Riccardo N. Barbagallo

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.

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.

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.

Use in Vivo of Natural Anti-browning Agents Against Polyphenol Oxidase Activity in Minimally Processed Eggplant

Plant polyhenol oxidase (PPO, EC 1.14.18.1) is responsible along with other oxidases for the enzymatic browning reaction occurring during handling, storage and processing of vegetables. This work aimed at assessing the efficacy in vivo of some natural anti-browning agents in minimally processed eggplants. They were collected, washed, diced, submitted to dipping with inhibitors (L-ascorbic, benzoic, citric, ferulic and L-glutamic acid) at three concentrations (0.2, 0.5 and 1 %) and packed in ordinary atmosphere bags (PET), covered by a double barrier film and refrigerated for 7 days (4.0 ± 0.5 °C, 95 % RH). The enzymatic activity was inhibited by addition of all anti-browning agents tested. At t=7 the greatest reduction in PPO activity was observed at the highest concentrations (0.5 and 1 %) of the inhibitors in the following order: ferulic acid (-43 %), L-glutamic acid (-32 %), citric acid (-27 %), L-ascorbic acid (-21 %) and benzoic acid (-15 %). These positive effects were also translated in terms of browning index, demonstrating the efficacy of the anti-browning treatments studied to extend the shelf-life of minimally processed eggplants.

A specific method for determination of pectin esterase in blood oranges

Pectin esterase (PE, E.C. 3.1.1.11) is implicated in the cloud loss in citrus juices. This study describes a specific method of extracting pectin esterase from blood oranges and developing, contemporarily, an enzyme assay suitable for the pH of such juices. The PE showed an optimum extraction at pH 7.0, without addition of surfactants. The enzyme assay was carried out at pH 3.6, a condition typical of the orange juices, increasing sensitivity in determining PE compared to other methods proposed up until now.

Effect of nitrogen fertilization on the overall quality of minimally processed globe artichoke heads

BACKGROUND Although nitrogen (N) fertilisation is essential for promoting crop yield, it may also affect the produce quality. Here, the influence of three N fertiliser rates (0 kg ha-1 as a control, 200 kg ha-1 and 400 kg ha-1 referred to as N0 , N200 and N400, respectively) on the overall quality of minimally processed globe artichoke heads was investigated during refrigerated storage for 12 days. RESULTS Throughout the storage time, N fertilised samples had higher inulin contents than those unfertilised. In addition, the respiratory quotient of N200 and N400 samples was 2-fold and 2.5-fold lower than N0 ones, whose values were close to the normal range for vegetables. All the samples reported good microbiological standards, although N200 and N400 achieved lower mesophilic and psychotropic counts than N0 throughout the storage time. After 8 and 12 days of refrigerated storage, the N200 samples showed the highest scores of positive sensory descriptors. CONCLUSION A fertiliser level of 200 kg N ha-1 is suitable for obtaining minimally processed globe artichoke heads with good nutritional, sensory and microbiological quality, characterised by low endogenous oxidase activities. Proper packaging systems and procedures are, however, crucial for extending the product shelf-life and, thus, promoting its exportation on a wider scale.

Inexpensive isolation of β-d-glucopyranosidase from α-l-arabinofuranosidase, α-l-rhamnopyranosidase, and o-acetylesterase

Abstractβ-d-Glucopyranosidase (βG, EC 3.2.1.21) has been isolated from some collateral activities, α-l-arabinofuranosidase (Ara, EC3.2.1.55), α-l-rhamnopyranosidase (Rha, EC 3.2.1.40), and o-acetylesterase (Est, EC 3.1.1.53), using a commercial enzyme preparation and a simple method economically sustainable for the food industry. The procedure comprises precipitation of extraneous substances by adding ethanol and CaCl2, ultrafiltration, and adsorption, first on bentonite and then on chitosan. The results obtained were the complete isolation of βG from the above-mentioned activities, a drastic reduction in extraneous compounds, such as brown substances and polysaccharides, and a slight increase in purification.

Effect of freezing/thawing process in different sizes of blue fish in the Mediterranean through lysosomal enzymatic tests.

The assessment of freshness of different sizes of blue fish (Engraulis encrasicolus 12 cm, Sardina pilchardus 15 cm, Trachurus trachurus 40 cm, Scomber japonicus colias 60 cm) was carried out using non-conventional enzymatic methods. The activities of the three lysosomal enzymes (α-glucosidase (AG), β-galactosidase (B-GAL) and β-N-acetylglucosamidase (B-NA)) in extracts of blue fish muscle were measured over a period of 21 days of storage. A significant increase (p<0.05) of AG activity was observed in all species, with a large increase seen after only one day of storage. B-NA activity increased slightly in sardines, horse mackerels and chub mackerel during frozen/thawed storage. Finally, the increase of B-GAL activity was significant (p<0.05) only in the samples of larger blue fish as horse mackerel and chub mackerel. All of these enzyme activities may be helpful predictive markers to limit fraud in these species.