Hypovitaminosis D is a common condition among elderly individuals in temperate-climate countries, with a clear seasonal variation on 25 hydroxyvitamin D [(25(OH)D] levels, increasing after summer and decreasing after winter, but there are few data from sunny countries such as Brazil. We aimed to evaluate 25-hydroxyvitamin D concentrations and its determining factors, in individuals in the city of São Paulo belonging to different age groups and presenting different sun exposure habits.591 people were included as follows: 177 were living in institutions , 243 were individuals from the community , 99 were enrolled in physical activity program designed for the elderly  and 72 were young . Ionized calcium, PTH, 25(OH)D, creatinine and albumin were evaluated. ANOVA, Mann–Whitney and Kruskal Wallis tests, Pearson Linear Correlation and Multiple Regression were used in the statistical analysis.-(vitD-12.5)/62.36 and the 25(OH)D value above which correlation with PTH is lost is 75.0 nmol/L. In a multiple regression analysis having 25(OH)D concentration as the depending variable, the determining factors were PTH, ionized calcium and month of the year (p < 0.05).25(OH)D mean values during winter for the different groups were 36.1 ± 21.2 nmol/L (NH), 44.1 ± 24.0 nmol/L (CD), 78.9 ± 30.9 nmol/L (PA) and 69.6 ± 26.2 nmol/L (YOUNG) (p < 0.001) while during summer they were 42.1 ± 25.9 nmol/L, 59.1 ± 29.6 nmol/L, 91.6 ± 31.7 nmol/L and 103.6 ± 29.3 nmol/L, respectively (p < 0.001). The equation which predicts PTH values based on 25(OH)D concentration is PTH = 10 + 104.24.eMuch lower 25(OH)D values were found for the older individuals when compared to younger individuals. This finding is possibly due to age and habit-related differences in sunlight exposure. The existence of seasonal effects on 25(OH)D concentration throughout the year was evident for all the groups studied, except for the nursing home group. According to our data, PTH values tend to plateau above 75 nmol/L. Hypovitaminosis D is well documented, mainly among elderly populations in countries from the northern hemisphere -4. For tSão PAulo Vitamin DEvaluation Study) and its main objective was to recognize the factors that contribute to determining blood 25(OH)D concentrations in four different population groups in the city of São Paulo, Brazil .This study was named SPADES D -10.The first population studied was called NURSING HOMES (NH) and it was composed of individuals who live in two nursing homes in the city of São Paulo. Mostly these individuals are from low income families that have little access to health services and for this reason their health conditions were generally poor. Individuals showing creatinine values above 2.0 mg/dL, hypercalcemia, hypocalcemia and those who were confined to a bed were excluded from the study. The final sample was represented by 177 individuals, ages varying from 60 to 100 years. Ethnic distribution: 90.9% white and 9.1% black. Blood samples were drawn in April  and July  of 2001 [The second population was called COMMUNITY DWELLINGS (CD) and it originated from a cohort study (EPIDOSO) developed with people from the district around UNIFESP , one of the 55 districts that compose the city of São Paulo. This region is predominantly composed by a Caucasian middle class population. We analyzed the data from 250 individuals, evaluated in 2001, ten years after the beginning of the study. From those, seven were excluded from the study . The final population studied was composed by 243 individuals, ages varying from 61 to 97 years . Ethnic The third population was called PHYSICAL ACTIVITY (PA) and was composed of 101 individuals (54 women and 47 men), enrolled in a physical activity program destined to the elderly . Each voThe fourth population was designated YOUNG and was composed of 72 individuals, age varying from 17 to 35 years. Ethnic distribution: 75% white, 19.4% Asian and 5.6% black. The blood samples were obtained between August 2002 and February 2003 . In thisThe final population of the SPADES study was composed of 591 individuals.The blood samples were taken after an eight-hour fast. Ionized calcium, creatinine, albumin, 25(OH)D and intact parathyroid hormone (PTH) were measured. All serum samples were collected into refrigerated tubes, processed in refrigerated centrifuges, and frozen at −20°C until measured. The ionized calcium was measured after centrifugation.Creatinine and albumin were measured by an automatic colorimetric method . The ionized calcium was measured using a specific ion-electrode method . PTH (parathyroid hormone) was measured by an immunofluorimetric in house method  . The creThe 25(OH)D concentrations were determined by an immunoradiometric assay . Intra-assay coefficient of variation was 4.8%, and inter-assay coefficient of variation was 16.0% for the lowest values (mean: 35.5 nmol/L) and 3.0% for the highest control (mean: 154.0 nmol/L).To certify that the radiation conditions were similar through the years, we compared ultraviolet radiation during 4 consecutive years, and the UV year-pattern was repetitive .The exponential formula that predicts PTH values from 25(OH)D was created using the software Origin 5.0 . This same program was used for generating Figure The Mann–Whitney test was used to evaluate the differences between men and women, and ANOVA and Kruskal Wallis test were used to check for the difference between the groups according to their origin. The correlation between 25(OH)D and PTH with the other quantitative variables was evaluated through the Pearson Linear Correlation and Multiple Regression. Data are reported as Mean ± SD (Standard Deviation). Differences were considered significant when p < 0.05.A total of 591 individuals were included as follows: NH, 177 individuals ; CD, 243 individuals ; PA, 99 individuals  and YOUNG, 72 individuals . In Tables The laboratory results obtained for the group as a total and also separated by gender and subgroup of origin are described in Table Only 6.3% of the total population studied had made use of vitamin D supplements (dose varying from 200 to 400 IU a day) and these had a 25(OH)D average blood concentration of 69.1 ± 34.9 nmol/L, while the group that had not taken a vitamin D supplement showed a significantly lower blood concentration .Using the Pearson´s linear correlation and considering only individuals who were not using vitamin D supplements, significant correlations were found between 25(OH)D and age , PTH , albumin , creatinine clearence , ionized calcium , month of the year  and BMI . Creatinine did not show any significant correlation . Using a model of multiple linear regression, setting the age as the predictive variable and adjusting by ethnicity and gender, the following correlations were still significant: PTH (p = 0.01), ionized calcium (p = 0.048) and month of the year (p = 0.01).Considering the PTH values, significant correlations were found with the following variables: age , 25(OH)D , albumin , creatinine clearence , creatinine , ionized calcium  and BMI . Using a model of multiple linear regression, setting the age as the predictive variable, 25(OH)D (p = 0.001), creatinine (p = 0.001) and ionized calcium (p = 0.011) were still significant.-(vitD-12.5)/62.36. We also defined the concentration of 75.0 nmol/L as the cut-off value for which 25(OH)D concentrations did not correlate with PTH levels D concentration is expressed by the formula PTH = 10 + 104.24.eIn Table Within each age group, as age advances, progressively lower 25(OH)D and higher PTH concentrations were observed. 25OHD concentrations lower than 75.0 nmol/L were found for 85.7% of those older than 71 years, while the incidence of secondary hyperparathyroidism was 57% among those Table .et al. and van der Wielen et al. demonstrated that countries from southern Europe show higher incidence of hypovitaminosis D when compared to northern countries [Several reports have previously shown that the prevalence of hypovitaminosis D is high among the elderly in the United States and Europe (< 30 ng/mL), but there are few data from countries in South America, where sunlight is abundant throughout the year, such as Brazil -4. The aountries -5. Some ountries ,14.The city of São Paulo is crossed by the Tropic of Capricorn (23°S) and presents relatively mild winters, with temperatures rarely achieving 0°C (32°F). It is a large urban center, being the largest one in South America, with a predominant Caucasian population. It has more than 10 million inhabitants and a large concentration of high-rise buildings and industries. During winter there is a significant concentration of air pollutants which can absorb solar UVB radiation and, consequently, reducing vitamin D formation . Our eldAge was the variable that most significantly correlated with 25(OH)D plasma concentrations, as was demonstrated by a multiple regression model applied. Younger individuals had higher 25(OH)D concentrations Table . For thiIt is known that the 25(OH)D plasma concentrations follow a seasonal variation, demonstrated in places of more temperate weather ,18,19. Oet al. demonstrated that there are influences from life habits on 25(OH)D concentration  [Gannagé-Yared aluated) . Other aaluated) ,21. In taluated) . An intealuated) -24. ThisIn our study, it was possible to observe the correlation that exists between PTH and 25(OH)D, which was expressed through an exponential equation Figure , the samFor the multiple regression analysis, setting PTH level as the depending variable and age as the adjusted predictive variable, the evaluated parameters that maintained the significance in the model were 25(OH)D, creatinine and ionized calcium, variables which are known to exert influence on PTH levels ,29. Tablet al., the supplementation with at least 700 IU of vitamin D/d was enough to reduce the risk of vertebral and non-vertebral fractures [The primary objective of hypovitaminosis D correction is promoting the maximum intestinal calcium absorption, reducing the frequency of falls and correcting the secondary hyperparathyroidism . Accordiractures . Besidesractures . In spitractures ,4. Thereractures .These results point out to the need for governmental public health action, which could implement measures such as vitamin D supplement distribution to nursing homes, food supplementation and medical education about the high prevalence of this condition among the elderly, considering the associated risk for fractures.Positive aspects of our paper include that we evaluated a large number of adults from different populations collected in the same city and analyzed by the same method. It was possible to demonstrate that hypovitaminosis D is a public health problem even in Brazil. However what is still lacking is the relationship with vitamin D status with the amount of sun exposure.Due to the high degree of miscegenation of the Brazilian population, it is always difficult to classify them ethnically. Nevertheless, most of the evaluated individuals were white (84.4%). According to the 2000 Brazilian Demographic Census for the city of São Paulo, the white population is the majority . However, our data cannot be extrapolated to all different regions of the country, because, besides different latitudes and weather conditions, the population presents different genetic backgrounds and habits, which are important factors for the serum vitamin D status.In addition, several other factors potentially affect vitamin D status such as genetic factors, adiposity, dietary intake, intestinal malabsorption and impaired hydroxylation in the liver and kidneys.We found very low 25(OH)D concentrations for the elderly when compared to younger people. This finding is possibly due to age and habit-related sunlight exposure differences. The existence of a seasonal variation over 25(OH)D concentrations is evident, as well as the influence of age is. Secondary hyperparathyroidism was frequent among the older individuals and was associated with 25(OH)D levels, creatinine and ionized calcium. According to our data, PTH values tend to plateau above 75.0 nmol/L. We found that 73.3% of the sample population studied is below this value.São PAulo Vitamin DEvaluation Study; 25(OH)D: 25 hidroxyvitamin D; NH: Nursing homes; CD: Community dwellings; PA: Physical activity; PTH: Parathyroid hormone; BMI: Body mass index; UVB: Ultraviolet radiation B; IBGE: Instituto Brasileiro de Geografia e Estatística (Brazilian Institute of Geography and Statistics).UNIFESP: Universidade Federal de São Paulo ; SPADES: The authors declare that they have no competing interests.SSM - main author. GLS – provided the data of NH and CD groups, participated in its design. ISK - carried out the immunoassays. LFH - carried out the immunoassays. MSC - provided the data of NH and CD groups, participated in its design. LRR - provided the data of NH and CD groups, participated in its design. MLC – conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.SSM – M.D., Ph.D. at the UNIFESP (Bone Metabolism section), and Professor at the Santa Casa de São Paulo Medical SchoolMLC – M.D., Ph.D. and Professor at the UNIFESP (Bone Metabolism section)The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6823/13/14/prepub


