A Maltooligosaccharides Producing α-Amylase from Bacillus subtilis SDP1 Isolated from Rhizosphere of Acacia cyanophylla Lindley

Maltooligosaccharides producing amylases are required in the food industry, especially in breadmaking. The Bacillus subtilis strain SDP1 amylase hydrolyses starch to produce maltotriose and maltotetraose along with maltose after prolonged reactions of 5 h. Bacillus subtilis strain SDP1 was isolated from the rhizosphere of Acacia cyanophylla Lindley from the Çukurova region of Turkey. The highest enzyme production was achieved with soluble starch as the carbon and yeast extract as the nitrogen source and at pH 7.0 and 37°C. Under optimized culture conditions, 68.49 U/mL activity was obtained. SDP1 α-amylase had molecular weight of 61 kD. The optimum pH of the enzyme was 7.0 and was highly active at pH ranging from 5.0 to 9.0. The optimum temperature of the crude enzyme was 60°C, and it retained 83% and 74% of its initial activity after 1 h and 2 h incubation periods, respectively, at 50°C. While, Mn⁺² has a stimulatory effect on the activity, Ca⁺², Mg⁺², Na⁺ did not effect the enzyme activity. Fe⁺³, Ni⁺², Cu⁺² and Co⁺² had an inhibitory effect on SDP1 amylase activity.     

Purification and Characterization of Polyphenol Oxidase from Hemşin Apple (Malus communis L.)

The polyphenol oxidase (PPO) enzyme was purified and characterized from Hem?in Apple (Malus communis L.), which was organically grown in Hem?in, in the Rize province of Turkey. Enzyme (PPO) activation was determined with catechol substrate. Apples were homogenized with homogenate buffer (pH 8.5). This process was followed by precipitation with (20–80%) saturated solid (NH₄)₂SO₄ and dialysis. Finally, purification with DE52-Cellulose ion-exchange and Sephadex G-25 columns was performed. Experiments were performed at an optimum pH (5.5) and optimum temperature (30–40°C). The kinetic and thermal parameters Km (3.40 mM), Vmax (333.3 EU/mL.min), Ea (3.57 kcal), ?H (2.968 kcal/mol), Q₁₀ (1.33), k_cat (24.57 min^?1) and V₀ (7.2x10³ mM^?1.min^?1) were assessed. Additionally, the effects of Mg²⁺, Pb ²⁺, Fe²⁺, Fe³⁺, Cd²⁺, Cu²⁺, Zn²⁺, Co²⁺, Al³⁺, Mn²⁺ and Na⁺ on enzyme activity was recorded, and the IC₅₀ values, K_? values and inhibition types were determined.     

Characterization of an acidophilic and thermostable α‐amylase from Alicyclobacillus sendaiensis NUST

This paper describes the characterization of an acidophilic and thermostable α‐amylase from Alicyclobacillus sendaiensis NUST. The MW of this enzyme was estimated to be 56 kDa by SDS–PAGE. The enzyme was stable over a range of pH from 2.5 to 5.5 with an optimum around 3.5. Maximum activity of the α‐amylase was observed at pH 3.5 and 85°C in the presence of soluble starch as substrate. The enzyme activity was decreased by Mg²⁺, Cu²⁺, Zn²⁺, Al³⁺, K⁺, Li⁺, Ag⁺, urea, EDTA, trichloroacetic acid and Tween 60 and inhibited by Hg²⁺, Ce²⁺ and SDS, whereas the activity was increased by Mn²⁺, DTT, and β‐mercaptoethanol. Ca²⁺and Fe²⁺ did not affect the enzyme activity.     

