Madalena Baron

Bioproduction of carotenoids: The comparative use of raw sugarcane juice and depolymerized bagasse by Phaffia rhodozyma

Brown sugar, raw sugarcane juice, and enzymatic hydrolyzates from amilaceous or ligno(hemi)cellulosic sources like cane bagasse turned out to be a minimal but satisfactory culture medium for the production of astaxanthin in Phaffia rhodozyma. The condition for this was a small supplementation (1 g/l) of a nitrogen source. Urea resulted in a preferential yeast biomass increase as compared to soya meal and tannery shavings, the major beneficial effect of which was in the pigment content of the yeast cells.

A factorial approach for a sugarcane juice-based low cost culture medium: increasing the astaxanthin production by the red yeast Phaffia rhodozyma

Abstract A sugarcane juice-based low cost culture medium was previously explored to produce the carotenoid pigment astaxanthin in liquid culture by the red yeast Phaffia rhodozyma (1300 μg astaxanthin/g of dry yeast and 6500 μg/l whole culture medium). Two peculiar limitations in Phaffia are growth temperature (<26 °C) and lack of sugar osmotolerance. Two advantages are the wide biochemical ability for the assimilation and metabolization of disaccharides and the prompt utilization of simple nitrogen sources. For instance, the sucrolytic/ureolytic enzymatic activities deserves exploration. In order to improve the culture medium composition and the conditions of fermentation for highly oxygenated carotenoids (e.g., astaxanthin) a study was carried out with a factorial design in two steps. As a first step, the production of astaxanthin was studied as a function of the nutrient concentration levels and their interactions. The production increase (μg/l) obtained was 23.0% but at the expense of 16.0% pigment content decrease (μg/g). In the second step, the variables pH and agitation level (OTR, oxygen transfer rate) were optimized and then, both goals were attained: the increase of pigment content (418 μg astaxanthin/g of yeast) as well as the absolute pigment production enhancement (1987 μg/l).

Acetobacter Cellulosic Biofilms Search for New Modulators of Cellulogenesis and Native Membrane Treatments

Since natural substances like pseudoxanthins exert a positive effect on the cellulogenic ability ofAcetobacter xylinum when producing cellulosic pellicles suitable for skin burn therapy, new defined and complex modulators were sought. Ca2+ and Mg2+ (4 mM) were strongly stimulatory. Na+ had no effect and K+ was inhibitory. Ammonium dihydrogen phosphate (0.12 g/L) ensured the same nitrogen supply as the same concentration of yeast extract as measured by cellomembrane dry wt./yield albeit higher yeast extract supplies produced thicker membranes. Corn steep liquor (CSL) was also progressively beneficial from 0.125 to 0.5 mL/L, and this yield could be further improved by the combination of CSL with a tea infusion (source of caffeine). Uridine (precursor for UDP-Glc, sugar donor in cellulose biosynthesis), guanine, guanosine, and its butirylated derivatives (precursors for the positive modulator of cellulose synthetase, di-cGMP) resulted in only moderate stimulation. Sodium phytate and betaine were also slightly stimulatory. The fibrilar product from a newAcetobacter isolate (Ax-M) was characterized as cellulose by comparison with the solid-state13C-NMR of algal cellulose. Its X-ray diffractogram was a confirmatory analysis. After incorporation of tamarind xyloglucan to previously air-dried cellulosic pellicles, diffractometry displayed only slight differences. Mercerized (5M NaOH) fresh cellulosic biofilms underwent drastic size reduction (3.5-fold), turning compact nut still flexible if maintained wet.

Culture of the Astaxanthinogenic Yeast Phaffia rhodozyma in Low-Cost Media

Growth of the yeastPhaffia rhodozyma was carried out in a simplified medium based on less expensive nutrient sources, such as diluted sugar cane juice, urea, and sodium phosphate. The usual content of the astaxanthin, an oxygenated pink carotenoid useful for fish flesh staining, was improved along with with good cell yields (respective values of >1300 μg/g cells and >5 g cells/L were observed). Yeast invertase and urease must therefore play an important role in the implementation of low-cost culture media.

