Production of d-galactose using β-galactosidase and Saccharomyces cerevisiae entrapped in poly(vinylalcohol) hydrogel

d-Galactose was produced from lactose (200 g l⁻¹) in the batch mode of a simultaneous saccharification and fermentation process (SSF). β-Galactosidases (from Kluyveromyces lactis and Aspergillus oryzae) and yeasts were immobilized in poly(vinylalcohol) hydrogel lens-shaped capsules – LentiKats^®. After 20 repeated batch runs with entrapped K. lactis, β-galactosidase and free Saccharomyces cerevisiae (10% v/v inoculum), galactose productivity decreased to 50% and 1.4 kg of galactose were prepared. Compared to this, just 20% decrease of galactose productivity and a 0.9 kg production of galactose were observed for the SSF process with β-galactosidase from A. oryzae after 15 repeated batches under the same conditions. In the process of SSF with co-immobilized enzyme from K.lactis and S.cerevisiae, the galactose productivity increased from 3 g l⁻¹ h⁻¹ to 4.1 g l⁻¹ h⁻¹, which reduced the time of preparation of d-galactose.     

The effect of gel composition on the adsorption of invertase on poly(acrylamide/maleic acid) hydrogels

The effects of external stimuli such as pH of solution, temperature, substrate concentration of solution and storage stability on the invertase adsorption capacity of poly(acrylamide/maleic acid) [P(AAm/MA)] hydrogels, synthesized by gamma irradiation of ternary mixtures of AAm/MA/Water, were investigated. The adsorption capacities of the hydrogels were found to increase from 4.0 to 13.3 mg invertase/g dry gel with increasing amount of MA in the gel system, while P(AAm) gel adsorbed only 3.1 mg invertase/g dry gel. Kinetic parameters were calculated as 20.6 mM for K_m and 6.44×10⁻⁵ mol/dm³ min for V_max for free enzyme and in the range of 23.6–57.7 mM for K_m and 8.62×10⁻⁵–1.05×10⁻⁴ mol/dm³ min for V_max, depending on the amount of MA in the hydrogel. Enzyme activities were found to increase from 50.0 to 74.0% with increasing amount of MA in the gel system and retained their activities for one month storage. The enzyme activities, after storage at 4°C for one month, were found to be 21.0 and 50.0–74.0% of the initial activity values for free and adsorbed enzyme, respectively. The optimal pH values for free and adsorbed enzymes were determined as 4.56 and 4.56–5.00. The optimum temperature for free and adsorbed enzymes was 55°C. Adsorption studies show that, not only gel composition but also the stimuli, temperature and pH of the solution, play important roles on the invertase adsorption capacity of poly(AAm/MA) hydrogels.     

Structural characterisation and antimutagenic activity of a novel polysaccharide isolated from Sepiella maindroni ink

A new heteropolysaccharide, named as SIP, was isolated from the ink of cuttlefish, Sepiella maindroni, by enzymolysis, anion-exchange and gel-permeation chromatography and tested for its antimutagenic activity. It was homogeneous with a molecular weight of 1.13 × 10⁴ Da by HPSEC–MALLS analysis. SIP contained glucuronic acid, mannose, N-acetylgalactosamine, and fucose in a molar ratio of 1:1:2:2. Its structural characteristics were investigated and elucidated by methylation analysis, GLC–MS, and NMR (¹H, ¹³C, H–H COSY, HMQC, HMBC, TOCSY and NOESY). The hexasaccharide repeating unit of SIP was found to be a backbone composed of fucose, N-acetylgalactosamine and mannose in a molar ratio of 2:2:1, and with a single branch of glucuronic acid at the C-3 position of mannose. According to the micronucleus test, SIP could significantly reduce the frequency of micronucleated cells in polychromatic erythrocytes and reticulocytes induced by cyclophosphamide in tumor-bearing mice, which revealed that SIP presented strong antimutagenic activity.     

