Pročišćavanje i karakterizacija ekstracelularne dekstran saharaze iz bakterije Pediococcus pentosaceus, izolirane iz tla sjeveroistočne Indije

The extracellular dextransucrase produced from Pediococcus pentosaceus, a new isolate from the soil in Assam, India, was purified and characterized. The enzyme activity of cell-free supernatant was 3.4 U/mL and specific activity was 0.6 U/mg. The crude enzyme was purified by a single-step fractionation using polyethylene glycols of different molecular mass. The specific activity achieved was 18 U/mg with 31-fold purification by PEG 400 and 26 U/mg with 45-fold purification by PEG 1500. The molecular mass of dextransucrase determined by non-denaturing SDS-PAGE was approx. 180 kDa. The dextran formation activity of the enzyme was confirmed by activity staining. Optimum conditions for dextransucrase activity were: pH=5.4, reaction temperature 30 °C, 5 % sucrose and 20 mM sodium acetate buffer. A concentration of 1 mM MgCl2 and 6 mM CaCl2 enhanced dextransucrase activity by 5 and 150 %, respectively. The chaotropic agent urea (7 M) and chelating agent EDTA (1 mM) resulted in the residual enzyme activity of 98 and 80 %, respectively. The organic solvents such as ethanol (50 %), DMSO (90 %), acetone (50 %) and acetonitrile (20 %) decreased the dextransucrase activity by 80, 91, 94 and 80 %, respectively.U radu je pročišćena i okarakterizirana dekstran saharaza iz bakterije Pediococcus pentosaceus, izolirane iz tla u gradu Assamu, Indija. Aktivnost je enzima u supernatantu bila 3,4 U/mL, a njegova je specifična aktivnost iznosila 0,6 U/mg. Sirovi je enzim pročišćen jednostupanjskim frakcioniranjem pomoću polietilen glikola različite molekularne mase. Utvrđena je specifična aktivnost enzima od 18 (pročišćenog 31 put pomoću PEG 400), odnosno 26 U/mg (pročišćenog 45 puta pomoću PEG 1500). Molekularna je masa dekstran saharaze određena pomoću SDS-PAGE, a iznosila je otprilike 180 kDa. Aktivnost je enzima potvrđena bojanjem nastalog dekstrana s Coomasie brilijant plavom bojom. Optimalni su uvjeti za aktivnost enzima bili: pH=5,4; temperatura reakcije od 30 °C; te dodatak 5 %-tne saharoze i acetatnog pufera (20 mM). Dodatak 1 mM MgCl2 i 6 mM CaCl2 povećali su aktivnost enzima za 5, odnosno 150 %. Inaktivirajući agensi, poput uree (7 M) i EDTA (1 mM) smanjili su aktivnost enzima na 98 odnosno 80 %. Organska su otapala također smanjila aktivnost enzima, i to: 50 %-tni etanol na 80 %, 90 %-tni DMSO na 91 %, 50 %-tni aceton na 94 % i 20 %-tni acetonitril na 80 %

Dextransucrase from the mutant of Pediococcus pentosaceus (PPm) is more stable than the wild type

A comparative study on both wild type and mutant of Pediococcus pentosaceus for dextransucrase activity, its stability, dextran synthesizing activity, antibiotic sensitivity and carbohydrate utilization was performed. The wild type P. pentosaceus had specific activity of 0.58 U/mg whereas the mutant showed that of 1.0 U/mg with 72% enhancement. The antibiogram of 27 antibiotics tested against mutant showed significant differences with 9 antibiotics when compared to wild type. In carbohydrate fermentation profile, trehalose, galactose, maltose, lactose and fructose are metabolized by both the strains, but weakly in case of mutant. Stabilization of purified dextransucrase from wild type and mutant with various stabilizers was studied at 30 and 4 °C. Both enzymes were more stable at 4 °C. Among various stabilizers such as dextran (100 kDa, 10 μg/ml), glycerol (0.5%, v/v), PEG 8000 (10 μg/ml) and Tween 80 (0.5%, v/v), Tween 80 provided maximum stabilization at 4 and 30 °C. The mutant showed better stabilization than that of the wild type at both 30 and 4 °C. The loss of activity at 30 °C after 24 h in wild type and mutant in the presence of Tween 80 was only 34 and 32%, respectively, whereas the loss of activity in control of wild type and mutant was 76 and 59%, respectively. After 15 days at 4 °C, the loss of activity in control of wild type and mutant in the presence of Tween 80 was only 15 and 8%, respectively, whereas at 30 °C, the loss of activity in control of wild type and mutant was 49 and 42% respectively. Half-life of the enzyme with Tween 80 was 28.5 and 33.5 h for wild type and mutant, respectively, at 30 °C and 52.1 and 106.6 days for wild type and mutant respectively, at 4 °C

