吡哆醇对鲁氏接合酵母高盐胁迫耐受的影响

为了提高鲁氏接合酵母抗高盐胁迫能力,作者探索了添加吡哆醇对鲁氏接合酵母高盐胁迫耐受的影响。通过测量甘油、海藻糖含量以及Na⁺/K⁺-ATP酶活性等一系列生理表征来验证鲁氏接合酵母高盐适应性的变化。实验结果表明,鲁氏接合酵母在高盐胁迫条件下外源添加吡哆醇,在延滞期时,菌株提前24 h积累细胞内甘油,并且加快了细胞内海藻糖分解代谢速率;在对数期时,细胞内钠钾比率（Na⁺/K⁺）降低82.3%,细胞膜上Na⁺/K⁺-ATP酶活性增加16.9%,2-苯乙醇产量（质量浓度）提高6.42倍;在稳定期时,生物量提高10.6%,乙醇产量（质量浓度）提高5%,2-苯乙醇产量（质量浓度）提高1.26倍。综上所述,吡哆醇的添加能有效提高鲁氏接合酵母的高盐胁迫耐受能力,进一步增强了鲁氏接合酵母在酱油等高盐发酵食品中的应用前景。     

Characterization of plasma membrane H+-ATPase from salt-tolerant yeast Candida versatilis

Plasma membrane was isolated from the salt-tolerant yeast Candida versatilis and the ATPase in plasma membrane was characterized. The ATPase was a typical H⁺-ATPase with similar properties to the Saccharomyces cerevisiae and Zygosaccharomyces rouxii enzymes. It was reacted with antibody (IgG) raised against S. cerevisiae plasma membrane H⁺-ATPase. The ATPase activity was not changed by adding NaCl and KCl to the assay solutions, but was increased by NH, especially by ammonium sulfate. In vivo stimulation of ATPase activity was observed by the addition of NaCl into the culture medium, as observed in Z. rouxii. No in vivo activation of H⁺-ATPase by glucose metabolism was observed in C. versatilis cells and the activity was independent of the growth phase, like Z. rouxii and unlike S. cerevisiae cells.     

Characterization of plasma membrane proton-ATPase from salt-tolerant yeast Zygosaccharomyces rouxii cells

Plasma membrane was isolated from the salt-tolerant yeast Zygosaccharomyces rouxii and from Saccharomyces cerevisiae. The ATPase in the plasma membrane of Z. rouxii cells was a typical proton-ATPase as judged by testing with various ATPase inhibitors. There were slight differences in the pH optima of activities and in the sensitivity to sodium chloride (NaCl) and potassium chloride (KCl) of the ATPase from Z. rouxii and S. cerevisiae. The specific ATPase activity from Z. rouxii was higher in cells grown in a medium containing 2 _M-NaCl than in those not containing NaCl. No in vivo activation by incubation with glucose was observed in Z. rouxii cells and the specific ATPase activity was independent of the growth phase, unlike S. cerevisiae cells.     

KCI, CaCI2, Na+ Binding, and Salt Taste of Gum Systems

Aqueous nonionic (0.3% w/v) and ionic (0.1% and 0.3% w/v) gum systems containing NaCl, or equal weights of NaCl plus KCl, or NaCl plus CaCl, were examined. At equivalent molar concentrations of added ions, ²³Na NMR transverse relaxation rates (R₂, set^?1) showed an increase in average Na⁺ mobility with the addition of K⁺ or Ca²⁺ to ionic gum systems. Correspondingly, salt taste increased with addition of KCl as determined by Decision Boundary modeling of subject identification data. Viscosity did not affect saltiness. Na⁺ was free to induce salt taste when K⁺ was bound to the gum. Enhancement of salt taste by KCl is due, in part, to competitive binding of Na⁺ and K⁺ in a system.     

Theasaponin E1 destroys the salt tolerance of yeasts

Cells of Zygosaccharomyces rouxii in a medium containing a high concentration of NaCl were killed during incubation for 2–4 h with a low concentration of a mixture of saponins from tea seeds (TSS). The higher the concentration of NaCl in the medium, the higher the inhibitory effect of TSS on the growth of the yeast. The above inhibitory effect of TSS on the growth of the yeast was not observed when cells were incubated in hypertonic media composed of nonionic substances such as sugars. The ATPase activity of plasma membrane preparations from the yeast cells was slightly affected by the addition of TSS. It is shown that TSS facilitates leakage of glycerol from the yeast cells under NaCl-hypertonic conditions. The major inhibitor in the mixture of saponins was isolated and identified as theasaponin E₁. Its isomer, theasaponin E₂, did not have any effect on the salt tolerance of Z. rouxii or Saccharomyces cerevisiae.     