Effect of cooling conditions on separation of volatile compounds in grappa using tray and packed columns without reflux

Marcs were distilled using an industrial batch distillation plant. The performance of a tray column and a packed column without reflux were studied under different conditions of vapour cooling obtained by changing the water flow rates (100–350 L h^?1). The temperature of cooling water was +12 °C. With this kind of plant the cooling conditions normally used for producing grappa in the tray column is a water flow of 200 L h^?1 and 250 L h^?1 in the packed column. Each distillate was collected in fractions and analysed for ethanol, lower volatile alcohols and esters. The results show that the tray column under a water flow rate of 200 L h^?1 has a strong rectification effect on the volatile compounds and it has a significant decrease in separation efficiency at 300 L h^?1. Instead, the packed column is poorly affected by the change of the water flow rates from 150 to 350 L h^?1 and a distillate very rich of volatile compounds can be obtained. It was concluded that in order to maintain the classical method of grappa production, which consists in cutting the initial and the final fractions of distillate, the packed column is the most suitable.     

Purification and biochemical properties of a fibrinolytic enzyme from Bacillus subtilis-fermented red bean

Natto-red bean with fibrinolytic activity was prepared by fermenting red beans with Bacillus subtilis. A fibrinolytic enzyme was purified from fermented natto-red bean by sequential steps of ammonium sulfate fractionation, Sephacryl S-200 HR gel filtration and PBE 94 chromatofocusing. Through these steps, the purity of the enzyme increased 291-fold with 1.5% activity recovery. SDS–PAGE and isoelectric focusing electrophoresis showed the molecular mass and pI of the purified enzyme to be 29.93 kDa and 6.35, respectively. When N-succinyl-Ala-Ala-Pro-Phe-ρNA was used as an enzyme substrate, the K_m, V_max, and optimal reaction pH and temperature were 0.59 mM, 79.4 μmole ρNA/min mg, 9 and 60 °C, respectively. Among the synthetic substrates, the most sensitive were N-succinyl-Ala-Ala-Pro-Phe-ρNA, followed by N-benzoyl-Val-Gly-Arg-ρNA. Chemical modifiers, such as phenylmethyl sulfonyfluoride, N-bromosuccinimide and N-ethyl-5-phenylisoxazolium-3′-sulfonate, almost completely inhibited the activity of the purified enzyme. These results indicated that the purified fibrinolytic enzyme was a subtilisin-like serine protease.     

High nisin-Z production during repeated-cycle batch cultures in supplemented whey permeate using immobilized Lactococcus lactis UL719

Nisin-Z production was studied during repeated-cycle pH-controlled batch (RCB) cultures using Lactococcus lactis subsp. lactis biovar. diacetylactis UL719 immobilized in κ-carrageenan/locust bean gum gel beads in supplemented whey permeate. After an initial colonization of gel beads during the first two cycles, nisin-Z production in bulk medium and gel beads was very similar for 1-h and 2-h cycle RCB cultures. A very high nisin-Z production (8200 IU mL⁻¹) was measured in the broth after the 1-h cycles, with a corresponding volumetric productivity of 5730 IU mL⁻¹ h⁻¹. This productivity is much higher than maximum nisin productivities reported in literature or maximum productivities obtained previously for free-cell batch cultures (850 IU mL⁻¹ h⁻¹), and free-cell (460 IU mL⁻¹ h⁻¹) or immobilized-cell (1760 IU mL⁻¹ h⁻¹) continuous cultures, using the same strain and fermentation conditions. The stability of RCB cultures was demonstrated for 24 and 36 1-h cycles carried out over 3 and 6-day periods, respectively. Changing environmental conditions during batch cultures resulted high nisin production.     