A novel potentiometric pH electrode based on sulfated natural Fe<Subscript>3</Subscript>O<Subscript>4</Subscript> and analytical application in food samples

A novel solid state potentiometric pH electrode based on sulfated natural Fe₃O₄ silicone was fabricated. The optimum potentiometric performances such as Nernstian response, response time, selectivity, life-time and reproducibility of pH electrodes were investigated by using a computer-controlled potentiometric device. Moreover, the potentiometric performance of the solid state pH electrodes was studied with different mixtures sulfated natural Fe₃O₄, silicone and graphite powder. The best potentiometric behavior of proposed pH electrode was obtained with a composition of 20% (w/w) sulfated natural Fe₃O₄, 40% (w/w) graphite powder and 40% (w/w) silicone. The sub-Nernstian response for pH electrode was exhibited with a slope of 30.8?±?1.4 mV/pH (r?=?0.9963) from pH 2 to pH 12. In addition, the dynamic response time was found as 10 s in acidic medium and further the proposed pH electrode can be used for at least 1 year without any significant slope of the pH–potential curve. The selectivity coefficient of pH electrode was interpreted according to fixed interference method in the presences of Na⁺, Li⁺, K⁺, NH₄⁺, SO₄^2?, CH₃COO^? and NO₃^? ions. The reproducibility of pH electrode was calculated in pH 4 and pH 6 phosphate buffers and it was found as 0.24 RSD (%) and 0.27 RSD (%) respectively. The proposed pH electrode was used to determine of pH in acid?bases titration compared with glass pH electrode and is highly stable in corrosive systems including HF solution. Terminally, the pH value was successfully determined in some soft drinks and milk samples by proposed solid state pH electrode at 95% confidence level with satisfactory agreement compared with glass pH electrode.     

Wheat-bug damage in New Zealand wheats: Some properties of a glutenin hydrolysing enzyme in bug-damaged wheat

Some properties of a glutenin hydrolysing enzyme present in bug (Nysius huttoni) damaged wheat (Triticum aestivum) were examined using a modified SDS sedimentation test reported previously. The enzyme appears to be a water-soluble alkaline protease with an activity optimum at pH 9.0. It is relatively heat stable, but the temperature optimum for activity is quite low (35–40°C). The enzyme is not inhibited by EDTA or N-ethylmaleimide, but is inhibited by the metal ions Co²⁺, Mn²⁺ and Fe²⁺.     

Substrate Specificity and Inhibition of Polyphenoloxidase (PPO) From a Dwarf Variety of Banana (Muss Cavendishii, L.)

Banana polyphenoloxidases oxidize specifically o-diphenols. The Km values were low for dopamine, epinephrine, and norepinephrine. It was lower for L-dopa as compared with D-dopa. The most effective inhibitor was ascorbic acid followed by cysteine and then sodium metabisulfite. Diethyldithiocarbamate, at pH 7.0, was not as effective as the above inhibitors. Ammonium ion and the oxidized β-nicotine adenine dinucleotide acted as activators whereas Fe⁺², Fe+³, Al⁺³, Ca⁺² and Zn⁺² showed various degrees of inhibition. On the other hand, C⁺¹, Cu⁺², Mg⁺² and Mn⁺² did not affect the enzyme activity. Mercaptoethanol (17 mM) completely inactivated the enzymes. On dialysis 30% of the activity was restored with regeneration of two isozymes.     

Effect of Acid Pretreatment on the Stability of Ascorbic Acid Complexes with Various Iron Sources in a Wheat Flake Cereal

The effect of acid incubation of ascorbic acid with each of five iron sources (ferrous sulfate, ferric chloride, ferric orthophosphate, hydrogen and electrolytically reduced iron) on iron solubilization in a wheat flake cereal was evaluated. Incubation produced more soluble iron at pH 2 but not necessarily at the endogenous pH of the cereal nor at pH 6. At pH 2, Fe⁺² rather than Fe ⁺³ was produced, apparently by a reduction of bound Fe ⁺³ and subsequent release of Fe⁺². At pH 6, the soluble iron was mainly in a complexed form. This indicates that acid incubation with ascorbate might facilitate bioavailability of iron if it were incorporated by fortification techniques.     