Physiological changes, phenolic content and antioxidant activity of Salvia officinalis L. grown under saline conditions

BACKGROUND: Hydroponic culture was used to investigate the effect of NaCl concentrations on the growth, nutrient uptake, phenolic content and antioxidant activity of Salvia officinalis L. leaves. The antioxidant capacity of the methanolic extract of S. officinalis was evaluated by using 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH) radical scavenging test and β‐carotene‐linoleic acid bleaching assay. Physiological and biochemical parameters of S. officinalis were assessed after 4 weeks of salt treatment with 0, 25, 50, 75 and 100 mmol L^?1 NaCl. RESULTS: Plant growth exhibited a reduction of 61% at 100 mmol L^?1 NaCl. Assessment of Na⁺, K⁺ and Ca²⁺ and water contents of shoots and roots showed that S. officinalis is able to regulate Na⁺ concentration by active compartmentation in vacuoles. Salvia officinalis phenolics were increased in response to salinity at the threshold of 75 mmol L^?1 NaCl. This herb was also found to be able to achieve important DPPH^? quenching activity and to inhibit the β‐carotene‐linoleic acid bleaching notably enhanced by salt treatment. It is interesting to highlight the correlation between the phenolic and antioxidant activity, suggesting the involvement of these compounds in this activity. CONCLUSION: Salvia officinalis treated with 75 mmol L^?1 NaCl constitutes a potential source for production of secondary metabolites useful in several applications. Copyright © 2011 Society of Chemical Industry     

H+‐ATPase‐Defective Variants of Lactobacillus delbrueckii subsp. bulgaricus Contribute to Inhibition of Postacidification of Yogurt during Chilled Storage

Continued acid production by Lactobacillus delbrueckii subsp. bulgaricus during the chilled storage of yogurt is the major cause of postacidification, resulting in a short shelf life. Two H⁺‐ATPase defective variants of L. delbrueckii subsp. bulgaricus were successfully isolated and their H⁺‐ATPase activities were reduced by 51.3% and 34.3%, respectively. It was shown that growth and acid production of variants were remarkably inhibited. The variants were more sensitive to acidic condition and had a significant rate for inactivation of H⁺‐ATPase by N, N‐dicyclohexylcarbodiimide (DCCD), along with a low H⁺‐extrusion, suggesting that H⁺‐ATPase is direct response for H⁺‐extrusion. In addition, the variants were also more sensitive to NaCl, while H⁺‐ATPase activities of variants and parent strain were significantly enhanced by NaCl stress. Obviously, H⁺‐ATPase might be involved in Na⁺ transportation. Furthermore, variants were inoculated in fermented milk to ferment yogurt. There was no significant difference in flavor, whereas the postacidification of yogurt during chilled storage was remarkably inhibited. It is suggested that application of L. delbrueckii subsp. bulgaricus with reduced H⁺‐ATPase activity in yogurt fermentation is one of effect, economic and simple avenues of inhibiting postacidification of yogurt during refrigerated storage, giving a longer shelf life. Practical Application: During yogurt fermentation, continued acid production by Lactobacillus delbrueckii subsp. bulgaricus during the chilled storage of yogurt leads to milk fermentation with high postacidification, resulting in a short shelf life. In this work, 2 acid‐sensitive variant strains of L. delbrueckii subsp. bulgaricus were isolated. The characteristics related to H⁺‐ATPase were compared and it was observed that milk fermented by the variants had lower postacidification, giving a longer shelf life. Application of L. delbrueckii subsp. bulgaricus with reduced H⁺‐ATPase activity in yogurt fermentation might be one of effect, economic and simple avenues of inhibiting yogurt postacidification during chilled storage, giving a longer shelf life.     

