Production Of FOS Research Articles

Production and characterization of an extracellular Mn2+ activated ß-D-fructofuranosidase from Aspergillus labruscus ITAL 28.255 with transfructosylating activity

Fungal ß-D-fructofuranosidases present biotechnological potential for use in food, beverage, and pharmaceutical industries. In light of this, the best culture conditions for the production of ß-D-fructofuranosidase by Aspergillus labruscus ITAL 28.255 were obtained with an M-5 medium maintained at 30°C and 100 rpm for 120 h. The use of a mixture of rye flour and sucrose (1.5:0.5, m/m) and 2% yeast extract as the carbon and nitrogen sources, respectively, proportioned the best production of the enzyme. The extracellular ß-D-fructofuranosidase was purified 17-fold, with 9% recovery, and was characterized as a glycoprotein with 67 kDa (SDS-PAGE) and 40.7% carbohydrate content. The best temperature and pH for enzyme activity were 55°C and 5.5, respectively. The enzyme activity was maintained around 75% at 40 and 50°C for 1 h, with a half-life of 5 min at 70°C. The hydrolytic activity was increased in the presence of Mn2+. The Vmax and KM were 4.74 mM and 140.3 U/mg using sucrose; while, in the presence of Mn2+, the KM was 2.17 mM and the Vmax was 292.9 U/mg. The enzyme performed the transfructosylation reaction, enabling the production of FOS, mainly 1-kestose. This is the first report on the potential of A. labruscus to produce β-fructofuranosidase with transfructosylation activity.

Heterofunctional epoxy support development for immobilization of fructosyltransferase from Pectinex® Ultra SP-L: batch and continuous production of fructo-oligosaccharides

The FTase from Pectinex® Ultra SP-L was immobilized using a newly developed epoxy-Purolite support with amino and epoxy groups. Continuous production of FOS in the air-lift reactor was established for 7 days (52.47% FOS of total carbohydrates).

Improving the fructooligosaccharides production by solid-state fermentation

The aim of the study was the fungal production of fructooligosaccharides from solid-state fermentation of different agro-industrial wastes (sugar cane bagasse , coffee husk, pineapple peel, prickle pear peel and banana peel) enriched with maguey sap. An exploratory statistical design with a factorial arrangement (2 3 ) was used to identify some parameters that are directly related to the production of FOS. The production was optimized and evaluated through fermentation kinetics in order to determine the time of maximum FOS production. Among the evaluated materials, sugar cane bagasse was the most promising substrate suited for the FOS production reaching a concentration of 7.64 g of FOS per liter of culture medium based on the initial sucrose concentration (21 g/L), a high Yp/s = 0.45 based on sucrose consumed was observed. The results showed that use of sugar cane bagasse enriched with aguamiel is the most economical and excellent alternative source for the FOS production by Aspergillus oryzae DIA-MF. • FOS were produced via Solid-State Fermentation (SSF) employing agro-wastes enriched with aguamiel. • Aspergillus oryzae DIA-MF was used as microorganism for SSF. • Sugar cane bagasse was the most promising substrate suited for the FOS production. • A FOS production of 7.64 g/L was obtained with a Yp/s = 0.45 based on sucrose consumed. • Use of sugar cane bagasse and aguamiel is an economical and excellent alternative source for the FOS production.

Purification and biochemical characterization of an extracellular fructosyltransferase-rich extract produced by Aspergillus tamarii Kita UCP1279

