Degradation of white wine haze proteins by Aspergillopepsin I and II during juice flash pasteurization

Bentonite is commonly used to remove grape proteins responsible for haze formation in white wines. Proteases potentially represent an alternative to bentonite, but so far none has shown satisfactory activity under winemaking conditions. A promising candidate is AGP, a mixture of Aspergillopepsins I and II.; a food grade, well characterized and inexpensive protease, active at wine pH and at high temperatures (60-80°C). AGP was added to two clarified grape juices with and without heat treatments (75°C, 1min) prior to fermentation. AGP showed some activity at fermentation temperatures (≈20% total protein reduction compared to control wine) and excellent activity when combined with juice heating (≈90% total protein reduction). The more heat stable grape proteins, i.e. those not contributing to wine hazing, were not affected by the treatments and therefore accounted for the remaining 10% of protein still in solution after the treatments. The main physicochemical parameters and sensorial characteristics of wines produced with AGP were not different from controls.     

Activity and stability enhancement of α-amylase treated with sub- and supercritical carbon dioxide

Various physical, chemical and genetic approaches have been applied in order to enhance enzyme stability and activity. In this study, the aim was to investigate the capability of sub- and supercritical carbon dioxide to alter the stability and activity of α-amylase as an alternative technique. The effects of operational parameters such as pressure (50-300 bar), temperature (28-80 °C), CO? flow (2-10 g min?1) and time (60-180 min) were evaluated in regard to the activity and stability of fungal based α-amylase from Aspergillus oryzea. The activity of untreated enzyme was determined as 17,726 μmol/ml/min. While both sub- and supercritical conditions enhanced the activity, the increase in flow rate had an adverse effect and the activity was decreased by 28.9% at a flow rate of 10 g min?1 under supercritical conditions. Nuclear magnetic resonance (NMR) spectra of untreated enzyme and treated samples exhibiting the lowest and the highest activities were almost identical except for the chemical shifts observed at the lowest activity sample from 4.0 to 4.4 ppm which were assigned to protons of hydrogen-bonded groups. Optimum conditions were determined as 240 bar, 41 °C, 4 g min?1 CO? flow and 150 min of process duration yielding 67.7% (29,728 μmol/ml/min) higher activity than the untreated enzyme providing fundamental basis for enzymatic applications.     

甜酒药中的菌群分析及保鲜甜酒制作

将传统生产的甜酒药进行微生物的种类分析,得出甜酒药中存在3大主要菌群:根霉3.6×107 CFU/g,细菌9.5x103 CFU/g和酵母茵1.7x 105 CFU/g.对分离出来的根霉进行淀粉水解酶的活性测定,得出甜酒药中根霉的淀粉水解酶活力为2380.6U.保鲜甜酒的制作最佳工艺为发酵时间为36h左右,发酵温度28...     

双孢菇菇柄酵素发酵条件优化及其抗氧化活性分析

本文以双孢菇菇柄为原料,以植物乳杆菌接种量、糖添加量和发酵时间为主要因素,总酚含量为评判指标,在单因素实验基础上,通过响应面试验研究优化了双孢菇菇柄酵素发酵工艺,并对酵素产品发酵前后及在4 ℃和25 ℃贮藏5 d的其抗氧化能力进行了研究。结果表明,菇柄酵素发酵的最佳条件为植物乳杆菌接种量3%、糖添加量9.50%、发酵时间24 h,酵素产品总酚含量值可达2.20 mg/mL,与预测值2.19 mg/mL相符;研究发现发酵后菇柄酵素的总酚、抗坏血酸含量分别为2.20 mg/mL与44.40 μg/mL,DPPH、ABTS自由基清除率、铁离子还原力和总抗氧化能力分别为51.93%、52.11%、0.70、28.09 U/mL,与发酵前比较有明显地提高。进一步对4 ℃和25 ℃贮藏条件下酵素的抗氧化活性分析表明,4 ℃贮藏的酵素,在贮藏0 d时DPPH、ABTS自由基清除能力、铁离子还原力和抗氧化能力均较强,在贮藏1 d后,酵素的抗氧化能力开始急剧下降,同时25 ℃条件下贮藏的酵素抗氧化能力显著低于4 ℃贮藏（P<0.05）。     