Saccharification resulted in glucan reductions of 28%, 45%, 76%, and 80% . Simultaneous saccharification and fermentation (SSF) trials were conducted at 5%, 10%, 15%, and 20% solids loading with Saccharomyces cerevisiae NRRL Y-2034 and Scheffersomyces stipitis NRRL Y-7124, with protein, fiber, and lipids analyzed at SSF 10% solids and saccharification trials. S. cerevisiae and S. stipitis produced ~3–12.5 g/L ethanol and ~2.5–8.6 g/L ethanol, respectively, on SB, SBM, and WF over all solid loading rates. SH resulted in higher ethanol titers for both S. cerevisiae (~9–23 g/L) and S. stipitis (~9.5–14.5 g/L). Protein concentrations decreased by 2.5–10% for the SB, SBM, and WF, but increased by 53%–55% in SH. Oil concentrations increased by ~50% for SB; by ~500%–1300% for the others.Carbohydrates in soybeans are generally undesirable due to their low digestibility and because they “dilute” more valuable components . To remove these carbohydrates and raise the titer of more valuable components, ethanol production was investigated. Commercial enzymes (Novozyme cellulase,  Soybeans are one of the most valuable crops in the world due to their high oil and protein content, which provides for a wide variety of uses. Soybean oil is used as a food and feed ingredient as well as in cosmetics –4 and biIn contrast to the oil and proteins, carbohydrates found in soybeans, (~10% dry weight), are largely undesirable due to their low digestibility . StachyoIn many countries, soybeans are “crushed” and then extracted with hexane or other solvents to separate soybean oil from the solids . SBM has a protein content of about 45% and is widely used as a livestock feed. Soybeans can be further processed via ethanol extraction to remove carbohydrates, resulting in soy protein concentrate (SPC) that contains at least 65% protein . This isAs an alternative to ethanol washing, we evaluated saccharification and bioconversion of soybean carbohydrates to ethanol. This would create an additional product to help offset processing costs, while making use of an underutilized material (soybean carbohydrates). Commercial hydrolytic enzymes and yeast were tested on soybeans and three fractions from the soybeanoil extraction facility. We hypothesized that much of the protein or lipids used by yeast as nutrients would be left in the final solids as yeast cell mass. Moreover, we expected an increase in protein content due to the conversion of carbohydrates into cell protein.Substrates tested included whole soybeans, soybean hulls, white flakes, and defatted soybean meal. These were obtained as a gift from South Dakota Soybean Processors, Volga, SD, USA. β-glucosidase with an activity of 250 CBU/g. NS 22016 is a pectinase cocktail with an activity of 3800 U/mL. Enzymes were stored at 4°C prior to use.Enzymes were obtained as a gift from Novozymes . NS 50013 (Celluclast 1.5 L) is a cellulase cocktail with an activity of 70 FPU/g. NS 50010 (Novozyme 188) is a Saccharomyces cerevisiae NRRL Y-2034 and Scheffersomyces stipitis NRRL Y-7124 were obtained from the USDA ARS Culture Collection . For short-term maintenance, cultures were grown on Potato Dextrose Agar (PDA) plates and slants for 72 h at 35°C and then stored at 4°C, with subculturing of the organisms every 4 weeks. Lyophilization in a 20% sucrose solution was used for long-term storage. S. cerevisiae, or 5% (v/v) for S. stipitis, from broth seed cultures stored at 4°C. Flasks for inoculum were incubated for 24 h at 35°C in a 250 rpm rotary shaker. Broth seed cultures were grown for 24 h at 250 rpm before refrigeration and used within 60 days to inoculate flasks for inoculum.Inoculum for all trials was prepared by aseptically inoculating sterile 5% glucose, 0.5% yeast extract broth (100 mL in a 250 mL Erlenmeyer flasks) with a 1% (v/v) aliquot for 2SO4. A stock solution of 10 mg/mL tetracycline HCl (70% ethanol and filter-sterilized) was prepared and stored at −20°C, from which 0.4 mL/100 mL of total trial volume was used to prevent bacterial contamination. A stock solution of 10 mg/mL cycloheximide (filter-sterilized) was prepared and stored at 4°C, from which 0.3 mL/100 mL of total trial volume was used for contamination control in saccharification trials only.Saccharification and SSF trials were conducted in a sterile 0.1 M sodium citrate buffer with the pH adjusted to 4.8 using concentrated H2SO4 or 12 M NaOH. The stoppers were pierced with 21 gauge syringe needles and Whatman 0.2 μm syringe filters. Enzyme dosages per gram of glucan included 23.2 FPU of NS 50013, 41 CBU of NS50010, and 500 U NS 22016. Saccharification trials were conducted by mixing 15 g of ground substrate with 75 mL of sterile 0.1 M sodium citrate buffer, along with tetracycline and cycloheximide solutions in 250 mL Erlenmeyer flasks fitted with rubber stoppers. The pH of the solutions was adjusted to 5.0 using concentrated Hμm syringe filters. The pH was adjusted to 5.0 using concentrated H2SO4 or 12 M NaOH. The substrates were not autoclaved in an effort to preserve protein and carbohydrate integrity by preventing the Maillard reaction [S. cerevisiae or S. stipitis was added. Flasks were incubated for 96 h in a 35°C reciprocating shaker set at 250 rpm. Control flasks without enzymes were also included to assess ethanol production from the free carbohydrates released from the substrates. These controls were prepared in the same manner as described above, except that the volumes of enzymes were replaced with sterile-deionized water.Ground substrates  were mixed with 150 mL of sterile 0.1 M sodium citrate buffer, along with an appropriate amount of tetracycline solution in 500 mL Erlenmeyer flasks fitted with rubber stoppers that were pierced with 21 gauge syringe needles and attached to Whatman 0.2 reaction  as well μm syringe filter into HPLC autosampler vials.Samples (5–10 mL) were drawn aseptically from the flasks at 0, 2, 4, 6, 12, 24, 48, 72, and 96 h. Samples were boiled for 5 minutes to inactivate enzymes and then centrifuged at 2400 ×g for 10 min. After freezing for 24 h at −20°C, samples were thawed, centrifuged again at 13,000 rpm for 15 min, and the supernatant was filtered through 0.2 2SO4 flowing at a rate of 0.6 mL/min, with the column at 65°C. Carbohydrates were analyzed using a Waters 1200 HPLC with a Waters Sugar-pak I column and refractive index detector. The mobile phase for the Sugar-pak I column was 0.0001 M calcium EDTA flowing at a rate of 0.5 mL/min, with the column at 65°C. Ethanol concentrations were determined using a Waters 717 HPLC with an Aminex HPX-87H column and Waters 2410 refractive index detector (RID). The mobile phase was 0.005 M HAt the end of saccharification and SSF trials, in the 10% solid loading rate experiments, the slurries from all replicates of a trial were combined and evaporated to dryness in an 80°C oven for 96 h. A proximate analysis was performed on the solids by Olson Agricultural Analytical Laboratory Services .Ethanol Productivity (g/L/h) = (Net Maximum Ethanol Concentration)/Time.Component Percent Difference (%) =  − )/% of original substrate.Saccharification trials were done in replicates of six, while the SSF trials were done in triplicate. Parameters analyzed included maximum ethanol concentration, ethanol productivity, and residual carbohydrates. The percent difference of the fiber, protein, and lipid content when compared to the original substrate was calculated using the formulas listed below. Residual carbohydrates were corrected by subtracting additional carbohydrate results that resulted from denatured enzymes or buffer. Graphs and calculations were made in Microsoft Excel 2007.As expected, the presence of enzymes resulted in higher soluble carbohydrate levels for each of the substrates, compared to control trials lacking enzymes. This difference was statistically significant for all substrates except white flakes and was also correlated with the reduction in fiber content. Whole beans contained the lowest concentration of fiber, due to the presence of both lipids and protein. Consequently, soluble carbohydrates were the lowest and only a moderate reduction in percent fiber was observed. Enzymatic saccharification efficiency in raw beans may have been reduced by the lack of any pretreatment effect that the typical soy processing operation provides. On the other hand, hulls contained the highest level of fiber and therefore responded most significantly to enzymatic hydrolysis, yielding the highest level of carbohydrates and greatest percent reduction in fiber.In the soybean crushing process, after oil extraction the solids are referred to as white flake. This material is then heated to drive off any residual hexane and inactivate certain antinutritional factors. Low levels of hulls then may be added to white flake to reduce the protein content to ~45%. This material is then called soybean meal. Thus, white flake and soybean meal are relatively similar in composition and we anticipated similar results upon saccharification. As can be seen in Protein and lipid levels in the samples were not expected to change significantly, since only small amounts of enzymes, buffers, and other components were added and the total solids were recovered. The only significant change expected was the conversion of fiber to soluble sugars as described above. Changes in relative protein levels varied from −14% to +11% and showed no significant trends. Lipid levels in whole beans increased from 4.5% to 12.9%, again likely due to greater solubilization during the saccharification process. Lipid levels in the other materials were very low, and therefore slight variability in values resulted in large percent changes. S. cerevisiae or S. stipitis. Enzyme dosages were normalized based on glucan levels and control trials lacking enzymes were also performed. Carbohydrate and ethanol titers were monitored throughout each 96 h SSF. After SSF, the replicates from the 10% solid loading rate treatments were combined and dehydrated for fiber, protein, and lipid analysis by Olson Agricultural Analytical Laboratory Services.Each substrate was subjected to SSF treatment using four different concentrations of substrate ) as well as either S. cerevisiae produced more ethanol than S. stipitis . However, the difference was only significant in the 15% SLR trial with enzymes and the 20% SLR trial without enzymes. The presence of enzymes