Monovalent cation fluxes and physiological changes of Debaryomyces hansenii grown at high concentrations of KCl and NaCl

Debaryomyces hansenii showed an increased growth in the presence of either 1m KCl or 1m NaCl and a low acidification of the medium, higher for the cells grown in the presence of NaCl. These cells accumulated high concentrations of the cations, and showed a very fast capacity to exchange either Na⁺ or K⁺ for the opposite cation. They showed a rapid uptake of ⁸⁶ Rb⁺ and ²² Na⁺ . ⁸⁶ Rb⁺ transport was saturable, with K_m and V_max values higher for cells grown in 1m NaCl. ²² Na⁺ uptake showed a diffusion component, also higher for the cells grown with NaCl. Changes depended on growth conditions, and not on further incubation, which changed the internal ion concentration. K⁺ stimulated proton pumping produced a rapid extrusion of protons, and also a decrease of the membrane potential. Cells grown in 1m KCl showed a higher fermentation rate, but significantly lower respiratory capacity. ATP levels were higher in cells grown in the presence of NaCl; upon incubation with glucose, those grown in the presence of KCl reached values similar to the ones grown in the presence of NaCl. In both, the addition of KCl produced a transient decrease of the ATP levels. As to ion transport mechanisms, D. hansenii appears to have (a) an ATPase functioning as a proton pump, generating a membrane potential difference which drives K⁺ through a uniporter; (b) a K⁺ /H⁺ exchange system; and (c) a rapid cation/cation exchange system. Most interesting is that cells grown in different ionic environments change their studied capacities, which are not dependent on the cation content, but on differences in their genetic expression during growth. © 1998 John Wiley & Sons, Ltd.     

Na+ Binding as Measured by 23Na Nuclear Magnetic Resonance Spectroscopy Influences the Perception of Saltiness in Gum Solutions

Binding of Na⁺ in aqueous gum systems as determined by ²³Na nuclear magnetic resonance (NMR) spectroscopy and its relations to perceived saltiness were examined. Two levels of NaCl (0.1% and 0.2%) were added to two concentrations (0.1% and 0.3%) of two ionic (xanthan and kappa carrageenan) and two non-ionic (locust bean and guar) gum solutions. Saltiness perception was affected by the ionic properties of the gums. NMR transverse relaxation rates (R₂, see^?l) indicated Na⁺ was less mobile in ionic than nonionic systems. Ionic gums correspondingly suppressed saltiness perception- compared to nonionic gums. As Na⁺ increased in both ionic and nonionic systems, R₂ values converged and perceived saltiness equalized. Food components that bind Na⁺ may suppress saltiness perception, which may be important in low-sodium foods.     

Production optimization,purification, and characterization of a novel acid protease from a fusant by Aspergillus oryzae and Aspergillus niger

The production of a novel acid protease was enhanced by 44 % through statistical optimization. The cultural parameters, such as inoculum size, temperature, moisture content, and incubation time, were 8.59 × 10⁵ g^?1 dry koji, 31 °C, 57 %, and 86 h, respectively. This novel acid protease was purified by 17 folds with a recovery yield of 33.56 % and a specific activity of 4,105.49 U mg^?1. Far-UV circular dichroic spectra revealed that this purified protease contained 7.1 % α-helix, 64.1 % β-sheet, and 32 % aperiodic coil. This novel acid protease was active over the temperature range of 35–55 °C with optimum temperature of 40 °C and was stable in the pH range of 2.5–6.5 with optimum pH of 3.5. Mn²⁺ enhanced its activity while Co²⁺ showed inhibitory effect. With casein as substrate, the kinetic parameters of K _m, V _max, energy of activation (E _a), and attenuation index of inactivation velocity by heat inducing (λ) were 0.96 mg mL^?1, 135.14 μmol min^?1 mg^?1, 64.11 kJ mol^?1, and 0.59, respectively.     