基于分子动力学模拟研究温度对乳酸脱氢酶活性的影响

乳酸脱氢酶是糖无氧酵解及糖异生的重要酶系之一，它能够催化丙酮酸形成乳酸，在食品发酵工业中具有很高的应用价值，但该酶易受高温影响，导致乳酸制品产量下降。为了研究不同温度对乳酸脱氢酶的构象及活性的影响，采用分子动力学模拟的方法，针对4种不同温度条件下（37、55、70和85 ℃）的乳酸脱氢酶分别进行了80 ns的计算模拟，分析了构象变化及酶活性中心的差异。研究发现，在37 和55 ℃条件下，乳酸脱氢酶比较稳定；当温度升高至70和85 ℃，乳酸脱氢酶的均方根误差、均方根波动、回旋半径值和溶剂可及表面积显著增加，而85 ℃ 时的蛋白二级结构已发生较大改变，这表明高温会导致蛋白质构象不稳定。对比37和85 ℃条件下该酶的底物丙酮酸的结合能力，发现高温会导致丙酮酸的结合位点残基之间的距离增大，进而破坏底物分子结合的微环境。因此，乳酸脱氢酶在温度超过70 ℃时发生变性，并随着温度的升高变性程度增加，进而导致酶活性丧失，不利于其在食品发酵等方面的应用。本研究在原子水平上分析了4种不同温度对乳酸脱氢酶的影响，揭示了其酶活性及构象变化的关键信息，为乳酸制品在发酵过程中选择合适温度提供了理论支撑。     

Changes in selected physical property and enzyme activity of rice and barley koji during fermentation and storage

Koji are solid-state fermentation products made by inoculating steamed grains with the spores of fungi, particularly Aspergillus spp. This research was undertaken to identify the fermentation and storage conditions optimal for the production and maintenance of selected hydrolytic enzymes, such as α-amlyase and protease, in koji. Steamed rice and barley were inoculated with 2 × 10 (11) Aspergillus oryzae spores per kilogram of grains and fermented for 118 h in a growth chamber at 28 to 32 °C with controlled relative humidities. Samples were drawn periodically during fermentation and storage at -20, 4, or 32 °C, and α-amylase and protease activity, mold counts, a(w), moisture contents, and pH of collected samples were determined. It was observed that the a(w), moisture contents, and pH of the koji were influenced by the duration of fermentation and temperature of storage. The α-amylase activity of both koji increased as the populations of A. oryzae increased during the exponential growth phase. The enzyme activity of barley koji was significantly higher than that of rice koji, reaching a peak activity of 211.87 or 116.57 U at 46 and 58 h, respectively, into the fermentation process. The enzyme activity in both products started to decrease once the mold culture entered the stationary growth phase. The protease activities of both koji were low and remained relatively stable during fermentation and storage. These results suggest that rice and barley koji can be used as sources of α-amylase and desired enzyme activity can be achieved by controlling the fermentation and storage conditions. PRACTICAL APPLICATION: Amylases and proteases are 2 important hydrolytic enzymes. In the food industry, these enzymes are used to break down starches and proteins while reducing the viscosity of foods. Although amylases and proteases are found in plants and animals, commercial enzymes are often produced using bacteria or molds through solid state fermentation, which is designed to use natural microbial process to produce enzymes in a controlled environment. A properly produced and maintained koji with a high hydrolytic enzyme activity can serve as an important source of the enzymes for the food industry.     