enhanced ethanol levels, but was only statistically significant in the 5% SLR trials. The high degree of variability in the 15% and 20% SLR trials was likely due to the high viscosity of these trials, which reduced mixing efficiency. S. cerevisiae had significantly higher productivities compared to S. stipitis. Ethanol productivities were actually higher in many of the enzyme-free trials , suggesting that enzymatic hydrolysis of the nonpretreated soybean was the rate limiting factor. S. cerevisiae can hydrolyze the fructose residue from both stachyose and raffinose by use of invertase [S. stipitis does not produce invertase and cannot catabolize either oligosaccharide. In most treatments, total residual carbohydrate levels were similar between yeasts; however, at the 10% and 20% SLR trials with enzymes, S. stipitis accumulated significantly higher carbohydrate levels than S. cerevisiae. Also expected were the higher carbohydrate levels in enzyme-hydrolyzed trials compared to enzyme-free trials. The highest carbohydrate concentration was 20% SLR with enzymes and S. stipitis (18.76 g/L ± 5.501), and the lowest was 5% SLR without enzymes with S. cerevisiae (1.96 g/L ± 0.661).nvertase , 28, butS. cerevisiae outperformed S. stipitis at the higher SLRs, perhaps due to increased ethanol tolerance. Maximum concentrations obtained were 23.177 g/L ± 10.148 for S. cerevisiae and 14.501 g/L ± 6.748 for S. stipitis. As with the beans, the hulls were highly viscous at the higher SLRs, making proper mixing of the slurry very difficult, adding to the variability of the trials. Figures S. cerevisiae). Ethanol productivities increased with the SLR , though the difference between the 15% and 20% SLR was not significant. As expected, residual carbohydrate levels were higher in trials with enzymes, compared to nonenzyme trials  and with enzymes (SBM), respectively. S. stipitis had a corresponding maxima of 8.59 g/L ± 0.24 and 7.67 g/L ± 0.51 at 20% SLR with enzymes for white flakes and SBM, respectivly. Viscosity and mixing became an issue with both substrates at the higher SLR's, again affecting variability between replications. Figures S. cerevisiae at 15% SLR without enzymes, and 0.18 g/L/h ± 0.01 for S. stipitis at 20% with enzymes for white flakes. In trials with SBM, maximum productivities were 0.23 g/L/h ± 0.11 for S. cerevisiae at 15% SLR without enzymes and 0.11 g/L/h ± 0.01 for S. stipitis at 20% SLR with enzymes. S. cerevisiae generally had higher ethanol productivities than S. stipitis, but high variability at the higher SLR's often prevented significant differences from being established.Figures S. cerevisiae typically had lower residual sugar levels than S. stipitis. Figures S. cerevisiae reduced the carbohydrate levels more than S. stipitis. In the trials without enzymes, the S. stipitis reduced the carbohydrates more than the S. cerevisiae, but only by very small amounts. These data suggest that S. stipitis was more susceptible to high osmotic pressure than S. cerevisiae, but was otherwise comparable to S. cerevisiae at consuming carbohydrates at a 10% SLR of any of these substrates. However, the ethanol titers for these trials also showed that S. cerevisiae was capable of producing more ethanol than S. stipitis. We had anticipated that reducing fiber levels (and converting some of the resultant sugars into biomass) would increase protein levels. In the case of enzyme-treated hulls, we observed an average 54.2% increase in protein content. Mielenz et al.  showed aLipid concentrations increased by an average of 37% in soybean trials, likely due to the conversion of fibers into sugars and then ethanol. The remaining samples all showed a large percentage increase in lipid (average ~764%), but these numbers are somewhat misleading unless one considers the low content of lipids in hulls, white flakes, and SMB (avg ≤ 1.5% oil) in these substrates. Lipids from yeast cell mass production likely accounted for this change.Ethanol production as a means of removing carbohydrates from soybean fractions to concentrate the remaining components was successful in some cases. For example, SSF treatment of soybeans reduced fiber levels by ~84% and concentrated oil by ~46% in the fermented solids. Similar treatment of soybean hulls reduced fiber levels by ~94% and concentrated protein by ~54%. Fiber levels were also significantly lowered in white flake and SBM fractions and oil levels were raised . These changes would have been more impressive if the yeasts were capable of utilizing the xylose present. β-glucosidase, and pectinase in a 13-day SSF process. They achieved ethanol titers of 32.5 g/L and increased the protein content by 2.5–3.0 times the original concentration of the substrate. However, Schirmer-Michel et al. [Candida guilliermondii NRRL Y-2075. As noted previously, Mielenz et al.  treated l et al.  were onlFuture research should seek to minimize enzyme dosage requirements, while using yeast strains able to convert all of the monosaccharides into ethanol. Minimizing soy protein catabolism by yeast is another critical parameter and this could be achieved by minimizing SSF time.


Paecilomyces sp. was studied in this work. It was found that the biomass was very efficient removing the metal in solution, using Atomic Absorption, reaching the next percentage of removals: 64.5%. The highest adsorption was obtained at pH 6.0, at 30°C after 24 hours of incubation, with 1 mg/L of modified fungal biomass.The biosorption of As(III) on iron-coated fungal biomass of  The distribution between dissolved As(III) and As(V) is dependent on redox potential and pH. Under oxidizing conditions, the predominant specie is As(V), which exists as deprotonated oxyanions of arsenic acid (HAsO32−) . The As2SO4, 0.50% NaCl, 0.25% glucose] supplemented with 500 mg/L K2CrO4; the pH of the medium was adjusted and maintained at 5.3 with 100 mMol/L citrate-phosphate buffer. The cultures were incubated at 28°C for 7 days. The strain was identified based on their morphological structures such as color, diameter of the mycelia, and microscopic observation of spores  [with 0.25% KHf spores . The fun2) of 1 mg/L were prepared, pH was adjusted with nitric acid and/or NaOH, and the quantity of biomass added to each flask was of 1 g/100 mL for the arsenic's solution. It took samples at different times, the biomass is removed for centrifugation (3000 rpm/5 min), and the supernatant is analyzed to define the ion metal concentration.For analysis, a series of solutions of Arsenic(III) (NaAsO3)3·9H2O was prepared, and 1.0 mL of 10 M NaOH was added to this solution and mixed thoroughly. A total 20 g of the fungal biomass powder was taken in a porcelain pot, a mixture of iron oxide and NaOH solution was added to the porcelain pot and homogenized and kept in an oven for 3 h, at 80°C. After 3 h the oven temperature was raised to 110°C and continued for 24 h. The coated biomass powder was separated by crushing with mortar and pestle [A total 80 mL of the 2 M Fe. Paecilomyces sp. biomass. It was found that the removal is very low in untreated biomass, because at 24 h of incubation and pH 7.0, there is an 8.4% (0.084 mg/L) of removal  [Acacia nilotica, 0.19 mg/L [2AsO−3 form at a pH of 6.0 or above 9.22 and form complexes with the iron oxide-coated biomass. At higher pH, OH− ions in the solution increase compete with the arsenate ions, and the adsorption of As(III) is reduced [A. niger [ removal , and the glabrum , which wbiomass) , and ste.19 mg/L . Structu reduced . Conside reduced . These r reduced , chemica reduced , iron oxA. niger , mixed mA. niger , and modA. niger .A. niger, 80.1 mg/L, 12 h) [Acacia nilotica,  [The biosorption of As(III) onto iron-coated biomass, with different time (0–24 h) As(III), solutions of 1 mg/L, at pH 6.0, and 1 g/L of dosage of biosorbent material, is shown in L, 12 h) , and witbiomass) .A. niger, [A. nilotica [Paecilomyces sp. fungus seems to be nondependent of the temperature tested (30–50°C). . niger, , and difnilotica . The temthe Paecilomyces sp. iron-coated biomass for the As(III) ions were studied as a function of the initial As(III) ions concentration between 1 and 5 mg/L in the biosorption medium -treated biomass of Staphylococcus Xylosus, Inonotus hispidus biomass, and Al/Fe modified montmorillonite exhibited higher biosorption capacities of As(III) at 67.2, 54.35, 51.9, and 18.19 mg/g, respectively, [Acidithiobacillus ferrooxidans BY-3, Bacillus sp. strain DJ-1, agricultural residues, modified fungal biomass of A. niger, and iron oxide-coated sand exhibited lower values of maximum uptake capacity at 277.22 μg/g, 6.14 μg/g, 138.88 μg/g, 75 μg/100 mg, and 41.1 μg/g, for As(III), respectively, [Paecilomyces sp. has been previously used for the effective removal of Cr(VI) [Biosorption capacities of n medium . The uptnilotica  and Hg , the resA. nilotica [The influence of biomass on the removal capacity of As(III) was depicted in nilotica  and for nilotica .A. nilotica, Ionic Exchange Resins, and Maracuyá [Finally, this study has demonstrated the potential of iron-coated fungal biomass for the removal of As(III) at different conditions. The most attractive proposition of the biosorbent material is that it can be grown in large quantities and is cheap. The biosorbent material was successfully used for the removal of As from water samples of pole water samples having 1.0 mg/L, precedent of Zimapan, Hidalgo state, México. The mean results of water quality before and after biosorption of studied water samples are shown in Maracuyá , 37, 38.Paecilomyces sp. is a very promising biomaterial for removal or recovery of the metal ion studied.In this study, As(III) uptake by iron-coated fungal biomass was investigated. The performance of the biosorbents was examined as a function of the operating conditions, in particular incubation time, pH and initial metal ion concentration, and fungal biomass. The experimental evidence shows a strong effect of the experimental conditions. Maximum biosorption capacity values showed that the modified biosorbent used is very effective in recovery or removal of As(III) ions from aquatic systems. When the ease of production and economical parameters is concerned, it was observed that


