Rag−mutations involved in glucose metabolism in yeast: Isolation and genetic characterization

Some natural isolates and many laboratory strains of the yeast Kluyveromyces lactis cannot grow on glucose when respiration is inhibited by antimycin A. The ability or inability to grow on glucose in the absence of mitochondrial respiration has been called Rag⁺ or Rag^? phenotype (resistance to antimycin on glucose, respectively). Rag^? strains, unable to grow on glucose in the presence of the respiratory drug, behave as if they were defective in fermentation. The Rag phenotype was first found to be determined by variant alleles of either of the two nuclear genes, RAG1 and RAG2, which code for a low-affinity glucose transport protein and for phosphoglucose isomerase, respectively. These findings suggested that the Rag^? phenotype can be used to obtain mutations of genes involved in glucose metabolism in K. lactis. We thus looked for other Rag^? mutants. Seventy-four mutants were isolated and genetically characterized. All of the mutations were nuclear recessive alleles, defining 11 new complementation groups, which we designate rag3 through rag13.     

Potential Prophylactic Properties of Apple and Characterization of Potent Bioactive from cv. “Granny Smith” Displaying Strong Antimutagenicity in Models Including Human Lymphoblast TK6+/− Cell Line

Potential prophylactic attributes in terms of antimutagenicity, antioxidant, and radioprotective properties were evaluated for 8 common apple cultivars namely “Fuji,” “Golden Delicious,” “Granny Smith,” “Ambri Kashmiri,” “Kinnaur,” “Red Delicious,” “Royal Gala,” and “Shimla,” where cultivar based significant variation was observed. Cv. “Granny Smith” displayed significantly higher and broad spectrum antimutagenicity in Escherichia coli rpoB based rifampicin resistance (Rif^R) assay, whereas, “Ambri Kashmiri,” “Royal Gala,” and “Shimla” showed lower antimutagenicity. Cultivars “Ambri Kashmiri,” “Kinnaur,” and “Red Delicious” exhibited strong antioxidant activity than cv. “Granny Smith” as assayed by radical scavenging, reducing potential and radioprotective property assays. The antioxidant and radioprotective properties were found to be better correlated than antimutagenicity. Suppression of error‐prone DNA repair pathway (such as E. coli SOS response) was found to be one of the possible mechanisms contributing to its antimutagenicity. Phenolic extract of “Granny Smithˮ showing higher antimutagenicity was HPLC purified and the bioactive fraction (t_R 35.4 min) contributing maximally (∼80%) to the observed antimutagenicity was identified as procyanidin dimer (PD) by ESI‐MS/MS. The above observed antimutagenicity in bacterial assay system was well reproduced in Thymidine Kinase Mutation (TKM) assay performed using human lymphoblast cell line (TK6^+/−) cell line making the findings more prophylactically relevant.     

Antioxidative activities and the chemical constituents of two Chinese teas,Camellia kucha and C. ptilophylla

Two kinds of green tea were prepared with the young shoots of Camellia kucha and C. ptilophylla, and antioxidative activities of the chemical constituents were determined. Levels of alkaloids, catechins, total polyphenols, flavonoids and amino acids were determined by high‐performance liquid chromatography and UV‐Vis spectrophotometer. Antioxidative activity was evaluated by applying 2,2‐diphenyl‐1‐picrylhydrazyl radical assay and ferric reducing antioxidant power assay. Our results showed that theobromine was the only alkaloid in C. ptilophylla, while there was theacrine in C. kucha, which was coexisting with the caffeine. We confirmed the high content of (?)‐gallocatechin gallate in C. ptilophylla was not the epimerisation of (?)‐epigallocatechin gallate during the heating procedure. These wild teas had higher content of tea polyphenols, water extracts, amion acids and stronger antioxidative activity compared with the longjing tea. They could be used for the development of noncaffeine beverages and new types of tea.     

O-dihydroxyphenoloxidase action on natural polyhydric phenolics and enzymic browning of edible yams

