Indole ring oxidation by activated leukocytes prevents the production of hypochlorous acid

Hypochlorous acid (HOCl) released by activated leukocytes has been implicated in the tissue damage that characterizes chronic inflammatory diseases. In this investigation, 14 indole derivatives, including metabolites such as melatonin, tryptophan and indole-3-acetic acid, were screened for their ability to inhibit the generation of this endogenous oxidant by stimulated leukocytes. The release of HOCl was measured by the production of taurine-chloramine when the leukocytes (2 x 10(6) cells/mL) were incubated at 37ºC in 10 mM phosphate-buffered saline, pH 7.4, for 30 min with 5 mM taurine and stimulated with 100 nM phorbol-12-myristate acetate. Irrespective of the group substituted in the indole ring, all the compounds tested including indole, 2-methylindole, 3-methylindole, 2,3-dimethylindole, 2,5-dimethylindole, 2-phenylindole, 5-methoxyindole, 6-methoxyindole, 5-methoxy-2-methylindole, melatonin, tryptophan, indole-3-acetic acid, 5-methoxy-2-methyl-3-indole-acetic acid, and indomethacin (10 µM) inhibited the chlorinating activity of myeloperoxidase (MPO) in the 23-72% range. The compounds 3-methylindole and indole-3-acetic acid were chosen as representative of indole derivatives in a dose-response study using purified MPO. The IC50 obtained were 0.10 ± 0.03 and 5.0 ± 1.0 µM (N = 13), respectively. These compounds did not affect the peroxidation activity of MPO or the production of superoxide anion by stimulated leukocytes. By following the spectral change of MPO during the enzyme turnover, the inhibition of HOCl production can be explained on the basis of the accumulation of the redox form compound-II (MPO-II), which is an inactive chlorinating species. These results show that indole derivatives are effective and selective inhibitors of MPO-chlorinating activity.

Identification and in vitro production of Lactobacillus antagonists from women with or without bacterial vaginosis

Lactobacilli isolated from the vaginal tract of women with and without bacterial vaginosis (BV) were identified and characterized for the production of antagonists. Bacterial samples were isolated from healthy women (N = 16), from patients with clinical complaints but without BV (N = 30), and from patients with BV (N = 32). Identification was performed using amplified ribosomal DNA restriction analysis. Production of antagonistic compounds was evaluated by the double-layer diffusion technique using Gram-positive (N = 9) and Gram-negative bacteria (N = 6) as well as yeast (N = 5) as indicator strains. Of a total of 147 isolates, 133 were identified as pertaining to the genus Lactobacillus. Lactobacillus crispatus was the species most frequently recovered, followed by L. johnsonii and L. jensenii. Statistical analysis showed that L. crispatus was more frequent in individuals without BV (P < 0.05). A higher production of antagonists was noted in L. crispatus isolates from healthy women (P < 0.05). More acidic local pH and higher H2O2 production by isolated lactobacilli from healthy women suggest these mechanisms as the possible cause of this antagonism. In conclusion, a significant correlation was detected between the presence and antagonistic properties of certain species of Lactobacillus and the clinical status of the patients.

Aging alters the production of iNOS, arginase and cytokines in murine macrophages

The limited amount of information on the primary age-related deficiencies in the innate immune system led us to study the production of inducible nitric oxide synthase (iNOS), arginase, and cytokines in macrophages of young (8 weeks old) and old (72 weeks old) female BALB/c mice. We first evaluated iNOS and arginase inducers on peritoneal (PMΦ) and bone marrow-derived (BMMΦ) macrophages of young BALB/c and C57BL/6 mice, and then investigated their effects on macrophages of old mice. Upon stimulation with lipopolysaccharide (LPS), resident and thioglycolate-elicited PMΦ from young mice presented higher iNOS activity than those from old mice (54.4%). However, LPS-stimulated BMMΦ from old mice showed the highest NO levels (50.1%). Identical NO levels were produced by PMΦ and BMMΦ of both young and old mice stimulated with interferon-γ. Arginase activity was higher in resident and elicited PMΦ of young mice stimulated with LPS (48.8 and 32.7%, respectively) and in resident PMΦ stimulated with interleukin (IL)-4 (64%). BMMΦ of old mice, however, showed higher arginase activity after treatment with IL-4 (46.5%). In response to LPS, PMΦ from old mice showed the highest levels of IL-1α (772.3 ± 51.9 pg/mL), whereas, those from young mice produced the highest amounts of tumor necrosis factor (TNF)-α (937.2 ± 132.1 pg/mL). Only TNF-α was expressed in LPS-treated BMMΦ, and cells from old mice showed the highest levels of this cytokine (994.1 ± 49.42 pg/mL). Overall, these results suggest that macrophages from young and old mice respond differently to inflammatory stimuli, depending on the source and maturity of the cell donors.