甘谷红芪酸性多糖的纯化及结构初探

本实验主要采用水提醇沉法提取甘谷红芪多糖,首先用DEAE-Sepharose Fast Flow 阴离子交换柱层析进行分离得到其中的一种酸性多糖,后者再经 Sepharose 6B Fast Flow凝胶层析纯化后浓缩、透析、冷冻干燥。琼脂糖凝胶电泳显色证明该组分为均一性多糖,再经过紫外、红外光谱分析以及酸水解产物薄层分析,分析结果表明该酸性多糖为杂多糖,其组成单糖可能分别为：木糖、葡萄糖醛酸或半乳糖醛酸、氨基葡萄糖盐酸盐或氨基半乳糖盐酸盐、葡萄糖醛酸内酯或半乳糖醛酸内酯,并且含有硫酸根。     

Adsorption of volatile aroma compound 2-phenylethanol from synthetic solution onto granular activated carbon in batch and continuous modes

This paper reports a study on the adsorption of the aroma component 2-phenylethanol from an aqueous solution onto granular activated carbon, derived from coconut husks, using batch and continuous (packed-bed column) systems. In the batch mode, the contact time required to attain the adsorption equilibrium was 90 min. The adsorption isotherms were evaluated at 20.0 °C, 30.0 °C and 40.0 °C. In the continuous mode, the effects of flow rate and bed length were studied. Experimental data showed that the breakthrough and exhaustion times decreased with higher flow rates and increased with higher packed beds. The adsorption in the fixed-bed column was more efficient under the following conditions: flow rate of 3.5 mL min⁻¹ and bed length of 11 cm. Based on the data acquired, adsorption appears to represent a promising method for the recovery of 2-phenylethanol lost during the instant coffee production process.     

Removal of methylene blue from aqueous media using activated carbon web

Activated carbon web is prepared by controlled pyrolysis of acrylic fibrous waste under the layer of charcoal using physical activation in high-temperature furnace. The carbonization was carried out at 1200 °C under different heating rate (i.e. 150 to 450 °C h^?1) with different holding time (i.e. 0 to 60 min) to decide optimum pyrolysis parameters. The heating rate of 300 °C h^?1 with no holding time revealed higher specific surface area of 280 m² g^?1. The prepared activated carbon web was later employed as adsorbent for removal of methylene blue from aqueous media. The effect of initial dye concentration, adsorbent dosage, stirring speed, and pH of solution was studied. The obtained results were later compared with adsorption isotherms (i.e. Langmuir and Freundlich). The Freundlich model was found to fit closely with results due to heterogeneous adsorption of dye molecules. Finally, virgin activated carbon and dye adsorbed activated carbon were tested for desorption behavior using differential scanning calorimetry and thermo gravimetric analysis. The significant reduction in desorption enthalpy from 172.46 to 52.43 J g^?1 is attributed to less adsorption energies of dye molecules on the surface of activated carbon due to nonhomogeneous distribution of active sites.     

Surface dilatational properties of whey protein and hydroxypropyl-methyl-cellulose mixed systems at the air–water interface

In this work we have studied the impact of the competitive adsorption of a whey protein concentrate (WPC) and three well characterized hydroxypropyl-methyl-cellulose (HPMCs), commercially known as E4 M, E50LV and F4 M, on the surface dilatational properties (surface dilatational modulus, E, surface dilatational elasticity, E_d, and loss angle tangent, tan δ) of mixed films adsorbed at the air–water interface. The increase in E_d values with adsorption time could be associated with biopolymer adsorption at the interface. The surface dilatational properties depend on the WPC and HPMC concentrations in the aqueous phase and on the WPC/HPMC ratio. Although the values of E_d were mainly determined by HPMC at short adsorption times, for mixed systems with the lowest protein concentration (1 × 10⁻⁴ wt%) the E_d values were close to those of HPMCs, even at long term adsorption. The values of tan δ indicate the formation of adsorbed mixed films with high viscoelasticity, with a gel structure, which in turn should be attributed to the association of biopolymer molecules occurring at the interface. Only one biopolymer is the dominant one in the solid character of these mixed systems. HPMC at high concentrations slightly reduced the long-term solid character of the films confirming the existence of competition for the air–water interface as expected with two surface-active biopolymers with high molecular weight.     