Klebsiella sp. In this paper, MFX is added to 1 mg/L sulfanilamide aqueous solution substrate, and the removal ratio is evaluated. According to literatures review, for MFX absorption of sulfanilamide, flocculant dosage, coagulant-aid dosage, pH, reaction time, and temperature are considered as influence parameters. The result shows that the optimum condition is 5 mg/L bioflocculant MFX, 0.5 mg/L coagulant aid, initial pH 5, and 1 h reaction time, and the removal efficiency could reach 67.82%. In this condition, MFX could remove 53.27% sulfamethoxazole in domestic wastewater, and the process obeys Freundlich equation. R2 value equals 0.9641. It is inferred that hydrophobic partitioning is an important factor in determining the adsorption capacity of MFX for sulfamethoxazole solutes in water; meanwhile, some chemical reaction probably occurs.Although the treatment technology of sulfamethoxazole has been investigated widely, there are various issues such as the high cost, inefficiency, and secondary pollution which restricted its application. Bioflocculant, as a novel method, is proposed to improve the removal efficiency of PPCPs, which has an advantage over other methods. Bioflocculant MFX, composed by high polymer polysaccharide and protein, is the metabolism product generated and secreted by  Klebsiella sp. into the extracellular environment, composed of macromolecular polysaccharide and protein. In this study, based on its physical and chemical property, the effective ingredient of MFX is extracted by water abstraction and alcohol precipitation, transformed into dry powder. And then the removal efficiency and mechanism of sulfamethoxazole in aqueous environment are researched. The study aims at developing an effective treatment method of sulfamethoxazole and expanding the applied range of bioflocculation.In recent decades, the use of pharmaceutical and personal care products (PPCPs) has increased dramatically , 2. TheyThe strain was screened by our laboratory from activated sludge in municipal wastewater treatment plants and preserved in China General Microbiological Culture Collection Center (CGMCC number 6243).4·7H2O 0.2, NaCl 0.1, K2HPO4 5, KH2PO4 2, H2O 1000 mL, pH 7.2~7.5.Peptone 10, NaCl 5, beef extract 3, agar 15~18, water 1000 mL, pH 7.0~7.2; Flocuclant fermentation medium (g/L): glucose 10, yeast extract 0.5, urea 0.5, MgSO2 liquid are added into a beaker, pH is adjusted to 7.2 by adding NaOH, then 10 mL flocculant is added, compared with control without flocculant addition. Flocculator is applied during the experiment, after 40 s fast mixing, and changed into slow mixing for 4 minutes, after 20 min settling, and the absorbance of the supernatant is measured under 550 nm by 721 UV spectrometer [A is turbidity of the supernatant in control (light transmittance); B is turbidity of the supernatant in sample. Flocculating rate: 5.0 g chemically pure kaolin clay, 1000 mL tap water, and 1.5 mL 10% CaCltrometer . The floC is the concentration in control and D is the sulfamethoxazole concentration after treatment: Polysaccharide measurement: Phenol-sulphuric acid method . Protein measurement: Coomassie light blue .The removal efficiency of sulfamethoxazole is calculated by the following equation:Add 2x volume absolute alcohol (precooled under 4°C) to fermentation liquid, and filter and collect the white flocs after mixing. Add 1x volume absolute alcohol to filtered liquid, and then collect the white flocs again. Add small amount of DI water to collected flocs, after uniformly dissolving, freeze the flocculants in the ultralow temperature freezer for 24 h, and then put them into freeze drying to change the flocculants into dry powder.μL; column temperature 30°C; wave length 265 nm [Chromatographic column: C18 ; mobile phase is formic acid water: Acetonitrlle (60 : 40 V/V); flow rate 1.0 mL/min; sample size 10 h 265 nm .Add flocculants into 1 mg/L sulfamethoxazothe liquid with dosage 0 mL, 1 mL, 3 mL, 5 mL, 7 mL, and 9 mL; set the coagulant aids dosage as 0 mL, 0.5 mL, 1 mL, 1.5 mL, and 2 mL; adjust pH value to 4, 5, 6, 7, and 8 under 5°C, 15°C, 25°C, 35°C, 45°C, and 55°C; change the reaction time as 0 h, 0.25 h, 0.5 h, 0.75 h, 1 h, 2 h, 4 h, 6 h, 8 h, 10 h, and 12 h; calculate the removal rate.Based on the preliminary obtained optimum condition, flocculant dosage, coagulant-aid dosage, pH, reaction time, and temperature are considered as influencing parameters. The design of experiment is shown in Mix the flocculant MFX and sulfamethoxazole with initial concentration as 0.8 mg/L, 1 mg/L, 1.2 mg/L, 1.4 mg/L, 1.6 mg/L, and 1.8 mg/L separately, under different temperature condition as 15°C, 35°C, put on 140 rpm shaking table with constant temperature, conduct adsorption isotherm experiment, and adsorption time is 1 h.Klebsiella. sp on the basis of the morphological characteristics and 16S rDNA sequence. The J1 showed a high yield of flocculant and good flocculation activity toward kaolin suspension. The active ingredients of bioflocculant MFX produced by J1 distributed mainly in the supernatant after the first centrifugation of fermentation broth, that is to say, the flocculation active extracellular secretions remain freely in fermentation broth. , and 11.4% (β-amylase) of the flocculation activity. On the contrary, proteins were removed, while polysaccharides remained. EPS has no flocculent activity , 61.9% , and results are shown in In order to evaluate the removal effect of bioflocculant MFX on sulfamethoxazole in actual wastewater, domestic wastewater is used, with sulfamethoxazole concentration as 23.26 ug/L. 9 sets of experiments are conducted according to the orthogonal design table L9 , while n is constant which is indicative of the adsorption energy and intensity:CS is mass concentration in solid phase after adsorption equilibrium (mg/g); CW is concentration in liquid phase after adsorption equilibrium (mg/L).The dry power of MFX is white, sparses and reticulate, while the aqueous solution is milky white, ropy, and turbid. The material of MFX adsorbent is glycoprotein which has hydrophobicity and displays a wide range of sorption behavior for hydrophobic organic compounds in aqueous solutions . The adsKf) of 176.6445 mg/g was obtained with MFX in 35°C. Compared n value, it is known that, under this temperature, the adsorptive property of MFX to sulfamethoxazole is high. However, MFX has poorer adsorption efficiency in 15°C. R2 value is only 0.882, while n value is lower than the former.The results show that MFX exhibited high-adsorption capacities for sulfamethoxazole in water. A maximum adsorption capacity (2+, sulfamethoxazole becomes unstable rapidly, passing into flocculation phase.This is due to the effect of temperature on chemical reaction and molecular movement, which finally promote or restrain flocculent effect. On the one hand, providing appropriate temperature, colloidal particle is bombarded intensively by molecules of flocculation to form a whole. If temperature is excessively low, molecular movement slows down, and reaction rate lessens, leading to bad adsorptive property. On the other hand, some active groups between bioflocculant and sulfamethoxazole start the chemical reaction to separate out of aqueous solution system. What is more, when hydrophobic chain polymer-bioflocculation MFX comes into sulfamethoxazole aqueous solution system, with the help of appropriate pH and CaDriven by such mechanism, the adsorption on MFX does not rely on a high porosity and a resultant high specific surface area to reach a high adsorption capacity, as for most activated carbon and polymeric adsorbents. This result implies that hydrophobic partitioning is an important factor in determining the adsorption capacity of MFX for sulfamethoxazole solutes in water; meanwhile, some chemical reaction probably occurs.The active flocculent constituents of MFX are EPS which is composed by polysaccharides and proteins fermented by J1. Proteins mainly accounted for the flocculation activity. The MFX displays great sorption behavior for sulfamethoxazole in aqueous solutions. The optimum condition is 5 mg/L bioflocculant, 0.5 mg/L coagulant aid, initial pH 5, and 1 h reaction time, and the removal efficiency could reach 67.82%. Using MFX, the removal rate of sulfamethoxazole in domestic wastewater can reach 53.27%. The effect size of ecological factor is as follow: pH > flocculant dosage > coagulant aid > reaction time.R2 value equals 0.9641. It is inferred that hydrophobic partitioning is an important factor in determining the adsorption capacity of MFX for sulfamethoxazole solutes in water.It is efficient for MFX to remove sulfamethoxazole in aqueous environment in 35°C. And the process obeys Freundlich equation. This work demonstrates the efficient removal of sulfamethoxazole from water using bioflocculant MFX as adsorbents. The findings are summarized as follows.The study shows that bioflocculation MFX can be used as an efficient alternative adsorbent for the removal of sulfamethoxazole in water, with high-adsorption capacities observed in actual wastewater. Further studies are underway to make mathematical models of the relationship between flocculant and contaminant. When many PPCPs coexist, the research on removal efficiency with bioflocculant is more significant.


Candida viswanathii strain were investigated. The highest lipase activity (101.1 U) was observed at 210 rpm, pH 6.0, and 27.5°C. Other fermentation parameters analyzed showed considerable rates of biomass yield (YL/S = 1.381 g/g), lipase yield (YL/S = 6.892 U/g), and biomass productivity (PX = 0.282 g/h). Addition of soybean lecithin increased lipase production in 1.45-fold, presenting lipase yield (YL/S) of 10.061 U/g. Crude lipase presented optimal activity at acid pH of 3.5, suggesting a new lipolytic enzyme for this genus and yeast in general. In addition, crude lipase presented high stability in acid conditions and temperature between 40 and 45°C, after 24 h of incubation in these temperatures. Lipase remained active in the presence of organic solvents maintaining above 80% activity in DMSO, methanol, acetonitrile, ethanol, acetone, 1-propanol, isopropanol, and 2-propanol. Effectiveness for the hydrolysis of a wide range of natural triglycerides suggests that this new acid lipase has high potential application in the oleochemical and food industries for hydrolysis and/or modification of triacylglycerols to improve the nutritional properties.Influences of environmental variables and emulsifiers on lipase production of a  Lipases  are responsible for the hydrolysis of triglyceride ester bonds into diglycerides, monoglycerides, fatty acids, and glycerol using a complex phenomenon of interfacial activation. Lipases present a number of unique characteristics, including substrate specificity, stereospecificity, and the ability of the resolution of racemic mixtures and synthesis of esters bonds in nonaqueous media –5.Lipases are ubiquitous and indispensable for triacylglycerols bioconversion in nature. They occur in plants, animals, and microorganisms , but theThe use of the submerged culture is advantageous because of the facility of sterilization and process control in these systems. Depending on the strain and the culture conditions, the enzyme can be constitutive or inducible, showing different production. Submerged fermentation processes for the lipolytic enzymes production usually employ renewable low-cost hydrophobic substrates such as vegetable oil, poultry fat, tallow, waste fats, soap stocks, rapeseed oil, and grease containing waste water . The useEnvironmental factors such as oxygen, agitation, temperature, and pH are important parameters that affect extracellular lipase production and must be considered in submerged process. Temperature and pH can be determined in shake flasks fermentations , 14, butCandida viswanathii was capable to produce lipase and biomass efficiently when cultured in a large diversity of natural triacylglycerols [In a previous study, a strain of lycerols . In thisC. viswanathii strain is available in the Culture Collection of the Environmental Studies Center, CEA/UNESP, Brazil. C. viswanathii was cultivated on malt extract agar (MEA) for 3 days, at 28°C, for inoculum preparation. Liquid medium was prepared using Vogel's medium, with 1.5% (w/v) olive oil and 0.2% (w/v) yeast extract as single carbon and nitrogen sources, respectively, according to the conditions established previously [7 cells/mL) and incubated at different conditions as indicated subsequently. All cultures were developed in triplicate and the results were assayed for biomass, maximum lipase activity, biomass yield on substrate, lipase yield on substrate, biomass productivity, and specific rate of lipase production.eviously . ErlenmeBiomasses were separated from the fermentation broth by filtration. Cell-free broth was used for lipase activity determination. Biomasses were dried at 105°C until constant mass.The culture was carried out in an orbital shake at 150 rpm, 180 rpm, and 210 rpm. Initial pH was adjusted to 6.0 and the incubation temperature was 27.5°C. Fermentation parameters were monitored in different intervals for 96 h.The effect of initial pH on lipase production was analyzed from 3.0 to 10.0. The initial medium pH was adjusted by the addition of NaOH 1 M or HCl 1 M. Cultures were carried out for 72 h, 210 rpm orbital shake, at 27.5°C.The temperature influence on lipase production was varied from 20 to 40°C, with intervals of 2.5°C. Cultures were carried out for 72 h, 210 rpm orbital shake, and pH 6.0.Different surfactants  and emulsifiers  on lipase production were added to the medium at 0.1% (w/v). Cultures were carried out for 72 h, 210 rpm orbital shake, and pH 6.0 at 27.5°C.