Selective Synthesis of Galactooligosaccharides Containing ß(1→3) Linkages with ß-Galactosidase from Bifidobacterium bifidum (Saphera).

The transglycosylation activity of a novel commercial ß-galactosidase from Bifidobacterium bifidum (Saphera) was evaluated. The optimal conditions for the operation of this enzyme, measured with o-nitrophenyl-ß-d-galactopyranoside, were 40 °C and pH around 6.0. Although at low lactose concentrations the property of this enzyme was basically hydrolytic, an increase of lactose concentration to 400 g/L resulted in a significant formation (107.2 g/L, 27% yield) of prebiotic galactooligosaccharides (GOS). The maximum amount of GOS was obtained at a lactose conversion of approximately 90%, which contrasts with other ß-galactosidases, for which the highest GOS yield is achieved at 40-50% lactose conversion. Using high-performance anion-exchange chromatography with pulsed amperometric detection, semipreparative high-performance liquid chromatography-hydrophilic interaction liquid chromatography, mass spectrometry, and 1D and 2D NMR, we determined the structure of most of the GOS synthesized by this enzyme. The main identified products were Gal-ß(1→3)-Gal-ß(1→4)-Glc (3'-O-ß-galactosyl-lactose), Gal-ß(1→6)-Glc (allolactose), Gal-ß(1→3)-Glc (3-galactosyl-glucose), Gal-ß(1→3)-Gal (3-galactobiose), and the tetrasaccharide Gal-ß(1→3)-Gal-ß(1→3)-Gal-ß(1→4)-Glc. In general, B. bifidum ß-galactosidase showed a tendency to form ß(1→3) linkages followed by ß(1→6) and more scarcely ß(1→4).

Prebiotic effect of xylooligosaccharides produced from birchwood xylan by a novel fungal GH11 xylanase.

A fungal endoxylanase belonging to the glycoside hydrolase gene family 11 (GH11) was obtained from the ascomycete Talaromyces amestolkiae. The enzyme was purified, characterized and used to produce a mixture of xylooligosaccharides (XOS) from birchwood xylan. A notable yield of neutral XOS was obtained (28.8%) upon enzyme treatment and the mixture contained a negligible amount of xylose, having xylobiose, xylotriose and xylotetraose as its main components. The prebiotic potential of this mixture was demonstrated upon analyzing the variations in microorganisms' composition and organic acids profile in breast-fed child faeces fermentations. The strong production of acetic and lactic acid, the decrease of potentially pathogenic bacteria and the increase of bifidobacteria, and possible beneficial commensals, confirmed the prebiotic value of these xylooligosaccharides.

Micro-scale procedure for enzyme immobilization screening and operational stability assays.

OBJECTIVE: A simple and inexpensive methodology, based on the use of micro-centrifuge filter tubes, is proposed for establishing the best enzyme immobilization conditions. RESULTS: The immobilized biocatalyst is located inside the filter holder during the whole protocol, thus facilitating the incubations, filtrations and washings. This procedure minimizes the amount of enzyme and solid carrier needed, and allows exploring different immobilization parameters (pH, buffer concentration, enzyme/carrier ratio, incubation time, etc.) in a fast manner. The handling of immobilized enzymes using micro-centrifuge filter tubes can also be applied to assess the apparent activity of the biocatalysts, as well as their reuse in successive batch reaction cycles. The usefulness of the proposed methodology is shown by the determination of the optimum pH for the immobilization of an inulinase (Fructozyme L) on two anion-exchange polymethacrylate resins (Sepabeads EC-EA and Sepabeads EC-HA). CONCLUSION: The micro-scale procedure described here will help to overcome the lack of guidelines that usually govern the selection of an immobilization method, thus favouring the development of stable and robust immobilized enzymes that can withstand harsh operating conditions in industry.

Síntesis enzimática de fructooligosacáridos estimuladores de la microbiología colónica / Enzymatic synthesis of fructooligosaccharides that stimulate the gut microbiota

El control de la microflora intestinal es uno de los objetivos de los alimentos funcionales y nutracéuticos. El equilibrio simbiótico puede lograrse mediante la ingesta de microorganismos vivos (probióticos) o de los denominados prebióticos (oligosacáridos no digeribles). Los prebióticos son fermentados selectivamente por la microbiota generando cambios específicos en su composición que producen un beneficio en la salud del hospedador. Entre los prebióticos los fructooligosacáridos (FOS) constituyen uno de los grupos más importantes. Las levansacarasas (EC 2.4.1.10) son una familia de enzimas que catalizan la transferencia de un grupo fructosilo desde una sacarosa a diferentes aceptores entre ellos otra molécula de sacarosa dando lugar a FOS sobre los que puede transferir otro grupo fructosilo para llegar a formar levano un polímero con aplicaciones en alimentación y biomedicina. Si el grupo fructosilo se transfiere sobre una molécula de agua da lugar a la hidrólisis de la sacarosa. En este trabajo se caracterizó una levansacarasa de Z. mobilis y los productos de reacción con sacarosa como sustrato se analizaron por cromatografía de intercambio aniónico con detector amperométrico de pulsos (HPAEC-PAD). Con objeto de optimizar el proceso biocatalítico la enzima se inmovilizó por atrapamiento en geles de alginato cálcico y las esferas resultantes se deshidrataron para formar DALGEEs (Dry ALGinate Entrapped Enzymes). Se probaron diferentes estrategias de inmovilización para minimizar la pérdida de la enzima por los poros. El efecto de la inmovilización en el comportamiento de la levansacarasa fue analizado

