Giovanni Spagna

A simple method for purifying glycosidases: α-l-rhamnopyranosidase from Aspergillus niger to increase the aroma of Moscato wine

alpha-L-rhamnopyranosidase (Rha, EC 3.2.1.40) is an enzyme of considerable importance in food technology for increasing the aroma of wines, musts, fruit juices and other alcoholic beverages. The aim of this research is to study the purification of Rha contained in a commercial preparation already used in the winemaking industry. With the procedure adopted, Rha recovery values were excellent (ca 85%), comparable with those we found in a previous paper on the purification of other glycosidases such beta-D-glucopyranosidase (betaG) and alpha-L-arabinofuranosidase (Ara) [1]. The Rha purification value (4.3) and drastic reduction in brown compounds (DeltaAbs 95%) represent other strengths of the proposed method that has proved inexpensive and simple to apply. In addition, purified Rha has shown itself to be more stable than other glycosidases. This had optimum effect at pH 4, while optimum temperature was 70 degrees C, greater than that found for other glycosidases. The purified enzyme was characterized in terms of the kinetic parameters K(m) (1.40 mM) and V(max) (1.30 U mg(-1) of protein) and subsequently used to increase aroma a model wine solution containing aromatic precursors extracted from the skins of Moscato grapes, with an increase in the content of total terpenols of ca 2.3 times.

A novel chitosan derivative to immobilize α-L-rhamnopyranosidase from Aspergillus niger for application in beverage technologies

alpha-L-rhamnopyranosidase (Rha, EC 3.2.1.40) is an enzyme of considerable importance to food technology in increasing the aroma of wines, musts, fruit juices and other beverages. The aim of this research is the immobilization of the Rha contained in a commercial preparation already used in the winemaking industry and purified in the manner described in a previous study [1]. The immobilization supports tested were chitin, chitosan and derivatized chitosan, diethylaminoethyl chitosan (DE-chitosan) never previously used for this type of application. Particularly, on DE-chitosan, the Rha was adsorbed and cross-linked with various bifunctional agents (glutaraldehyde, diepoxyoctane, suberimidate and carbodiimide), whose best results (immobilization yields and activity) were obtained with carbodiimide (EDC) that allowed a reduction in the involvement of the enzyme amine groups that are probably important in catalytic mechanism. In addition, the use of rhamnose and a succinimide (NHS) during cross-linking enhanced the action of the EDC and so increased the immobilization yield and activity. The immobilized Rha retained the kinetic parameters (K(m) and V(max)) of the free enzyme and increased stability. Moreover, this biocatalyst allowed an increase in the aroma in a model wine solution containing glicosidic precursors with a marked reduction in specificity toward tertiary monoterpenols as compared to the free enzyme.

A mixture of purified glycosidases from Aspergillus niger for oenological application immobilised by inclusion in chitosan gels

Abstract Several glycosidases (β-D-glucopyranosidase, α-L-arabinofuranosidase, α-L-rhamnopyranosidase), purified [1] , [2] from an Aspergillus niger enzyme preparation were immobilised contemporarily with a method in view of its possible application in the wine-making and fruit-juice processing industry. Immobilisation of the three enzymes was carried out by inclusion using chitosan gels and subsequent cross-linking with glutaraldehyde. This was followed by addition of various agents in order to improve the gel’s physical and mechanical properties, reduce enzyme release phenomena and increase immobilisation yields and operational stability. The best additives proved is be gelatine and silica gel, of which a more in-depth study was made. Finally, the immobilised glycosidases were used to increase the aroma in a model wine solution.

Preliminary characterization of wild lactic acid bacteria and their abilities to produce flavour compounds in ripened model cheese system

Aims:  The aim of this work was to preliminary characterize wild lactic acid bacteria (LAB), previously isolated during artisanal Pecorino Siciliano (PS) cheese‐making for technological and flavour formation abilities in a model cheese system.

Study and characterization of polyphenol oxidase from eggplant (Solanum melongena L.).

In this study the catecholase and cresolase activities of eggplant polyphenol oxidase (PPO) were investigated. Enzyme activity was determined by measuring the increase in absorbance using catechol as substrate and 3-methyl-2-benzothiazolinone hydrazone (MBTH) as coupled reagent. The effects of substrate specificity, heat inactivation, temperature, pH, and inhibitors were investigated to understand the enzymatic alteration of ready-to-eat preparations. Browning of vegetables was determined through a colorimeter. Decrease of lightness (L*) and increase of color difference values (ΔE*) were correlated with tissue browning. Antibrowning agents were tested on PPO under the same conditions. The enzyme activity was strongly inhibited by 0.4 M citric acid. Under natural pH conditions, the enzyme was also inhibited by tartaric acid and acetic acid. All of the results were used to understand the best conditions for food transformation (ready-to-eat and grilled eggplant slices).