ρ-nitrophenyl-palmitate (ρNPP) as substrate [ρNPP was first dissolved in 0.5 mL of dimethyl sulfoxide and then diluted to 50 mM with 50 mM sodium phosphate buffer pH 7.0 containing 0.5% Triton X-100. The hydrolysis of ρNPP was determined discontinuously at 37°C by releasing ρ-nitrophenol (ρNP). After 5 min of preincubation of 0.9 mL of this substrate solution in water bath, the reaction was started by addition of 0.1 mL of appropriately diluted sample. The reaction was stopped at different intervals (1 and 2 min) by heat shock , followed by addition of 1 mL of saturated sodium tetraborate solution. The absorbance was measured at 405 nm and the activity was determined according to the standard curve carried out with ρ-nitrophenol (ρNP molar extinction coefficient: 1.8 × 104 M−1 cm−1). Controls were prepared without enzyme. One unit of enzyme activity was defined as the amount of enzyme that releases 1 μmol of ρNP per mL per min.Lipase activity was assayed with ubstrate . ρNPP waThe protein was determined by the Lowry method , using bEnzyme activity was measured at 37°C in different pH values using glycine-HCl 0.05 M buffer from 2.0 to 3.0 and McIlvaine buffer from 3.0 to 8.0. Enzyme stability was carried out with the same buffers, except from 8.6 to 10.0 when glycine-NaOH was used. Enzyme preparation was diluted in each buffer  and incubated for 24 h at 10°C. The optimum temperature was determined by incubation of the reaction mixture from 25 to 60°C, in the McIlvaine buffer pH 4.0. For thermal stability, the enzyme was incubated for different periods of time at 40, 45, 50 and 60°C in McIlvaine buffer pH 4.0 and the residual activity was determined in McIlvaine buffer pH 3.5 at 40°C.Methanol, ethanol, acetone, isopropanol, acetonitrile, 1-propanol, 2-propanol, and dimethylsulfoxide (DMSO) were added at 1% (v/v) and 10% (v/v) to the reaction medium. The relative activities were expressed as a percentage of the control. 4, MgSO4, NaCl, BaCl2, CoCl2, NH4Cl, Pb(CH3COO)2, CaCl2, and EDTA at final concentration of 10 mM and 20 mM. Crude enzyme was incubated at 40°C for 1 h in the presence of substances ZnSOμmol of fatty acid per mL per min. The results were expressed as percentage of triolein hydrolyzed.Hydrolysis of pure  or natural oils and fats  was developed at 40°C by following the titration of fatty acids released. The oils  were emulsified in McIlvaine buffer pH 4.0, containing 5% (w/v) Triton X-100. The reaction was started by adding 1 mL of sample to 5 mL of this emulsion and then maintained for 30 min at 300 rpm orbital agitation. The reaction was interrupted by adding 16 mL of an acetone : ethanol solution  to the mixture. The fatty acids released were titrated to pH 11 with a 0.05 M NaOH solution. One unit of enzyme activity was defined as the amount of enzyme that releases 1 C. viswanathii growth and lipase production, and specific activity was determined. Three agitation speeds were assayed, and lipase production was found to be growth-associated with maximum after 72 h cultivation  of culture in stationary cultures. The subsequent experiments evaluated the agitation effect on the  Figures . The pea Figures . The decYX/S) 1.456 g/g, lipase yield (YL/S) 6.638 U/g, biomass productivity (PX) 0.303 g/h, and specific rate of lipase production (qL) 0.062 U of lipase/g of biomass·h . An optimal initial pH for lipase production by the yeast Pichia lynferdii was found at 7.0 [Bacillus pumilus was 9.0 [The initial pH of the fermentation medium is an important physical parameter that affects microbial growth and enzymes synthesis by  strains , 30. Culwanathii . Changesd at 7.0  and for  was 9.0 .YX/S = 1.312 g/g and YL/S = 6.807 U/g, resp.) as well as specific rate lipase production (qL = 0.079 U of lipase/g of biomass·h). Ali et al. [C. lipolytica in this same pH. An initial pH of 4.5 was reported as optimum for lipase production by continuous fermentation of Yarrowia lipolytica [The highest specific activity (13.8 U/mg protein) and biomass (18.9 g/L) were also verified in these culture conditions. Fermentation parameters analyzed showed high biomass and lipase yields in pH 6.0 , were also observed at 27.5°C (YX/S = 1.381 g/g and YL/S = 6.892 U/g), (qL = 0.079 U of lipase/g of biomass·h) although operational cost is inferior at temperature next to room temperature.The cultivation temperature ranging from 20 to 40°C and its effects in the cell growth and lipase production were also analyzed, maintaining the conditions previously established . When the incubation temperature was increased, lipase production, specific activity, and cell growth also increased up to 27.5°C . The mic92 U/g), . HoweverC. viswanathii was sensitive to variations of 2.5°C for enzyme production and growth. In other studies, several microorganisms were investigated to produce lipase at different temperatures [These results are consistent with those observed by Lin et al.  which deeratures , 34 and eratures , 35, 36.YL/S = 10.061 U/g) and specific rate of lipase production (qL = 0.114 U of lipase/g of biomass·h), were considerably superior to the results observed without emulsifier (control). The parameters biomass yield (YX/S = 1.226 g/g) and biomass productivity (PX = 0.250 g/h) decreased with the addition of this emulsifier. Soybean lecithin is composed of specific phospholipids, triglycerides and other nonphospholipids nontoxic compounds [Y. lipolytica, but Pogori et al. [Rhizopus chinensis lipase production decreased when surfactants were added to the culture medium.Surfactant or emulsifier addition during the fermentation process has often been shown to enhance extracellular lipase production due to changes in the permeability of the cell or surfactant effects on cell-bound lipase. Nevertheless, surfactants and emulsifiers do not always increase lipase production, and their effects appear to depend on both surfactant and the strain studied . The infompounds , which mompounds  reportedi et al.  reportedThe effects of pH on lipase activity and stability are shown in C. viswanathii lipase activity is different from other Candida lipases that presented optimal pH range in alkaline conditions. Candida rugosa lipase had optimal activity at pH 7.5 [Candida antarctica showed the highest activity at pH 8.0 and the enzyme was stable at pH range of 7.0–9.0 after 24 h [Candida cylindracea lipases A and B showed similar pH profiles using tributyrin as substrate, with optimum activity around pH 7.0. However, the decrease in activity at pH 8.0 was more pronounced in lipase B, whereas lipase A was less active at pH 5.0 [Aspergillus niger NCIM 1207 [Pseudomonas gessardii lipase produced with beef tallow was observed at pH 5.0; however above pH 7.0, the activity rapidly decreased, retaining 33% of its activity at pH 9.0 [The optimum acid pH of t pH 7.5 . Lipase ter 24 h . Candidat pH 5.0 . An acidCIM 1207 . The lipt pH 9.0 . This ent pH 9.0 .C. viswanathii lipase activity increased with temperature from 25°C up to 40°C  and 10% (v/v) (C. viswanathii lipase retained approximately 90% of its activity. At 10% (v/v), methanol and DMSO weakly affected the lipase activity and ethanol had a moderate effect under this condition. However, isopropanol, acetone, 2-propanol, 1-propanol, and acetonitrile drastically reduced the lipase activity. Ogino and Ishikawa [Lipase stability in organic solvents is an essential prerequisite for lipase applications in organic synthesis , since s0% (v/v) . At 1% . Tween 20 at 1% and 5% inhibited the activity to 88.2% and 52.5%, respectively. SDS completely inactivated the enzyme and Triton X-100 was a strong lipase inhibitor, reducing the activity to 30.3% and 26.9% at both concentrations. The lipase from tergents .C. viswanathii lipase showed the lowest activity on tributyrin (45.5%). These results suggest that this enzyme is more active on triacylglycerol with long chain fatty acids being capable of hydrolyzing a broad spectrum of esters of fatty acid chain lengths. Similar results were found for lipases from Penicillium camembertii [Colletotrichum gloeosporioides [ Pseudomonas gessardii [The hydrolytic activity of the lipase on pure and natural triacylglycerols is shown in embertii , Colletoorioides , and Pseessardii .C. viswanathii. Agitation speed, temperature, and initial pH are essential to produce 101 U/L of crude lipase. The lipase production was even more increased by supplementation with soybean lecithin. The biochemical characterization of the crude C. viswanathii lipase revealed that this enzyme shows different properties when compared to those from other Candida spp. Optimal activity at acid pH of 3.5 suggests a new lipolytic enzyme for this genus and for yeast in general. In addition, crude lipase presented high stability in acid conditions and was highly stable at 40 and 45°C, remaining active in the presence of organic solvents as DMSO and methanol. The enzyme was not activated by metal ions and the activity was preserved in the presence of gum Arabic for 1 h at 40°C. C. viswanathii lipase presented a broad specificity for triacylglycerols hydrolysis suggesting that this enzyme can be applied in lipid digestion and biotransformation.The present study demonstrated the importance of controlling physical parameters for lipase production by


Adult healthy volunteers from 18 to 68 years of age underwent extensive clinical and investigational procedure and were included in the study. Vitamin C and α-tocopherol were estimated using simple Spectrophotometric method. Of the 71 healthy Bengali volunteers participated, 31 were males and 40 were females. The mean concentration of plasma vitamin C was found to be 0.65 mg/dL. The mean α-tocopherol was found 6.35 mg/L (14.74 μmol/L) in the study population higher than the normal threshold value for α-tocopherol but lower than other populations. The study data enabled us to determine the gender nonspecific coverage interval of antioxidant vitamins, and the intervals were lower than the established reference interval in other populations.Reference interval of all haematological and biological analytes should be measured for every population because of the huge diversity in genetic make-up, dietary habits. The coverage interval of antioxidant vitamins (vitamin C and  Coverage interval where it can be stated with a given degree of confidence that the interval contains a given fraction of the population of reference values [In recent years, with the increased dependence on laboratory tests, the sound understanding of reference intervals is vital as diagnosis of disease frequently depends on results of laboratory analytes measured from blood, urine, cerebrospinal fluid, and so forth. Reference interval denotes normative values related to laboratory parameters used by the clinical laboratories for clinical diagnosis . The cone values . The covIn both health and disease, increasing interest is being directed to the significance of optimal vitamin and antioxidant status . Antioxiα-tocopherol in serum has been done by Baker and Frank method (1968) [Seventy one healthy Bengalis, thirty one males (M = 31) and forty females (F = 40), in the age group between 18 to 68 years (mean 28.49 years) were enrolled. The study subjects had ordinary eating habits with none on a special diet or vitamin supplements. The staple diet consists of rice, vegetables, egg, and fish. Fruits were not very prominent but there was ample consumption of vegetables like pumpkin and carrot. The