The control of the intestinal flora is one of the targets of the functional foods and nutraceuticals. A symbiotic equilibrium can be achieved by the intake of live microorganisms (probiotics) or by the so-called prebiotics (non-digested oligosaccharides). Prebiotics are selectively fermented by the human microbiota allowing specific changes and conferring benefits upon host well-being and health. Among prebiotics fructooligosaccharides (FOS) constitute one of the most established groups. Levansucrases (EC 2.4.1.10) are a family of enzymes that catalyse the transfer of the fructosyl moiety from sucrose to different acceptors such as: (1) sucrose -yielding FOS that can be further fructosylated forming levan a polymer with food and biomedical applications -; (2) water resulting in sucrose hydrolysis. In this work a levansucrase from Zymomonas mobilis was characterized and the reaction products using sucrose as substrate were analysed by High-Performance Anion-Exchange Chromatography (HPAEC-PAD). The number of FOS synthesized by the soluble enzyme was significantly higher compared with previous reports. In order to optimize the biocatalytic process the enzyme was further immobilized by entrapment in calcium alginate gel and the resulting beads were dehydrated to obtain DALGEEs (Dry ALGinate Entrapped Enzymes). Different immobilization strategies were studied to minimize enzyme loss (lixiviation) throughout the pores. The effect of enzyme immobilization on levansucrase behaviour was also analysed

Galactooligosaccharides formation during enzymatic hydrolysis of lactose: towards a prebiotic-enriched milk.

The formation of galactooligosaccharides (GOS) in skim milk during treatment with several commercial ß-galactosidases (Bacillus circulans, Kluyveromyces lactis and Aspergillus oryzae) was analysed in detail, at 4 and 40°C. The maximum GOS concentration was obtained at a lactose conversion of approximately 40-50% with B. circulans and A. oryzae ß-galactosidases, and at 95% lactose depletion for K. lactis ß-galactosidase. Using an enzyme dosage of 0.1% (v/v), the maximum GOS concentration with K. lactis ß-galactosidase was achieved in 1 and 5h at 40 and 4 °C, respectively. With this enzyme, it was possible to obtain a treated milk with 7.0 g/L GOS - the human milk oligosaccharides (HMOs) concentration is between 5 and 15 g/L--and with a low content of residual lactose (2.1g/L, compared with 44-46 g/L in the initial milk sample). The major GOS synthesised by this enzyme were 6-galactobiose [Gal-ß(1 → 6)-Gal], allolactose [Gal-ß(1 → 6)-Glc] and 6'-O-ß-galactosyl-lactose [Gal-ß(1 → 6)-Gal-ß(1 → 4)-Glc].

Analysis of fermentation selectivity of purified galacto-oligosaccharides by in vitro human faecal fermentation.

The in vitro fermentation of several purified galacto-oligosaccharides (GOS), specifically the trisaccharides 4'-galactosyl-lactose and 6'-galactosyl-lactose and a mixture of the disaccharides 6-galactobiose and allolactose, was carried out. The bifidogenic effect of GOS at 1% (w/v) was studied in a pH-controlled batch culture fermentation system inoculated with healthy adult human faeces. Results were compared with those obtained with a commercial GOS mixture (Bimuno-GOS). Changes in bacterial populations measured through fluorescence in situ hybridization and short-chain fatty acid (SCFA) production were determined. Bifidobacteria increased after 10-h fermentation for all the GOS substrates, but the changes were only statistically significant (P<0.05) for the mixture of disaccharides and Bimuno-GOS. Acetic acid, whose formation is consistent with bifidobacteria metabolism, was the major SCFA synthesized. The acetate concentration at 10 h was similar with all the substrates (45-50 mM) and significantly higher than the observed for formic, propionic and butyric acids. All the purified GOS could be considered bifidogenic under the assayed conditions, displaying a selectivity index in the range 2.1-3.0, which was slightly lower than the determined for the commercial mixture Bimuno-GOS.