Abstract The market for prebiotics is gaining strength and is growing greatly in recent years since the consumers demand are changing and are highly influenced by the increasing consumption of healthier products. The fructosyltransferase (FTase) mainly produced by filamentous fungi is responsible for the synthesis of fructooligosaccharides which are widely used as a bioactive ingredient in functional foods. The objectives of this work were to select the best Aspergillus strains fructosyltransferase producer, purify FTase using ion-exchange chromatography coupled to FPLC under Superdex-G75 and to produce fructooligosaccharides. Six strains of Aspergillus sp. were studied, and A. tamarii Kita was highlighted because of the greater fructosyltransferase activity. At the production stage, the maximum activity of crude extract (1629.03 U/gds) was obtained under the following conditions: wheat bran 3.0g, sucrose concentration at 20%, 106 spore/mL, 70% moisture, 12h in the presence of light, at 30 °C for 48h. Superdex-G75 was able to purify the enzyme with high activity, and after SDS-PAGE under zymography conditions, one band of protein was observed at 89.7 kDa. The specific activity of the final purified material was 112,629.03 U/gds with purification ratio of 49 times and yield of 36%. FTase presented optimum temperature at 60 °C and pH 5.0, respectively. Together, these results indicated that the FTase of Aspergillus tamarii Kita is an excellent candidate for the industrial production of FOS.

Technological Aspects of the Production of Fructo and Galacto-Oligosaccharides. Enzymatic Synthesis and Hydrolysis.

Fructo- and galacto-oligosaccharides (FOS and GOS) are non-digestible oligosaccharides with prebiotic properties that can be incorporated into a wide number of products. This review details the general outlines for the production of FOS and GOS, both by enzymatic synthesis using disaccharides or other substrates, and by hydrolysis of polysaccharides. Special emphasis is laid on technological aspects, raw materials, properties, and applications.

PRODUCCIÓN DE FRUCTOOLIGOSACÁRIDOS CON DOS NUEVAS ENZIMASβ-FRUCTOFURANOSIDASA RECOMBINANTES DE LEVADURAS AISLADAS DELA FERMENTACIÓN DEL MEZCAL

The gene Cap inv32 from Candida apicola and Tde inv32 from Torulaspora delbrueckii were identified as β-fructofuranosidase belonging to glycoside hydrolases family 32, with an opening reading frame of 1593 base pairs and 1743 base pairs, and a molecular weight of 59 kDa and 65 kDa, for Cap Inv32 and Tde Inv32 correspondingly. These enzymes contained the motif characteristics of the GH32 and showed an identity of 40 % (Cap Inv32) and 52.4 % (Tde Inv32) with an invertase of S. cerevisiae. The fructosyltransferase activity was tasted obtaining for Cap Inv32 the quantity of FOS 12.4 gL-1 and 9.03 gL-1 with 420 gL-1 and 700 gL-1, the production of FOS with Tde Inv32 was 14.61 gL-1 (420 gL-1) and 13.3 gL-1 (700 gL-1). Cap Inv32 and Tde Inv32 are β-fructofuranosidase with fructosyltransferase activity, with potential to industrial applications, focused in FOS production.

Immobilisation of endoinulinase on polyhydroxybutyrate microfibers

Due to the health benefits associated with the consumption of prebiotic short-chain fructooligosaccharides (sc-FOS) and inulooligosaccharides (IOS), there is increased interest in the use of these compounds in food products. We have developed a new biocatalyst for the production of FOS and IOS by inulin hydrolysis. Endoinulinase from Aspergillus niger Inulinase<sup>® </sup>Novozym 960 (Novozymes) was immobilised on polyhydroxybutyrate (PHB) nanofibres and microfibres by hydrophobic interactions. The PHB fibres were prepared by centrifugal spinning. FOS and IOS profiles were determined by ion-exchange chromatography with the Rezex RSO-Oligosaccharide column. The biocatalyst had very good activity and stability after repeated applications. It can be used in biocatalytic membrane reactors for the production of prebiotic oligosaccharides.

Tofu Whey Permeate Is an Efficient Source To Enzymatically Produce Prebiotic Fructooligosaccharides and Novel Fructosylated α-Galactosides.