Effect of TNF-α production inhibitors on the production of pro-inflammatory cytokines by peripheral blood mononuclear cells from HTLV-1-infected individuals

Human T lymphotropic virus type 1 (HTLV-1) is the causal agent of myelopathy/tropical spastic paraparesis (HAM/TSP), a disease mediated by the immune response. HTLV-1 induces a spontaneous proliferation and production of pro-inflammatory cytokines by T cells, and increasing interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) levels are potentially involved in tissue damage in diseases related to HTLV-1. This exaggerated immune response is also due to an inability of the natural regulatory mechanisms to down-modulate the immune response in this group of patients. TNF-α inhibitors reduce inflammation and have been shown to improve chronic inflammatory diseases in clinical trials. The aim of this study was to evaluate the ability of pentoxifylline, forskolin, rolipram, and thalidomide to decrease in vitro production of TNF-α and IFN-γ in cells of HTLV-1-infected subjects. Participants of the study included 19 patients with HAM/TSP (mean age, 53 ± 11; male:female ratio, 1:1) and 18 HTLV-1 carriers (mean age, 47 ± 11; male:female ratio, 1:2.6). Cytokines were determined by ELISA in supernatants of mononuclear cell cultures. Pentoxifylline inhibited TNF-α and IFN-γ synthesis with the minimum dose used (50 µM). The results with forskolin were similar to those observed with pentoxifylline. The doses of rolipram used were 0.01-1 µM and the best inhibition of TNF-α production was achieved with 1 µM and for IFN-γ production it was 0.01 µM. The minimum dose of thalidomide used (1 µM) inhibited TNF-α production but thalidomide did not inhibit IFN-γ production even when the maximum dose (50 µM) was used. All drugs had an in vitro inhibitory effect on TNF-α production and, with the exception of thalidomide, all of them also decreased IFN-γ production.

Production of L1 protein from different types of HPV in Pichia pastoris using an integrative vector

Human papillomavirus (HPV) infection is the most common sexually transmitted disease in the world and is related to the etiology of cervical cancer. The most common high-risk HPV types are 16 and 18; however, the second most prevalent type in the Midwestern region of Brazil is HPV-33. New vaccine strategies against HPV have shown that virus-like particles (VLP) of the major capsid protein (L1) induce efficient production of antibodies, which confer protection against the same viral type. The methylotrophic yeast Pichia pastoris is an efficient and inexpensive expression system for the production of high levels of heterologous proteins stably using a wild-type gene in combination with an integrative vector. It was recently demonstrated that P. pastoris can produce the HPV-16 L1 protein by using an episomal vector associated with the optimized L1 gene. However, the use of an episomal vector is not appropriate for protein production on an industrial scale. In the present study, the vectors were integrated into the Pichia genome and the results were positive for L1 gene transcription and protein production, both intracellularly and in the extracellular environment. Despite the great potential for expression by the P. pastoris system, our results suggest a low yield of L1 recombinant protein, which, however, does not make this system unworkable. The achievement of stable clones containing the expression cassettes integrated in the genome may permit optimizations that could enable the establishment of a platform for the production of VLP-based vaccines.

The superoxide anion donor, potassium superoxide, induces pain and inflammation in mice through production of reactive oxygen species and cyclooxygenase-2

It is currently accepted that superoxide anion (O2•−) is an important mediator in pain and inflammation. The role of superoxide anion in pain and inflammation has been mainly determined indirectly by modulating its production and inactivation. Direct evidence using potassium superoxide (KO2), a superoxide anion donor, demonstrated that it induced thermal hyperalgesia, as assessed by the Hargreaves method. However, it remains to be determined whether KO2 is capable of inducing other inflammatory and nociceptive responses attributed to superoxide anion. Therefore, in the present study, we investigated the nociceptive and inflammatory effects of KO2. The KO2-induced inflammatory responses evaluated in mice were: mechanical hyperalgesia (electronic version of von Frey filaments), thermal hyperalgesia (hot plate), edema (caliper rule), myeloperoxidase activity (colorimetric assay), overt pain-like behaviors (flinches, time spent licking and writhing score), leukocyte recruitment, oxidative stress, and cyclooxygenase-2 mRNA expression (quantitative PCR). Administration of KO2 induced mechanical hyperalgesia, thermal hyperalgesia, paw edema, leukocyte recruitment, the writhing response, paw flinching, and paw licking in a dose-dependent manner. KO2 also induced time-dependent cyclooxygenase-2 mRNA expression in the paw skin. The nociceptive, inflammatory, and oxidative stress components of KO2-induced responses were responsive to morphine (analgesic opioid), quercetin (antioxidant flavonoid), and/or celecoxib (anti-inflammatory cyclooxygenase-2 inhibitor) treatment. In conclusion, the well-established superoxide anion donor KO2 is a valuable tool for studying the mechanisms and pharmacological susceptibilities of superoxide anion-triggered nociceptive and inflammatory responses ranging from mechanical and thermal hyperalgesia to overt pain-like behaviors, edema, and leukocyte recruitment.