New applications of pHluorin—measuring intracellular pH of prototrophic yeasts and determining changes in the buffering capacity of strains with affected potassium homeostasis

pHluorin is a pH‐sensitive variant of green fluorescent protein for measuring intracellular pH (pH_in) in living cells. We constructed a new pHluorin plasmid with the dominant selection marker KanMX. This plasmid allows pH measurements in cells without auxotrophic mutations and/or grown in chemically indefinite media. We observed differing values of pH_in for three prototrophic wild‐types. The new construct was also used to determine the pH_in in strains differing in the activity of the plasma membrane Pma1 H⁺‐ATPase and the influence of glucose on pH_in. We describe in detail pHluorin measurements performed in a microplate reader, which require much less hands‐on time and much lower cell culture volumes compared to standard cuvettes measurements. We also utilized pHluorin in a new method of measuring the buffering capacity of yeast cell cytosol in vivo, shown to be ca. 52 mM /pH for wild‐type yeast and moderately decreased in mutants with affected potassium transport. Copyright © 2010 John Wiley & Sons, Ltd.     

Partial characterization of β‐ d‐xylosidase from wheat malts

Arabinoxylans (AXs) from wheat malts potentially affect beer quality and production. β‐ d ‐Xylosidase is a key enzyme that degrades the main chains of AXs to produce xylose. This study performed a partial characterization of β‐ d ‐xylosidase from wheat malts. The optimal temperature was 70 °C and the enzyme exhibited excellent thermostability, that is, residual activities were 92.6% at 60 °C for 1 h. The enzyme was stable over a pH range of 3.0–6.0 and showed optimum activity at pH 3.5 and 4.5. Kinetic parameters K_m and V_max of wheat malt β‐ d ‐xylosidase against p‐nitrophenyl‐xyloside were 1.74 mmol L⁻¹ and 0.76 m m min⁻¹, respectively. The enzyme activity was severely inhibited by Cu²⁺, moderately inhibited by Mn²⁺, Mg²⁺, Al³⁺, Ca²⁺, Ba²⁺ and Na⁺ and mildly inhibited by Fe³⁺ and Fe²⁺. The partial enzymatic characterization achieved in this study can be used as a theoretical basis for purifying β‐ d ‐xylosidase from wheat malts. Copyright © 2015 The Institute of Brewing & Distilling     

Optimization of Trace Metals Concentration on Citric Acid Production by Aspergillus niger NRRL 2001

Citric acid is nowadays produced by submerged fermentation of Aspergillus niger. The process yield depends on the composition of the medium, as well as on the microorganism strain. In this work, the effect of Fe⁺³, Zn⁺², and Mn⁺² on citric acid production by A. niger NRRL 2001 is presented. The culture medium composition was glucose (120 g/L) KH₂PO₄ (1.0 g/L); K₂HPO₄ (1.0 g/L), MgSO₄.7H₂O (0.5 g/L), (NH₄)₂SO₄ (3.0 g/L). The ions Fe⁺³, Zn⁺², and Mn⁺² had their concentrations changed according to an experimental design. The experiments were carried out in an orbital shaker at 200 rpm and 30°C. The strain produced an extracellular polysaccharide that was also quantified. The optimum experimental condition was found using 7.0 mg/L of Fe⁺³ and 6.5 mg/L of Zn⁺² in absence of Mn⁺². No oxalic acid formation was observed using this experimental condition. Metal contents were not significant for the production of the polysaccharide. The highest production rate (2.95 g L⁻¹ day⁻¹) was reached after 10 days of fermentation. After this period, the productivity decreased slightly. In 20 days, the citric acid production rate (2.44 g L⁻¹ day⁻¹) was 82% of the highest productivity. The conversion into citric acid increased continuously, yielding 45.8% in 20 days of fermentation.     

EFFECT OF ACID PRETREATMENT ON THE STABILITY OF CITRIC AND MALIC ACID COMPLEXES WITH VARIOUS IRON SOURCES IN A WHEAT FLAKE CEREAL1

