Integration of gas chromatography mass spectrometry methods for differentiating ricin preparation methods

The investigation of crimes involving chemical or biological agents is infrequent, but presents unique analytical challenges. The protein toxin ricin is encountered more frequently than other agents and is found in the seeds of Ricinus communis, commonly known as the castor plant. Typically, the toxin is extracted from castor seeds utilizing a variety of different recipes that result in varying purity of the toxin. Moreover, these various purification steps can also leave or differentially remove a variety of exogenous and endogenous residual components with the toxin that may indicate the type and number of purification steps involved. We have applied three gas chromatography-mass spectrometry (GC-MS) based analytical methods to measure the variation in seed carbohydrates and castor oil ricinoleic acid, as well as the presence of solvents used for purification. These methods were applied to the same samples prepared using four previously identified toxin preparation methods, starting from four varieties of castor seeds. The individual data sets for seed carbohydrate profiles, ricinoleic acid, or acetone amount each provided information capable of differentiating different types of toxin preparations across seed types. However, the integration of the data sets using multivariate factor analysis provided a clear distinction of all samples based on the preparation method, independent of the seed source. In particular, the abundance of mannose, arabinose, fucose, ricinoleic acid, and acetone were shown to be important differentiating factors. These complementary tools provide a more confident determination of the method of toxin preparation than would be possible using a single analytical method.     

AOT/Triton X-100混合反胶束体系中假丝酵母脂肪酶催化蓖麻油水解的活性

研究了二(2-乙基己基)琥珀酸磺酸钠(AOT)/Triton X-100混合反胶束体系中假丝酵母脂肪酶(candida rugosa lipase)催化蓖麻油水解的反应. 考察了Triton X-100占总表面活性剂的摩尔分数(x(Triton X-100))、水与总体表面活性剂的摩尔比(ω0)、pH值、反应温度以及底物蓖麻油的浓度等因素对酶活性的影响. 研究结果表明, 加入非离子表面活性剂Triton X-100可以使假丝酵母脂肪酶的活性得到显著提高, 但是当底物蓖麻油的浓度大于0.24 mol·L-1时, 会对假丝酵母脂肪酶产生抑制作用.     

Optimized enzymatic synthesis of hesperidin fatty acid esters in a two-phase system containing ionic liquid

Response surface methodology (RSM) based on a five-level, three-variable central composite design (CCD) was employed for modeling and optimizing the conversion yield of the enzymatic acylation of hesperidin with decanoic acid using immobilized Candida antarctica lipase B (CALB) in a two-phase system containing [bmim]BF(4). The three variables studied (molar ratio of hesperidin to decanoic acid, [bmim]BF(4)/acetone ratio and lipase concentration) significantly affected the conversion yield of acylated hesperidin derivative. Verification experiments confirmed the validity of the predicted model. The lipase showed higher conversion degree in a two-phase system using [bmim]BF(4) and acetone compared to that in pure acetone. Under the optimal reaction conditions carried out in a single-step biocatalytic process when the water content was kept lower than 200 ppm, the maximum acylation yield was 53.6%.     

Lipase Production in Solid-State Fermentation Monitoring Biomass Growth of Aspergillus niger Using Digital Image Processing

The aim of this study was to monitor the biomass growth of Aspergillus niger in solid-state fermentation (SSF) for lipase production using digital image processing technique. The strain A. niger 11T53A14 was cultivated in SSF using wheat bran as support, which was enriched with 0.91% (m/v) of ammonium sulfate. The addition of several vegetable oils (castor, soybean, olive, corn, and palm oils) was investigated to enhance lipase production. The maximum lipase activity was obtained using 2% (m/m) castor oil. In these conditions, the growth was evaluated each 24 h for 5 days by the glycosamine content analysis and digital image processing. Lipase activity was also determined. The results indicated that the digital image process technique can be used to monitor biomass growth in a SSF process and to correlate biomass growth and enzyme activity. In addition, the immobilized esterification lipase activity was determined for the butyl oleate synthesis, with and without 50% v/v hexane, resulting in 650 and 120 U/g, respectively. The enzyme was also used for transesterification of soybean oil and ethanol with maximum yield of 2.4%, after 30 min of reaction.     