Ten cultivars of five edible yam species were examined for o-dihydroxyphenoloxidase (o-DPOase) activity against yam extracts containing phenolic compounds, and for their propensity to brown when cut slices are exposed to air. (+)-Catechin levels in Dioscorea cayenensis (260 mg kg^?1) and D. bulbifera (240 mg kg^?1) were similar to those in D. dumetorum (270 mg kg^?1), but the latter contained less o-DPOase and showed less tendency to browning. D. alata contained more (+)-catechin than other yams, and one cultivar with a higher (+)-catechin content (660 mg kg^?1) showed more browning than another cultivar containing 430 mg kg^?1 (+)-catechin. Five cultivars of D. rotundata showed less tendency to brown, and had lower (+)-catechin content (90–190 mg kg^?1) than the other yams examined, but showed marked variation in o-DPOase activity. o-DPOase activity, assayed by recording the rate of oxygen consumption, varied between 75 units (1 unit=μmol O₂ consumed min^?1 100 g^?1 fresh yam tissue) in D. dumetorum and 2380 units in one D. rotundata cultivar when a crude extract of phenolic compounds diluted to contain 10 mM (+)-catechin was used as substrate and from 60 to 3480 units for these same two enzyme extracts when assayed using crude extracts of phenolic compounds (also adjusted to contain 10 mM (+)-catechin) prepared from the same yam as the enzyme extract. However, in general, the o-DPOase activities recorded using the D. alata extract showed little correlation with the activity when the phenolic extract was prepared from the same yam as the enzyme extract. Incubation of crude extracts containing phenolics with crude extracts of o-DPOase at 20°C for 24 h resulted in a decrease in the quantity of (+)-catechin estimated by h.p.l.c. Cyanidin released by hydrolysis with HCl under mild conditions was also measured. The level in D. alata was sufficiently high to account for ‘pinking’ when this species is boiled. It is concluded that the o-DPOase-catalysed oxidation of (+)-catechin is largely responsible for the browning of yams. The possible influence of other factors, including the reducing agent ascorbic acid in moderating the rate and extent of browning observed, is discussed.     

Sensory evaluation of bitterness and astringency of 3R(−)-epicatechin and 3S(+)-catechin

Bitterness and astringency of the monomeric flavan-3-o!s ( + )-catechin and (?)-epicatechin were rated using time-intensity (T-I) methodology. Three concentration levels (0.5. 0.9 and 1.2 g liter^?1) oi each compound were assessed, and the parameters time to maximum intensity (TMAX), intensity at the maximum (IMAX) and total duration (TTOT) were extracted from the T-I curves. No differences in TMAX for either bitterness or astringency were found between these chiral isomers or as a function of concentration within a compound. Epicatechin had a significantly higher bitter IMAX than catechin at all three concentration levels, and had a significantly longer TTOT at the two higher concentrations. Epicatechin was more astringent than catechin. but this was only significant at one concentration. Astringency TTOT was longer for epicatechin, although this was nonsignificant at the lowest concentration. The three concentrations of catechin were significantly different for both bitterness and astringency IMAX and TTOT. Epicatechin showed evidence for the astringency response plateauing above the 0.9 g litre^?1 level. n-Propylthiouracil status had no effect on perception of either bitterness or astringency.     

The formation of thearubigin-like substances by in-vitro polyphenol oxidase-mediated fermentation of individual flavan-3-ols

An enzymic in-vitro model tea fermentation system has been used in combination with gradient elution reversed-phase high-performance liquid chromatography to study the formation from individual fiavan-3-ols of resolvable and unresolvable thearubigin-like substances. It has been found that in 30 min at pH 5.6 tea polyphenol oxidase (EC 1.14.18.1) produces a distinctive range of resolvable thearubigin-like products from each flavan-3-ot substrate examined ((+)-catechin, (?)-epicatechin, (?)-epigallocatechin, (?)-epicatechin gallate and (?)-epigallocatechin gallate). However, (?)-epicatechin, and to a much lesser extent (+ )-catechin, were the only flavan-3-ols converted to an unresolvable thearubigin-like hump under these conditions. In addition, it was demonstrated that varying the pH of incubation in the range 5.0–6.0 had little effect on the profile of products obtained from the simple catechins, but a pronounced effect in the case of the gallocatechins. It is concluded that the combination of (?)-epicatechin and polyphenol oxidase is the simplest model of tea fermentation that can be used to generate in quantity thearubigin-like materials for structure elucidation.     

Interaction of β‐casein with (−)‐epigallocatechin‐3‐gallate assayed by fluorescence quenching: effect of thermal processing temperature

The interactions between the flavan‐3‐ol (?)‐epigallocatechin‐3‐gallate (EGCG) and bovine β‐casein in phosphate‐buffered saline (PBS) of pH 6.5 subjected to thermal processing at various temperatures (25–100 °C) were investigated using fluorescence quenching. The results indicated that different temperatures had different effects on the structural changes and EGCG‐binding ability of β‐casein. At temperatures below 60 °C, the β‐casein–EGCG interaction changed little (P > 0.05) with increasing temperature. At temperatures above 80 °C, native assemblies of β‐casein in solution dissociated into individual β‐casein molecules and unfolded, as demonstrated by a red shift of the maximum fluorescence emission wavelength (λ_max) of up to 8.8 nm. The highest quenching constant (K_q) and the number of binding sites (n) were 0.92 (±0.01) × 10¹³ m ^?1 s^?1 and 0.73 (±0.02) (100 °C), respectively. These results provide insight into the potential of interactions between β‐casein–EGCG that may modulate bioactivity or bioavailability to be altered during thermal process.     