An in vitro incubation at pH 2 of citric and malic acids with each of five iron sources [(hydrogen (HRI) and electrolytically reduced elemental iron (ERI), ferric chloride (FeCl₃), ferrous sulfate (FeSO₄) and ferric orthophosphate (FOP)] at a 10:1 molar ratio (acid:iron) was evaluated for its effect on iron solubiliza-tion in a wheat flake cereal subjected to a sequential gastrointestinal pH treatment from endogenous pH (E) to 2 to 6. Citric acid maintained significantly more complexed and ionic iron (Fe⁺³) in solution than malic acid, with and without incubation, through the final stage of the sequential pH treatment with each of the five iron sources. However, the malate treated samples were affected less than the citrate by acidification from pH E to 2, with less of the soluble complexed iron being insolubilized and significantly more ionic iron (Fe⁺²) being produced. Incubation significantly enhanced the iron solubilizing capacity of citrate at all stages of the sequential pH treatment with HRI and at pH E with ERI, FeCl₃ and FeSO₄, As well, incubation increased the iron solubilization properties of malate at all stages with FeSO₄ and at E and 2 with HRI and ERI. These results indicate that acid incubation to form an organic acid-iron chelate, particularly with elemental iron, has the potential to improve cereal iron fortificant bioavail-ability.     

Purification and some properties of α-amylase from post-harvest Pachyrhizus erosus L. tuber

α-Amylase, a starch splitting enzyme, was purified to homogeneity from post-harvest Pachyrhizus erosus L. tuber by successive chromatography on DEAE- and CM-cellulose columns. Purification achieved was 110 fold from the crude extract with a yield of 22.8%. SDS-PAGE showed a molecular weight of 40 kDa for the enzyme. The enzyme is of α-type as it lost total activity in the presence of EDTA, a chelating agent. It is a glycoprotein that contains 2.6% sugar as estimated by the phenol-sulfuric acid method. The enzyme displayed optimum activity at pH 7.3 and 37 °C with an apparent K_m value of 0.29% for starch as substrate. The enzyme was strongly inhibited by Cu²⁺, Fe²⁺ and Zn²⁺, moderately by Li²⁺, Hg⁺ and Cd²⁺ and slightly by Ag⁺, Mg²⁺ and K⁺. Calcium ion almost doubled the activity whereas Fe³⁺, Mn²⁺ and Na⁺ enhanced it appreciably.     

Comparison of the thermal characteristics of connective tissue in loose structured and normal structured porcine M. semimembranosus

The aim of the present study was to characterise the thermal properties of connective tissue in loose structured porcine SM muscles in comparison to normal looking SM muscles and to see whether the meat quality traits were related to the properties of connective tissue. Samples from the muscles with loose structure and light colour were selected by visual assessment and normal looking SM muscles were used as a control (n = 25 loose structured + 25 control). The loose structured muscles had lower ultimate pH (pH_u) than the control muscles. The onset and peak temperatures of thermal shrinkage of intramuscular connective tissue (T_o and T_p, respectively) in loose structured muscles were similar to those of control muscles when the full set of data (25 loose structured + 25 control) were analysed. When the T_o and T_p data from muscles the exhibiting ten lowest and ten highest pH_u values were analysed, the low pH_u muscles (all classified as loose structured) had lower T_o and T_p than the high pH_u muscles (all classified as control) (p < 0.05). Drip loss of loose structured SM muscles (11.1%) was dramatically higher than that of control muscles (3.9%). Collagen content and collagen solubility were similar in loose structured and control muscles. It seemed that changes in the properties of intramuscular connective tissue were more easier found in porcine SM muscles with low pH_u than in SM muscles with high pH_u.     

Production,Characterization and Application of a Xylanase from Streptomyces sp. AOA40 in Fruit Juice and Bakery Industries

Streptomyces sp. AOA40, which produces halotolerant and thermotolerant xylanase, was isolated from Mersin soil. Various carbon sources were tested for xylanase production with selected fermentation medium. The best carbon source was selected as corn stover. The effect of corn stover concentration and particle size, composition of fermentation medium, fermentation condition such as initial pH and agitation rate on xylanase production was determined. After production, xylanase was partially purified with ion-exchange chromatography and gel filtration chromatography for characterization of xylanase and application in fruit juice and dough improvement. The optimum pH for the activity of xylanase occurred at pH 6.0 in phosphate buffer, while the optimum temperature was 60°C. The relative xylanase activity in the pH ranges of 4–9 remained between 59.93 and 54.43% of the activity at pH 6.0 (100.00%). The xylanase activity showed a half-life of 172 min at 70°C, which was reduced to 75 min at 80°C. The enzyme was highly inhibited by 10–100 mM of Hg⁺², EDTA, Mg⁺², SDS and 100 mM Cu⁺². Clarity of fruit juices increased after enzymatic treatment of apple (17.85%), grape (17.19%) and orange juice (18.36%) with partially purified xylanase and also reducing sugar concentrations of these fruit juices were improved by 17.21, 16.79 and 19.57%, respectively. Also, dough volume was raised 17.06% with using partially purified Streptomyces sp. AOA40 xylanase in bread making.     

