Transglycosylation of a β-galactosyl radical, in the course of enzymic hydrolysis of lactose, in the presence of selected polyhydroxyalcohols

Sorbitol, xylitol, erythritol and lactitol were used as the acceptors of galactosyl radicals, in the process of transgalactosylation accompanying the hydrolysis of lactose, conducted with -galactosidase (4.0 ml Lactozym 3000 was added to 430 ml 1.45 M lactose with 0.95 M polyhydroxyalcohols). The following concentrations of galactosyl derivatives of polyols were obtained after hydrolysis for 4 h at 40°C: 0.31 M Gal-erythritol, 0.22 M Gal-xylitol, 0.18 M Gal-sorbitol and 0.14 M Gal-lactitol. A quadruple increase of xylitol content in dry matter of a solution (from 11.5% (w/w) to 44.5% (w/w) brought about a 2.3-fold increase of the product content in the solution (15.2% (w/w) of dry matter).     

Antagonistic activity of lactic acid bacteria as probiotics against selected bacteria of the Enterobaceriacae family in the presence of polyols and their galactosyl derivatives

Probiotic lactic acid bacteria were grown on erythritol, xylitol, sorbitol or lactitol and produced various derivatives: gal-erythritol, gal-xylitol, and gal-sorbitol as prebiotics. Galactosyl derivatives of erythritol, xylitol and sorbitol were metabolised by Lactobacillus spp. This resulted in their antagonistic activity against the test microflora. No activity was observed in the presence of xylitol and erythritol. Gal-sorbitol obtained by enzymatic transglycosylation from lactose had the same abilities of inducing the antagonistic activity of lactic acid bacteria that lactitol had.     

Kinetic characterization of galacto‐oligosaccharide (GOS) synthesis by three commercially important β‐galactosidases

Many β‐galactosidases show large differences in galacto‐oligosaccharide (GOS) production and lactose hydrolysis. In this study, a kinetic model is developed in which the effect of lactose, glucose, galactose, and oligosaccharides on the oNPG converting activity of various β‐galactosidases is quantified. The use of oNPG as a competing substrate to lactose yields more information than can be obtained by examining only the conversion of lactose itself. The reaction rate with lactose or oligosaccharides as substrate relative to that with water as acceptor is much higher for the β‐galactosidase of Bacillus circulans than the β‐galactosidases of Aspergillus oryzae and Kluyveromyces lactis. In addition, the β‐galactosidase of B.circulans has a high reaction rate with galactose as acceptor, in contrast to those of A. oryzae and K. lactis. The latter two are strongly inhibited by galactose. These differences explain why β‐galactosidase of B. circulans gives higher yields in GOS production than other β‐galactosidases. Many of the reaction rate constants for the β‐galactosidase isoforms of B. circulans increase with increasing molecular weight of the isoform. This indicates that the largest isoform β‐gal‐A is most active in GOS production. However, its hydrolysis rate is also much higher than that of the other isoforms, which results in a faster hydrolysis of oligosaccharides as well. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:38–47, 2014     

Biochemical characterization of a strictly specific beta-galactosidase from the digestive juice of the palm weevil Rhynchophorus palmarum larvae

