Manuela Rollini

Biosynthesis and biotechnological production of statins by filamentous fungi and application of these cholesterol-lowering drugs.

Abstract. Hypercholesterolemia is considered an important risk factor in coronary artery disease. Thus the possibility of controlling de novo synthesis of endogenous cholesterol, which is nearly two-thirds of total body cholesterol, represents an effective way of lowering plasma cholesterol levels. Statins, fungal secondary metabolites, selectively inhibit hydroxymethyl glutaryl-coenzyme A (HMG-CoA) reductase, the first enzyme in cholesterol biosynthesis. The mechanism involved in controlling plasma cholesterol levels is the reversible inhibition of HMG-CoA reductase by statins, related to the structural similarity of the acid form of the statins to HMG-CoA, the natural substrate of the enzymatic reaction. Currently there are five statins in clinical use. Lovastatin and pravastatin (mevastatin derived) are natural statins of fungal origin, while symvastatin is a semi-synthetic lovastatin derivative. Atorvastatin and fluvastatin are fully synthetic statins, derived from mevalonate and pyridine, respectively. In addition to the principal natural statins, several related compounds, monacolins and dihydromonacolins, isolated fungal intermediate metabolites, have also been characterized. All natural statins possess a common polyketide portion, a hydroxy-hexahydro naphthalene ring system, to which different side chains are linked. The biosynthetic pathway involved in statin production, starting from acetate units linked to each other in head-to-tail fashion to form polyketide chains, has been elucidated by both early biogenetic investigations and recent advances in gene studies. Natural statins can be obtained from different genera and species of filamentous fungi. Lovastatin is mainly produced by Aspergillus terreus strains, and mevastatin by Penicillium citrinum. Pravastatin can be obtained by the biotransformation of mevastatin by Streptomyces carbophilus and simvastatin by a semi-synthetic process, involving the chemical modification of the lovastatin side chain. The hypocholesterolemic effect of statins lies in the reduction of the very low-density lipoproteins (VLDL) and LDL involved in the translocation of cholesterol, and in the increase in the high-density lipoproteins (HDL), with a subsequent reduction of the LDL- to HDL-cholesterol ratio, the best predictor of atherogenic risk. The use of statins can lead to a reduction in coronary events related to hypercholesterolemia, but the relationship between benefit and risk, and any possible interaction with other drugs, must be taken into account.

Production and purification of statins from Aspergillus terreus strains

Lovastatin, mevastatin, pravastatin and monacolin J were produced using Aspergillus terreus strains. Mevastatin (170 mg/l) was obtained at 14 days from the A1 strain, lovastatin (256 mg/l) at 21 days from the A2 strain and pravastatin (270-300 mg/l) at 14 days from both the A1 and A2 strains grown on defatted soybean flour. Similar yields of monacolin J (5-10 mg/l) were detected for both strains. Fermentation carried out by adding glycerol to A1 7-d old cultures gave 244 mg lovastatin/l at 14 days employing whole soybean flour. A new extraction procedure was applied to an A2 19-d old culture on the mycelium and the culture filtrate separately. Recovery yield showed that 83% lovastatin was associated with the mycelium and 17% was free in the culture filtrate.

Development of a novel antimicrobial film based on chitosan with LAE (ethyl-Nα-dodecanoyl-l-arginate) and its application to fresh chicken