Enhancement of dextransucrase activity of <i style="">Pediococcus</i> <i style="">pentosaceus</i> mutant SPAm1 by response surface methodology

346-351In the present investigation, an optimal fermentation medium for the production of dextransucrase from mutant SPAm1 of a natural isolate of Pediococcus pentosaceus (GenBank Acc. No. EU569832) was developed following response surface method. A two-level Plackett-Burman design (PBD) and five-level Central composite design (CCD) was combined to optimize the medium composition for enhancement of the dextransucrase activity of SPAm1. A second-order model equation was suggested and validated experimentally. The model adequacy was very satisfactory as the coefficient of determination was 0.958. The optimum values for the tested variables were; sucrose 55.2 g/L; beef extract 2.3 g/L and Tween 80 8.3 mL/L. The predicted dextransucrase activity after response optimization was 15.9 U/mL, whereas the experimental value was 15.6 U/mL at 16 h of incubation, which showed a 3.2-fold enhancement over the enzyme activity (4.9 U/mL) of mutant SPAm1 given by the unoptimized medium. However, the increase in dextransucrase activity by SPAm1 after optimization of the medium was an outstanding 4.6-fold higher over that of the wild-type P. pentosaceus from the unoptimized medium (3.4 U/mL)

Purification and Characterization of an Extracellular Dextransucrase from Pediococcus pentosaceus Isolated from the Soil of North East India

The extracellular dextransucrase produced from Pediococcus pentosaceus, a new isolate from the soil in Assam, India, was purified and characterized. The enzyme activity of cell-free supernatant was 3.4 U/mL and specific activity was 0.6 U/mg. The crude enzyme was purified by a single-step fractionation using polyethylene glycols of different molecular mass. The specific activity achieved was 18 U/mg with 31-fold purification by PEG 400 and 26 U/mg with 45-fold purification by PEG 1500. The molecular mass of dextransucrase determined by non-denaturing SDS-PAGE was approx. 180 kDa. The dextran formation activity of the enzyme was confirmed by activity staining. Optimum conditions for dextransucrase activity were: pH=5.4, reaction temperature 30 °C, 5 % sucrose and 20 mM sodium acetate buffer. A concentration of 1 mM MgCl2 and 6 mM CaCl2 enhanced dextransucrase activity by 5 and 150 %, respectively. The chaotropic agent urea (7 M) and chelating agent EDTA (1 mM) resulted in the residual enzyme activity of 98 and 80 %, respectively. The organic solvents such as ethanol (50 %), DMSO (90 %), acetone (50 %) and acetonitrile (20 %) decreased the dextransucrase activity by 80, 91, 94 and 80 %, respectively

Antioxidant Properties and Kidney Cell Protection by the Extracts of Curcuma longa, Artemisia princeps, Salicornia herbacea, and Schisandra chinesis

This study evaluated the antioxidant properties and kidney cell protection of medicinal plants as natural medicaments. A total of four medicinal plants including turmeric (TM), gangwha mugwort (GM), glasswort (GW), and omija (OM) were selected and fermented. Hot water extracts (HWE) and ethanol extracts (EE) of the plants were prepared before and after fermentation and tested in experiments in vitro and in vivo. Total polyphenol contents (TPC) and total flavonoid content (TFC) of GM were the highest among them. The TPC based HWE decreased after fermentation except OM; in contrast, TFC from HWE increased. The DPPH radical scavenging activity and ABTS value from HWE increased after fermentation, especially OM, which showed significant differences, while DPPH and ABTS from EE were decreased. The cell viability was not changed after addition of these plants extracts below 50 &mu;g/mL; however, TM from HWE significantly decreased. The protective effect on kidney cells against cisplatin showed a 60% range of cell viability in each plant extract. In the in vivo experiment, the protective effect on kidney cells by the supplemented plant extracts was demonstrated by the serum creatinine and BUN level. During experimental periods, the serum creatinine and BUN level of GW and GM-treated mice decreased with significant differences compared to the adenine control group. As a result, these plant extracts had no cytotoxicity and maintained a protective effect as well as antioxidant activity. These results suggest that plants such as gangwha mugwort (GM) and glasswort (GW) may be good extracts for kidney cell protection and antioxidant agents

The Genus Allium as Poultry Feed Additive: A Review

The genus Allium, belonging to the family Amaryllidaceae has been known since ancient times for their therapeutic potentials. As the number of multi-drug resistant infections has increased due to in-feed antibiotic usage in poultry, the relevance of alliums as feed additives has been critically assessed. Garlic and the other Allium species, such as onions, leek, shallot, scallion, and chives, have been characterized to contain a plethora of bioactive compounds such as organosulfur compounds, polyphenols, saponins, fructans, and fructo-oligosaccharides. Consequently, alliums have been validated to confer antioxidant, antibacterial, antiviral, immunostimulatory, gut homeostasis, and lipid- as well as cholesterol-lowering properties in poultry. This review intends to summarize recent progress on the use of edible alliums as poultry feed additives, their beneficial effects, and the underlying mechanisms of their involvement in poultry nutrition. Perspectives for future research and limitations are also briefly discussed