This work addresses a novel and efficient bioconversion method for the utilization of tofu whey permeate (TWP), an important byproduct from the soybean industry, as a precursor of high value-added ingredients such as prebiotic fructooligosaccharides and novel fructosylated α-galactosides. This process is based on the high capacity of the commercial enzyme preparation Pectinex Ultra SP-L to transfructosylate the main carbohydrates present in TWP as sucrose, raffinose, and stachyose to produce up to a maximum of 164.2 g L(-1) (equivalent to 57% with respect to initial sucrose, raffinose, and stachyose contents in TWP) of fructooligosaccharides and fructosylated α-galactosides in a balanced proportion. Raffinose- and stachyose-derived oligosaccharides were formed by elongation from the nonreducing terminal fructose residue up to three fructosyl groups bound by β-(2→1) linkages. These results could provide new findings on the valorization and upgrading of the management of TWP and an alternative use of raw material for the production of FOS and derivatives.

Neural correlates of nesting behavior in zebra finches (Taeniopygia guttata)

Nest building in birds involves a behavioral sequence (nest material collection and deposition in the nest) that offers a unique model for addressing how the brain sequences motor actions. In this study, we identified brain regions involved in nesting behavior in male and female zebra finches (Taeniopygia guttata). We used Fos immunohistochemistry to quantify production of the immediate early gene protein product Fos (a molecular indicator of neuronal activity) in the brain correlated this expression with the variation in nesting behavior. Using this technique, we found that neural circuitry involved in motor sequencing, social behavior, reward and motivation were active during nesting. Within pairs of nesting birds, the number of times a male picked up or deposited nesting material and the amount of time a female spent in the nest explained the variation in Fos expression in the anterior motor pathway, social behavior network, and reward neural circuits. Identification of the brain regions that are involved in nesting enables us to begin studying the roles of motor sequencing, context, and reward in construction behavior at the neural level.

The effect of ultrasonication on the release of fructosyltransferase from Aureobasidium pullulans CFR 77

Aureobasidium pullulans has been a major source of fructosyltransferase (EC 2.4.1.9) for the production of fructooligosaccharides from sucrose. The organism is known to produce both intra and extra cellular FTase. The present study examines the effect of ultrasonication on the release of FTase from A. pullulans CFR 77, and subsequently on the enzymatic production of fructooligosaccharides. The acoustic power (W), and the processing time (min) were the chosen key elements to study their effect on the release of the enzyme. The release of the enzyme (FTase) was monitored by determining the OD at 275 nm, and the protein content of the fractions. The intracellular FTase obtained after the sonication was used for the production of FOS. The amount of FOS produced ranged from 57 to 59%. Based on the energy cost of the system, and the maximum amount of FOS produced, ultrasonication of the cells at acoustic power of 20 W for 9 min was found to be optimum. Thus, the study demonstrates the potential role of ultrasonication in efficient release of the intracellular FTase which can be used for the production of FOS, an industrially important prebiotic.

Activation of spinally projecting and nitrergic neurons in the PVN following heat exposure

The present study investigated the effect of acute thermal stimulation in conscious rats on the production of Fos, a marker of increased neuronal activity, in spinally projecting and nitrergic neurons in the hypothalamic paraventricular nucleus (PVN). The PVN contains a high concentration of nitrergic neurons, as well as neurons that project to the intermediolateral cell column (IML) of the spinal cord that can directly influence sympathetic nerve activity (SNA). During thermal stimulation, the PVN is activated, but it is unknown whether spinally projecting PVN neurons and the nitrergic neurons are involved. Compared with controls, rats exposed to an environmental temperature of 39 degrees C for 1 h had a 10-fold increase in the number of cells producing Fos in the PVN (133 +/- 23 vs. 1,336 +/- 43, respectively, P < 0.0001). Of the spinally projecting neurons in the PVN of heated rats (98 +/- 10), over 20% expressed Fos. Additionally, of the nitrergic neurons (NADPH-diaphorase positive) located in the parvocellular PVN (723 +/- 17), 40% also expressed Fos (P < 0.0001 compared with controls). Finally, there was a significant increase in the number of spinally projecting neurons in the PVN that were nitrergic and expressed Fos after heat exposure (12%) compared with controls (0.1%) (P < 0.0001). These results suggest that spinally projecting and nitrergic neurons in the PVN may contribute to the central pathways activated by thermal stimulation.