A beta‐galactosidase from the digestive juice of the palm weevil Rhynchophorus palmarum L. larvae was purified by chromatography on ion exchange, gel filtration, and hydrophobic interaction columns. The preparation was shown to be homogeneous on polyacrylamide gel. Beta‐galactosidase was a monomeric protein with a molecular weight of 62 kDa based on its mobility in sodium dodecyl sulfate–polyacrylamide gel electrophoresis and 60 kDa based on gel filtration. Maximal enzyme activity occurred at 55°C and pH 5.0. The purified beta‐galactosidase was stable at 37°C and its pH stability was in the range of 4.6–6.0. Beta‐galactosidase was highly specific for the beta‐d ‐galactosyl residue and beta‐(1‐4) linkage. The catalytic efficiency (V_max/K_m) values for p‐nitrophenyl‐beta‐d ‐galactopyranoside, beta‐d ‐galactosyl(1‐4)‐d ‐glucose (lactose), beta‐d ‐galactosyl(1‐4)‐d ‐galactose and beta‐d ‐galactosyl(1‐4)‐beta‐d ‐galactosyl(1‐4)‐d ‐glucose were, respectively, 72.95, 10.97, 20.74 and 12.73. 5,5‐Dithio‐bis(2‐nitrobenzoate) and sodium dodecyl sulfate inhibited completely the beta‐galactosidase activity. The enzyme was capable of catalyzing transgalactosylation reactions. The yield of galactosylation of 2‐phenylethanol (43%), catalyzed by the beta‐galactosidase in the presence of lactose as galactosyl donor, is higher than those reported previously with conventional sources of beta‐galactosidases. In addition, the optimum pH is different for the hydrolysis (pH 5.0) and transgalactosylation reactions (pH 6.0).     

The effects of organic solvents on the efficiency and regioselectivity of N‐acetyl‐lactosamine synthesis,using the β‐galactosidase from Bacillus circulans in hydro‐organic media

The enzymatic synthesis of N‐acetyl‐lactosamine (LacNAc) by the transgalactosylation of N‐acetyl‐D ‐glucosamine (GlcNAc), catalyzed by the β‐galactosidase from Bacillus circulans (BcβGal), was studied in hydro‐organic media, starting from o‐nitrophenyl‐β‐D ‐galactopyranoside (oNPG) as a galactosyl donor. Thermal stability and synthesis activity of BcβGal were shown to depend on the organic solvent polarity, characterized by its Log P value. BcβGal was thus most stable in 10% (v/v) t‐BuOH, an organic solvent found to have a stabilizing and/or weakly denaturing property, which was confirmed for high t‐BuOH concentrations. In the same manner, the optimal synthesis yield increased as the Log P value of the organic solvent increased. The best results were obtained for reactions carried out in 10% (v/v) pyridine or 2‐methyl‐2‐butanol, which gave 47% GlcNAc transgalactosylation yield based on starting oNPG, of which 23% (11 mM; 4.3 g/L) consisted in LacNAc synthesis. Furthermore, it was also established that both the GlcNAc transgalactosylation yield and the enzyme regioselectivity depended on the percentage of organic solvent used, the optimal percentage varying from 10 to 40% (v/v), depending on the solvent. This phenomenon was found to correlate mainly with the thermodynamic activity of water (a_w) in the aqueous organic solvent mixture, which was found to be optimal when close to 0.96, whatever the organic solvent used. Finally, this study highlighted the fact that the regioselectivity of BcβGal for 1‐4 linkage formation could be advantageously managed by controlling the a_w parameter. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Fed‐batch synthesis of galacto‐oligosaccharides with Aspergillus oryzae β‐galactosidase using optimal control strategy

Fed‐batch synthesis of galacto‐oligosaccharides (GOS) from lactose with β‐galactosidase from Aspergillus oryzae was evaluated experimentally and reaction yield was maximized via optimal control technique. The optimal lactose and enzyme feed flow rate profiles were determined using a model for GOS synthesis previously reported by the authors. Experimentally it was found that fed‐batch synthesis allowed an increase on the maximum total GOS concentration from 115 (batch synthesis) to 218 g L^?1 as consequence of the increase in total sugars concentration from 40 to 58% w/w. Such high concentration of total sugars was not attainable in batch operation because of the low solubility of lactose at the reaction temperature (40°C). Simulations predicted a GOS yield of 32.5 g g^?1 in fed‐batch synthesis under optimal conditions, while experimentally the same yield as in batch synthesis was obtained (28 g g^?1). Besides, an enrichment of total oligosaccharides in GOS with a high polymerization degree (GOS‐5 and GOS‐6) was observed in the fed‐batch synthesis. Experimental profiles for all sugars were similar to the ones predicted by simulation, which supports the use of this methodology for the optimization of GOS synthesis. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:59–67, 2014     

Improvement of covalent immobilization procedure of β‐galactosidase from Kluyveromyces lactis for galactooligosaccharides production: Modeling and kinetic study