Screening of Bothrops snake venoms for L-amino acid oxidase activity

Toxins, enzymes, and biologically active peptides are the main components of snake venoms from the genus Bothrops. Following the venom inoculation, the local effects are hemorrhage, edema, and myonecrosis. Nineteen different species of Brazilian Bothrops were screened for protein content and L-amino acid oxidase activity. B. cotiara, formerly found in the South of Brazil, is now threatened with extinction. Its venom contains a highly hemorrhagic fraction and, as expected from the deep yellow color of the corresponding lyophilized powder, a high L-amino acid oxidase CLAO) activity was also characterized. Flavin adenine dinucleotide (FAD) is its associate coenzyme. B. cotiara venom LAO catalyzed the oxidative deamination of several L-amino acids, and the best substrates were methionine, leucine, tryptophan, and phenylalanine, hence, its potential application for the use in biosensors for aspartame .determination and for the removal of amino acids from plasma. High levels for LAO were also found in other species than B. cotiara. In addition, the technique of isoelectric focusing (IEF) was employed as a powerful tool to study the isoor multienzyme distribution for LAO activity in the B. cotiara snake venom. Index Entries: Bothrops cotiara venom; LAO/L-amino acid oxidase; hemorrhagic activity; LAO isoelectrofocusing; LAO zymogram. *Author to whom all correspondence and reprint requests should be addressed. Applied Biochemistry and Biotechnology ] 9 7 WoL 51/52, 1995

Astaxanthinogenesis in the yeast Phaffia rhodozyma - optimization of low-cost culture media and yeast cell-wall lysis

Astaxanthin is a diketo-dihydroxy-carotenoid produced byPhaffia rhodozyma, a basidiomicetous yeast. A low-cost fermentation medium consisting of raw sugarcane juice and urea was developed to exploit the active sucrolytic/urelolytic enzyme apparatus inherent to the yeast. As compared to the beneficial effect of 0.1 g% urea, a ready nitrogen source, mild phosphoric pre inversion of juice sucrose to glucose and fructose, promptly fermentable carbon sources, resulted in smaller benefits. Corn steep liquor (CSL) was found to be a valuable supplement for both yeast biomass yield (9.2 g dry cells/L) and astaxanthin production (1.3 mg/g cells). Distillery effluent (vinace), despite only a slightly positive effect on yeast growth, allowed for the highest pigment productivity (1.9 mg/g cells). Trace amounts of Ni2 (1 mg/L, as a cofactor for urease) resulted in controversial effects, namely, biomass decrease and astaxanthin increase, with no effect on the release (and uptake) of ammonium ion from urea. Since the synthesized astaxanthin is associated with the yeast cell and the pigment requires facilitated release for aquaculture uses (farmed fish meat staining), an investigation of the yeast cell wall was undertaken using detergent-treated cells. The composition of the rigid yeast envelope was found to be heterogeneous. Its partial acid or enzymatic depolymerization revealed glucose and xylose as common monomeric units of the cell-wall glycopolymers. Yeast cell-wall partial depolymerization with appropriate hydrolases may improve the pigment bioavailability for captive aquatic species and poultry.Astaxanthin is a diketo-dihydroxy-carotenoid produced by Phaffia rhodozyma, a basidiomicetous yeast. A low-cost fermentation medium consisting of raw sugarcane juice and urea was developed to exploit the active sucrolytic/urelolytic enzyme apparatus inherent to the yeast. As compared to the beneficial effect of 0.1 g% urea, a ready nitrogen source, mild phosphoric pre inversion of juice sucrose to glucose and fructose, promptly fermentable carbon sources, resulted in smaller benefits. Corn steep liquor (CSL) was found to be a valuable supplement for both yeast biomass yield (9.2 g dry cells/L) and astaxanthin production (1.3 mg/g cells). Distillery effluent (vinace), despite only a slightly positive effect on yeast growth, allowed for the highest pigment productivity (1.9 mg/g cells). Trace amounts of Ni2 (1 mg/L, as a cofactor for urease) resulted in controversial effects, namely, biomass decrease and astaxanthin increase, with no effect on the release (and uptake) of ammonium ion from urea. Since the synthesized astaxanthin is associated with the yeast cell and the pigment requires facilitated release for aquaculture uses (farmed fish meat staining), an investigation of the yeast cell wall was undertaken using detergent-treated cells. The composition of the rigid yeast envelope was found to be heterogeneous. Its partial acid or enzymatic depolymerization revealed glucose and xylose as common monomeric units of the cell-wall glycopolymers. Yeast cell-wall partial depolymerization with appropriate hydrolases may improve the pigment bioavailability for captive aquatic species and poultry.

Novel d-glucans obtained by dimethyl sulfoxide extraction of the lichens Letharia vulpina, Actinogyra muehlenbergii, and an Usnea SP☆

Abstract Extraction of certain lichens with cold dimethyl sulfoxide provided a β- d -glucan virtually free of contaminating α- d -glucan and galactomannan. Applied to Letharia vulpina , the method gave β- d -glucan, and extraction of the residue with hot water followed by cooling gave α- d -glucan. From Usnea sp. a β- d -glucan was isolated, but little α- d -glucan was present. Extraction of Actinogyra muehlenbergii provided a (1→6)-linked β- d -glucopyranan containing one acetyl group for every 8–9 glucosyl units, being present almost exclusively as monosubstituent at O-2, O-3, and O-4.