Investigation of the interaction between (−)‐epigallocatechin‐3‐gallate with trypsin and α‐chymotrypsin

Tea polyphenol (TP) inhibits digestive enzymes and reduces food digestibility. To explore the interaction between TP with digestive enzymes, bindings of ‐epigallocatechin‐3‐gallate (EGCG) to trypsin and α‐chymotrypsin were studied in detail using fluorescence, resonance light‐scattering, circular dichroism, fourier transform infrared spectroscopy methods and protein‐ligand docking. The binding parameters were calculated according to Stern–Volmer equation, and the thermodynamic parameters were determined by the van't Hoff equation. The results indicated that EGCG was capable of binding trypsin and α‐chymotrypsin with high affinity, resulting in a change of native conformation of these enzymes. EGCG had a greater influence on the structure of α‐chymotrypsin than trypsin. This study can be used to explain the binding interaction mechanism between TP and digestive enzymes.     

Fermentative production of (R)-(-)-3-hydroxybutyrate using 3-hydroxybutyrate dehydrogenase null mutant of Ralstonia eutropha and recombinant Escherichia coli

Two systems, one using an (R)-(-)-3-hydroxybutyrate dehydrogenase (BDH) null mutant of Ralstonia eutropha and the other using a recombinant Escherichia coli strain containing a synthetic poly[(R)-(-)-3-hydroxybutyrate] (PHB) operon and an extracellular PHB depolymerase gene, were used for the fermentative production of (R)-(-)-3-hydroxybutyrate (3HB). The concentration of 3HB in the culture supernatant of the mutant R. eutropha system reached about 30 mM after 5 d under anaerobic conditions, although it was about 4-10 mM under aerobic conditions. On the other hand, the 3HB concentration in the culture supernatant of the recombinant E. coli system reached about 70 mM after 4 d, indicating that about 70% of the glucose added was converted to 3HB.     

Enhanced preservation effects of sugar apple fruits by salicylic acid treatment during post‐harvest storage

BACKGROUND: Salicylic acid (SA) is recognised as an endogenous signal, mediating in plant defense and against pathogens. It has been reported that SA treatment can reduce decay and extend storage life of various fruit, such as bananas, peaches and apples. RESULTS: Physiological and biochemical responses in harvested sugar apple fruit (Annona squamosa L.) to SA at 0.4, 0.8 and 1.2 mmol L^?1 were investigated during post‐harvest storage. Results indicated that SA treatments lowered respiration, increased activities of antioxidant enzymes of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) and ascorbate peroxidase (APX), decreased lipoxygenase (LOX) activity and correspondingly lowered Malondialdehyde (MDA) contents in treated fruits was observed as compared to the control. Moreover, production of superoxide free radical (O₂^{?, ?}) and ethylene was significantly decreased in the treated fruits. Total soluble solids, total soluble sugar, softness and decay rate were significantly lowered in treated fruits, and in turn a delay in the fruits ripening process was achieved after 10 days of storage. CONCLUSION: SA has positive effects in maintaining membrane integrity and in delaying fruit ripening process, which results in improved storability of sugar apple. Copyright © 2008 Society of Chemical Industry     

Polyphenol–cellulose interactions: effects of pH,temperature and salt

Response surface methodology was employed to investigate the effects of common food and digestive tract conditions (pH, temperature, sodium chloride) on the adsorption of polyphenols to cellulose. Major dietary polyphenols selectively bound to cellulose to different extents and exhibited different binding behaviour changes with the environment. Three factors were studied, of which pH (3–7) was found to be the most influential factor, followed by temperature (4–37 °C), while NaCl (0–100 mm ) had no significant effect on polyphenol adsorption. Polyphenol adsorption was sensitive to variation in physicochemical parameters: cyanidin‐3‐glucoside > ferulic acid > (+/?)‐catechin. Second‐order polynomial equation was a significant and adequate model to express the interaction relationships between polyphenol adsorption and changes in the binding conditions, as the experimental test values agreed with predicted adsorption values under optimised binding conditions. These findings contribute to knowledge on the mechanism of polyphenol–cellulose interactions, important for both food quality and nutritional value.     