dietary pattern was enquired by a less accurate model questionnaire. Subjects with hypertension, hypercholesterolemia, diabetes, obesity, genetically determined risks, habits of smoking, alcohol consumption, any history of drug intake for treatment of disease or suffering like antiepileptic, ATD (anti-tubercular drugs), Chemotherapy, OCP , any variation in physiological status like pregnancy, stress, and excessive exercise were excluded from the study. Following overnight fasting, peripheral venous samples were taken into evacuated tubes, centrifuged, and plasma and sera were separated. The analysis of the antioxidant vitamins were done in accordance with the standards of the Ethical Committee of Medical College, Kolkata. Estimation of d (1968) . Plasma d (1968) . In spitα tocopherol the calculated Bias was −5.29%. The HPLC method for α-tocopherol was having the following performance criteria as linearity: 0.2–50 mg/L, recovery rate: 92–98%, lower detection limit: 0.30 mg/L, lower determination limit: 0.40 mg/L, interassay coefficient of variance: 3.23% and reference interval = 5–18 mg/L. Similarly, for the vitamin C the bias and exactness were −1.79% and 98.21%, respectively, after comparing the value of samples from the study population with an established HPLC method from an accredited laboratory.Moreover, the traceability of the methodology was calculated by comparing with an established HPLC from an accredited laboratory. For this purpose, randomised samples from the study population were measured in parallel and compared with an established HPLC method from an accredited laboratory, for The values of the antioxidant vitamins were analysed using IBM SPSS 16 statistical software. The Kolmogorov-Smirnov test was used to check for normal distribution. Both “parametric” and “nonparametric” coverage intervals were calculated. The mean age and weight of the study population 28.49 ± 9.49 years and 62.9 ± 11.58 Kilograms, respectively.It is evident from α-tocopherol was found 6.35 mg/L (14.74 μmol/L) in the study population. The cut-off value for serum α-tocopherol ≥ 11.6 μmol/L, subjects with serum concentration less than this are considered deficient. Comparison with studies done on Spanish, Italian, American, Japanese, Hispanic, Swiss, and Kuwaiti population suggested that the mean concentration of α-tocopherol of these study subjects was much lower. We observed the mean concentration of α-tocopherol to be 28.2 μmol/L, 26.2 μmol/L, 25.7 μmol/L, 24.4 μmol/L, 29.2 μmol/L, 29.3 μmol/L, and 20.0 μmol/L in Spanish [From  Spanish , Italian Spanish , America Spanish , Japanes Spanish , Swiss [ Spanish , and Kuw Spanish  populatiα-tocopherol showed greatest association with the lipid, an observation confirmed by the Swiss study of Winklhofer-Roob et al. [α-tocopherol is found to be more concentrated in the hydrophobic plasma membrane and phospholipid coats of lipoproteins. α-tocopherol has an well-established role against LDL oxidation. As the plasma concentration of α-tocopherol varies with cholesterol profile it is rationalised to estimate lipid standardised Vitamin E and express Vitamin E as μmol/L of tocopherol/mmol/L of cholesterol [μmol tocopherol/mmol total lipids were calculated as the threshold of deficiency equivalent to 0.8 mg tocopherol/g total lipid established by Horwitt et al.Moreover, b et al. . The conlesterol . Using rα tocopherol was found to be 3–11.9 mg/L and 3.3–13.3 mg/L, respectively. This is not in agreement with the established reference intervals found in the literature, that is, 5–18 mg/L in adults. The oxidative stress varies with age. In this study, the age group was 18–68 years, so naturally the concentration of α-tocopherol varied in accordance with age and corresponding oxidative stress. The literature survey revealed that all the studies done on Swiss, Kuwaitis, and Spanish population employed HPLC, the method of choice for quantification. According to the most recent and extensive listing of biological goals provided by Ricós et al., 13.8% within subject biological variation, 15% between subject biological variation, 6.9% imprecision, 5.1% bias, and 16.5% allowable total error are permissible for serum α tocopherol [From α tocopherol in most instances specific to Bengali population. To establish more credible “coverage interval” for these antioxidant vitamins as a replica of reference interval, a similar study with improved method of estimation like HPLC is warranted. It was established that western normative laboratory values of vitamin C and Vitamin E are not applicable to Bengali population. This finding provides an insight that not only methods of estimation, but also on different ethnicity, racial, genetic make-up, sociodemographic pattern and dietary habits.This study indicates that the coverage interval obtained from the Bengali population can be used to define antioxidant reference thresholds. In spite of the limitation imposed by cost and methodology like HPLC, the study has demonstrated that the plasma concentration of vitamin C and


Ulva prolifera and Ulva linza to Cd2+ stress were studied. We found that the relative growth rate (RGR), Fv/Fm, and actual photochemical efficiency of PSII (Yield) of two Ulvaspecies were decreased under Cd2+ treatments, and these reductions were greater in U. prolifera than in U. linza. U. prolifera accumulated more cadmium than U. linza under Cd2+ stress. While U. linza showed positive osmotic adjustment ability (OAA) at a wider Cd2+ range than U. prolifera. U. linza had greater contents of N, P, Na+, K+, and amino acids than U. prolifera. A range of parameters , proline, organic acids and soluble protein, Fv/Fm, Yield, OAA, and K+/Na+) could be used to evaluate cadmium resistance in Ulva by correlation analysis. In accordance with the order of the absolute values of correlation coefficient, contents of Cd2+ and K+, Yield, proline content, Fv/Fm, FAA content, and OAA value of Ulva were more highly related to their adaptation to Cd2+ than the other eight indices. Thus, U. linza has a better adaptation to Cd2+ than U. prolifera, which was due mainly to higher nutrient content and stronger OAA and photosynthesis in U. linza.Responses of  Heavy metal contamination is an environmental problem in the margin sea . As the Cd, an oxophilic and sulfophilic element, forms complexes with various organic particles and thereby triggers a wide range of reactions that collectively put the aquatic ecosystems at risk. Cadmium also poses a serious threat to human health due to its accumulation in the food chain , 6. It hUlva, have been considered as monitors of heavy metals in estuaries  × 100%, where M0 and Mt are the fresh weights (g) at days 0 and 7, respectively [Fresh weight was determined by weighing the algae after blotting by absorbent paper. RGR was calculated according to the formula RGR . OAA was calculated by the following equation:π100μ was the π100 of control seaweeds, and π100s was the π100 of Cd2+-stressed seaweeds.Saturated osmotic potential was measured by the freezing-point depression principle. Seaweeds were placed in double-distilled water for 8 h and then rinsed 5 times with double-distilled water. After blotting dry with absorbent paper, seaweeds were dipped into liquid nitrogen for 20 min. The frozen seaweeds were thawed in a syringe for 50 min, and the seaweed sap was then collected by pressing the seaweed in the syringe . The π1Determination of Chl and Car was carried out by the method of Häder et al. . Weighedin vivo chlorophyll fluorescence from chlorophyll in photosystem II (PSII) using different experimental protocols [A PHYTO-PAM Phytoplankton Analyzer  was used to determine rotocols . Before Dried samples were ground in a mortar and pestle. Total N in seaweed tissue was analyzed by an N gas analyzer using an induction furnace and thermal conductivity. Total P in seaweed tissue was quantitatively determined by Inductively Coupled Plasma Atomic Emission Spectrometry  following nitric acid/hydrogen peroxide microwave digestion. The total amounts of N and P in the seaweed tissue were calculated by multiplying N and P contents in tissue as a proportion of dry weight by the total dry weight of the sample . 3 : HClO4  and diluted to 50 mL with distilled water. The contents of Cd, Na, K, Ca, and Mg were determined by Inductively Coupled Plasma Atomic Emission Spectrometry  [3) [μL) for 20 min. The nitrate concentrations in the samples were measured spectrophotometrically at 540 nm.After 7 d, seaweeds were harvested, washed, and oven-dried at 65°C for 3 d. A 50 mg sample was ashed in a muffle furnace. The ash was dissolved in 8 mL of HNOUSA) [3) . Total nUSA) [3)  with nit−1 anthrone-ethyl acetate and 5 mL H2SO4. The mixture was heated at 100°C for 1 min, and its absorbance at 620 nm was read after cooling to room temperature. A calibration curve with sucrose was used as a standard. Soluble proteins (SPs) were measured by Coomassie Brilliant Blue G-250 staining [−1 NaOH, and 5 mL of Bradford reagent was added. Absorbance was recorded at 595 nm using bovine serum albumin as a standard. Free amino acids (FAAs) were extracted and determined following the method of Zhou and Yu [−1 NaOH titration method, with phenolphthalein as indicator [Soluble sugars (SS) determination was carried out by the anthrone method . Water estaining . Fresh su and Yu . A totalndicator .P ≤ 0.05. Statistical analysis on two-way variance analysis (ANOVA), and correlation coefficient was performed using Microsoft Excel. All experiments were performed in three replicates. The data are presented as the mean ± SD. Data were analyzed using SPSS statistical software. Significant differences between means were determined by Duncan's multiple range test. Unless otherwise stated, differences were considered statistically significant when μmol L−1 Cd2+ for 7 d did not change RGR of U. linza, but significantly decreased RGR of U. prolifera. The RGR of both Ulva species was significantly decreased as Cd2+ concentration increased. After 7 d exposure to 10, 20, 40, 80; or 120 μmol L−1 Cd2+, RGR of U. linza decreased by 53, 75, 116, 177, and 277%, respectively; U. prolifera decreased by 93, 139, 271, and 357%, respectively. U. prolifera died at 120 μmol L−1 Cd2+ on day 7  at 80 μmol L−1 Cd2+. However, in U. linza, 5 and 10 μmol L−1 Cd2+ had no significant influence on Ca2+ contents, whereas 20, 40, and 80 μmol L−1 Cd2+ increased Ca2+ content by 22, 39, and 50%, respectively  content appeared to have an ascending trend after an initial decline in both Ulva species. In U. prolifera, 40 μmol L−1 Cd2+ did not change the SS content, and 80 μmol L−1 Cd2+ increased SS concentration by 27% compared to the control. However, in U. linza, 40 and 80 μmol L−1 Cd2+ increased SS content by 40 and 90%, respectively  content by 25 and 16%, respectively. However, 10 μmol L−1 Cd2+ had no obvious change on FAA contents of the two Ulva species. Treatments with 20, 40, and 80 μmol L−1 Cd2+ significantly decreased FAA content by 52, 79, and 87% in U. prolifera and by 2, 25, and 43% in U. linza . Furthermore, this reduction was found to be more pronounced in U. prolifera than U. linza. After 7 d, U. prolifera died at 120 μmol L−1 Cd2+, whereas U. linza was still alive  through increased net concentrations of solutes (osmotic adjustment), which is a common response to water stress and an important mechanism for maintaining cell water content and, thus, turgor [U. linza had positive values in the treatments with 5, 10, or 20 μmol L−1 Cd2+, whereas U. prolifera had positive OAA only at 5 and 10 μmol L−1 Cd2+  decreased N and P contents in both Ulva species. The maintenance of total N and total P was more pronounced in less Cd-sensitive U. linza than Cd-sensitive U. prolifera  concentration at low Cdosmotica . The acctoxicity . U. prolifera and U. linza declined with increasing Cd2+ treatment concentrations. Free amino acid (FAA) contents in both Ulva species first increased and then declined, with such a decline more pronounced in U. prolifera than in U. linza. The decreased protein content together with the increased free amino acid content suggest that the protein synthesizing machinery was impaired due to the Cd2+ effect [Soluble protein (SP) content in organisms is an important indicator of metabolic changes and responds to a wide variety of stresses . In this+ effect . 2+-treated Ulva, its absolute amount was relatively low. Under assumed localization of inorganic ions in the vacuole and organic solutes in the cytoplasm, FAA and PRO may be mainly in the cytoplasm, accounting for about 5%–10% volume in mature cells [PRO accumulation in plant tissues in response to a number of stresses, including drought, salinity, extreme temperatures, ultraviolet radiation, or heavy metals, is well documented . In thisre cells . A smallre cells . It has re cells . In addire cells . U. prolifera and U. linza to different concentrations of Cd2+ resulted in the changes in growth, pigment content, chlorophyll fluorescence parameters, Cd accumulation, OAA, and concentration of N, P, main inorganic ions, and organic solutes. These changes make U. linza better adapted to withstanding Cd2+ stress in comparison with U. prolifera. Our results highlight the role of osmotic adjustment in Ulva during Cd2+ stress as an important mechanism enabling Ulva to maintain photosynthetic activity and, thus, growth under Cd2+ stress. In conclusion, exposing