Detailed analysis of galactooligosaccharides synthesis with ß-galactosidase from Aspergillus oryzae.

The synthesis of galactooligosaccharides (GOS) catalyzed by ß-galactosidase from Aspergillus oryzae (Enzeco) was studied. Using 400 g/L of lactose and 15 U/mL, maximum GOS yield, measured by HPAEC-PAD, was 26.8% w/w of total carbohydrates, obtained at approximately 70% lactose conversion. No less than 17 carbohydrates were identified; the major transgalactosylation product was 6'-O-ß-galactosyl-lactose, representing nearly one-third (in weight) of total GOS. In contrast with previous reports, the presence of at least five disaccharides was detected, which accounted for 40% of the total GOS at the point of maximum GOS concentration (allolactose and 6-galactobiose were the major products). A. oryzae ß-galactosidase showed a preference to form ß(1→6) bonds, followed by ß(1→3) and ß(1→4) linkages. Results were compared with those obtained with ß-galactosidases from Kluyveromyces lactis and Bacillus circulans. The highest GOS yield and specific productivity were achieved with B. circulans ß-galactosidase. The specificity of the linkages formed and distribution of di-, tri-, and higher GOS varied significantly among the three ß-galactosidases.

Galacto-oligosaccharide synthesis from lactose solution or skim milk using the ß-galactosidase from Bacillus circulans.

The synthesis of galacto-oligosaccharides (GOS) catalyzed by a novel commercial preparation of ß-galactosidase from Bacillus circulans (Biolactase) was studied, and the products were characterized by MS and NMR. Using 400 g/L lactose and 1.5 enzyme units per milliliter, the maximum GOS yield, measured by HPAEC-PAD analysis, was 165 g/L (41% w/w of total carbohydrates in the mixture). The major transgalactosylation products were the trisaccharide Gal-ß(1→4)-Gal-ß(1→4)-Glc and the tetrasaccharide Gal-ß(1→4)-Gal-ß(1→4)-Gal-ß(1→4)-Glc. The GOS yield increased to 198 g/L (49.4% w/w of total carbohydrates) using a higher enzyme concentration (15 U/mL), which minimized the enzyme inactivation under reaction conditions. Using skim milk (with a lactose concentration of 46 g/L), the enzyme also displayed transgalactosylation activity: maximum GOS yield accounted for 15.4% (7.1 g/L), which was obtained at 50% lactose conversion.

Analysis of neofructooligosaccharides production mediated by the extracellular ß-fructofuranosidase from Xanthophyllomyces dendrorhous.

The extracellular ß-fructofuranosidase Xd-INV from the yeast Xanthophyllomyces dendrorhous mainly synthesizes the neo-fructooligosaccharides (neo-FOS) neokestose and neonystose. This enzyme is a glycoprotein with a content of 59-67% N-linked carbohydrates and an estimated molecular mass of 160-200 kDa. The extent level of glycosylation affects the thermal behaviour of the enzyme but not its hydrolase and transferase activities, which are optimal at 60-70 °C. The neo-FOS yield of this enzyme was increased from 40 to 168 g/L when the sucrose concentration increased from 420 to 600 g/L and when the reaction was carried out at 60 °C. The neo-FOS levels obtained (168 g/L) in this work are the largest reported for any microbial ß-fructofuranosidase.

Production of Galacto-oligosaccharides by the ß-Galactosidase from Kluyveromyces lactis : comparative analysis of permeabilized cells versus soluble enzyme.

The transgalactosylation activity of Kluyveromyces lactis cells was studied in detail. Cells were permeabilized with ethanol and further lyophilized to facilitate the transit of substrates and products. The resulting biocatalyst was assayed for the synthesis of galacto-oligosaccharides (GOS) and compared with two soluble ß-galactosidases from K. lactis (Lactozym 3000 L HP G and Maxilact LGX 5000). Using 400 g/L lactose, the maximum GOS yield, measured by HPAEC-PAD analysis, was 177 g/L (44% w/w of total carbohydrates). The major products synthesized were the disaccharides 6-galactobiose [Gal-ß(1→6)-Gal] and allolactose [Gal-ß(1→6)-Glc], as well as the trisaccharide 6-galactosyl-lactose [Gal-ß(1→6)-Gal-ß(1→4)-Glc], which was characterized by MS and 2D NMR. Structural characterization of another synthesized disaccharide, Gal-ß(1→3)-Glc, was carried out. GOS yield obtained with soluble ß-galactosidases was slightly lower (160 g/L for Lactozym 3000 L HP G and 154 g/L for Maxilact LGX 5000); however, the typical profile with a maximum GOS concentration followed by partial hydrolysis of the newly formed oligosaccharides was not observed with the soluble enzymes. Results were correlated with the higher stability of ß-galactosidase when permeabilized whole cells were used.