Reactivation of the cellulose acetate gel bead column used for the removal of limonin from citrus juices

Simple water washes reactivated a cellulose acetate gel bead column after its use to remove the bitter principle limonin from citrus juices on a pilot-plant scale, preferably after the passage of 10 bed volumes (BV) of juice. Initially, washes with water equal in volume to the treated juice were effective because they removed inhibitors of limonin adsorption but the column would eventually become poisoned. A regime was established for long term operation of the column from the results of monitoring the limonin content and chemical oxygen demand of the wash waters. These results could be fitted to standard mathematical equations for extraction processes and these equations were used, in conjunction with previously developed equations for the adsorption of limonin from citrus juices, to calculate the conditions for the steady-state operation of the column. A three-column unit is suggested as a suitable basis for the adsorptive treatment of juice within a continuous processing line. Ten BV of juice would be passed through two of the columns in series at 2.5 BV h^?1 reducing the limonin content by about 50-70% while the third column was being reactivated by the passage of 16 BV of warm water at 3.8 BV. After 4 h^?1, the first of the two linked columns would be reactivated and the second would become the first of the new pair of linked columns with the newly reactivated column as the second component.     

Purification and characterization of trypsin from the pyloric caeca of brownstripe red snapper (Lutjanus vitta)

Trypsin was purified from the pyloric caeca of brownstripe red snapper (Lutjanus vitta) by ammonium sulphate (40–60% saturation) precipitation, soybean trypsin inhibitor (SBTI)-Sepharose 4B column and DEAE-Sephacel column chromatography. Purified trypsin showed a single band on sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS–PAGE) and native-PAGE. A yield of 4.9% with the purification-fold of 20 was obtained. Trypsin had an apparent molecular weight of 23 kDa. SBTI and N-ρ-tosyl-l-lysine-chloromethylketone (TLCK) showed a strong inhibitory effect on the purified trypsin, while other protease inhibitors exhibited negligible inhibition. Trypsin had maximal activity at pH 8.5 and 60 °C for the hydrolysis of α-N-benzoyl-dl-arginine-ρ-nitroanilide (BAPNA). It was stable within the temperature range of 25–55 °C and pH range of 7.0–10.0. Purified trypsin had a Michaelis–Menten constant (K_m) and catalytic constant (k_cat) of 0.507 mM and 4.71 s⁻¹, respectively, when BAPNA was used as the substrate. For the hydrolysis of α-N-ρ-tosyl-l-arginine methyl ester (TAME), K_m and k_cat were 0.328 mM and 112 s⁻¹, respectively.     

Purification and characterization of transglutaminase from a newly isolated Streptomyces hygroscopicus

Transglutaminase (TGase, EC 2.3.2.13) from a Streptomyces hygroscopicus strain isolated from soil was purified from culture broth by ethanol precipitation, followed by successive chromatographies on CM-cellulose and Sephadex G-75 columns with a yield and purification-fold of 21.1% and 30%, respectively. The enzyme’s molecular weight was estimated as 38,000 Da by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The purified microbial transglutaminase (MTG) exhibited optimum activity at 37–45 °C and in a range of pH 6.0–7.0 for hydroxamate formation from N-carboxybenzoyl-l-glutaminyl-glycine and hydroxylamine. The enzyme was not stable above 50 °C and was stable within a pH range of 5.0–8.0 at lower temperature. The MTG was not inhibited by Ca²⁺ and ethylenediaminetetraacetic acid, suggesting it was calcium-independent. Purified MTG was strongly inactivated by 5,5′-dithiobis (2-nitrobenzoic acid), Cu²⁺, Zn²⁺, Pb²⁺, and Hg²⁺, suggesting that this enzyme could possess a thiol group at the active site. The MTG stability was strongly affected by ethanol concentration. The enzyme activity was slightly elevated at a lower concentration of ethanol at 25 °C.     