Q10 increased. Our findings indicate that a loss of BSCs cover will lead to greater soil C loss through respiration. Given these results, understanding the disturbance sensitivity impact on Rs could be helpful to modify soil management practices which promote carbon sequestration.Soil respiration (Rs) is a major pathway for carbon cycling and is a complex process involving abiotic and biotic factors. Biological soil crusts (BSCs) are a key biotic component of desert ecosystems worldwide. In desert ecosystems, soils are protected from surface disturbance by BSCs, but it is unknown whether Rs is affected by disturbance of this crust layer. We measured Rs in three types of disturbed and undisturbed crusted soils , as well as bare land from April to August, 2010, in Mu Us desert, northwest China. Rs was similar among undisturbed soils but increased significantly in disturbed moss and algae crusted soils. The variation of Rs in undisturbed and disturbed soil was related to soil bulk density. Disturbance also led to changes in soil organic carbon and fine particles contents, including declines of 60–70% in surface soil C and N, relative to predisturbance values. Once BSCs were disturbed,  BSCs erosion . In tota erosion , with 56 regions . These n regions . Soil st regions . Most st regions .Studies on disturbance of BSCs have focused on soil nutrient losses, microbial communities, surface hydrology, erosion dynamic, and recovery rate , 11. TheThe landscape of the study area is characterized by a mosaic distribution of shrubs, BSCs, and bare land. Characteristics of the site are long dry periods and very infertile soils. Efforts to mitigate anthropogenic disturbance through fencing continue, but disturbance, such as human and livestock trampling, off-road driving, and annual grasses invasion, has increased. The objectives of the present study were to (1) compare Rs in undisturbed crusted soil with that in disturbed crusted soil of different types under drought conditions to allow estimate on changes in Rs following disturbance and (2) identify the controls which result in the difference of Rs between disturbed and undisturbed crusted soil.−3. All meteorological data were provided by the meteorological station of Yanchi County. Natural vegetation in the area is dominated by Artemisia ordosica. The soil surface between the shrubs is commonly covered by algae, lichen, and moss crusts which are mainly composed of Microcoleus vaginatus, Oscillatoria chlorine, Collema tenax, and Bryum argenteum. Species composition of BSCs were identified using the Opton West Germany 475200-9901 and Optex BK5000 microscopic technique and an extensive reference collection of field samples .The research was conducted in the Yanchi Research Station , at the south edge of the Mu Us desert, China. An elevation is 1550 m a.s.l. The prevailing climate is of the temperature arid and semiarid type, with average rainfall and temperature of 287 mm (62% of which fell between July and September) and 7.6°C, respectively. The soil has a bulk density of 1.61 g cmArtemisia ordosica shrub were chosen, including algae crusted soil, lichen crusted soil, moss crusted soil, and bare land as control. For each cover type above, five 2 × 3 m plots at least 3 m apart were randomly selected as independent replicates. In each plot, two 3 m2 quadrats were established. One was scraped to 0% BSC cover ; the other was undisturbed with intact BSCs . The reestablished BSCs within all disturbed crusted soil quadrats were carefully removed every two weeks after being scraped. The uppermost sediments at each plot were similar before disturbance because the geomorphology of the sites and chemical and textural properties of deeper (25 cm soil depth) soil samples were similar. In the bare land type, we selected five plots as independent replicates for Rs measurement. At each quadrat and plot in bare land, 8 cm tall, circular 80 cm2 PVC soil respiration collars were permanently inserted 5 cm into soils before one month of Rs measurement. Collars were placed >3 m apart from plants to minimize potential risk on Rs from roots and mycorrhiza.Four most frequent soil cover types in 2 to collect soil samples. In each disturbed quadrat and bare land plot, samples were collected by inserting three 5.4 cm diameter cutting rings into soil about 5 cm deep, two were carefully removed to the aluminium containers to measure soil water content (SWC), soil organic carbon (SOC), total nitrogen content (TNC), and soil particle content, and the other cutting ring was put by a hole round cover with filter paper to examine soil bulk density (SBD). In each undisturbed soil quadrat, the BSC layer was carefully removed using a small shovel before sample being collected (as above) and then the removed BSC layer was carefully collected to examine SBD.During each measurement, we used aluminium containers with a surface area of 22.89 cm3) connected to the portable Li-6400 . Measurements were performed at 2 h intervals on clear days from 7:00 a.m. to 19:00 p.m. on April 28, May 7, May 16, May 25, June 4, June 11, July 7, July 13, and July 25, 2010, during dry conditions and SWC was below 0.07 mm and photosynthesis of BSCs was limited Rs(Tγs is soil bulk density (g·cm−3), same below; g1 is sample weight (g); g2 is sample weight completely wrapped by wax (g); g3 is original reading of electronic balance (g); g4 is reading of electronic balance with sample (g); ρ1 is specific gravity of water, ρ1 = 1.0 g·cm−3; ρ2 is specific gravity of wax, ρ2 = 0.9 g·cm−3; W is water content of sample:f is soil total porosity (%); ρs is soil particle density (g·cm−3), ρs = 2.65 g·cm−3.The sample was oven-dried at 105°C to determine SWC. The samples were air-dried and homogenized, then passed through a 2 mm sieve, and analyzed for SOC by potassium dichromate oxidation-outer heating, TNC by semimicro-Kjeldahl method, soil particle content by a laser particle analyzer , and SBD of disturbed and bare land soil by a Soil Moisture Equipment  model 0200 soil core sampler. SBD and total porosity of BSCs layer were determined by the wax seal method  and SBD P values of <0.05. Prior to these analyses, data were tested for assumptions of normality and homogeneity of variances and were log-transformed when necessary. All the regression and ANOVA analyses were performed using the SPSS 15.0 statistical software .Mean diurnal cycles of Rs and Ts were computed by averaging the hourly means for each time of day. Daily mean values were computed as the average of the hourly means. The daily mean values were used to examine the seasonal responses of Rs to Ts. Exponential equations  were useP < 0.05). Temperature was not significantly different in the microsites of the three crusted soil cover areas and their mean values are those reported in P > 0.05).Rs showed signal peak curves at a day scale in four soil cover areas, with the highest values occurring in 13:00–15:00 Figures . Rs was μmol m−2 s−1 . Interactions between microsites and measurement time had significant effects on Rs (P < 0.001). In lichen crusted soil, RM ANOVA showed that daily mean Rs was not significantly different among HLC, NLC, and BL (P > 0.05).In the BL, daily mean Rs ranged from 0.41 ± 0.04 to 1.19 ± 0.73  m−2 s−1 . In alga1 in NLC . In moss1 in NMC . In algaP < 0.001) higher than that of HMC and HAC, respectively, which is consistent with the result studied in Kalahari [P < 0.001)  . Similaremission .There were no differences of carbon release observed in HMC, HLC, HAC, and BL under dry conditions, regardless of the strong difference in organism. This result indicated that respiration of the organisms is not the main driver factor under dry condition. Consistent with our study, the same difference was observed between the two crusted (moss and cyanobacteria/lichen crusted soils) soils and bare land in the Gurbantünggüt desert, north China , and no R2 = 0.94, Q10 = 1.45, P < 0.01; NAC: R2 = 0.78, Q10 = 1.67, P < 0.01; BL: R2 = 0.78, Q10 = 1.38, P < 0.01; HLC: R2 = 0.86, Q10 = 1.65, P < 0.01; NLC: R2 = 0.87, Q10 = 1.84, P < 0.01; HMC: R2 = 0.42, Q10 = 1.16, P < 0.01; NMC: R2 = 0.50, Q10 = 1.34, P < 0.01)  or by SOC quality variation in the disturbed and undisturbed crusted soil [In disturbed and undisturbed crusted soil and bare land, daily mean Rs fluctuated exponentially with Ts at seasonal scale (HAC:  Figures . The aveosystems , 24. Howosystems  and Thomosystems  in dry sted soil , 27, whi2 and N2 [Without protective cover from BSC, wind preferentially removes fine-grained material relative to coarse-grained material , which a2 and N2 . The finC losses in gases respired and C input in soils under dry condition. The results suggest that crusted soils protect strictly to sequester CO2 and can rapidly become a net source of CO2 if disturbed in dry condition. Therefore, disturbance of BSCs soil should be a consideration for management related to the long-term sustainability in dry-land environments.In conclusion, disturbance of BSCs will potentially lead to greater