Production of yeast extracts from whey for food use: market and technical considerations

Whey is produced in large amounts by cheese industries. This by-product can be used for biomass production by yeast cultivation, resulting in commercially attractive products. The use of yeast extracts as source of flavour enhancer consists of an expansible market, encouraged by costumer's choice for natural additives. The development of a suitable and economically viable project for the generation of valued-added by-products, may allow the dairy industry to diversify their portfolio and increase their rentability.

Biotechnological production of bioflavors and functional sugars

Bioflavors and oligosaccharides are two classes of substances that may be produced biotechnologically through microbial bioprocesses. These compounds have attracted the interest of pharmaceutical and food industries not only due to their technological properties (sweetening/fiber or flavoring, respectively), but also as a consequence of other functional properties such as, for example, health promoting benefits. The use of agro-industrial residues as substrates in biotechnological processes seems to be a valuable alternative in helping to overcome the high manufacturing costs of industrial fermentations. This manuscript reviews the most important advances in biotechnological production of bioflavors and oligosaccharides. The use of some agro-industrial residues in such processes is also cited and discussed, showing that this is a rising trend in biotechnology.

Production of glucose and fructose syrups from cassava (Manihot esculenta Crantz) starch using enzymes produced by microorganisms isolated from Brazilian Cerrado soil

The high demands for sugars and the development of enzymatic technology have increased the production of sweeteners, especially for glucose and fructose syrups. This work describe a technology for glucose and fructose syrups from Brazilian cassava starch using enzymes produced by soil microrganisms isolated from the Brazilian Cerrado soil. Firstly, Aspergillus niger and Streptomyces sp. were isolated from the soil and used as glucoamylase (GA) and glucose isomerase (GI) producer sources. After characterization, GA and GI exhibited optimum pH 4.5 and 8.0, respectively. GA showed maximum activity at 60 ºC and GI at 85 ºC. GA and GI retained 65 and 80%, respectively, of initial activity after 180 minutes of incubation at 60 ºC. The kinetic parameters Km and Vmáx were 0.476 (mg.mL-1) and 8.58 (µmol/minute) for GA and 0.082 (M) and 48.20 (µmol/minute) for GI. The maximum glucose syrups production occurred after 24 hours of reaction with a 98% yield. The production of fructose syrups with 42% (w/v) was reached after 96 hours of reaction.

Production of isomaltulose obtained by Erwinia sp. cells submitted to different treatments and immobilized in calcium alginate

In recent decades, there has been an increase in the studies of isomaltulose obtainment, due to its physicochemical properties and physiological health benefits. These properties, which include low cariogenicity, low glycemic index and greater stability, allow the use of this sweetener as a substitute for sucrose in foods; besides the fact that it can be converted to isomalt, a dietary non-cariogenic sugar alcohol used in pharmaceuticals as well as in the food industry. Isomaltulose (6-O-α-D-glucopyronosyl-1-6-D-fructofuranose) is a disaccharide reducer obtained by the enzymatic conversion of sucrose - the α-glucosyltransferase enzyme. Different treatments were performed for the preparation of whole cells; lysed cells; and crude enzyme extract of Erwinia sp. D12 strain immobilized in calcium alginate. The packed bed column of granules, containing Erwinia sp. cells sonicated and immobilized in calcium alginate (CSI), reached a maximum conversion of 53-59% sucrose into isomaltulose and it presented activity for 480 hours. The converted syrup was purified and the isomaltulose crystallization was performed through the lowering of temperature. The isomaltulose crystals presented purity of 96.5%.

Evaluation of process parameters in the industrial scale production of fish nuggets

This work reports the use of experimental design for the assessment of the effects of process parameters on the production of fish nuggets in an industrial scale environment. The effect of independent factors on the physicochemical and microbiological parameters was investigated through a full 24 experimental design. The studied factors included the temperature of fish fillet and pulp in the mixer, the temperature of the added fat, the temperature of water and the ratio of protein extraction time to emulsion time. The physicochemical analyses showed that the higher temperature of the pulp and fillet of fish, the lower the protein in the final product. Microbiological analyses revealed that the counting of Staphylococcus coagulase positive, total and thermo-tolerant coliforms were in accordance with the current legislation.