Screening ofBothrops snake venoms forl-amino acid oxidase activity

Toxins, enzymes, and biologically active peptides are the main components of snake venoms from the genusBothrops. Following the venom inoculation, the local effects are hemorrhage, edema, and myonecrosis.Nineteen different species of BrazilianBothrops were screened for protein content andl-amino acid oxidase activity.B. cotiara, formerly found in the South of Brazil, is now threatened with extinction. Its venom contains a highly hemorrhagic fraction and, as expected from the deep yellow color of the corresponding lyophilized powder, a highl-amino acid oxidase (LAO) activity was also characterized. Flavin adenine dinucleotide (FAD) is its associate coenzyme.B. cotiara venom LAO catalyzed the oxidative deamination of severall-amino acids, and the best substrates were methionine, leucine, tryptophan, and phenylalanine, hence, its potential application for the use in biosensors for aspartame determination and for the removal of amino acids from plasma. High levels for LAO were also found in other species thanB. cotiara. In addition, the technique of isoelectric focusing (IEF) was employed as a powerful tool to study the iso-or multienzyme distribution for LAO activity in theB. cotiara snake venom.

Selective Polarity- and Adsorption-Guided Extraction/Purification of Annona sp. Polar Acetogenins and Biological Assay Against Agricultural Pests

Annonaceae acetogenins (AG) comprise a family of natural chemical modifications of long-chain fatty acids (C35-37) bearing one to several hydroxyls (less often oxo), middle-chain tetrahydrofuran rings, and a 7-lactonized, α/β-unsaturated carboxyl group. Acetogenins’ strong biological activity as larvicides, pesticides, and antitumorals is dependent on these structural variations. The hydroxylation degree is particularly important for these etfects. Seeds, albeit rich in fats (mostly triacylglycerols, [TAG]), are a nonpredatory source of these drugs as compared to other botanical parts such as roots and stems. Conventional lipid extractions lead to quantitative lipid recovery and then the unfavorable natural ratio of TAG:AG in the range >90:<0.1 These extracts thus require, for instance, partitions and extensive silica gel column Chromatographic steps, in order to enrich or purify the AG fraction(s). Great operational difficulties result from the similar polarity and mol. wt. range of TAG and AG when carrying out these purification steps. An alternative fast two-step procedure to obtain polar acetogenin (pAG)-enriched preparations was developed. The extraction procedure for Annona spp. seeds pAG was carried out with acetonitrile (E° = 0.65; log P = - 0.33) as a polar organosolvent, followed by the adsorption of the solvent-free extract on activated charcoal, then washed with hexane and/or chloroform (E° = 0.0 and 0.40: log P = 3.5 and 2.0) for most of the contaminating TAG removal, and then with acetone (E° = 0.56; log P = - 0.23) to the desorption of an enriched pAG fraction. An alternative procedure for pAG extraction was supercritical fluid extraction (SFE) at moderate thermopressurization conditions (65-82°C; 120-130 atm) using CO2 with 10% acetonitrile as the polarity modifier. The pAG fractions’ bioactivity was evaluated with the brine-shrimp test (BST), and for feed deterrance, growth inhibition, and lethality against the high-impact agricultural pests Anticarsia gemmatalis and Pseudaletia sequax caterpillars feeding on soya or grass leaves sprayed with a 10% alcohol-stabilized emulsion of pAG.

The cuticle of the cactusCereus peruvianus as a source of a homo-α-d-galacturonan

The waxy pecto-cellulosic cuticle of cladodes of the columnar cactusCereus peruvianus (19% of the whole phytobiomass; dry wt) is a source of an α-d-polygalacturonic or pectic acid (35–40% yield, on a dry wt based on the wax-free pectocellulose layer). Warm EDTA/oxalate or room temperature strong acid/alkali cycles are efficient for pectic acid extraction, since divalent cation (mainly Ca2+) is a barrier to be removed within the native and compact architecture of the cuticle. Despite some molecular dispersion arising from the application of strong mineral acid in the first extraction step, the pectic material appears to be quite homogeneous and, on acid or enzymatic analyses, was shown to contain onlyd-galacturonic acid as its monomer.Cereus cuticle pectate (sodium salt) tends to gel above a concentration of 1%, a useful property that can be more easily obtained by the inclusion of sucrose, light addition of calcium salt, and/or mild acidification.