Catalysis in Organic Solvents with Lipase Immobilized by Sol-Gel Technique

A series of immobilized lipases were obtained by sol-gel process, using silica prepolymers prepared from tetramethoxysilane, methyltrimethoxysilane, propyltrimethoxysilane and (3-aminopropyl)triethoxysilane. The activities of these biocatalysts were compared with the lipase adsorbed on poly(methylhydroxysiloxane) and encapsulated into a silicone rubber, lipase entrapped in nanoporous silica matrix and commercial sol-gel lipase. Model reactions were the esterification of stearic acid and Corey lactone bisalcohol (an intermediate of prostaglandin synthesis). The positive effect of hydrophobic-hydrophilic interface, created by the addition of organosilanes, on the activity of biocatalysts was partially reduced by decreasing specific surface of mesopores. Hydrophobic solvents increased the activity of the lipase entrapped in tetramethoxysilane–methyltrimethoxysilane prepolymer in the sequence acetone < toluene < benzene < decane < hexane. The activity of silicone rubber-encapsulated biocatalysts was proportional to polymer swelling in organic solvents (hexane > toluene > acetone).     

Comparison of fatty acid profile and antioxidant potential of extracts of seven Citrus rootstock seeds

The extracts of seven Citrus rootstock seeds have been compared regarding fatty acid profile and antioxidant potential. Sour orange (Citrus aurantium L.) was found to contain the highest oil amount (34%), while the Poncirus trifoliata cultivars contained the highest percentage of unsaturated fatty acids (84-87%). In addition, the antioxidant properties of the extracts from defatted seeds have been evaluated by measuring their radical scavenging activity against 2,2'-diphenyl-1-picrylhydrazyl. The highest antioxidant activities were observed in the case of the acetone extract of sour orange and Citrumelo Swingle (76% and 75%, respectively), at a concentration of 0.17?mg?mL(-1). Moreover, the total phenolic content of the extracts, determined using the Folin-Ciocalteau reagent, was found to be correlated with the radical scavenging activity results. The acetone extracts of sour orange and Citrumelo Swingle exhibited the highest phenolic content [112.3 and 103.4?mg gallic acid equivalent?g(-1) dry sample weight, respectively].     

Phenolic content and antioxidant properties of soybean (Glycine max (L.) Merr.) seeds

The contents and antioxidant ability of various classes of phenolic compounds present in the seeds of twenty soybean hybrids were evaluated. Total phenolics, tannins and proanthocyanidins were determined spectrophotometrically, after extraction of seeds with 70% aqueous acetone. In addition, the flavonoid contents were determined. The antioxidant activity of aqueous acetone extracts was evaluated by the 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging activity assay. The highest contents of total phenolics were found in Serbian cultivar 1511 and Chinese cultivar LN92-7369, which also displayed the highest total antioxidant activity. Conversely, genotypes poor in phenolics also showed low levels of DPPH-radical scavenging activity. The results suggested that besides protein and oil contents, the phenolic contents should be also considered as an important characteristic feature of soybean seeds, and as a potential selection criterion for antioxidant activity in soybean.     

Concentration,Partial Characterization,and Immobilization of Lipase Extract from <Emphasis Type="Italic">P. brevicompactum</Emphasis> by Solid-State Fermentation of Babassu Cake and Castor Bean Cake

One relevant limitation hindering the industrial application of microbial lipases has been attributed to their production cost, which is determined by the production yield, enzyme stability among other. The objective of this work was to evaluate the concentration and immobilization of lipase extracts from Penicillium brevicompactum obtained by solid-state fermentation of babassu cake and castor bean cake. The precipitation with ammonium sulfate 60% of saturation of crude extract obtained with babassu cake as raw material showed an enhancement in hydrolytic and esterification activities from 31.82 to 227.57 U/g and from 170.92 to 207.40 U/g, respectively. Concentrated lipase extracts showed preference to medium-chain triglycerides and fatty acids. It is shown that the enzyme activity is maintained during storage at low temperatures (4 and −10°C) for up to 30 days. Higher esterification activities were achieved when the lipase extract was immobilized in sodium alginate and activated coal.     