Isolation of immunoglobulin in yolk (IgY) and rabbit serum immunoglobulin G (IgG) specific against bovine lactoferrin by immunoaffinity chromatography

Hens were intramuscularly immunized and rabbits were subcutaneously immunized once every two weeks for 6 weeks using bovine lactoferrin (LF) as antigen. Antibody titers of both yolk (IgY) and rabbit serum (IgG) were as high as 1.68×10⁸ at the 6th and 8th weeks, respectively, after the initial immunization treatment. However, antibody titer against LF in yolk was 9.4×10⁷ at 16 weeks. While antibody titer of rabbit serum declined sharply to 2.1×10⁷ at the 12th week and to 2.6×10⁶ at the 13th week after the initial immunization. The purification efficiency (specific activity of purified antibody against LF/specific activity of the corresponding antiserum or yolk against LF) of rabbit serum IgG purified by laboratory-prepared LF-Sepharose 4B immunoaffinity column (0.05 mg LF/ml wet gel) was about 2400, similar to that of IgY purified by LF-Sepharose 4B immunoaffinity column. Different amounts (0–15.0 mg) of IgY purified by LF-Sepharose 4B immunoaffinity chromatography were applied to the same column to determine the binding capacity (q_m) and dissociation constant (K_d) of LF-Sepharose 4B immunoaffinity gel for IgY specific against LF. It was found that q_m was 0.81 mg IgY/ml wet gel (1.620 mg IgY/mg LF) and K_d was 6.4×10⁻⁶ M as determined by Langmuir-type adsorption isotherms.     

Fe~(3+)修饰磁性纳米粒子的制备与表征及对卵黄高磷蛋白的吸附作用

通过化学共沉法和表面功能化修饰得到硫酸软骨素钠和Fe~(3+)负载的磁性纳米粒子Fe_3O_4-CS@Fe~(3+)(CMNP@Fe~(3+)),并对该粒子的形貌等特性进行分析。透射电子显微镜观察显示CMNP@Fe~(3+)呈尺寸为20 nm的圆球形,分散性较好;磁滞回线结果表明该粒子具有超顺磁性;傅里叶变换红外光谱测定证明硫酸软骨素钠和Fe~(3+)已成功修饰在Fe_3O_4表面。利用磁性纳米粒子表面Fe~(3+)与卵黄高磷蛋白的强结合力,建立从蛋黄中磁性分离卵黄高磷蛋白的新方法,并对吸附过程的影响因素进行研究。结果发现当溶液pH 4.0、底物初始质量浓度10 mg/m L、吸附时间180 min时,磁性纳米粒子的吸附能力最强。利用动力学模型和等温吸附模型进行拟合,确定CMNP@Fe~(3+)吸附卵黄高磷蛋白的过程符合伪二级动力学模型和Freundlich等温吸附模型,并通过模型计算得到吸附平衡时卵黄高磷蛋白的理论吸附量为625.00 mg/g。该研究结果为鸡蛋中蛋白质实现磁性分离提供了依据。     

Phage adsorption on Enteropathogenic and Shiga Toxin-Producing Escherichia coli strains: Influence of physicochemical and physiological factors

Bacteriophages have proved to be useful tools against pathogenic Escherichia coli strains. The first step in the phage life cycle is the adsorption to the host cell surface. In the present work, three Myoviridae phages (DT1, DT5 and DT6) were used to characterize the adsorption process on three pathogenic E. coli strains, namely two Shiga-toxin producing E. coli (STEC) and one enteropathogenic E. coli (EPEC), in several conditions found on food. The influence of Na⁺, Mg^2 +, temperature, pH, periodate, proteinase K and physiological cell state on phage adsorption was investigated. The three phages evaluated showed high adsorption rates at pH 7.5 and 5.7 while they were moderate at the lowest pH evaluated (4.5). Sodium or magnesium ions were not indispensable for the adsorption of the three phages evaluated. Specifically, phage particles were adsorbed either in the presence or absence of Mg^2 +, while increasing Na⁺ concentration resulted in lower adsorption values for all the systems evaluated. Regarding temperature, phage adsorption was slightly affected at 4 °C and 50 °C, while it reached its maximum at 37 °C. Adsorption rates decreased after the thermal inactivation of cells, though, when chloramphenicol (as protein-synthesis inhibitor) was used, adsorption values on treated and untreated cells were similar. In addition, periodate was able to decrease phage adsorption, thus suggesting the receptors were carbohydrates in nature. All these results showed that the adsorption process was only partially affected and most conditions are suitable for the completion of the first step in the phage life cycle. Therefore, phages evaluated in this study can be used to prevent foodborne diseases on several food matrices since they are active in a wide range of environmental conditions.     