Recent trends in the microbial production, analysis and application of Fructooligosaccharides

This review focuses on the recent developments in the area of FOS research—its microbial production, functional properties and applications and an overview of the different analytical methods for the determination of the FTase. Different microbial sources of FTase reported in literature to produce FOS with different linkages to form 1-kestose, 6-kestose and neokestose in varying yields based on initial sucrose concentration is discussed. Different fermentative methods have been used for production of FOS. SSF has been used for the production of a value added product FOS utilizing various agroindustrial byproducts. The nutritional and culture parameters when optimized, the FOS yields and productivity could be improved. The use of immobilized enzymes and cells has led to the development of effective and economic methods for large-scale production of FOS. Forced flow Membrane reactor systems, biocatalyst system with a bioreactor equipped with a microfiltration systems, have been used for production of high content FOS by removing the released glucose and unreacted sucrose from the reaction mixture resulting in up to 98% FOS. The use of mixed enzyme system of Fructosyl Transferase and glucose oxidase or glucose dehydrogenase, could produce highly concentrated FOS up to 90–98%. Nano-filtration for removing glucose resulted in FOS of 90% concentration. The purified enzyme was found to produce kestose and nystose unlike the crude enzyme which produced GF5 and GF6 oligosaccharides Kinetic parameters (Vm, Km, and Ki) of the enzyme were determined from experimental data on the transfructosylation rate at various substrate concentrations with and without addition of glucose Techniques like HPLC, using polar-bonded phase and resin-based HPLC columns are commonly used for separation of oligosaccharides with refractive Index Detector or pulsed amperometric detector and annular size exclusion chromatography for large scale and continuous fractionation. Other techniques like gas liquid chromatography, thin layer chromatography, NMR and Mass Spectrometry have been used for structure analyses. The functional properties like use as prebiotics, dietary fiber, role in absorption and defense/Immunity, lipid metabolism control of diabetics have been discussed. A variety of applications in food formulations are also discussed.

Fos, RVLM-projecting neurons, and spinally projecting neurons in the PVN following hypertonic saline infusion.

Hypertonic saline (HTS; 1.7 M) infused intravenously into conscious rats increases the production of Fos, a marker of cell activation, in the hypothalamic paraventricular nucleus (PVN). The parvocellular PVN contains subpopulations of neurons. However, which subpopulations are activated by HTS is unknown. We determined whether PVN neurons that innervate the rostral ventrolateral medulla (RVLM) or the spinal cord (important autonomic sites) expressed Fos following HTS. Experiments were performed 24-96 h after chronic implantation of an intravenous cannula. HTS significantly increased the number of Fos-positive cells. In the parvocellular PVN, the maximum number of Fos-positive cells occurred rostral of the anterior-posterior level at which the number of neurons that projected to the medulla or spinal cord peaked. Compared with controls, HTS did not significantly increase the number of double-labeled neurons. These findings demonstrate that an elevation in plasma osmolality activates PVN neurons but not the subgroups of PVN neurons with projections to the RVLM or to the spinal cord.