Galactooligosaccharides (GOS) are prebiotics produced from lactose through an enzymatic reaction. Employing an immobilized enzyme may result in cost reductions; however, the changes in its kinetics due to immobilization has not been studied. This study experimentally determined the optimal reaction conditions for the production of GOS from lactose by β‐galactosidase (EC 3.2.1.23) from Kluyveromyces lactis covalently immobilized to a polysiloxane‐polyvinyl alcohol (POS‐PVA) polymer activated with glutaraldehyde (GA), and to study the transgalactosylation kinetics. Yield immobilization was 99 ± 1.1% with 78.5 ± 2.4% enzyme activity recovery. An experimental design 24 with 1 center point and 2 replicates was used. Factors were lactose [L], enzyme concentration [E], pH and temperature (T). Response variables were glucose and galactose as monosaccharides [G1], residual lactose [Lac]r and GOS as disaccharides [G2] and trisaccharides [G3]. Best conditions were pH 7.1, 40 °C, 270 gL^?1 initial lactose concentration and 6 U mL^?1 enzyme concentration, obtaining 25.46 ± 0.01 gL^?1 yield of trisaccharides. Although below the HPLC‐IR detection limit, tetrasaccharides were also identified after 115 min of reaction. The immobilization protocol was then optimized by diminishing total reactant volumes : support ratio, resulting in improved enzyme activity synthesizing 43.53 ± 0.02 gL^?1 of trisaccharides and 13.79 ± 0.21 gL^?1 of tetrasaccharides, and after four cycles remaining relative activity was 94%. A reaction mechanism was proposed through which a mathematical model was developed and rate constants were estimated, considering a pseudo steady‐state hypothesis for two concomitant reactions, and from this simplified analysis, the reaction yield could eventually be improved. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1568–1578, 2017     

Modelling and parameter estimation of the enzymatic synthesis of oligosaccharides by β‐galactosidase from Bacillus circulans

The aim of this research is to develop a model to describe oligosaccharide synthesis and simultaneously lactose hydrolysis. Model A (engineering approach) and model B (biochemical approach) were used to describe the data obtained in batch experiments with β‐galactosidase from Bacillus circulans at various initial lactose concentrations (from 0.19 to 0.59 mol·kg⁻¹). A procedure was developed to fit the model parameters and to select the most suitable model. The procedure can also be used for other kinetically controlled reactions. Each experiment was considered as an independent estimation of the model parameters, and consequently, model parameters were fitted to each experiment separately. Estimation of the parameters per experiment preserved the time dependence of the measurements and yielded independent sets of parameters. The next step was to study by ordinary regression methods whether parameters were constant under the altering conditions examined. Throughout all experiments, the parameters of model B did not show a trend upon the initial lactose concentration when inhibition was included. Therefore model B, a galactosyl‐enzyme complex‐based model, was chosen to describe the oligosaccharide synthesis, and one parameter set was determined for various initial lactose concentrations. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 64: 558–567, 1999.     

Intergeneric yeast fusants with efficient ethanol production from cheese whey powder solution: Construction of a Kluyveromyces marxianus and Saccharomyces cerevisiae AY‐5 hybrid

Due to its high content of lactose and abundant availability, cheese whey powder (CWP) has received much attention for ethanol production in fermentation processes. However, lactose‐fermenting yeast strains including Kluyveromyces marxianus can only produce alcohol at a relatively low level, while the most commonly used distiller yeast strain Saccharomyces cerevisiae cannot ferment lactose since it lacks both β‐galactosidase and the lactose permease system. To combine the unique aspects of these two yeast strains, hybrids of K. marxianus TY‐22 and S. cerevisiae AY‐5 were constructed by protoplast fusion. The fusants were screened and characterized by DNA content, β‐galactosidase activity, ethanol tolerance, and ethanol productivity. Among the genetically stable fusants, the DNA content of strain R‐1 was 6.94%, close to the sum of the DNA contents of TY‐22 (3.99%) and AY‐5 (3.51%). The results obtained by random‐amplified polymorphic DNA analysis suggested that R‐1 was a fusant between AY‐5 and TY‐22. During the fermentation process with CWP, the hybrid strain R‐1 produced 3.8% v/v ethanol in 72 h, while the parental strain TY‐22 only produced 3.1% v/v ethanol in 84 h under the same conditions.     