Purification and Properties of Beta-galactosidase from Streptococcus thermophilus

A β-galactosidase from Streptococcus thermophilus was purified to homogeneity by ammonium sulfate and acetone fractionation, gel filtration on Sephadex G-200, and ion exchange chromatography on DEAE-Sephadex A-50. The purified enzyme preparation exhibited an optimum pH at 6.6–7.0 and an optimum temperature of 57°C. The enzyme was stable at pH 6.8–7.0. Km and V_max for the enzyme, using ortho-nitrophenyl β-D-galactopyranoside as the substrate, were 0.25 mM and 83 μmoles/mg protein/min, respectively. It was strongly inhibited by Hg⁺⁺, Ag⁺, and Cu⁺⁺ as well as pchloro-mercuri benzoate. The enzyme had a molecular weight of about 6 × 10⁵ and was highly specific for β-galactoside bonds.     

Purification of New β-Galactosidase from Enterococcus faecium MTCC 5153 with Transgalactosylation Activity

A new β-galactosidase (β-gal) was purified from a lactic acid bacterial strain of Enterococcus faecium MTCC5153 by chromatographic techniques. The purified enzyme had a specific activity of 24.06 U/mg of protein with k _m and V_max values of 2 mM and 18.2 mM/min/mg of protein, respectively. The yield of purified β-gal was 10.65% and estimated molecular weight found to be ～90 kDa, consisting of two homodimeric subunits of 43kDa. The enzyme was stable in pH range of 8.0–9.0 with an optimum pH of 8 and the optimum temperature of 40°C. The enzyme was activated in the presence of metal ions such as Mg⁺², Mn⁺², Ca⁺², K⁺ and Na⁺ and was inhibited by Zn⁺², Co⁺² and Cu⁺². Chemical modifiers (N-bromosuccinamide and Diethylpyro carbonate) inactivated the enzyme indicating the role of tryptophan and histidine moieties for activity. The purified β-gal was able to synthesize oligosaccharides from lactose. This study suggests that the β-gal of Enterococcus faecium MTCC5153 could be applied in dairy industry for hydrolysis of lactose and to improve its digestibility. β-gal of probiotic cultures are of particular interest due to their transgalactosylation properties.     

Voluminosity of some food proteins in aqueous dispersions at various pH and ionic strengths

Commercial casein and whey protein and laboratory preparation of soybean protein of various pH were dispersed in distilled water at the initial concentration of 4.0% w/v and then diluted with NaCl solutions of known ionic strengths (μ). Viscosity was measured and voluminosity (V_e=ml/g) was calculated using Lee’s equation relating volume fraction (φ_v) to relative viscosity: η/η_o=1+2.5φ_v+7.031φ²_v+37.371φ³_v. The combined effects of pH and μ on V_e were determined from regression analysis. V_e was inversely related to μ between 0.005 and 0.07 M and increased with pH between 6 and 8 and decreased at higher pH. The results are discussed in terms of the relationship between the protein electrostatic charges and structure. It is concluded that in associative proteins V_e is controlled by the number of electrostatic charges and their strength.     