Lipase Immobilized in Organic-Inorganic Matrices

Enzyme lipase was immobilized with ferrite powder and deposited in layers on glass slides from lipase to a solution of silicone alkoxides. The highest hydrolytical activity was observed with the magnetic lipase prepared by mixing the paste of ferrite powder and lipase with tetramethoxysilane, 3-aminopropyltriethoxysilane and propyltrimethoxysilane. In a mixed reactor, the particles of the magnetic lipase were desintegrated by mechanical stirring which caused loosing the lipase linked to magnetic material and resulted in a significant drop of activity after magnetic separation. Transparent layers were prepared by dip- or spin-coating from partially hydrolyzed tetraethoxysilane and solutions containing methyltriethoxysilane with 3-aminopropyltriethoxysilane or tetraethoxysilane with 3-mercaptopropyltriethoxysilane. The lipases immobilized in films with magnetic particles were active in tests with 4-nitrophenyl butyrate and were not inhibited by 0,0-dimethyl-0-(2,2-dichlor-vinyl)-phosphate.     

A New Lipase Isolated from Oleaginous Seeds from <Emphasis Type="Italic">Pachira aquatica</Emphasis> (Bombacaceae)

A new lipase from seeds of Pachira aquatica was purified to homogeneity by SDS-PAGE obtaining an enzyme with a molecular weight of approximately 55 kDa. The purified lipase exhibited maximum activity at 40 degrees C and pH 8.0, for an incubation time of 90 min. Concerning temperature stability, at the range from 4 to 50 degrees C, it retained approximately 47% of its original activity for 3 h. The enzyme activity increased in the presence of Ca(++) and Mg(++), but was inhibited by Hg(++), Mn(++), Zn(++), Al(+++) and various oxidizing and reducing agents. The lipase was highly stable in the presence of organic solvents, and its activity was stimulated by methanol. The values of K(m) and V(max) were 1.65 mM and 37.3 micromol mL(-1) min(-1), respectively, using p-nitrophenylacetate as substrate. The enzyme showed preference for esters of long-chain fatty acids, but demonstrated significant activity against a wide range of substrates.     

Antiatherogenic properties of acetone extract of Alpinia zerumbet seeds

Oxidation of low-density lipoprotein (LDL) is the principal risk factor for the development of atherosclerosis. In this study, we used several methods to investigate the ability of the acetone extract from rhizomes, stems, leaves, flowers, pericarps and seeds of Alpinia zerumbet to inhibit atherosclerosis in vitro. The seed extract had the strongest activity against tyrosinase, pancreatic lipase (PL), 15-lipoxygenase (15-LO) and LDL oxidation activities (IC?? = 2.30 ± 0.02, 5.00 ± 0.07, 1.29 ± 0.07 and 15.40 ± 0.86 μg/mL, respectively), amongst all different parts. It also had similar effects to the positive controls. Most of the extracts showed partial agonistic properties towards estrogenic activity. Cholest-4-ene-3,6-dione, a steroid present only in the seed extract seems to be the compound responsible for these activities. The results showed that cholest-4-ene-3,6-dione had similar ability to curcumin and quercetin against PL and LDL oxidation (IC?? = 19.50 ± 1.17 and 16.12 ± 1.43 μg/mL, respectively). Furthermore, cholest-4-ene-3,6-dione (IC?? = 34.21 ± 1.31 μg/mL) had higher inhibition against 15-LO than quercetin (IC?? = 54.79 ± 1.12 μg/mL).     