Effect of temperature on the fate of genes encoding tetracycline resistance and the integrase of class 1 integrons within anaerobic and aerobic digesters treating municipal wastewater solids

The objective of this research was to investigate the ability of anaerobic and aerobic digesters to reduce the quantity of antibiotic resistant bacteria in wastewater solids. Lab-scale digesters were operated at different temperatures (22 °C, 37 °C, 46 °C, and 55 °C) under both anaerobic and aerobic conditions and fed wastewater solids collected from a full-scale treatment facility. Quantitative PCR was used to track five genes encoding tetracycline resistance (tet(A), tet(L), tet(O), tet(W), and tet(X)) and the gene encoding the integrase (intI1) of class 1 integrons. Statistically significant reductions in the quantities of these genes occurred in the anaerobic reactors at 37 °C, 46 °C, and 55 °C, with the removal rates and removal efficiencies increasing as a function of temperature. The aerobic digesters, in contrast, were generally incapable of significantly decreasing gene quantities, although these digesters were operated at much shorter mean hydraulic residence times. This research suggests that high temperature anaerobic digestion of wastewater solids would be a suitable technology for eliminating various antibiotic resistance genes, an emerging pollutant of concern.     

Novel low-temperature-active, salt-tolerant and proteases-resistant endo-1,4-β-mannanase from a new Sphingomonas strain

Sphingomonas sp. JB13, isolated from slag of a >20-year-old phosphate rock-stacking site, showed the highest 16S rDNA (1343bp) identity of 97.2% with Sphingomonas sp. ERB1-3 (FJ948169) and <97% identities with other identified Sphingomonas strains. A mannanase-coding gene (1191bp) was cloned and encodes a 396-residue polypeptide (ManAJB13) showing the highest amino acid sequence identities of 56.2% with the putative glycosyl hydrolase (GH) family 26 endo-1,4-β-mannanase from Rhodothermus marinus (YP_004824245), and 44.2% with the identified GH 26 endo-1,4-β-mannanase from Cellvibrio japonicus (2VX5_A). The recombinant ManAJB13 (rManAJB13) was expressed in Escherichia coli BL21 (DE3). Purified rManAJB13 displayed the typical characteristics of low-temperature-active enzymes: showing apparent optimal at 40°C, ~55% of the maximum activity at 20°C and ~20% at 10°C, and thermolability at 45°C (~15min half-life). The potential mechanism for low-temperature-activity of GH 26 endo-1,4-β-mannanases might be ascribed to the more hydrophobic residues (AILFWV) and less polar residues (NCQSTY) compared with typical thermophilic and mesophilic counterparts. The purified rManAJB13 exhibited >85% mannanase activity at the concentration of 0-4.0M NaCl. No loss of enzyme activity was observed after incubating the enzyme with 1M or 2M NaCl, or trypsin or proteinase K at 37°C and pH 6.5 for 1h. The K(m), V(max) and k(cat) values were 5.0mgml(-1), 277.8μmol min(-1)mg(-1), and 211.9s(-1), respectively, using locust bean gum as the substrate.     

Gene cloning and biochemical characterization of a catalase from Gluconobacter oxydans

Gluconobacter oxydans has a large number of membrane-bound dehydrogenases linked to the respiratory chain that catalyze incomplete oxidation of a wide range of organic compounds by oxidative fermentation. Because the respiratory chain is a primary site of reactive oxygen species (ROS) production, the bacterium is expected to have a high capacity to detoxify nascent ROS. In the present study, a gene that encodes a catalase of G. oxydans, which might act as a potential scavenger of H(2)O(2), was cloned, and the expression product (termed rGoxCat) was characterized biochemically. rGoxCat is a heme b-containing tetrameric protein (molecular mass, 320 kDa) consisting of identical subunits. The recombinant enzyme displayed a strong catalase activity with a k(cat) of 6.28×10(4) s(-1) and a K(m) for H(2)O(2) of 61 mM; however, rGoxCat exhibited no peroxidase activity. These results, along with the phylogenetic position of the enzyme, provide conclusive evidence that rGoxCat is a monofunctional, large-subunit catalase. The enzyme was most stable in the pH range of 4-9, and greater than 60% of the original activity was retained after treatment at pH 3.0 and 40°C for 1h. Moreover, the enzyme exhibited excellent thermostability for a catalase from a mesophilic organism, retaining full activity after incubation for 30 min at 70°C. The observed catalytic properties of rGoxCat, as well as its stability in a slightly acidic environment, are consistent with its role in the elimination of nascent H(2)O(2) in a bacterium that produces a large amount of organic acid via oxidative fermentation.     