Purification and Characterization of a Halotolerant Alkaline Serine Protease from Penicillium citrinum YL-1 Isolated from Traditional Chinese Fish Sauce

A halotolerant alkaline serine protease from Penicillium citrinum YL-1 which was isolated from traditional Chinese fish sauce was purified by ammonium sulfate precipitation, dialysis, and DEAE 52-Cellulose column, thereby resulting in a 4.66-fold increase in specific activity (110.68 U/mg). The molecular weight (MW) was estimated to be 32.27 kDa using SDS-PAGE analysis. The protease exhibited optimal activity toward the substrate casein at pH 8.0 at 40°C and was stable at pH 6.0–8.0 and 4–30°C. Activity was inhibited by NaCl and retained at 28.3, 21.4 and 18.1% of the initial activity after incubation for 6 h at 20, 25 and 30% NaCl concentrations, respectively. The enzyme was stimulated by Mn²⁺ and inhibited by K⁺, Ca²⁺, Zn²⁺, Mg²⁺, Fe²⁺, and Fe³⁺. K_m and V_max of the protease for casein were 1.93 mg/ml and 56.81 μg/(min·ml), respectively. Protease activity was strongly inhibited by phenylmethyl sulfonylfluoride (PMSF), which confirmed the serine protease nature of the enzyme. The protease can hydrolyze tilapia protein in the absence or presence of NaCl (5–30%), thus suggesting that this protease is more halotolerant than the protease from other bacteria with high salinity resistance based on the current literature. These properties make the halotolerant alkaline serine protease a suitable candidate enzyme for fish protein hydrolysis during fish sauce fermentation.     

The effect of interaction between genotype CAST/RsaI (calpastatin) and MYOG/MspI (myogenin) on carcass and meat quality in pigs free of RYR1(T) allele

The purpose of the studies was to demonstrate to what degree genotypes of calpastatin (CAST/RsaI) and myogenin (MYOG) genes as well as the interaction between them may affect the carcass and meat quality of pigs. The investigations were conducted on 397 stress resistant pigs (free of RYR1^T allele). It was demonstrated that the favourable effect of the variants of CAST and MYOG genes on carcass quality traits depends on the cut. The gene variant favourably affecting the weight of ham simultaneously had a negative effect on the weight of the loin. It was also shown that the interaction between CAST and MYOG genotypes has a significant effect on backfat thickness. The effect of a given combination of CAST and MYOG genotypes on carcass traits is related to the weight of a substantial cut (ham, loin).Genotypes at loci CAST/RsaI and MYOG have a significant effect on the value of certain traits and parameters of meat quality and its technological value (genotype CAST on pH at 35 min and 2, 3, 24, 48, 96, 144 h post-mortem (pH₃₅, pH₂, pH₃, pH₂₄, pH₄₈, pH₉₆, pH₁₄₄, respectively), R₁ (IMP/ATP), electrical conductivity at 3 and 4 h post-mortem (EC₃, EC₄), technological yield of meat in curing and thermal processing (TY) and protein content in the muscle tissue, while genotype MYOG on pH₄₈, EC₃₅, EC₃, EC₂₄ and dry matter content).     

PURIFICATION AND PROPERTIES OF CYCLODEXTRIN GLUCANOTRANSFERASE FROM AN ALKALOPHILIC BACILLUS sp. 7-12

Cyclodextrin glucanotransferases (EC 2.4.1.19) (CGTase) are industrially important enzymes for production of cyclodextrin (CD) from starch. γ‐CD yield of CGTase from alkalophilic Bacillus species is usually much lower than β‐CD, while from alkalophilic Bacillus sp. 7‐12. γ‐CD yield is close to β‐CD. A CGTase from alkalophilic Bacillus sp. 7‐12 was purified and characterized. When purified by ammonium sulfate fractionation, DEAE‐cellulose column chromatography and Sepharose CL‐6B column chromatography, the enzyme obtained consisted of a single band that did not dissociate into subunits by SDS polyacrylamide gel electrophoresis. Molecular weight of the purified enzyme was determined to be 69,000 Da by SDS‐PAGE. The enzyme showed a K_mof 1.24 mg/mL and V_max0.101 µM/min when potato starch was used as substrate. The enzyme was stable below 70C with an optimum activity at 60C, and stable at pH range 6–10 with an optimum pH at 8.5. The enzyme activity was strongly inhibited by Ag⁺, Cu²⁺, Mg²⁺, Al³⁺, Co²⁺, Zn²⁺, Fe²⁺and slightly inhibited by Sn²⁺, Mn²⁺. The ions Ca²⁺and K⁺, EDTA and DTT had no influence on the enzyme activity.