Identification and characterization of cellular locus of limonin biotransforming enzyme in Pseudomonas putida

Limonin is a highly oxygenated triterpene derivative of class of limonoids which causes delayed bitterness in citrus. Pseudomonas putida (P. putida) is able to reduce 47% and 63% of limonin from juice serum extract and standard limonin, respectively. Biochemical studies with P. putida indicate that probably two metabolic pathways viz. 17‐dehydrolimonoid and deoxylimonoid pathway exists in the test organism. Experimental results indicate that the enzyme limonoate dehydrogenase which is found to be localized in periplasmic space of P. putida plays a major role in conversion of limonin to 17‐dehydrolimonoate A‐ring lactone. Enzymatic studies have shown a 72% reduction in limonin. The experimental results show 9 folds reduction in limonin content in presence of NAD as cofactor. The molecular weight of one of its polypeptide is found to be 66 kDa. The optimum pH and temperature for enzyme activity is 8.5 and 30 °C, respectively.     

The effects of caffeine intake on weight loss: a systematic review and dos-response meta-analysis of randomized controlled trials

Abstract

This systematic review and meta-analysis of randomized controlled trials (RCTs) was performed to summarize the effect of caffeine intake on weight loss. We searched the following databases until November 2017: MEDLINE, EMBASE, Web of Science, and Cochrane Central Register of Controlled Trials. The relevant data were extracted and assessed for quality of the studies according to the Cochrane risk of bias tool. We estimated an intake-status regression coefficient (Beta) for each primary study and estimated the overall pooled Beta and SE using random effects meta-analysis on a double-log scale. Heterogeneity between studies was assessed by the Cochran Q statistic and I-squared tests (I²). Thirteen RCTs with 606 participants were included in the meta-analyses. The overall pooled Beta for the effect of caffeine intake was 0.29 (95%CI: 0.19, 0.40; Q = 124.5, I²?=?91.2%) for weigh, 0.23 (95%CI: 0.09, 0.36; Q = 71.0, I²?=?93.0%) for BMI, and 0.36 (95% CI: 0.24, 0.48; Q = 167.36, I²?=?94.0%) for fat mass. For every doubling in caffeine intake, the mean reduction in weight, BMI, and fat mass increased 2 Beta-fold (20.29 = 1.22, 20.23 = 1.17, and 20.36 = 1.28), which corresponding to 22, 17, and 28 percent, respectively. Overall, the current meta-analysis demonstrated that caffeine intake might promote weight, BMI and body fat reduction.     

进口肉类中恶臭假单胞菌的分离与鉴定

目的选择具有较高选择性的培养基,分离与鉴定进口肉类中恶臭假单胞菌。方法参照GB4789.4-2010《食品安全国家标准食品微生物学沙门氏菌检验》、SN/T2099-2008《进出口食品中绿脓杆菌检测方法》,根据VITEK2显示的结果做生化鉴定。结果在选择性较高的沙门氏菌显色培养基上生长的菌落形态与沙门氏菌极为相似,VITEK2显示结果为恶臭假单胞菌,后经生化鉴定,结果与VITEK2一致,为恶臭假单胞菌。结论沙门氏菌显色培养基难以区分假单胞菌,但是亚硫酸铋琼脂(bismuth agar,BS)却可以明显区分,所以在日常检测过程中建议不要省去BS平板,在配制BS平板时需严格按照说明进行,以保证实验效果。     

Optimisation of a CE method for caffeine analysis in decaffeinated coffee

Multiple response simultaneous optimisation was used to develop a micellar electrokinetic chromatography method for caffeine determination in decaffeinated coffees. Buffer composition and voltage were optimised using a central composite design. Six responses were evaluated and each set of response values was regressed on the factor levels of the experimental design using linear and quadratic models. The regression models, correlation coefficients and a principal component analysis (PCA) were used to determine the optimum conditions. Successful results were obtained using a buffer containing 10 mmol L⁻¹ sodium carbonate and 50 mmol L⁻¹ sodium dodecylsulfate, 15 kV voltage, 25 °C temperature, 48 cm × 50 μm fused-silica capillary, hydrodynamic injection of 50 mBar during 7 s and detection at 206 nm. This optimised method was applied for caffeine analysis in 45 samples of commercial decaffeinated coffee.     

