by lactic acid- than by ethanol production. Up-regulation of the total capacity for CO2 fixation in the ethanol-producing strain was due to hierarchical- rather than metabolic regulation. Furthermore, plasmid-based expression of heterologous gene(s) may induce genetic instability. For selected, limited, number of genes a striking correlation between the respective mRNA- and the corresponding protein expression level was observed, suggesting that for the expression of these genes regulation takes place primarily at the level of gene transcription.
Microbiological Analyses of Traditional Alcoholic Beverage (Chhang) and its Starter (Balma) Prepared by Bhotiya Tribe of Uttarakhand, India.
Bhardwaj, K. N., Jain, K. K., Kumar, S. & Kuhad, R. C. (2016). Indian Journal of Microbiology, 56(1), 28-34.
Present article depicts microbiology of starter (Balma) used in traditional solid-state fermentation of alcoholic beverage (Chhang) by Bhotiya tribe of Uttarakhand. It also highlights the importance of herbs in Balma preparation and kinetics of lactic acid and ethanol fermentation under Chhang preparation using Balma. Balma contains 214 × 106 cfu/g yeasts, 2.54 × 106 cfu/g lactic acid bacteria (LAB) and 1.4 × 106 cfu/g other mesophilic bacteria. ITS sequence analysis revealed a rich diversity of yeast comprising of Saccharomyces cerevisiae, Saccharomycopsis fibuligera and Saccharomycopsis malanga in Balma. 16S rDNA sequence analysis revealed Lactobacillus pentosus and Pediococcus pentosaceus among LAB, while amylolytic Bacillus subtilis and B. aerophilus among other bacteria in Balma. Based on the results, it is speculated that herbs such as Inula cuspidata, Micromeria biflora, Origanum vulgare, Rubus sp. and Thymus linearis used earlier by Bhotiya in Balma preparation contribute as a source of yeasts, LAB and amylolytic bacilli. Study also demonstrates that Bhotiya tribe is rational in preparation of starter as they have circumvented the need of plants by using previous year Balma as inoculum and possibility of deficient quality of Balma due to weak colonization of phyllosphere and rhizosphere microbiota. Results suggest that simultaneous saccharification and lactic acid-ethanol fermentation take place in traditional cereal based Chhang fermentation system of Bhotiya.
Thermal inactivation kinetics of surface contaminating Listeria monocytogenes on vacuum-packaged agar surface and ready-to-eat sliced ham and sausage.
Wang, X., Uyttendaele, M., Geeraerd, A., Steen, L., Fraeye, I. & Devlieghere, F. (2016). Food Research International, 89, 843-849.
The aim of this work was to study thermal inactivation kinetics of Listeria monocytogenes on vacuum-packaged food surfaces. The kinetics were first determined on model agar systems (BHI agar plates), mimicking cooked meat products, which have the same characteristics (pH, sodium chloride (NaCl) or sodium lactate (NaL) content and thickness) as the cooked meat products. Then, in order to validate how well the thermal inactivation on the model agar system simulated inactivation on real products, inactivation kinetics of L. monocytogenes on slices of cooked ham and cooked sausage were examined. BHI agar plates (pH 6.2 or 7.2) were prepared with and without the addition of 3% NaCl or NaL. They were initially inoculated with approximately 109 CFU/plate culture, aseptically packaged in linear low-density polyethylene pouches, and vacuum-sealed. Thermal treatments were performed by submerging packages in a water bath maintained at 60°C. For most of the conditions studied, the inactivation curves were linear; shoulders were only observed for curves at conditions of pH 6.2 with 3.0% NaL. The t4D values (time needed to obtain an inactivation of four-log reduction) were calculated based on the best fitting models included in GInaFit. The observed t4D values for L. monocytogenes on agar surfaces ranged from 6.8 (pH 6.2) to 13.7 min (pH 7.2 with 3.0% NaCl). At pH 6.2 addition of NaCl or NaL significantly increased the heat resistance of L. monocytogenes while at pH 7.2 this effect was not significant. NaL seemed to affect the heat resistance to the same extent as NaCl. Inactivation curves of L. monocytogenes on slices of cooked ham at pH 6.2 with or without addition of NaCl or NaL appeared to be log-linear in shape. However, the curves obtained from cooked sausages were markedly concave and the Weibull model was used for fitting. Concerning heat resistance of L. monocytogenes on meat products, t4D values increased approximately two-fold compared to those corresponding on model agar surfaces. The addition of 3.0% NaCl and NaL in cooked ham increased t4D values of L. monocytogenes from 11.8 to 24.9 min and 24.3 min, respectively. Similar effects were observed on cooked sausage. Survival on the cooked sausage, containing about 33% of fat, was not significantly different from that on cooked ham. Meanwhile, the addition of NaCl or NaL decreased the average proportions of injured cells substantially. The results of this study can be used by food processors to validate thermal processes with regard to the expected inactivation of L. monocytogenes post-contaminating meat product surfaces.
The antioxidant uncoupling protein 2 stimulates hnRNPA2/B1, GLUT1 and PKM2 expression and sensitizes pancreas cancer cells to glycolysis inhibition.