Family B DNA polymerase from a hyperthermophilic archaeon Pyrobaculum calidifontis: cloning, characterization and PCR application

The 2352 bp gene coding for 783 amino acid family B DNA polymerase from Pyrobaculum calidifontis was cloned and expressed in Escherichia coli. Expression of the gene resulted in the production of Pca-Pol in soluble fraction. After heat denaturation of the host proteins, the Pca-Pol was further purified by ion exchange and hydrophobic interaction chromatographies. Activity gel analysis showed the presence of a catalytically active polypeptide of about 90 kDa. The mass of the protein, determined by Matrix-Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry was found to be 89,156 Da. The isoelectric point of the enzyme was found to be 6.13. The optimal pH and magnesium ion concentration for the enzyme activity were 8.5 and 4mM, respectively. Unlike other commercially available DNA polymerases the enzyme activity of Pca-Pol was inhibited by monovalent cations such as ammonium and potassium. The half-life of the polymerase at 95 °C and 100 °C was 4.5h and 0.5h, respectively. The enzyme possessed 3'→5' exonuclease activity and was able to amplify, under suitable conditions, up to 7.5 kb DNA fragments by polymerase chain reaction which makes it a potential candidate for amplification of long DNA fragments.     

Effect of high pressure on the regulation of phosphorylase activity in pre-rigor rabbit muscle

The effects of high pressure (150 MPa) on the regulation of phosphorylase activity in pre-rigor rabbit muscles have been studied at 35° and 0°C. At 35°C muscle contracts, phosphorylase is activated and the muscle pH falls to 5·8 in 2 min. Coinciding with these changes, phosphorylase phosphatase activity falls rapidly, while phosphorylase kinase, although active for longer, loses its activity as the pH falls. Both of these enzymes are completely inactivated after 5 min under pressure, while phosphorylase still retains 80% of its activity under these conditions. The effects of pressure on the activities of these enzymes in white and red muscles of rabbits were compared, with a greater effect being observed in white muscles. At 0°C, muscles subjected to high pressure did not contract, but at this temperature the three enzyme activities (phosphorylase, phosphorylase kinase and phosphorylase phosphatase) were all lost at a greater rate than at 35°C, although the pH of the muscle did not fall below 6·5. The effects of high pressure treatment on isolated phosphorylase a and b and phosphorylase kinase were also studied at both 0° and 35°C and the results obtained closely paralleled those observed in whole muscle.     

An instant measurement of oxidoreductase activity above 100 degrees C by monitoring the absorbance change

A conventional absorbance monitoring method using a cuvette covered with a tight rubber cap was found to be applicable for measuring oxidoreductase activity at temperatures up to 115 degrees C. Using this method, the optimal temperatures of the enzymes, including oxygen-sensitive enzymes from a hyperthermophilic archaeon Thermococcus profundus, were determined.     