General characterization of noncommercial microbial lipases in hydrolytic and synthetic reactions

Fourteen noncommercial preparations of microbial lipases were investigated with respect to their catalytic activity for hydrolysis and synthesis of ester bonds. Six of the lipases were derived from microorganisms that have not previously been described as lipase producers, and another four were characterized for the first time. The synthetic reactions were carried out in two solvents of different polarities (n-heptane and acetone) using a series of fatty acids and primary and secondary alcohols with different chain lengths. Under the culture conditions employed, Pseudomonas cepacia produced more active enzyme than the other microorganisms. The lipase preparations produced using Ovadendron sulphureo-ochraceum, Monascus mucoroides, Monascus sp., Fusarium oxysporum, Penicillium chrysogenum, Rhodotorula araucariae, Pseudomonas cepacia, Streptomyces halstedii, and Streptomyces sp. were the most efficient catalysts for hydrolysis at lipid-water interfaces. Enzyme preparations from P. cepacia, Streptomyces sp., S. halstedii, and R. araucariae were good biocatalysts for esterification in the polar medium (acetone). When the lipase preparations with the greatest activity for hydrolytic, reactions were excluded, regression analysis of the data for the hydrolytic and synthetic activities of the remaining lipase preparations yielded high multiple correllation coefficients for these reactions in both n-heptane and acetone (R=0.82 and 0.91, respectively).     

Studies on the factors that affect stereoselective esterification of (R,S) 2-octanol with octanoic acid catalyzed by lipase in organic solvent

Stereoselective esterification of(R, S) 2-octanol with octanoic acid catalyzed byCandida Sp lipase (CSL) was carried out in cyclohexane. We have studied the effects of factors, such as temperature and the microenvironment of lipase, on this reaction. The results showed that CSL favoredR enantiomer of(R, S) 2-octanol, and that the esterification activity and stereoselectivity of the lipase were dependent on these factors. The higher the temperature, the greater the esterification activity of CSL. A slight increase in stereoselectivity can be seen with temperature decrease. The optimal range of pH value for this reaction was 4.9–6.2. When the salt concentration was between 0 and 0.05 mol/L, CSL showed high activity. The salt concentration in the reaction system and the pH value at which CSL powder was prepared from the aqueous solution had no evident effect on the stereoselectivity of CSL. The optimal range of the water content in the reaction system was 0.4–1.6%. The esterification activity and the stereoselectivity of CSL were enhanced 1.4-fold and 2.0-fold, respectively, by immediately removing the produced water. (S) 2-octanol with 95.2% enantiomeric excess (ee) was prepared. Based on these results, we have discussed why that all these factors affected this reaction.     

Relationship between Phenolic Compounds,Antioxidant Properties,and the Allergenic Protein Mal d 1 in Different Selenium-Biofortified Apple Cultivars (Malus domestica)

Notable parts of the population in Europe suffer from allergies towards apples. To address this health problem, the analysis of the interactions of relevant allergens with other substances such as phenolic compounds is of particular importance. The aim of this study was to evaluate the correlations between the total phenolic content (TPC), polyphenol oxidase (PPO) activity, antioxidant activity (AOA), and the phenolic compound profile and the content of the allergenic protein Mal d 1 in six apple cultivars. It was found that the PPO activity and the content of individual phenolic compounds had an influence on the Mal d 1 content. With regard to the important constituents, flavan-3-ols and phenolic acids, it was found that apples with a higher content of chlorogenic acid and a low content of procyanidin trimers and/or epicatechin had a lower allergenic potential. This is probably based on the reaction of phenolic compounds (when oxidized by the endogenous PPO) with proteins, thus being able to change the conformation of the (allergenic) proteins, which further corresponds to a loss of antibody recognition. When apples were additionally biofortified with selenium, the composition of the apples, with regard to TPC, phenolic profile, AOA, and PPO, was significantly affected. Consequently, this innovative agronomic practice seems to be promising for reducing the allergenic potential of apples.     