Antifungal Activity of <Emphasis Type="Italic">Lactobacillus pentosus</Emphasis> ŁOCK 0979 in the Presence of Polyols and Galactosyl-Polyols

The antifungal activity of Lactobacillus pentosus ?OCK 0979 depends both on the culture medium and on the fungal species. In the control medium, the strain exhibited limited antagonistic activity against indicator food-borne molds and yeasts. However, the supplementation of the bacterial culture medium with polyols (erythritol, lactitol, maltitol, mannitol, sorbitol, xylitol) or their galactosyl derivatives (gal-erythritol, gal-sorbitol, gal-xylitol) enhanced the antifungal properties of Lactobacillus pentosus ?OCK 0979. Its metabolites were identified and quantified by enzymatic methods, HPLC, UHPLC-MS coupled with QuEChERS, and GC-MS. The presence of polyols and gal-polyols significantly affected the acid metabolite profile of the bacterial culture supernatant. In addition, lactitol and mannitol were used by bacteria as alternative carbon sources. A number of compounds with potential antifungal properties were identified, such as phenyllactic acid, hydroxyphenyllactic acid, and benzoic acid. Lactobacillus bacteria cultivated with mannitol synthesized hydroxy-fatty acids, including 2-hydroxy-4-methylpentanoic acid, a well-described antifungal agent. Scanning electron microscopy (SEM) and light microscopy confirmed a strong antifungal effect of L. pentosus ?OCK 0979.     

Improved Properties of Bacillus coagulans β‐Galactosidase through Immobilization

Thermostable β‐galactosidase from Bacillus coagulans RCS3 was purified by successive column chromatography using DEAE‐cellulose and Sephadex G‐50. Immobilization of the purified enzyme was studied with DEAE‐cellulose and calcium alginate. The efficiency of β‐galactosidase retention was 87 % with DEAE‐cellulose (17 mg protein/mL of matrix) and 80 % with calcium alginate (2.2 mg protein/g bead). Comparative studies of immobilization displayed a shift in the optimum temperature from 65 °C to 70 °C provoked by DEAE‐cellulose, although no effect was observed with calcium alginate. The heat inactivation curve revealed an improvement in the stability (t_1/2 of 14.5 h for the immobilized enzyme as compared to 2 h for the free enzyme at 65 °C) in a calcium alginate system. This immobilized enzyme has a wide pH stability range (6.5–11). β‐Galactosidase immobilized by DEAE‐cellulose and calcium alginate allowed a 57 and 70 % lactose hydrolysis, respectively, to be achieved within 48 h after repeated use for twenty times.     

Control of erythritol dehydrogenase in Schizophyllum commune

Summary An NAD-dependent erythritol dehydrogenase was detected in cell-extracts of basidiospore germinants of Schizophyllum commune following culture on either meso-erythritol or glycerol as sole carbon sources. Induction of erythritol dehydrogenase was also observed in purely vegetative mycelium (str. 845 or str. 699). Erythritol dehydrogenase was not observed in ungerminated basidiospores or germinants which arose on d-glucose, d-mannitol, sorbitol, ribitol, xylitol, d-arabitol or l-arabitol. NAD-coupled polyol dehydrogenases for all the latter sugar alcohols were observed in ungerminated basidiospores, germinants, and vegetative mycelium of S. commune cultured on d-glucose. Basidiospore germination on d-glucose plus meso-erythritol led to a 90% decrease in erythritol dehydrogenase and the specific activity of ribitol dehydrogenase was directly comparable to that seen in d-glucose germinants. Storage experiments of crude extracts of meso-erythritol germinants indicated differential enzyme decay of dehydrogenases for d-mannitol, sorbitol and erythritol while the respective enzymes could be further distinguished by heat-stability as well as preferential utilization of analogues of NAD. DEAE-cellulose column chromatography led to separation of sorbitol dehydrogenase which was also active with xylitol, erythritol dehydrogenase, and mannitol dehydrogenase which was also active with d-arabitol.     