Optimization of extrusion process for production of nutritious pellets

A blend of 50% Potato Starch (PS), 35% Quality Protein Maize (QPM), and 15% Soybean Meal (SM) were used in the preparation of expanded pellets utilizing a laboratory extruder with a 1.5 × 20.0 × 100.0 mm die-nozzle. The independent variables analyzed were Barrel Temperature (BT) (75-140 °C) and Feed Moisture (FM) (16-30%). The effect of extrusion variables was investigated in terms of Expansion Index (EI), apparent density (ApD), Penetration Force (PF) and Specific Mechanical Energy (SME), viscosity profiles, DSC, crystallinity by X-ray diffraction, and Scanning Electronic Microscopy (SEM). The PF decreased from 30 to 4 kgf with the increase of both independent variables (BT and FM). SME was affected only by FM, and decreased with the increase in this variable. The optimal region showed that the maximum EI was found for BT in the range of 123-140 °C and 27-31% for FM, respectively. The extruded pellets obtained from the optimal processing region were probably not completely degraded, as shown in the structural characterization. Acceptable expanded pellets could be produced using a blend of PS, QPM, and SM by extrusion cooking.

Production of pizza dough with reduced fermentation time

The aim of this study was to reduce the fermentation time of pizza dough by evaluating the development of the dough during fermentation using a Chopin® rheofermentometer and verifying the influence of time and temperature using a 2² factorial design. The focus was to produce characteristic soft pizza dough with bubbles and crispy edges and soft in the center. These attributes were verified by the Quantitative Descriptive Analysis (QDA). The dough was prepared with the usual ingredients, fermented at a temperature range from 27 to 33 ºC for 30 to 42 minutes, enlarged, added with tomato sauce, baked, and frozen. The influence of the variables time and temperature on the release of carbon dioxide (H'm) was confirmed with positive and significant effect, using a rheofermentometer, which was not observed for the development or maximum height of the dough (Hm). The same fermentation conditions of the experimental design were used for the production of the pizza dough in the industrial process; it was submitted to Quantitative Descriptive Analysis (QDA), in which the samples were described by nine attributes. The results showed that some samples had the desired characteristics of pizza dough, demonstrated by the principal component analysis (PCA), indicating a 30 % fermentation time reduction when compared to the conventional process.

An alternative method for screening lactic acid bacteria for the production of exopolysaccharides with rapid confirmation

The accumulation of exopolysaccharides (EPS) produced by microorganisms occurs in the presence of excess substrate and limiting conditions of elements that are essential to growth, such as nitrogen, phosphorus, sulfur, and magnesium. The presence of EPS produced by bacterial cells contributes to slime colonies formation in solid medium and increased viscosity in liquid medium. This paper proposes an alternative method for screening EPS-producing lactic acid bacteria using solid medium-containing discs of filter paper that are saturated with active cultures. The screening was carried out under different culture conditions varying the type of sugar, pH, and temperature. EPS production was visualized by the presence of mucoid colonies on the discs, which was confirmed by the formation of a precipitate when part of this colony was mixed with absolute alcohol. The established conditions for obtaining a high number of isolates producing EPS were 10% sucrose, pH 7.5 and 28 ºC. This method proved to be effective and economical because several strains could be tested on the same plate, with immediate confirmation.

Optimization of extrusion variables for the production of snacks from by-products of rice and soybean

This study aimed to define the process conditions to obtain snacks from the by-products of rice and soybean with physical characteristics suitable for marketing. Therefore, the effects of moisture and extrusion temperature on the expansion and color of the products obtained experimentally obtained were evaluated, and the proximate composition of the by-products and that of the snack with greater desirability were determined. Response surface methodology and rotational central composite design were used, and desirability test based on the regression models adjusted was applied. The most desirable snack, with the highest expansion index (3.39), specific volume (13.5 mL.g-1), and the chromaticity coordinate a* (2.79), was obtained under 12 g.100 g-1 moisture and 85ºC of temperature in the third zone of the extruder. The snack produced under these conditions attained content of protein and lipid content 41 and 64% higher than that of the traditional corn snack. It can be concluded that producing extruded snack made form a mixture of broken grains, rice bran, and soybean okara (81:9:10) is technologically feasible, enabling the development of a new product with good nutritional value that can improve the diet of children, the main consumers of this type of food.