The effect of pomegranate seed powder addition on radical scavenging activity determined by TLC–DPPH test and selected properties of gluten-free pasta

The aim of the present work was to study the effect of pomegranate seeds powder (PSP) supplementation to gluten-free (GF) sheeted pasta on cooking properties, sensory characteristics and antioxidants properties using TLC-DPPH test. Five levels of pomegranate seed powder were used (2.5, 5.0, 7.5, 10.0 and 12.5%) on formula replacement basis. Antioxidant potential of GF pasta increased with the addition of pomegranate seeds powder. The GF pasta without additives and with 2.5% concentration of PSP did not reveal ability to scavenge free radicals. The highest aforementioned activity was observed for crude pomegranate seed extract followed by GF pasta with 12.5, 10.0, 7.5 and 5.0% addition of PSP. The total dietary fibers content of pasta increased from 5.68 to 14.80?g/100?g with the increase in the incorporation of PSP from 0 to 12.5%. The results revealed that cooking loss of gluten-free pasta increased from 9.09 to 10.18%, whereas pasta firmness decreased from 381.43 to 366.30?N, upon incorporation of PSP. Pomegranate seeds powder decreased the lightness of the pasta from 82.26 to 57.27. Sensory analysis suggested that control pasta (without PSP) and pasta supplemented with low levels of PSP have the most acceptable quality. In conclusion, incorporating PSP at low levels enhanced the nutritional quality of pasta without a significant adverse effect on its cooking, textural and sensory properties.     

Solid-State Treatment of Castor Cake Employing the Enzymatic Cocktail Produced from <Emphasis Type="Italic">Pleurotus djamor</Emphasis> Fungi

In this work, the enzymatic cocktail produced by Pleurotus djamor fungi extracted at pH of 4.8 and 5.3 was employed for castor cake solid-state treatment. Proximal, X-ray powder diffraction and scanning electron microscopy analysis of the pristine castor cake were carried out. First, Pleurotus djamor stain was inoculated in castor cake for the enzymatic production and the enzymatic activity was determined. The maximum enzymatic activity was identified at days 14 (65.9 UI/gss) and 11 (140.3 UI/gss) for the enzymatic cocktail obtained at pH 5.3 and 4.8, respectively. Then, the enzymatic cocktail obtained at the highest enzymatic activity days was employed directly over castor cake. Lignin was degraded throughout incubation time achieving a 47 and 45% decrease for the cocktail produced at pH 4.8 and 5.3, correspondingly. These results were corroborated by the SEM and XRD analysis where a higher porosity and xylan degradation were perceived throughout the enzymatic treatment.     

Isolation, Characterization, and Catalytic Properties of a Novel Lipase Which Is Activated in Ionic Liquids and Organic Solvents

A novel extracellular lipase with organic solvent tolerance was isolated from a local Pseudomonas species. The lipase gene was cloned and expressed in Escherichia coli as a heterologous host and purified by affinity chromatography. The activity of purified lipase was investigated in the presence of imidazolium-based ionic liquids (ILs) such as EMIM[Cl], BMIM[Cl], and HMIM[Cl]. It has been found that the activity of treated lipase with ILs was higher than untreated control in the hydrolysis reaction. Also, the results indicated that the enzymatic activity strongly depends on IL concentration in reaction media. The best concentration of the IL was 30%, 45%, and 50% (v/v) for HMIM[Cl], BMIM[Cl], and EMIM[Cl], respectively. Additionally, the enzyme exhibited excellent stability in the presence of 25% of n-hexane, toluene, acetone, and t-butanol. The optimum values of pH and temperature were determined 10 and 55 °C, respectively. The K (m) and V (max) values were calculated 0.4 mM and 1.92 U/ml, respectively, using p-nitrophenyl palmitate as substrate. With respect to the biochemical properties of the newly isolated lipase such as high-level stability and noticeable activity in the presence of organic solvents and ionic liquids, the newly isolated lipase seems to be a good candidate for environmental and industrial processes carried out in non-aqueous media.     