Inactivation of yeast and filamentous fungi by the lactoperoxidase-hydrogen peroxide-thiocyanate-system

The antifungal activity of the lactoperoxidase (LPO) system with glucose oxidase (GOD) as source of hydrogen peroxide was determined in salt solution and in apple juice. The test organisms Rhodutorula rubra and Saccharomyces cerevisiae were cultivated aerobically in apple juice, Mucor rouxii was grown on wort agar adjusted to pH 4.5. Aspergillus niger and Byssochlamys fulva were kept on malt extract agar. Spores of the filamentous fungi were harvested by suspension in salt solution supplemented with Tween 80® and checked microscopically. The antifungal activity of the combined enzyme system was tested with initial counts of approx. 10⁵ cfu · ml^?1 (yeast cells or spores) suspended in salt solution supplemented with 25 mg · l^?1 thiocyanate and 20 g · l^?1 glucose or in apple juice supplemented with the same amount of thiocyanate. The tests were performed with 25 ml of the medium in 100 ml Erlenmeyer flasks shaken at 28 °C under aerobic conditions. Inactivation was achieved for all test organisms in both media. The yeast strains were found to be least stable while B. fulva was most resistant. A combination of 5 U · ml^?1 LPO with 0.5 to 1 U · ml^?1 GOD was sufficient for complete inactivation of this mold in salt solution within 2 h. The enzyme system also showed antifungal activity in apple juice at acid pH (3.2), although its effectiveness was reduced. In this medium, B. fulva was inactivated by 20 U · ml^?1 LPD and 1 U · ml^?1 GOD within 4 h. R. rubra and S. cerevisiae were unable to survive in apple juice at 5 U · ml^?1 LPO combined with 1 U · ml^?1 GOD. For inhibition by GOD alone, higher amounts of this enzyme were needed and even then only M. rouxii and R. rubra have been affected within the concentration range tested (maximum 3 U · ml^?1).     

Proteolysis of αs1-Casein by Papain in a Complex Environment. Influence of Ionic Strength on the Kinetic Constants

The influence of α_s1-casein concentration on the hydrolytic activity of papain was studied. High substrate concentration was inhibitory. In presence of NaCl, the papain affinity for α_s1-casein increased (the apparent Michaelis constant (K_m. app.) decreased from 9.4 10^?5 to 5.6 10^?5 M) and was lower for 0.08M NaCl. The maximum velocity (V_max app-) was independant from NaCl concentration below 0.34M but decreased above. The enzyme-substrate affinity was increased by the addition of urea, but the V_max app. decreased. The inhibitory effect of excess substrate was more important with the presence of both urea and NaCl. Acetylation of α_s1-casein showed that K_m. app. (21.2 10^?5M) was independant of salt concentration, while the V_max app. varied, and the substrate inhibitory activity was suppressed.     

Isolation of Gellan Gum from Foods by Use of Monovalent Cations

Gellan gum added to foods was recovered by precipitation with 2M NaCl. The precipitate was trapped on glass wool, washed with 1M salt solution and eluted with boiling 40% H₂SO₄ or boiling 2 × 10^?4N NaOH. Elution with dilute NaOH was as effective as use of 40% H₂SO₄, except with dog food, mayonnaise and salad dressing. The hydrocolloid content of the eluate was determined by the carbazole test. Recoveries from milk, beverages, salad dressing and other foods ranged from 75–94% (reproducibility 2–4%). The preferred cations for precipitation were Na⁺ > K⁺ > Li⁺ > NH₄⁺. For the anions, the order was CO₃^? > SO₄^? > Cl^?.     

Production of trehalose by intramolecular transglucosylation of maltose catalysed by a new enzyme from Thermus thermophilus HB-8

Thermus thermophilus HB-8 is a source of trehalose synthase (GTase), which catalyses conversion of maltose into trehalose. Specific activity of maltose transglucosylation by cell-free extracts of the bacteria was about 0.1 U mg⁻¹ protein and precipitation at 28% saturation of ammonium sulphate caused 3.5-fold enzyme purification. The optimum temperature for conversion of maltose into trehalose was 65 °C with about 27% of maximum activity at 85 °C. The highest GTase productivity was achieved at cultivation temperature over 60 °C and at NaCl concentration range of 0.1–0.5% (w/v). However, larger concentrations of sodium chloride in the growth media caused a remarkable decrease of GTase synthesis. The results, of ammonium sulphate fractionation and activity towards maltotriose (0.028 U mg⁻¹), maltotetraose (0.16 U mg⁻¹) and GlcαpNp (0.27 U mg⁻¹), show that trehalose synthase and α-glucosidase activities reside in separate protein fractions of cell-free extracts from T. thermophilus cells.     