Adolescent development alters stressor-induced Fos immunoreactivity in rat brain

Adult-typical behavioural responses to environmental challenges as well as the stressor responsiveness of several neural systems emerge over adolescent development. The present study was undertaken to determine whether stressors might activate different neural populations in adult vs juvenile male rats. Fos-immunoreactivity was determined in various forebrain nuclei following 15 min or 2 h of restraint in 28- and 60-day-old male rats (representing late juvenile and young adult ages, respectively) and compared to non-restrained control animals at each age. Few Fos-positive cells were identifed in unrestrained controls at either age. Restraint, however, induced the production of Fos in several areas. Fos immunoreactivity was marked in parvocellular regions of the paraventricular nucleus of the hypothalamus following both restraint periods and at both ages, an observation consistent with previous observations that restraint increases plasma corticosterone at both ages. And at both ages, Fos immunoreactivity was evident in magnocellular regions of the hypothalamus only following the longer restraint period. Fos immunoreactivity, however, clearly varied as a function of adolescent age in several regions. Moderate to intense Fos immunoreactivity was observed in adults in all divisions of the anterior olfactory nucleus, cortical and medial amygdaloid nuclei, pyriform cortex and tenia tecta. In contrast to the adult, only a few Fos positive cells were observed in any of these regions in juveniles. Exposure to the same stressor induced Fos in a broader spectrum of neurons in young adult than in juvenile male rats. The lack of Fos-positive cells in specific areas of juveniles may relate to maturation in specific amygdaloid nuclei, which project to many of the other regions that showed age-related differences in Fos production. The emergence over adolescence of Fos-positive cells in specific areas in response to stressors may underlie the emergence of adult-typical behavioural and neural stressor-responsiveness.

Effect of intravenous captopril on c-fos expression induced by sodium depletion in neurons of the lamina terminalis.

Peripheral administration of the angiotensin converting enzyme (ACE) inhibitor, captopril, and the central infusion of sarile, an angiotensin II (Ang II) receptor antagonist, were used to evaluate the role of renal and brain generated Ang II in sodium depletion-induced production of Fos in cells of the subfornical organ (SFO) and organum vasculosum lamina terminalis (OVLT). Pretreatment with intravenous captopril (100 mg/kg) significantly inhibited the c-fos expression induced by sodium depletion in the SFO and OVLT. In contrast, continuous intracerebroventricular infusion of sarile (22.5 micrograms/4.5 h, 5 microliters/h) did not affect the expected pattern of c-fos expression observed in both nuclei, 4 h after peritoneal dialysis. These results show that systemic interference with the angiotensin system of renal origin by captopril inhibited the production of Fos induced by sodium depletion in cells of the SFO and OVLT. These findings are consistent with the hypothesis that a rise in peripheral Ang II levels, triggered by sodium deficiency, could be an important mediator of the physiological and behavioral responses that lead to the restoration of sodium balance. In addition, this study suggests that increased circulating Ang II levels in response to body sodium deficit can directly stimulate neural pathways in the SFO and OVLT.

A nitric oxide synthesis inhibitor (L-NAME) reduces licking behavior and Fos-labeling in the spinal cord of rats during formalin-induced inflammation

Formalin injected subcutaneously into the hindpaw of the rat produces an animal model of inflammation that exhibits a phasic component and a tonic component of pain. We evaluated the effects of a nitric oxide synthase inhibitor, Nω-nitro- l-arginine methyl ester (L-NAME), on a formalin-induced behavior, hindpaw licking, and on Fos-labeling of nuclei in the fifth lumbar spinal segment. Our results demonstrated that pretreatment with intrathecal doses of 0.3 and 1.0 mg of L-NAME significantly reduced licking behavior associated with injection of formalin into the left hindpaw of the rat. In addition, these same doses of L-NAME reduced formalin-induced Fos-labeling in the ipsilateral dorsal gray matter (as compared to the contralateral gray matter). Qualitative assessment suggested that the reduction in labeling occurred primarily in the superficial dorsal horn. The stereoisomer, D-NAME, administered at the same doses had little to no effect on either formalin-induced licking or Fos-labeling. Finally, our results revealed that total licking time was related to Fos-labeling. Rats that spent less time licking the hindpaw exhibited a smaller increase in Fos-labeling. Our results suggest that the production of nitric oxide is associated with licking behavior resulting from formalin injection into the hindpaw of rats. Our results also suggest that the production of nitric oxide and Fos are associated. Indeed, these substances may be involved in spinal pathways associated with nociception.