Optimization of pH,Temperature and Inoculum Ratio for the Production of β‐D‐Galactosidase by Kluyveromyces marxianus Using a Lactose‐free Medium

The pH, temperature and inoculum ratio for the production of β‐galactosidase by Kluyveromyces marxianus CDB 002 were optimized using sugar‐cane molasses (100 g/l) in a lactose‐free medium. The temperature optimum was evaluated in the range from 28–37 °C. Lactase production was initiated after substrate consumption indicating a reversible enzyme inhibition or catabolic repression. The specific enzyme activity after 45 h was between 456.3 U/g cell mass (37 °C) and 733.3 U/g (34 °C), whereas the highest volumetric activity was obtained at 30 °C: 21.8 U/ml. This is generally consistent with results from other authors that used whey as a carbon source. Ethanol as a by‐product reached its maximum concentration after 10–14 h (31.1–40.5 g/l), but was completely consumed afterwards. A pH of 5.5 without further control gave the best production rate for lactase (484.4 U/l × h). In this process, the pH was stable during cell growth at 5.5 and then went up to pH 7.2 after 45 h. At a fixed pH of 5.5 or 6.5, the production rates achieved 313.3 U/l × h and 233.3 U/ l × h, respectively. These results differed from those of other authors, who suggested a fixed pH at 7.0 using whey as a carbon source. There were no significant differences between inoculum ratios of 1% [v/v] and 10% [v/v] so that 1% is the preferable ratio as it is cheaper. Yeast extract (10 g/l) and peptone (20 g/l) were used as the vitamin and nitrogen source, respectively, for the studies of temperature and pH. These were substituted by corn steep liquor (100 g/l) for inoculum ratio experiments. Production of lactase using sugar cane molasses in a lactose‐free medium gave better enzyme productivity rates than obtained by other authors using whey. The optimum conditions for β‐galactosidase synthesis were a temperature of 30–34 °C and an inoculum ratio of 1% [v/v], an initial pH of 5.5 without any further control or a control of 5.5 during cell growth. Then the pH was raised up to 7.     

The activity of beta-galactosidase and lactose metabolism in Kluyveromyces lactis cultured in cheese whey as a function of growth rate

Aims: Kluyveromyces lactis was cultured in cheese whey permeate on both batch and continuous mode to investigate the effect of time course and growth rate on β‐galactosidase activity, lactose consumption, ethanol production and protein profiles of the cells. Methods and Results: Cheese whey was the substrate to grow K. lactis as a batch or continuous culture. In order to precise the specific growth rate for maximum β‐galactosidase activity a continuous culture was performed at five dilution (growth) rates ranging from 0·06, 0·09, 0·12, 0·18 to 0·24 h^?1. The kinetics of lactose consumption and ethanol production were also evaluated. On both batch and continuous culture a respirofermentative metabolism was detected. The growth stage for maximum β‐gal activity was found to be at the transition between late exponential and entrance of stationary growth phase of batch cultures. Fractionating that transition stage in several growth rates at continuous culture a maximum β‐galactosidase activity at 0·24 h^?1 was observed. Following that stage β‐gal activity undergoes a decline which does not correlate to the density of its corresponding protein band on the gel prepared from the same samples. Conclusion: The maximum β‐galactosidase activity per unit of cell mass was found to be 341·18 mmol ONP min^?1 g^?1 at a dilution rate of 0·24 h^?1. Significance and Impact of the Study: The physiology of K. lactis growing in cheese whey permeate can proven useful to optimize the conversion of that substrate in biomass rich in β‐gal or in ethanol fuel. In addition to increasing the native enzyme the conditions established here can be set to increase yields of recombinant protein production based on the LAC4 promoter in K. lactis host.     