SARCOPLASMIC RETICULUM Ca2+-ATPase ACTIVITY IN COD (GADUS MORHUA) MUSCLE MEASURED IN CRUDE HOMOGENATES

An assay for sarcoplasmic reticulum Ca²⁺‐ATPase in an unfractionated homogenate from cod(Gadus morhua) muscle has been established. Specificity of the assay was demonstrated by the Ca²⁺ dependence of the enzyme and the effect of a specific inhibitor, thapsigargin. Stimulation and inhibition of enzyme activity was observed at low and high Ca²⁺ concentration, respectively. Half the maximal activity was obtained at 0.2 μM free calcium and no activity could be detected above 20 mM free Ca²⁺. Thapsigargin inhibited 92% of enzyme activity. Sarcoplasmic reticulum Ca²⁺‐ATPase showed maximal activity around pH 7 and 25C. A nonlinear Arrhenius plot was found with pronounced changes in the slope in the temperature interval 6–15C. The activation energies obtained from the approximately linear parts above 15C and below 6C were 25 kJ mot^?1 and 172 kJ mol^?1, respectively.     

Salt-induced modulation in inorganic nutrients, antioxidant enzymes, proline content and seed oil composition in safflower (Carthamus tinctorius L.)

BACKGROUND: Safflower (Carthamus tinctorius L.) has gained considerable ground as a potential oil‐seed crop. However, its yield and oil production are adversely affected under saline conditions. The present study was conducted to appraise the influence of salt (NaCl) stress on yield, accumulation of different inorganic elements, free proline and activities of some key antioxidant enzymes in plant tissues as well as seed oil components in safflower. Two safflower accessions differing in salt tolerance (Safflower‐33 (salt sensitive) and Safflower‐39 (salt tolerant)) were grown under saline (150 mmol L^?1) conditions and salt‐induced changes in the earlier‐mentioned physiological attributes were determined. RESULTS: Salt stress enhanced leaf and root Na⁺, Cl^? and proline accumulation and activities of leaf superoxide dismutase, catalase and peroxidase, while it decreased K⁺, Ca²⁺ and K⁺/Ca²⁺ and Ca²⁺/Na⁺ ratios and seed yield, 100‐seed weight, number of seeds, as well as capitula, seed oil contents and oil palmitic acid. No significant effect of salt stress was observed on seed oil α‐tocopherols, stearic acid, oleic acid or linoleic acid contents. Of the two safflower lines, salt‐sensitive Safflower‐33 was higher in leaf and root Na⁺ and Cl^?, while Safflower‐39 was higher in leaf and root K⁺, K⁺/Ca²⁺ and Ca²⁺/Na⁺ and seed yield, 100‐seed weight, catalase activity, seed oil contents, seed oil α‐tocopherol and palmitic acid. Other attributes remained almost unaffected in both accessions. CONCLUSION: Overall, high salt tolerance of Safflower‐39 could be attributed to Na⁺ and Cl^? exclusion, high accumulation of K⁺ and free proline, enhanced CAT activity, seed oil α‐tocopherols and palmitic acid contents. Copyright © 2011 Society of Chemical Industry     

Characterization of Na+/H+-antiporter gene closely related to the salt-tolerance of yeast Zygosaccharomyces rouxii

In order to clarify the relationship between salt-tolerance of Zygosaccharomyces rouxii and the function of Na⁺/H⁺-antiporter, a gene was isolated from Z. rouxii which exhibited homology to the Na⁺/H⁺-antiporter gene (sod2) from Schizosaccharomyces pombe. This newly isolated gene (Z-SOD2) encoded a product of 791 amino acids, which was larger than the product encoded by its Sz. pombe homologue. The predicted amino-acid sequence of Z-Sod2p was highly homologous to that of the Sz. pombe protein, but included an extra-hydrophilic stretch in the C-terminal region. The expression of Z-SOD2 was constitutive and independent of NaCl-shock. Z-SOD2-disruptants of Z. rouxii did not grow in media supplemented with 3 M -NaCl, but grew well in the presence of 50% sorbitol, indicating that the function of Z-SOD2 was closely related to the salt-tolerance of Z. rouxii. Several genes are also compared and discussed in relation to the salt-tolerance of Z. rouxii. The nucleotide sequence data reported in this paper will appear in the GSDB, DDBJ, EMBL and NCBI nucleotide sequence databases with the following accession number: D43629.