Optimization of Chinese Chive Juice as a Functional Feed Additive

Allium tuberosum, commonly known as the Chinese chive (CC) is often used as a traditional medicine in East Asia for its health benefits. To explore the potential of CC as a functional feed additive, antibacterial and antioxidant assays, untargeted metabolomics, and a 2 &times; 3 &times; 3 fractional factorial design (FFD) were conducted. In the present study, CC displayed stable DPPH radical scavenging activity with constant total phenolic content, however, the total flavonoid contents and the antibacterial activities were attenuated following heat treatment. The FFD results identified the solid content (SBM) as the main determinant of the antibacterial activity and moisture content of the CC products along with two other factors: drying time and temperature. Two CC products manufactured with 30% (w/v) SBM with 3 h drying at 80 &deg;C and 20% (w/v) SBM with 8 h drying at 60 &deg;C obtained the maximum antibacterial activity and least moisture content (&lt;5%). Liquid chromatography-tandem mass spectrometry based multivariate analysis revealed 14 changed compounds in the non-heated and heated CC including flavonols, sinapinic acid, and lysophospholipids, which might affect the functionality. In conclusion, we propose an empirical approach to the pre-processing of CC juice that is suitable for blending in feed and simultaneously retaining its bioactivities

Optimized endodextranase-epoxy CIM ® disk reactor for the continuous production of molecular weight-controlled prebiotic isomalto-oligosaccharides

International audienceAn epoxy-activated monolithic Convective Interaction Media (CIM®) disk was used for the immobilization of endodextranase D8144 from Penicillium sp. (EC 3.2.1.11) in order to produce on-line isomalto-oligosaccharides (IMOs) from Dextran T40. Enzymatic parameters, molecular weight of IMOs and performance of the IMmobilized Enzymes Reactor (IMER) were investigated. The immobilization yield of enzymes was about 45.3% (w/w), and the real specific activity close to 3.26 U mg−1. The Km values did not significantly change between free (12.8 g L−1) and immobilized enzymes (14.2 g L−1), due to the absence of diffusional limitation. The IMER system presented more than 80% of its residual activity after 5000 column volumes, highlighting the high stability of the immobilized endodextranases. Response surface methodology was used to enhance the performance of the IMER. Depending on dextran concentrations and flow rates, specific patterns of IMOs distributions were observed during the enzymatic hydrolysis. Finally, prebiotic activity was also investigated on IMOs produced by medium conditions (flow rate 0.3 mL min−1 and dextran concentrations 4% w/w) against Lactobacillus rhamnosus GG (ATCC 53103). Their scores were at least as good as two commercialized fructo-oligosaccharides (FOS), Fibrulose® F97 and Orafti® P95

Evaluation of Non-Fermented and Fermented Chinese Chive Juice as an Alternative to Antibiotic Growth Promoters of Broilers

The present study explores the application of CC juice as a suitable feed additive and alternative to conventional antibiotics. We performed a comparative study to investigate the effects of non-fermented and fermented CC juice on broiler productivity, meat quality, blood characteristics, intestinal characteristics, and microbiota associated with intestinal characteristics. A total of 800 one-day-old Ross 308 broiler chickens were randomly assigned to one of the four dietary treatment groups: (1) basal diet (negative control; NC); (2) basal diet + 0.01% enramycin (positive control; PC); (3) basal diet + 3% non-fermented CC juice (NCC; CC juice 10%, water 90%); and (4) basal diet + 3% fermented CC juice (FCC; CC juice 10%, water 90%, Lactobacillus plantarum SK4719). Feed and water were provided ad libitum. Intriguingly, all treatments showed similar results in terms of broiler productivity and chicken meat quality. Considering organ characteristics, the FCC group showed a low spleen weight and lower (p &lt; 0.05) blood levels of AST and total cholesterol (TCHO). Regarding intestinal characteristics, the CC feed additive (NCC and FCC) resulted in a heavier intestinal weight (p &lt; 0.05) without affecting the length ratio of the villi or the crypt compared to the control (NC or PC). NCC and FCC lowered the growth of intestinal pathogens (p &lt; 0.01). In summary, the addition of FCC can maintain poultry health by improving blood compositions and inhibiting the growth of intestinal pathogens, leading to a productivity comparable to that of poultry treated with growth-promoting antibiotics