Focused microwaves-assisted extraction of theobromine and caffeine from cacao

With the aim of improving the extraction efficiency of Theobromine (TB) and Caffeine (CF) from cacao, a focused microwaves assisted method was developed. To optimize the extraction a 2³ factorial design was used with optimum values: 5 min of irradiation time, 210 W of power and 100 mL of the extractant (water). To quantify TB, a study about the reagents that must be added to the extracts (Carrez reagents: (Zn(CH₃COO)₂ and K₄Fe(CN)₆) and a Pb(CH₃COO)₂ solution) in order to eliminate interferences (molecular weight high compounds) was carried out. The extraction method was applied to cacao seeds, cacao powder and cacao peels. The focused microwave method was more efficient compared with a standard mechanic stirring method: for TB from 15.0% (in cacao seeds) to 72% (in cacao peels) and from 36% (in cacao peels) to 153% (in cacao seeds) for CF.     

恶臭假单胞杆菌肌酐酶在大肠杆菌中的表达及其酶学特性分析

肌酐酶(Creatininase)是肌酐酶法检测的一个关键酶,将恶臭假单胞菌(Pseudomonas putida)肌酐酶基因(Cre)克隆至原核表达载体p ET-28a(+),通过IPTG的诱导,实现了肌酐酶Cre在大肠杆菌(Escherichia coli)BL21(DE3)的可溶性表达。表达产物经60℃加热处理、Ni-NTA亲和层析和Sephadex G-200分子筛层析,分离纯化出重组肌酐酶,回收率为29.3%,其比酶活达到489.17 U/mg,是已有文献中报道中的最高水平。进一步进行酶学特性分析,结果表明其最适反应温度为60℃,50℃以下可以稳定保存,具有良好的热稳定性;最适p H值为7.0,在p H 7.0~8.0条件下比较稳定;Cu2+会抑制肌酐酶的活性;Mn2+、Zn2+和Co2+对肌酐酶有明显的激活作用;EDTA、SDS、Tween-20、Tween-80和Trinton-100几乎对酶活力没影响;Na N3不影响酶的活力。以肌酸为底物时,酶动力学常数Km值为47.38 mmol/L。本研究在大肠杆菌系统成功实现肌酐酶的可溶性表达,进行分离纯化并测定其酶学性质,为肌酐酶的表达和潜在工业化应用提供理论基础。     

Identification of endophytic bacterial strain MGP1 selected from papaya and its biocontrol effects on pathogens infecting harvested papaya fruit

BACKGROUND: Traditional approaches to the control of diseases of papaya fruit rely on the use of synthetic chemicals, which can cause serious human health and environmental problems. Endophytes might be used as an alternative to chemicals to effectively control diseases of harvested papaya fruit. RESULTS: MGP1 was one of the biological control agents that was selected from the pericarp of papaya and identified as Pseudomonas putida biovar A. The bacterium was able to colonise in the lamina, leafstalk, pericarp and pulp of papaya and strongly inhibit ten kinds of phytopathogen. Positive control effects were achieved when fruits were challenged with Phytophthora nicotianae at 24 and 48 h after MGP1 treatment. The control effect of MGP1 on anthracnose of harvested papaya fruit reached 54%. The application of MGP1 at five preharvest stages of papaya significantly reduced the disease index of anthracnose, with the best control effect reaching 63% after application at the florescence stage. However, the rate of latent infection of Colletotrichum gloeosporioides was significantly reduced only after application at the florescence stage. CONCLUSION: The results indicated the powerful ability of MGP1 as a biological agent. Copyright © 2009 Society of Chemical Industry     

恶臭假单胞菌产精氨酸脱亚胺酶发酵条件及特性的研究

对恶臭假单胞菌N0705产精氨酸脱亚胺酶的发酵条件及部分酶学性质进行了研究.结果表明,在培养温度28℃,培养基初始pH值为6.6,接种量4%,装液量40mL/250mL,发酵时间50h时,精氨酸脱亚胺酶酶活最大.精氨酸脱亚胺酶发酵粗酶液在温度37℃及pH 6.0～7.0时有较好的稳定性.     

Fast chromatographic determination of caffeine in food using a capillary hexyl methacrylate monolithic column

Caffeine beverages are widely consumed in the world, so new analytical techniques providing fast and reliable data are essential for a rapid and accurate evaluation of food quality. A capillary hexyl methacrylate monolithic column was designed and prepared for simple, rapid, economical and sensitive high performance liquid chromatography method for the determination of caffeine in food, using a water/acetonitrile (90:10, v/v) mobile phase with ultraviolet (UV) detection. The method was validated over the range 0.16–250 μg/mL of caffeine concentration and found to be linear (r > 0.995, n = 5) with relative standard deviation (RSD) less than 4.0%.     