Effect of pre-emulsified fish oil - as source of PUFA n-3 - on microstructure and sensory properties of mortadella, a Spanish bologna-type sausage

Conventional and low-fat cooked sausages (cursive) were prepared with fish oil as a source of n−3 polyunsaturated fatty acids. Fish oil was incorporated pre-emulsified with caseinates and water. Pre-emulsion was added in sufficient amounts to give fish oil levels of 1–6% in the final product. Lipid oxidation was determined (TBARs index) and instrumental measures of colour and texture were carried out. A sensory analysis was performed in which sensory parameters were evaluated by a hedonic test. There were no significant changes in TBAR values of final products being similar in both control and fish oil added sausages. In general terms, a slight increase in hardness and work of shearing were observed at the higher fish oil levels independently of the fat content. Sausages were analysed after 90 days of storage under refrigeration and similar results were observed. Microstructure of sausages was also studied by scanning electron microscopy (SEM) and a more compact structure was observed when the high levels were assayed. However, all sausages were well accepted when sensory quality was evaluated. Fat composition showed an increase in n−3 PUFA yielding a n−6/n−3 ratio close to 2, which is considered as healthier. For this, the incorporation of fish oil pre-emulsified seems to be a good option to include n−3 PUFA in cooked meat products, obtaining sensorially adequate and healthier stable sausages.     

Effect of a linseed diet on lipid composition, lipid peroxidation and consumer evaluation of French fresh and cooked pork meats

Twenty castrated pigs [(Large-White × Landrace) × (Pietrain)] (52.9 ± 5.1 kg initial body weight) were fed a control or a linseed diet containing 4.2% of extruded linseed. Animals were slaughtered at 106.6 ± 3.7 kg live weight. There was no effect of diet on pig performance. Feeding the linseed diet increased the contents of n−3 polyunsaturated fatty acids (PUFA) in chops (raw and cooked), chitterlings sausages, country style pâté, garlic sausages, liver pâté, and smoked belly. However, docosahexaenoic acid (DHA) level was not affected by the linseed diet. The linseed diet produced a robust decrease in the n−6/n−3 and linoleic acid (LA)/α-linolenic acid (ALA) ratios (<4). Feeding pigs with a high n−3 PUFA diet led to a decrease in the oxidative stability of chops, in contrast to smoked bellies for which thiobarbituric acid-reactive substances (TBARS) values were not affected by the diet. However, there was no deleterious effect on consumer overall appreciation of the meat.     

Effect of storage conditions on lipid components and color of Mugil cephalus processed roes

The salted and semidried mullet (Mugil cephalus) ovary product (bottarga) is proposed as an important source of the long-chain n-3 polyunsaturated fatty acids (PUFA), eicosapentaenoic acid, and docosahexaenoic acid. In this work, we investigated the extent of lipid oxidation and browning of grated bottarga samples during 7 mo of storage at -20 °C, 2 to 3 °C in the absence of light, and at room temperature in the presence or absence of light. Modifications of the levels of total choline (as index of phospholipid breakdown), total sugars, and free amino acids such as lysine, methionine, and tryptophan (involved in nonenzymatic browning) were also studied at different storage conditions. Storage of bottarga did not significantly affect the n-3 PUFA and cholesterol levels with respect to the control; nevertheless, a significant hydroperoxide increase was observed during 7 mo in bottarga samples at all the storage conditions, while low malondialdeyde levels were measured. Samples placed at room temperature in the absence and in the presence of light showed over time a marked browning process, lipid breakdown, a sensible decrease in the levels of total sugars, tryptophan, and methionine with respect to control and samples stored at -20 °C and 2 to 3 °C. The resistance against the oxidation of the isolated bottarga lipids was also assessed in dry state at several temperatures (37, 75, and 140 °C). PRACTICAL APPLICATION: We evaluated the change in lipid compounds and color of dried and salted mullet roes under different storage conditions. The obtained results suggest the importance of the low temperatures to preserve the nutritional properties of this fish product during long storage.     

The use of proline as a nitrogen source causes hypersensitivity to, and allows more economical use of 5FOA in Saccharomyces cerevisiae.

The use of proline as a nitrogen source causes hypersensitivity to 5-fluoro-orotic acid (5FOA) and allows up to 40-fold less of this drug to be used to select for the loss of URA3 function in Saccharomyces cerevisiae. 5FOA hypersensitivity is presumably due to the absence of nitrogen catabolite repression when proline is substituted for (NH4)2SO4 as a nitrogen source. There are two constraints to the use of the proline-5FOA combination: (1) S288c genetic background strains are hypersensitive to 5FOA when grown in proline as a nitrogen source but at least one other genetic background is resistant to low levels of 5FOA under these conditions. (2) The addition of some nutritional supplements confers phenotypic resistance to the 5FOA-proline combination.