产酮基还原酶基因工程菌培养温度和转速条件的优化

考察了27 ℃、30 ℃、33 ℃、37 ℃条件下酮基还原酶基因工程菌生长与产酶的影响。结果发现,37 ℃以下随着温度的升高,菌体产酶活性得到提升和提前,37 ℃条件下10 h最大酶活达到了157.5 U/mL。但是所有温度条件下的发酵后期,酶活都出现了下降,发酵后期转速的下降可能是一个原因。因此对37 ℃下不同恒定转速200 r/min、300 r/min、400 r/min、480 r/min的发酵条件进行了实验。结果发现,转速的恒定对于菌体酶活的积累具有重要意义,恒定转速480 r/min条件下酶活逐渐升高,20 h的酶活为217.1 U/mL。此研究确定了酮基还原酶基因工程菌发酵产酶的最佳温度和转速,并对酮基还原酶进一步的补料分批发酵优化具有指导意义。     

Modelling post-mortem tenderisation-I: Texture of electrically stimulated and non-stimulated beef

Texture in electrically stimulated and non-stimulated beef M Pectoralis profundus, stored under a range of temperatures from 0 to 30°C, while avoiding muscle shortening, was measured from 1 to 21 days after stunning. The pre-rigor temperature (from 0 to 30°C), maintained until the pH had fallen to 6·4 and then held at 15°C, had no effect on the toughness nor on the rate of tenderisation after rigor. Modelling toughness prior to 24 h suggested that toughness of all muscles could be rationalised and that first-order tenderisation began when the muscles reached pH 6·1 when the toughness of all the muscles was projected to be 12·5 kg. After pH 6·1, the rate of tenderisation at 30°C was 10-fold higher than at 1°C and was not affected by variations in pH from 6·1 to 5·5. At the higher temperatures, the ultimate toughness of aged meat was slightly higher than at the lower temperatures.     

MACERATION ACTIVITY OF AN ENDOPOLYGALACTURONASE FROM CANDIDA MACEDONIENSIS

The yeast Candida macedoniensis produces constitutively an extracellular pectinolytic enzyme with a high maceration activity; the culture filtrate is free from foreign enzyme activity. The enzyme formation is optimal under strictly anaerobic conditions with N₂ gassing. The culture medium for optimal enzyme recovery is a nutrient solution containing 1% yeast extract, 2% peptone and 10% sucrose, at pH 3, 28°C. Characterization of the enzyme showed it to be an endopolygalacturonase. The pH and temperature optima of the enzyme differ for pectic acid cleavage and maceration activity, these being 4.5 and 50—53°C and 2.5 and 40°C, respectively. The enzyme activities could not be separated from one another by protein chemical methods (analytical and preparative isoelectric focussing). Dialysis of the enzyme-containing culture filtrate did not decrease the enzyme activity. The endopolygalacturonase from Candida macedoniensis leads to the release of plant cells from the tissue, without destroying the cells by lysing the cell walls. After two hours incubation of the substrate (carrot slices), the tissue mass consisted of cell clumps of up to 15 cells.     

Inactivation of Bacillus amyloliquefaciens spores by a combination of sucrose laurate and pressure-assisted thermal processing

The aim of this research was to study the effect of sucrose laurate ester (SL) on enhancing pressure-assisted thermal processing (PATP) inactivation of Bacillus amyloliquefaciens Fad 82 spores. B. amyloliquefaciens spores (～10? CFU/ml) were suspended in deionized water, solutions of 0.1, 0.5, and 1.0% SL, and mashed carrots without or with 1% SL. Samples were treated at 700 MPa and 105°C for 0 (come-up time), 1, 2, and 5 min and analyzed by pour-plating and most-probable-number techniques. Heat shock (80°C, 10 min) was applied to untreated and treated samples to study the germination rates. Results were also compared against samples treated by high pressure processing (700 MPa, 35°C) and thermal processing (105°C, 0.1 MPa). Among the combinations tested, SL at concentrations of 1.0% showed the best synergistic effect against spores of B. amyloliquefaciens when combined with PATP treatments. In the case of high pressure and thermal processing treatments, SL did not enhance spore inactivation at the conditions tested. These results suggest that SL is a promising antimicrobial compound that can help reduce the severity of PATP treatments.     