Column-chromatographic extraction and separation of polyphenols, caffeine and theanine from green tea

A highly efficient column-chromatographic extraction (CCE) followed by sequential adsorption to extract and separate bioactive compounds from green tea was developed. Tea powder was loaded into columns with 4-fold solvents and eluted through a cyclic CCE. High-quality tea extracts with greater than 90% extraction efficiencies of polyphenols, epigallocatechin-3-gallate, caffeine, theanine and polysaccharides were obtained with 4-fold water circulated five times among different columns at 70 °C. Similar results, except for low polysaccharide extraction (35.5%), were obtained with 4-fold 30% ethanol circulated three times at room temperature. The highly concentrated water extraction was directly passed through columns of polyamide, DM130 macroporous and 732 ion exchange resins, resulting in high-purity polyphenols (99%), caffeine (98%) and theanine (98%) after simple purification of the eluates from each column. This method uses simple equipment, minimum solvents and can be used for both quantitative analysis and continuous preparation of high-quality tea extracts and bioactive compounds.     

Selective elution of tea catechins and caffeine from polyvinylpolypyrrolidone

Desorption of catechins and caffeine from polyvinylpolypyrrolidone (PVPP) was comprehensively investigated. The result showed that caffeine could easily be desorbed from PVPP by the tested solvents except n‐hexane, while catechins could only be thoroughly done by dimethyl sulphoxide (DMSO) and N, N‐dimethylformamide (DMF). Excellent desorption efficiency of DMSO and DMF might be attributed to their high dipole moments and H‐bond potentials. Addition of ethanol was recommended considering the elution efficiency and fluidity, but ethanol volume should be <40% (v/v) for DMSO or 20% (v/v) for DMF. Desorption would get to equilibrium within 1 h and followed the pseudo‐second‐order model. Caffeine and catechins could be separately desorbed through two‐stage elution procedure, that is, water or 20% aqueous ethanol for desorbing caffeine and part of nongalloylated catechins and DMSO/ethanol (8/2, v/v) for eluting the remaining catechins. Highly purified catechins (~95%) with high level (~70%) galloylated catechins would be achieved when the desorption procedure was applied in column chromatograph.     

牡蛎冷冻干燥后复水特性及微观结构的研究

研究牡蛎冷冻干燥后的复水特性,将新鲜牡蛎分为直接冻干组(Fresh+FD组)、预煮处理后冻干组(Boiled+FD组)和自然干燥组(Fresh+ND组),考察干燥后牡蛎的干燥比、复水比、复水率和微观结构的差异。结果表明:两冻干组牡蛎的含水量远低于自然干燥组,在常温和沸水中的复水比和复水速率均大于自然干燥组,冷冻干燥更有利于牡蛎中水分的去除和复水;干燥后牡蛎闭壳肌微观形态优劣程度依次为Fresh+FD组>Boiled+FD组>Fresh+ND组,其中Fresh+FD组闭壳肌纤维细长、舒展,间隙均匀,Fresh+ND组闭壳肌纤维束堆叠,组织致密;常温复水80min,两冻干组牡蛎的复重系数和复水率均高于自然干燥组。     

Determination of theobromine,theophylline and caffeine in cocoa samples by a high-performance liquid chromatographic method with on-line sample cleanup in a switching-column system

A simple reversed-phase high-performance liquid chromatographic method was developed for the determination of theobromine, theophylline and caffeine in cocoa samples. In the sample cleanup step, the procedure involves an on-line solid-phase extraction of analytes from cocoa samples into a home-made dry-packed pre-column with ODS-C₁₈ using a column-switching system. The separation was performed on a C₁₈ Nova-Pak column (150 mm × 3.9 mm, 4 μm) using a mobile phase consisting of a solution of 20% of methanol in water under isocratic conditions, at a flow-rate of 1.4 ml/min. The validation method revealed quantitative recoveries (>95.0%) with a coefficients of variation <3.2% and it also provided a good precision for data validation. The overlap of sample cleanup, analysis and recondition of the precolumn increases the sample throughput to 8 samples/h. Furthermore, the proposed method was successfully applied to analysis of cocoa samples “Trinitario”, “Forastero” and “Criollo” grown in different seasons of the year and fermented for 3 and 7 days, respectively. The results showed a slight reduction in the theobromine and caffeine content according to the fermentation times. In the same way, the theobromine/caffeine ratio was assessed, with the purpose of establishing a correlation with the genotype of the studied samples.     