Objective. The aim of this work is to study and compare the antioxidant properties and phenolic contents of aqueous leaf extracts of Juniperus thurifera, Juniperus oxycedrus, Juniperus Phoenicea, and Tetraclinis articulata from Morocco. Methods. Antioxidant activities of the extracts were evaluated by 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical-scavenging ability, Trolox equivalent antioxidant capacity (TEAC), and ferric reducing antioxidant power (FRAP) assays. Also the total phenolic and flavonoids contents of the extracts were determined spectrophotometrically. Results. All the extracts showed interesting antioxidant activities compared to the standard antioxidants (butylated hydroxytoluene (BHT), quercetin, and Trolox). The aqueous extract of Juniperus oxycedrus showed the highest antioxidant activity as measured by DPPH, TEAC, and FRAP assays with IC50 values of 17.91 ± 0.37 μg/mL, 19.80 ± 0.55 μg/mL, and 24.23 ± 0.07 μg/mL, respectively. The strong correlation observed between antioxidant capacities and their total phenolic contents indicated that phenolic compounds were a major contributor to antioxidant properties of these plants extracts. Conclusion. These results suggest that the aqueous extracts of Juniperus thurifera, Juniperus oxycedrus, Juniperus phoenicea, and Tetraclinis articulata can constitute a promising new source of natural compounds with antioxidants ability. Researchers are now looking for natural antioxidants which do not have any side effects on human health. The search is underway to find out newer, effective, and safe antioxidants, in order to use them in foods and pharmaceutical preparations to replace the synthetic ones.Medicinal plants are the major source of chemical compounds exhibiting antioxidant activity. Several studies have reported the interesting composition of Moroccan medicinal plants, including phenolic acids, flavonoids, and tannins, which are known for their health benefits as antioxidants –3.The Moroccan flora contains more than 4200 vascular plant species. However, few ones were screened for pharmacological or chemical properties [Juniperus thurifera, Juniperus phoenicea, Juniperus oxycedrus, and Tetraclinis articulata), it is interesting to evaluate the chemotaxonomic and pharmacological differences characterizing this plant family. These species have many uses in traditional medicine in several parts of the world. Juniper berries are used as a spice, particularly in European cuisine, which are the only spice derived from conifers. In Morocco, Cupressaceae tar, leaves, and fruits are widely used to treat different hair and skin problems like dandruff, eczema, itchiness, and fungal infections [Recent investigation has reaffirmed the validity of many of their traditional uses and reported that the extracts from Cupressaceae contain most of the important phenolic compounds, especially flavonoids, neolignans, and phenylpropanoids –7. In thfections . Additiofections , 10. Juniperus thurifera, Juniperus phoenicea, Juniperus oxycedrus, and Tetraclinis articulata using DPPH free radical-scavenging activity (DPPH), Trolox equivalent antioxidant capacity (TEAC), and ferric reducing antioxidant power (FRAP) assays and moreover to determine their total phenolic contents and investigate the relationship between total phenolic content and antioxidant activity.Therefore, the present study aims to determine and compare the antioxidant potential of aqueous leaf extracts of J. thurifera, J. Phoenicea, J. oxycedrus, and T. articulata were collected from wild populations located in Oukaimeden, Essaouira, Ourika, and Ait Issi Ihahan regions, respectively. The characteristics of collection sites are shown in J. thurifera), 79592 (J. phoenicea), 79588 (J. oxycedrus), and 79589 (T. articulata). The collected plant material was air-dried at room temperature (20–24°C). Leaves of dried plant were separated from plant and stored in tight-seal dark containers until use.In February 2014, leaves ofTo prepare the water extract, 10 g of dry powdered plant material was soaked in 100 mL of boiling distilled water and left to stand at room temperature for 30 min. The hot infusions were then filtered using Whatman filter paper and concentrated under vacuum on a rotary evaporator at 60°C and stored at 4°C for further use. J. thurifera, J. Phoenicea, J. oxycedrus, and T. articulata to scavenge the DPPH radical was estimated using the method described by Şahin et al. [μL of various sample concentrations was added to a volume of 2 mL from the DPPH methanolic solution (60 μM). The reaction mixture was well shaken and incubated for 20 min at room temperature in the dark and the absorbance was recorded at 517 nm. The blank was constituted by methanol instead of the extract. Butylated hydroxytoluene (BHT) and quercetin were used as positive controls. The percentage inhibition of the DPPH radical by the samples was calculated using the following equation:A0 is the absorbance of control sample and A1 is the absorbance of the test sample. The sample concentration providing 50% of inhibition (IC50) was determined from the plotted curve of inhibition using several concentrations.The ability of water extracts ofn et al. . An aliqμL of appropriately diluted samples was added to 2 mL of ABTS solution and the absorbance was recorded at 734 nm after 1 min of incubation at room temperature. A standard curve was obtained by using Trolox standard solution at various concentrations (ranging from 0 to 0.24 μg/mL). The scavenging activity of different concentrations of extracts and fractions against ABTS radical were also measured to calculate IC50, and the procedure was similar to the DPPH scavenging method described above.The ABTS free radical-scavenging activity of each sample was determined according to the method described by Loizzo et al. . The ABT3Fe(CN)6 (1%) were mixed and incubated at 50°C for 20 min, to reduce ferricyanide into ferrocyanide. The reaction was stopped by adding 2.5 mL of 10% (w/v) trichloroacetic acid followed by centrifugation at 1000 rpm for 10 min. Finally, 2.5 mL of the upper layer was mixed with 2.5 mL of distilled water and 0.5 mL of FeCl3 (0.1%) and the absorbance was measured at 700 nm. The sample concentration providing 0.5 of absorbance (IC50) was calculated by plotting absorbance against the corresponding sample concentration. BHT and quercetin were used as a reference compound.The ferric reducing capacity of extracts was investigated by using the potassium ferricyanide-ferric chloride method . BrieflyμL aliquot of extract solution was mixed with 1.16 mL of distilled water and 100 μL of Folin-Ciocalteu reagent, followed by the addition of 300 μL of Na2CO3 solution (20%). After 30 min of incubation at 40°C, the absorbance of the reaction mixture was measured at 760 nm. Gallic acid was used as a reference standard, and the results were expressed as microgram gallic acid equivalent (μg GAE)/mg dry weight of plant extract (edw).The total phenolic contents (TPC) were determined spectrophotometrically according to the Folin-Ciocalteu colorimetric method . Briefly3. After 1 hour at room temperature, the absorbance was measured at 420 nm. Total flavonoid content was expressed as microgram quercetin equivalent (μg QE)/mg dry plant extract (edw).Total flavonoid content was measured by the aluminum chloride colorimetric assay . 0.5 mL All the assays were carried out in triplicate. The means and standard deviation (SD) were determined using SPSS version 20, the results of antioxidant activities and phenolic compounds composition are expressed as mean values ± SD, and the analysis of variance was performed to determine significant differences. J. thurifera, J. phoenicea, J. oxycedrus, and T. articulata infusions: DPPH free radical-scavenging activity, Trolox equivalent antioxidant capacity (TEAC), and reducing power assays.Antioxidant activity is a complex procedure usually happening through several mechanisms and is influenced by many factors, which cannot be fully described with one single method. Therefore, it is essential to perform more than one type of antioxidant capacity measurement to take into account the various mechanisms of antioxidant action –18. In t J. oxycedrus, followed by J. thurifera, T. articulata, and J. oxycedrus.As summarized in  J. oxycedrus had the strongest free radical-scavenging activity with IC50 value of 17.91 ± 0.37 μg/mL ( J. phoenicea water extract (IC50 = 30.74 ± 0.11 μg/mL).All extracts were able to reduce the stable, purple-coloured radical DPPH into yellow-coloured DPPH-H. The water extract obtained from37 μg/mL . On the 50 = 4.20 ± 0.02 μg/mL) and quercetin (IC50 = 1.29 ± 0.01 μg/mL). Similarly, J. oxycedrus extract exhibited the best performance in ABTS and reducing power assays with IC50 = 19.80 ± 0.55 μg/mL and 24.23 ± 0.07 μg/mL, respectively.All water extracts were less effective than the synthetic antioxidant BHT  μg GAE/mg edw, followed by J. thurifera (193.79 ± 6.47) μg GAE/mg edw, T. articulata (175.67 ± 10.21) μg GAE/mg edw, and J. phoenicea (116.35 ± 9.71) μg GAE/mg edw.The aqueous extracts ofompounds . The higμg (QE)/mg edw /mg edw in J. thurifera and T. articulata, respectively. The lowest flavonoid concentration was determined for J. phoenicea (6.69 ± 0.22 μg (QE)/mg edw).Concentration of flavonoids in investigated species ranged from 6.70 to 20.81 )/mg edw . A high R2 values were positive at P < 0.01 significance level, indicating that the values of antioxidant capacities assayed by the three different methods were highly correlative. DPPH values were strongly correlated with the ABTS activities (R2 = 0.951) and reasonably well correlated with the FRAP values (R2 = 0.957). The correlation between the ABTS and FRAP values was also highly significant (R2 = 0.848).To evaluate the suitability and reliability of the three assay methods used to determine the total antioxidant capacities of the four Cupressaceae species, we performed correlation analysis of the values of total antioxidant capacity obtained by these methods. As shown in R2 = 0.986) and total flavonoids concentration (R2 = 0.986). The total phenolic and flavonoid content also correlated well with the ABTS  and FRAP values .The correlation between antioxidant capacity and phenolic content of the four Moroccan Cupressaceae samples is described in Oxidative stress has been implicated in several diseases including diabetes, rheumatoid arthritis, cardiovascular diseases, atherosclerosis, neurodegenerative diseases , cancer, and aging –21. NatuIn fact the used methods have different reaction mechanisms . For insFurthermore, well-pronounced correlations were observed between these methods which confirm that the three assays were all suitable and reliable for assessing total antioxidant capacities of the plant extracts. J. oxycedrus have the strongest scavenging activity against DPPH (17.91 ± 0.37 μg/mL), ABTS (19.80 ± 0.55 μg/mL), and the best performance reducing power activity (24.23 ± 0.07 μg/mL) followed by the leaf extract of J. thurifera (24.85 ± 0.42 μg/mL (DPPH); 30.36 ± 0.24 μg/mL (ABTS); 35.83 ± 0.37 μg/mL (FRAP)), T. articulata (27.38 ± 0.02 μg/mL (DPPH); 32.92 ± 0.56 μg/mL (ABTS); 47.12 ± 0.15 μg/mL (FRAP)), and J. phoenicea (30.74 ± 0.11 μg/mL (DPPH); 47.37 ± 0.59 μg/mL (ABTS); 46.85 ± 0.42 μg/mL (FRAP)) (The results of our screening on the four Cupressaceae species growing in Morocco showed that the leaf water extracts of (FRAP)) . This wo J. phoenicea showing a strong antioxidant activity in ABTS and DPPH radical-scavenging activity test afforded several phenolics , which were stated to be the free radical scavengers in this plant [ Juniperus [ J. oxycedrus were examined using two different tests of the ferric reducing antioxidant power assay and DPPH radical-scavenging activity and most of them showed a potent antioxidant effect under those systems, which is in accordance with our data. High DPPH scavenging activity of Tetraclinis articulata leaves collected in Tunisia was also reported [There have been diverse reports on antioxidant activity of a number of Cupressaceae species. The leaves and berries ofis plant . In a stuniperus , the aqureported .The strong correlations between the total antioxidant capacity assayed by DPPH, ABTS, and FRAP methods and the phenolic content  indicate J. oxycedrus presented the highest TPC, TFC, and antioxidant capacity values. In addition, there was a good correlation between phenolic content and antioxidant capacity of the leaves extracts. These results suggest that the water infusion from the leaves of J. thurifera, J. oxycedrus, J. phoenicea, and T. articulata constitutes a valuable source of antioxidant metabolites. Further investigations are required to identify their active metabolites.The findings obtained in this study support the traditional uses of those plant species as therapeutic agents. The higher antioxidant potential of those Moroccan Cupressaceae is conferred by their high phenolic content.