Chitosan (CS) films incorporating the antimicrobial compound ethyl-N(α)-dodecanoyl-l-arginate (LAE) were developed for food packaging applications. Cast chitosan films were made with 1, 5 or 10% LAE and 20% glycerol in the film forming solution. Optical properties, release of LAE and antimicrobial activity of developed films was determined. The minimum inhibitory concentration (MIC) and the minimum biocide concentration (MBC) of LAE were determined. CS films with LAE were transparent and uniform, without discontinuities or visible particles and no visual differences could be perceived between CS and CS-LAE films. When in contact with an aqueous food simulant, the agent was fully released following a Fickian behavior in a few hours at 4 and 28°C. Antimicrobial activity of films against mesophiles, psychrophiles, Pseudomonas spp., colifoms, lactic acid bacteria, hydrogen sulfide-producing bacteria, yeast and fungi, was evaluated at two, six and eight days for its application on chicken breast fillets. Films were active against bacteria, yeasts and fungi in liquid and solid media. CS films evidenced antimicrobial activity in the range 0.47-2.96 log reductions, while CS-5%LAE film produced 1.78-5.81 log reduction. Results highlighted that LAE incorporation in a chitosan-based packaging structure may provide a relevant antimicrobial activity that could improve the stability of fresh poultry products.

Acetic acid bacteria as enantioselective biocatalysts

Acetic acid bacteria (five strains of Acetobacter and five strains of Gluconobacter) were used for the biotransformation of different primary alcohols (2-chloropropanol and 2-phenylpropanol) and diols (1,3-butandiol, 1,4-nonandiol and 2,3-butandiol). Most of the tested strains efficiently oxidized the substrates. 2-Chloropropanol and 1,3-butandiol were oxidized with good rates and low enantioselectivity (enantiomeric excess = 18-46% of the S-acid), while microbial oxidation of 2-phenylpropanol furnished (S)-2-phenyl-1-propionic acid with enantiomeric excess (e.e.) >90% with 10 strains. The dehydrogenation of 2,3-butandiol was strongly dependent on the stereochemistry of the substrate; the meso form gave S-acetoin with all the tested strains, the only exception being a Gluconobacter strain. The formation of diacetyl was observed only by using R,R-2,3-butandiol with Acetobacter strains. Oxidation of 1,4-nonandiol gave γ-nonanoic lactone in one step, although with moderate enantioselectivity. 2002 Elsevier Science B.V. All rights reserved.

Biotransformation of d-galactitol to tagatose by acetic acid bacteria

Abstract Tagatose, a ketohexose C-4 fructose epimer present in nature in low concentration, is a potential low calorie bulking sweetener that can be obtained by the microbial oxidation of the corresponding polyalcohol galactitol. The screening of strains belonging to the acetic acid bacteria resulted in 100–160 mg tagatose/l, produced at 24 h in non growing conditions, while 260–340 mg tagatose/l was obtained at 48 h with growing cells of Gluconobacter strains, with a specific activity rate of tagatose production, 1.4×10−3 l/h. After galactitol adaptation the Gluconobacter oxydans DSM 2343 strain gave a notable increase in tagatose yield, reaching 3160 mg/l with a corresponding 6.6×10−3 l/h specific activity rate at 24 h of reaction. Preliminary enzyme characterisation experiments indicated that the dehydrogenase activity may be attributable to a sorbitol dehydrogenase (SDH).

Isolation and characterization of the exopolysaccharide produced by Daedalea quercina

The production of exopolysaccharide (EPS) by a strain of the basidiomycete Daedalea quercina was investigated. Of seven different carbon sources, glucose and dextrins gave the highest crude polysaccharide yield (4.7–5 g l−1, 55–60% carbohydrate content) in shake-flask cultures, at 14 days of fermentation. Experiments carried out in a 10 l fermenter, at two different agitation speeds, gave the best results at 300 rpm, resulting in 12–14 g l−1 of crude exopolysaccharide in 9–11 days. Fractionation of the EPS samples, carried out by tangential flow ultrafiltration, evidenced a single EPS fraction (MW >30 000 Da) in samples from glucose, while two fractions (MW > 30 000 Da and 30 000 > MW > 10 000 Da) were present in samples from dextrins. Fractions characterization by HPLC and proton NMR spectroscopy revealed diversity in composition and structure in the obtained EPS: from glucose mainly an α-linked mannan, and from dextrins mainly an α- and β-linked glucan.