弱酸特性D-来苏糖异构酶分子改造及D-甘露糖生产

本文旨在对来源于Thermoprotei archaeon菌株的D-LI（最适pH6.5）进行弱酸特性改造,以期该酶在pH5.5条件下生产D-甘露糖,从而抑制原有的美拉德反应,减少分离成本。基于蛋白序列比对及酸碱氨基酸置换策略,首先设计了8个单点突变,而后从中选择3个优良单点突变体E82K、P105K、E165K进一步进行两两组合双点突变。结果表明双点突变体E82K/P105K较野生酶的最适pH由6.5迁移至6.0,且在pH5.5条件下的酶活是原始酶的3.4倍。该突变体在以D-果糖和D-甘露糖为底物时的动力学参数K_m值分别为78.77 mmol/L和328.12 mmol/L,k_cat/K_m值分别为15.37 mmol/L?1·min?1和48.92 mmol/L?1·min?1。以80 g/L的D-果糖为底物反应10 h后,双点突变体E82K/P105K在pH5.5反应时的转化率较接近于原始酶在pH6.5时的转化率,美拉德反应程度较原始酶降低了约3~4倍,为工业上利用D-LI生产D-甘露糖提供了可行的酶制剂。     

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.     

SCREENING FOR PECTINOLYTIC CANDIDA YEASTS: OPTIMIZATION AND CHARACTERIZATION OF THE ENZYMES

In screening 72 Candida strains for extracellular enzymes, one acidic and seven alkaline proteases, one amylase and six pectinases were found. Candida kefyr, Candida macedoniensis and four strains from Candida pseudotropicalis were pectinolytically active; Candida kefyr showed considerable activity also under aerobic conditions but the highest activities were attained under strictly anaerobic conditions with N₂ gassing. YNB and peptone were the best nitrogen sources as regards enzyme production. The highest enzyme activities were achieved with succinic acid (aerobic) and inulin (anaerobic) as carbon sources. Enzyme production under aerobic conditions was considerably increased at pH 3.0. The addition of inducers (pectins or pectin ballast substances) led only to very slightly increased enzyme production. From kinetic studies of the enzyme, optimum activity was found to lie at pH 5.0 and 50°C. Subsequent characterization of the enzyme showed it to be an endo-polygalacturonase. Eight to ten bands could be resolved by analytical isoelectric focussing. The K_m value was found to be 1.14 × 10^?5 mol. The maceration test, using potato, carrot and apple tissue as substrate, showed considerable maceration activity, especially in the case of apple.     

Extraction, partial characterization, and storage stability of β-glucosidase from propolis

Extraction and assay conditions for β-glucosidase from propolis were optimized. Highest enzyme activity was obtained in a citric acid-disodium hydrogen phosphate buffer at pH 6.0 with 2.5% insoluble polyvinylpyrrolidone at incubation temperature of 57 °C. β-Glucosidase activities were found in all freshly harvested propolis while β-glucosidase activities were scarcely present in the randomly bought propolis. Propolis was stored at -20 °C and 4 °C for 3 mo with almost no loss of β-glucosidase activity, but at room temperature the activity decreased exponentially with the increase of storage time. These results indicated that the activity of β-glucosidase could be a candidate for propolis-freshness index. β-Glucosidase from propolis was capable of hydrolyzing p-nitrophenyl-β-D-glucoside and p-nitrophenyl-β-D-galactoside, but lacked activity toward p-nitrophenyl-β-D-glucuronide, p-nitrophenyl-β-D-cellobioside, amygdalin, cellobiose, and gentiobiose. These results were consistent with the hypothesis that flavonoid glucosides were hydrolyzed by β-glucosidase during propolis collection and processing and provided a possible explanation for why some flavonoid biosides (that is, rutin and isorhamnetin-3-O-rutinoside) exist in propolis. Practical Application: β-Glucosidase activity was detected and partial characterization of the enzyme was determined in propolis. The enzyme activity decreased exponentially with the increase of storage time at room temperature, which suggested that the activity of β-glucosidase could be regarded as a freshness index of propolis. The research will be useful for studying the chemical constituents of propolis.