罗非鱼加工副产物不同部位硫酸软骨素的制备、理化性质及结构表征

采用双酶酶解、氯化十六烷基吡啶法从罗非鱼副产物不同部位（鱼头、鱼脊骨、鱼鳍、鱼尾）制备硫酸软骨素,利用紫外光谱、醋酸纤维电泳、红外光谱和高效液相色谱对其理化性质和结构特征进行分析。结果表明,鱼头、鱼尾所含硫酸软骨素主要为C型（Δdi6S）,含量分别为46.10%和41.01%;鱼脊骨、鱼鳍所含硫酸软骨素主要为A型（Δdi4S）,含量分别为71.86%和69.59%。纯度从高到低依次为鱼头90.37%、鱼尾83.33%、鱼脊骨59.76%、鱼鳍52.01%。鱼头、鱼脊骨、鱼鳍、鱼尾硫酸软骨素的单糖组成主要由不同比例的葡萄糖醛酸、葡萄糖和氨基半乳糖构成,mn依次为51 422、18 402、19 481、76 371。不同部位提取的硫酸软骨素的种类和组成有明显差异,其中罗非鱼头、鱼尾提取的硫酸软骨素与鲨鱼软骨来源硫酸软骨素结构相似,有望作为代替鲨鱼硫酸软骨素的潜在来源。     

Enhanced pH and thermal stabilities of invertase immobilized on montmorillonite K-10

Invertase was adsorbed onto micro-porous acid-activated montmorillonite clay (K-10) by two procedures, namely adsorption and covalent binding. The immobilized enzymes were characterized by XRD, surface area measurements and ²⁷Al NMR. XRD measurements revealed an expansion of clay layers due to immobilization which suggests that intercalation had taken place. Surface area measurements also support this observation. ²⁷Al NMR showed that interaction of enzyme with tetrahedral and octahedral Al changes with the immobilization procedure. Sucrose hydrolysis was performed in a batch reactor. The immobilized enzymes showed enhanced pH and thermal stabilities. Optimum pH and temperature were found to increase upon immobilization. The effectiveness factor (η) and Michaelis constant (K_m) suggest that diffusional resistances play a major role in the reaction. The immobilized invertase could be stored in buffer of pH 5 and 6 at 5 °C without any significant loss in activity for 20 days.     

鸡胸软骨多糖组分的分级及自由基清除活性研究

直接用木瓜蛋白酶水解鸡胸软骨,经三氯乙酸除蛋白质、乙醇沉淀、干燥得多糖粗品,采用DEAESepharoseFast Flow 离子交换柱色谱和Sepharose 6B Fast Flow 分离纯化粗多糖,并进行光谱学分析和自由基清除活性研究。结果显示：乙醇沉淀多糖的自由基清除活性显著高于初步透析后的粗多糖,纯化后的硫酸软素的活性最小。粗多糖经DEAE-Sepharose Fast Flow 离子交换柱层析和Sepharose 6B Fast Flow 凝胶柱层析后,得到硫酸软骨素和其他5 种非糖胺聚糖类多糖。5 种多糖的自由基清除活性均显著大于硫酸软骨素。     

正交实验法优化牛鼻软骨中硫酸软骨素的醇沉工艺

研究牛鼻软骨碱提液中硫酸软骨素的醇沉工艺。通过单因素和正交实验对醇沉工艺进行优化,确定的最佳条件是:乙醇浓度65%,乙酸钠浓度4.5%,pH6.0。在该条件下,提取率为14.81%,葡萄糖醛酸含量33.36%,硫酸软骨素纯度达85.9%。紫外光谱分析:样品在196nm处有多糖的吸收峰,未见蛋白质(280nm)与核酸(260nm)的特征吸收峰。