Enzymatic and metabolic activities of four anaerobic sludges and their impact on methane production from ensiled sorghum forage

Biochemical methane potential (BMP) tests were run on ensiled sorghum forage using four inocula (urban, agricultural, mixture of agricultural and urban, granular) and differences on their metabolic and enzymatic activities were also discussed. Results indicate that no significant differences were observed in terms of BMP values (258±14NmLCH4g(-1)VS) with a slightly higher value when agricultural sludge was used as inoculum. Significant differences can be observed among different inocula, in terms of methane production rate. In particular the fastest biomethanization occurred when using the urban sludge (hydrolytic kinetic constant kh=0.146d(-1)) while the slowest one was obtained from the agricultural sludge (kh=0.049d(-1)). Interestingly, positive correlations between the overall enzymatic activities and methane production rates were observed for all sludges, showing that a high enzymatic activity may favour the hydrolysis of complex substrate and accelerate the methanization process of sorghum.

Sodium hydroxide pretreatment of ensiled sorghum forage and wheat straw to increase methane production.

The aim of this study was to determine the effect of sodium hydroxide pretreatment on the chemical composition and the methane production of ensiled sorghum forage and wheat straw. NaOH pretreatment was conducted in closed bottles, at 40 °C for 24 h. Samples were soaked in a NaOH solution at different dosages (expressed in terms of total solids (TS) content) of 1 and 10% gNaOH/gTS, with a TS concentration of 160 gTS/L. At the highest NaOH dosage the reduction of cellulose, hemicelluloses and lignin was 31, 66 and 44%, and 13, 45 and 3% for sorghum and wheat straw, respectively. The concentration of soluble chemical oxygen demand (CODs) in the liquid phase after the pretreatment was also improved both for wheat straw and sorghum (up to 24 and 33%, respectively). Total sugars content increased up to five times at 10% gNaOH/gTS with respect to control samples, suggesting that NaOH pretreatment improves the hydrolysis of cellulose and hemicelluloses. The Biochemical Methane Potential (BMP) tests showed that the NaOH pretreatment favoured the anaerobic degradability of both substrates. At 1 and 10% NaOH dosages, the methane production increased from 14 to 31% for ensiled sorghum forage and from 17 to 47% for wheat straw. The first order kinetic constant increased up to 65% for sorghum and up to 163% for wheat straw.

Glutathione-enriched baker's yeast: production, bioaccessibility and intestinal transport assays

A glutathione (GSH) yeast‐based biomass (Saccharomyces cerevisiae) was used to investigate GSH stability, solubilization during gastrointestinal digestion and GSH intestinal transport.

Sakacin-A antimicrobial packaging for decreasing Listeria contamination in thin-cut meat: preliminary assessment.

Abstract BACKGROUND Minimally processed ready‐to‐eat products are considered a high‐risk food because of the possibility of contamination with pathogenic bacteria, including Listeria monocytogenes from the animal reservoir, and the minimal processing they undergo. In this study, a sakacin‐A anti‐Listeria active package was developed and tested on thin‐cut veal meat slices (carpaccio). RESULTS Enriched food‐grade sakacin‐A was obtained from a cell‐free supernatant of a Lactobacillus sakei culture and applied (0.63 mg cm−2) onto the surface of polyethylene‐coated paper sheets to obtain an active antimicrobial package. The coating retained antimicrobial features, indicating that the process did not affect sakacin‐A functionality, as evidenced in tests carried out in vitro. Thin‐cut veal meat slices inoculated with Listeria innocua (a surrogate of pathogenic L. monocytogenes) were laid on active paper sheets. After 48 h incubation at 4 °C, the Listeria population was found to be 1.5 log units lower with respect to controls (3.05 vs 4.46 log colony‐forming units (CFU) g−1). CONCLUSION This study demonstrates the possibility of using an antimicrobial coating containing sakacin‐A to inhibit or decrease the Listeria population in ready‐to‐eat products, thus lowering the risk of food‐related diseases.