Cloning of the mink plasminogen activator inhibitor type-1 messenger RNA: An mRNA with a short half life

In mink lung CCL64 epithelial cells the rate of synthesis of plasminogen activator inhibitor type I (PAI-1) increases 10–100-fold within 3 h in response to 12-O-tetradecanoyl phorbol-13-acetate (PMA). The PAI-1 gene is regulated transcriptionally. Parallel studies of the time-courses of PAI-1 synthesis and secretion and of mRNA accumulation indicate that the amount of secreted PAI-1 produced by the cells is tightly coupled to the level of its transcript. The half-life of the PAI-1 mRNA was found to be 25 min which is much shorter than previously reported for PAI-1 in other cells. Actinomycin D, which is commonly used to determine mRNA half-life, stabilized the PAI-1 mRNA. Cycloheximide also stabilized the mRNA. The short half-life and the superinducibility of PAI mRNA are properties shared with rapidly degraded mRNAs encoding protooncoproteins. A 2.97-kb cDNA clone containing the entire coding sequence of PAI-1 was isolated from a cDNA library made from mink lung CCL64 epithelial cells stimulated with PMA. The PAI-1 cDNA contains a long 3′-untranslated region ( UTR) of 1720 bp whose sequence is highly conserved among PAI-1 mRNAs from different species. The PAI-1 mRNA also contains several AUUUA pentamer sequences which are the features of an A + U-rich regulatory element such as is found on the fos protooncogene mRNA. Upstream of one of these AUUUA pentamers are several highly conserved sequences that are also found in the 3′ UTR of the fos and integrin receptor α-subunit mRNAs. These are mRNAs that encode proteins which are involved in the cellular response to tissue wounds. Thus, it is possible that these conserved 3' sequences may be important for regulating the production of Fos, the integrin α-subunit and PAI-1 during wound healing.

Sodium depletion induces Fos immunoreactivity in circumventricular organs of the lamina terminalis

Acute sodium depletion by peritoneal dialysis (PD) induces c- fos expression in the subfornical organ (SFO) and organum vasculosum laminae terminalis (OVLT), in conscious rats. Fos immunoreactive (Fos-ir) neurons detected by immunohistochemistry first appeared in these nuclei 60 min after PD, increased gradually in the next 4 h and remained high for 27 h following PD. Fos-ir cells were distributed throughout the body of SFO, being the core of the posterior sections preferentially activated, whereas Fos-ir neurons occurred around the periphery of OVLT (annular disposition). When rats were allowed to drink sodium salt (1.8% NaCl) 24 h after PD, there was a marked reversion of the c- fos expression in the OVLT and a comparatively smaller effect in the SFO. Intracerebroventricular infusion of hypertonic CSF (170 mM NaCl) from 30 min before and during 4 h after PD, significantly inhibited the c- fos expression in both nuclei. These results demonstrate that an acute body sodium deficit induces c- fos activity in SFO and OVLT neurons, indicating the special role of these structures in sodium balance regulation. They also show that the sodium-depletion-induced production of Fos in neurons of the lamina terminalis can be modulated by central or systemic reposition of sodium.

Infusion into the brain of an antisense oligonucleotide to the immediate-early gen c-fos suppresses production of FOs and produces a behavioral effect

Infusion into the brain of an antisense oligonucleotide to the immediate-early gen c-fos suppresses production of FOs and produces a behavioral effect

Expression of c-fos in the brain stem of rats during hypercapnia.

Expression of the proto-oncogene c-fos is widely used as a marker of metabolic activation of individual neurones (1,2,12). Expression of this immediate early gene results in the production of the nuclear protein FOS, which forms a heterodimer complex with JUN, the protein product of the immediate early gene c-jun. By acting as a third messenger, the FOS-JUN complex regulates the expression of specific target genes in various types of cells. It is thought that in this way FOS and the products of other early response genes contribute to the genetically determined functional adaptation of the cell to altered stimulus environments (7,15). Various hormones, growth factors and neurotransmitters, as well as depolarisation and voltage gated Ca2+ entry into the cell have been shown to induce a rapid but transitional transcription of the c-fos gene in various types of neurones (6,8). Once FOS is produced in sufficient amounts depending on the duration and intensity of the activating stimulus, a FOS-like immunoreactivity (FOS-LI) can be demonstrated in the nuclei of these cells.