Effects of Organic Solvents on Immobilized Lipase in Pectin Microspheres

Lipase from Brevibacillus agri 52 was found stable up to 90% diethylenglycol (DEG), glycerol (GLY), and 1,2 propanediol (1,2 PRO) at 37 degrees C for 1 h and the stability was reduced only approximately 20% after 12 h incubation, but in 40% dimethylsulfoxide (DMSO), lipase activity was stable only for 1 h. Inhibition of the biocatalysts with dimethylformamide (DMF) was detected at 20% solvent concentration. In water immiscible systems, the stability of lipase in n-hexane, n-tetradecane and n-heptane resembles the water activity, but in the presence of isobutanol, 1-hexanol, and butylbutirate, the stability was significantly reduced. Lipase 52 precipitates in the presence of 50% acetone or ethanol/water mixtures, but enzymatic activity was partially recovered by adding 20% GLY, DEG, 1,2 PRO, or DMSO to the reaction mixture. Furthermore, by increasing DEG in 70% DMF/DEG mixtures, the lipase activity was protected. Encapsulation of lipase in pectin gels cross-linked with calcium ions brings three to four times more enzymatic activity in 70% water miscible organic solvents compared to aqueous systems.     

L-asparaginase fromErwinia carotovora

A large-scale process was developed to purify L-asparaginase from submerged cultures of Erwinia carotovora. Cells from 880 L of fermentation broth were harvested and washed using a plate and frame type filter press. A cellular acetone powder was prepared from the washed cells by suspending the cells twice in acetone and the residual acetone was removed by washing the acetone powder in the filter press with 10 mM phosphate buffer (pH 7.0). The cellular acetone powder was extracted with 10 mM borate buffer at pH 9.5. The enzyme-rich borate extract was recovered by filtration and clarified by an in-line bag filter. The filtrate was adjusted to pH 7.5 and filtered through a 1-micron bag filter precoated with Celite and then through a 0.22-micron cartridge filter. The cell-free extract, containing 21 x 10(6) IU of enzyme and 448 g of total protein, was applied to an L-asparagine Sepharose 6 Fast Flow affinity column (9 L) using a bag filter loaded with Cell Debris Remover as an in-line prefilter. The affinity gel was prepared by coupling L-Asn at pH 9.0 to epoxy-activated Sepharose 6 Fast Flow beads. A total of 14 x 10(6) IU of enzyme (35 g protein) was eluted at pH 9.0 in 10.5 L. The eluted enzyme was determined to be greater than 90% pure using sodium dodecyl sulfate polyacrylamide gel electrophoresis. The total process time from whole broth to affinity column elution was 68 h and the enzyme yield was 38%. This improved process for the 880 L fermentation broth produced a cell-free extract of high specific activity, shortened the process time, increased the column capacity, and yielded a product with high purity.     

Anti-obesity effects of escins extracted from the seeds of Aesculus turbinata BLUME (Hippocastanaceae)

To investigate the anti-obesity effects of escins extracted from the seeds of Aesculus turbinata BLUME, anti-obesity models in vitro and in vivo were employed. In a preliminary experiment, different solvent fractions of Aesculus turbinata BlUME as well as two isolated compounds were tested for their effects on pancreatic lipase (PL) in vitro. Subsequently, female ICR mice were fed a high fat diet with or without different concentrations of total escins for 11 weeks to examine body weight, parametrial adipose tissue weight, and hepatic triacylglycerol (TG) and total cholesterol (TC) contents. Plasma triacylglycerol levels (TG) after oral administration of lipid emulsions to rats were also investigated. The results showed that total escins (1 mg/ml) as well as two compounds isolated from total escins, namely escin Ib and IIa, showed inhibitory effects on PL activity. In vivo, total escins suppressed the increase in body weight, parametrial adipose tissue weight, TG content, and TC content in mice's liver; TG content in rat plasma was also reduced at 1, 2 and 3 h after oral administration of the lipid emulsion plus different concentrations of escins compared to those in the lipid emulsion groups. Meanwhile, mice fed a high fat diet plus 2% total escins for 3 d had an increased TG level in the feces compared to the HF group. The reason for this may be due to a delay in the intestinal absorption of dietary fat by inhibiting PL activity.