Storage stability of a tropical fruit (cashew apple,acerola, papaya,guava and passion fruit) mixed nectar added caffeine

Nectars are formulated beverages with a fruit juice or pulp as its main component. A blend of fruits can also be used, with the advantage of combining sensory and nutritional properties of different fruits. The beverages may be added with some energetic component, as caffeine, known by its stimulating effect on central nervous system. The objective of this study was to develop mixed nectar of cashew apple, papaya, guava, acerola fruit and passion fruit added caffeine. The beverage was prepared with 35% juice blend and 10% sugar, added with 100 mg of caffeine L^?1 and 60 mg of sodium metabisulfite (SO₂) L^?1 and 500 mg of sodium benzoate L^?1 as preservatives, packed in cleaned pasteurised bottles and heat processed at 90 °C for 30 s. Physicochemical, microbiological and sensory analyses of the beverage were performed initially (time zero) and during 6 months of storage at room temperature (about 25 °C). The products were microbiologically stable during storage. The vitamin C content decreased significantly throughout storage time, although it has remained relatively high. The product presented good sensory acceptance, which suggests its potential for market.     

Antimicrobial packaging of raw beef,pork and turkey using silver‐zeolite incorporated into the material

This study determined the efficacy of silver‐zeolite impregnated into wrapping paper to reduce the bacterial growth on raw beef, pork and turkey cuts. This was compared with that of regular butcher paper. The samples were inoculated with Pseudomonas putida (psychrotrophic spoilage bacterium) and stored on 4% silver‐zeolite and regular butcher paper for up to 4 days at 4 °C and 10 °C. Results showed that P. putida on the beef, pork and turkey samples did not increase in numbers after exposure to all paper packaging at 4 °C during the 4 days of storage. At 10 °C storage temperature, logarithmic growth patterns for the organisms were seen on all paper packaging. However, the growth rate was slower for the organisms on the silver‐zeolite paper. Storage on the silver‐zeolite paper accounted for one log cfu/sample mean reduction in viable cell count for the beef, pork and turkey samples when compared with the samples stored on the butcher paper at 3 days.     

1株产烟酸羟基化酶海洋细菌H9的分离鉴定及培养条件优化

利用96孔板和紫外显影相结合的定向快速筛选方法,从烟台渤海湾采集的腐败鱼尸上分离筛选得到1株高产烟酸羟基化酶的菌株H9,通过形态、生理生化特征和16S r DNA系统发育分析鉴定为恶臭假单胞菌(Pseudomonas putida),对其产酶培养条件进行优化,最适培养基和培养条件为:可溶性淀粉15 g/L、玉米浆15 g/L、烟酸10 g/L、K_2HPO_4 1.0 g/L、初始pH 7.0、培养温度25℃、接种量7%、500 mL三角瓶装液量50 mL。优化后烟酸羟基化酶活力达到0.37 U/mL,比优化前提高了68%,采用静息细胞转化生产6-羟基烟酸产率达到124.77 g/L,具有工业化生产6-羟基烟酸的应用前景。     

Simultaneous spectrophotometric determination of caffeine and theobromine in Iranian tea by artificial neural networks and its comparison with PLS

In order to determine the amount of caffeine and theobromine, spectrophotometry was used as a simple, rapid and economical method. Because of severe overlapping between these components, artificial neural network was used. The 230–300 nm spectral window with 1 nm interval was used for data acquisition. An artificial neural network (5-5-3) with linear transfer function between input-hidden and hidden-output layers was trained and applied for prediction of concentration of these methylxanthines in four Iranian tea samples. The model was compared with PLS modeling method. HPLC technique was used as a standard method.     

A new method based on electrospray ionisation ion mobility spectrometry (ESI-IMS) for simultaneous determination of caffeine and theophylline

In this work for the first time, simultaneous analysis of caffeine and theophylline was done by ion mobility spectrometry (IMS) only, without a powerful separation technique (e.g., HPLC). Ion mobility spectrometry with low cost, inexpensive maintenance and very fast analysis makes an attractive technique for the simultaneous determination of the caffeine and theophylline in foodstuff samples and biological matrices. In this study, the extraction protocol using molecular imprinted polymer-solid phase extraction (MIP-SPE) was successfully used to directly extract caffeine and theophylline from real samples. The results obtained provided the detection limits of 0.2 and 0.3 μg mL⁻¹ for caffeine and theophylline, respectively. The linear dynamic range of about two orders of magnitude was obtained for these compounds. Also, the proposed method was used to analyse various real samples of green tea and spiked human plasma, and the obtained results confirmed the capability of ESI-IMS for simultaneous detection of caffeine and theophylline.