Toward shrimp consumption without chemicals: Combined effects of freezing and modified atmosphere packaging (MAP) on some quality characteristics of Giant Red Shrimp (Aristaeomorpha foliacea) during storage.

The combined effects of freezing and modified atmosphere packaging (MAP) (100% N2 and 50% N2+50% CO2) on some quality characteristics of Giant Red Shrimp (GRS) (Aristaeomorpha foliacea) was studied during 12-month storage. In particular, the quality characteristics determined proximal and gas compositions, melanosis scores, pH, total volatile basic-nitrogen (TVB-N), thiobarbituric acid (TBA) as well as free amino acid (FAA). In addition, the emergent data were compared to those subject to vacuum packaging as well as conventional preservative method of sulphite treatment (SUL). Most determined qualities exhibited quantitative differences with storage. By comparisons, while pH and TVB-N statistically varied between treatments (P<0.05) and TBA that ranged between ∼0.15 and 0.30 mg MDA/kg appeared least at end of storage for 100% N2 treated-group, the latter having decreased melanosis scores showed such treatments with high promise to keep the colour of GRS sample hence, potential replacement for SUL group. By comparisons also, while some individual FAA values showed increases especially at the 100% N2-treated group, the total FAAs statistically differed with storage (P<0.05). The combination of freezing and MAP treatments as preservative treatment method shows high promise to influence some quality characteristics of GRS samples of this study.

An alkaline β-glucosidase isolated from an olive brine strain of Wickerhamomyces anomalus.

An efficient β-glucosidase (βG)-producing strain, Wickerhamomyces anomalus BS81, was isolated from naturally fermented olive brine and identified based on PCR/restriction fragment length polymorphism of the rDNA internal transcribed spacer and sequence analysis of the D1/D2 region of the 26S rRNA gene. The hydrolytic activity of the βG had an optimum pH of 8.5 and an optimum temperature of 35 °C. The enzyme had high substrate specificity and high catalytic efficiency (K(m) 0.99 mM, V(max) 14 U g(-1) of cells) for p-nitrophenyl-β-d-glucopyranoside. The enzyme was activated by increasing concentrations of NaCl, with maximum activity at 150 g L(-1) NaCl. Although βGs have been purified and characterized from several other sources, the W. anomalusβG is unique among βGs because its relative maximum activity occurs at alkaline pH and 35 °C. Moreover, the yeast strain has esterase activity that acts synergistically with βG to degrade oleuropein to debitter table olives and olive oil.

Stabilization of a β-glucosidase from Aspergillus niger by binding to an amine agarose gel

Abstract β- d -glucopyranosidase (βG, EC 3.2.1.21) is an enzyme of considerable importance in food technology for increasing the aroma of wines, musts, fruit juices and alcoholic beverages. In this research we have studied the stabilization of a commercial βG preparation, by covalent immobilization of its carbohydrate moiety to an amine agarose gel. The findings showed total adsorption of the enzyme, previously purified [G. Spagna, D. Romagnoli, A. Martion, G. Bianchi, P.G. Pifferi, Enzyme Microb. Technol., 22 (1998) 298], on the matrix, its low reduction in activity and finally a high stabilization over time.

Polyphenol Oxidase Activity from Three Sicilian Artichoke [Cynara cardunculus L. Var. scolymus L. (Fiori)] Cultivars: Studies and Technological Application on Minimally Processed Production

Several papers helped with the development of more methods to control browning, or study thermal polyphenol oxidase (PPO) inactivation, but did not provide any solutions to technological process problems and food process improvement. Artichokes [ Cynara cardunculus L. var. scolymus L. (Fiori)] are susceptible to browning; this alteration could affect and reduce the suitability for its use, fresh or processed. Within this study, the catecholase and cresolase activities of PPO from three different Sicilian artichokes cultivar were characterized with regard to substrate specificity and enzyme kinetics, optimum pH and temperature, temperature and pH stability, and inhibitor test; all of the results were used for technological purposes, particularly to optimize minimally processed productions (ready-to-eat and cook-chilled artichokes).

Partial sequencing of the β-glucosidase-encoding gene of yeast strains isolated from musts and wines

The aim of the present work was the identification of the gene encoding for β-glucosidase and its partial sequencing in the strainsPichia anomala AL112,Hanseniaspora uvarum Y8 andSaccharomyces cerevisiae AL41. To this aim degenerated primers, designed on the basis of aminoacid similarities of four known yeast β-glucosidases, have been used in PCR amplifications. An expected fragment of about 200 bp was amplified from all the DNAs, cloned and sequenced. Sequence homology demonstrated for the first time the presence of a β-glucosidase encoding gene inHanseniaspora uvarum andSaccharomyces cerevisiae.