Brandi, J., Cecconi, D., Cordani, M., Torrens-Mas, M., Pacchiana, R., Dalla Pozza, E., Butera, G., Manfredi, E., Marengo, E., Oliver, J., Roca, P., Dando, I., Donadelli, M. & Roca, P. (2016). Free Radical Biology and Medicine, 101, 305-316.
Several evidence indicate that metabolic alterations play a pivotal role in cancer development. Here, we report that the mitochondrial uncoupling protein 2 (UCP2) sustains the metabolic shift from mitochondrial oxidative phosphorylation (mtOXPHOS) to glycolysis in pancreas cancer cells. Indeed, we show that UCP2 sensitizes pancreas cancer cells to the treatment with the glycolytic inhibitor 2-deoxy-D-glucose. Through a bidimensional electrophoresis analysis, we identify 19 protein species differentially expressed after treatment with the UCP2 inhibitor genipin and, by bioinformatic analyses, we show that these proteins are mainly involved in metabolic processes. In particular, we demonstrate that the antioxidant UCP2 induces the expression of hnRNPA2/B1, which is involved in the regulation of both GLUT1 and PKM2 mRNAs, and of lactate dehydrogenase (LDH) increasing the secretion of L-lactic acid. We further demonstrate that the radical scavenger N-acetyl-L-cysteine reverts hnRNPA2/B1 and PKM2 inhibition by genipin indicating a role for reactive oxygen species in the metabolic reprogramming of cancer cells mediated by UCP2. We also observe an UCP2-dependent decrease in mtOXPHOS complex I (NADH dehydrogenase), complex IV (cytochrome c oxidase), complex V (ATPase) and in mitochondrial oxygen consumption, suggesting a role for UCP2 in the counteraction of pancreatic cancer cellular respiration. All these results reveal novel mechanisms through which UCP2 promotes cancer cell proliferation with the concomitant metabolic shift from mtOXPHOS to the glycolytic pathway.
Reagent-Less and Robust Biosensor for Direct Determination of Lactate in Food Samples.
Bravo, I., Revenga-Parra, M., Pariente, F. & Lorenzo, E. (2017). Sensors, 17(1), 144.
Lactic acid is a relevant analyte in the food industry, since it affects the flavor, freshness, and storage quality of several products, such as milk and dairy products, juices, or wines. It is the product of lactose or malo-lactic fermentation. In this work, we developed a lactate biosensor based on the immobilization of lactate oxidase (LOx) onto N, N′-Bis(3,4-dihydroxybenzylidene)-1,2-diaminobenzene Schiff base tetradentate ligand-modified gold nanoparticles (3,4DHS-AuNPs) deposited onto screen-printed carbon electrodes, which exhibit a potent electrocatalytic effect towards hydrogen peroxide oxidation/reduction. 3,4DHS-AuNPs were synthesized within a unique reaction step, in which 3,4DHS acts as reducing/capping/modifier agent for the generation of stable colloidal suspensions of Schiff base ligand-AuNPs assemblies of controlled size. The ligand-in addition to its reduction action-provides a robust coating to gold nanoparticles and a catalytic function. Lactate oxidase (LOx) catalyzes the conversion of L-lactate to pyruvate in the presence of oxygen, producing hydrogen peroxide, which is catalytically oxidized at 3,4DHS-AuNPs modified screen-printed carbon electrodes at +0.2 V. The measured electrocatalytic current is directly proportional to the concentration of peroxide, which is related to the amount of lactate present in the sample. The developed biosensor shows a detection limit of 2.6 μM lactate and a sensitivity of 5.1 ± 0.1 µA·mM-1. The utility of the device has been demonstrated by the determination of the lactate content in different matrixes (white wine, beer, and yogurt). The obtained results compare well to those obtained using a standard enzymatic-spectrophotometric assay kit.
Monascus ruber as cell factory for lactic acid production at low pH.
Weusthuis, R. A., Mars, A. E., Springer, J., Wolbert, E. J., van der Wal, H., de Vrije, T. G., Levisson, M., Leprince, A., Houweling-Tan, G. B., Moers, P. H. A., Hendriks, N. A., Mendes, O., Griekspoor, Y., Werten, M. W. T., Schaap, P. J. der Oost, J., Eggink, G. & Hendriks, S. N. (2017). Metabolic Engineering, 42, 66-73.
A Monascus ruber strain was isolated that was able to grow on mineral medium at high sugar concentrations and 175 g/l lactic acid at pH 2.8. Its genome and transcriptomes were sequenced and annotated. Genes encoding lactate dehydrogenase (LDH) were introduced to accomplish lactic acid production and two genes encoding pyruvate decarboxylase (PDC) were knocked out to subdue ethanol formation. The strain preferred lactic acid to glucose as carbon source, which hampered glucose consumption and therefore also lactic acid production. Lactic acid consumption was stopped by knocking out 4 cytochrome-dependent LDH (CLDH) genes, and evolutionary engineering was used to increase the glucose consumption rate. Application of this strain in a fed-batch fermentation resulted in a maximum lactic acid titer of 190 g/l at pH 3.8 and 129 g/l at pH 2.8, respectively 1.7 and 2.2 times higher than reported in literature before. Yield and productivity were on par with the best strains described in literature for lactic acid production at low pH.