Senescence‐associated β‐galactosidase activity marks the visceral endoderm of mouse embryos but is not indicative of senescence

Summary: Senescence‐associated β‐galactosidase (SA‐β‐gal) activity is widely used as a marker of cellular senescence and as an indicator of organismal aging. Here, we report that SA‐β‐gal activity is present in the visceral endoderm layer of early postimplantation mouse embryos in predictable patterns that vary as the embryo progresses in development. However, determination of the mitotic index and analysis of the expression of Cdkn1a (p21), a marker of senescent cells, do not indicate cellular senescence. Instead, analysis of embryos in culture revealed the presence of SA‐β‐gal activity in apical vacuoles of visceral endoderm cells likely a reflection of acidic β‐galactosidase function in these organelles. SA‐β‐gal serves as a practical marker of the dynamics of the visceral endoderm that can be applied to developmental as well as functional studies of early mammalian embryos. genesis 52:300–308, 2014. © 2014 Wiley Periodicals, Inc.     

Perspectives of engineering lactic acid bacteria for biotechnological polyol production

Polyols are sugar alcohols largely used as sweeteners and they are claimed to have several health-promoting effects (low-caloric, low-glycemic, low-insulinemic, anticariogenic, and prebiotic). While at present chemical synthesis is the only strategy able to assure the polyol market demand, the biotechnological production of polyols has been implemented in yeasts, fungi, and bacteria. Lactic acid bacteria (LAB) are a group of microorganisms particularly suited for polyol production as they display a fermentative metabolism associated with an important redox modulation and a limited biosynthetic capacity. In addition, LAB participate in food fermentation processes, where in situ production of polyols during fermentation may be useful in the development of novel functional foods. Here, we review the polyol production by LAB, focusing on metabolic engineering strategies aimed to redirect sugar fermentation pathways towards the synthesis of biotechnologically important sugar alcohols such as sorbitol, mannitol, and xylitol. Furthermore, possible approaches are presented for engineering new fermentation routes in LAB for production of arabitol, ribitol, and erythritol.     

A pseudo steady‐state model for galacto‐oligosaccharides synthesis with β‐galactosidase from Aspergillus oryzae

A pseudo steady‐state model for the kinetically controlled synthesis of galacto‐oligosaccharides (GOS) with Aspergillus oryzae β‐galactosidase is presented. The model accounts for the dynamics of lactose consumption and production of galactose, glucose, di, tri, tetra, and penta‐oligosaccharides during the synthesis, being able to describe the total GOS content in the reaction medium at the experimental conditions evaluated. Experimental results show that the formation of GOS containing only galactose residues is significant at high conversions of substrate, which was taken into account in the model. The formation of enzyme transition complexes was considered and reasonable assumptions were made to reduce the number of parameters to be determined. The model developed has 8 parameters; 2 of them were experimentally determined and the other 6 were estimated by fitting to the experimental data using multiresponse regression. Temperature effect on kinetic and affinity constants was determined in the range from 40 to 55°C, and the data were fitted to Arrhenius type equation. Parameters of the proposed model are independent from the enzyme load in the reaction medium and, differently from previously reported models, they have a clear biochemical meaning. The magnitude of the kinetic and affinity constants of the enzyme suggests that the liberation of galactose from the galactosyl–enzyme complex is a very slow reaction and such complex is driven into GOS formation. It also suggests that the affinity for sugars of the galactosyl–enzyme complex is higher than that of the free enzyme. Biotechnol. Bioeng. 2011;108: 2270–2279. © 2011 Wiley Periodicals, Inc.     

Crystal structure of a GH1 β‐glucosidase from Hamamotoa singularis

A GH1 β‐glucosidase from the fungus Hamamotoa singularis (HsBglA) has high transgalactosylation activity and efficiently converts lactose to galactooligosaccharides. Consequently, HsBglA is among the most widely used enzymes for industrial galactooligosaccharide production. Here, we present the first crystal structures of HsBglA with and without 4′‐galactosyllactose, a tri‐galactooligosaccharide, at 3.0 and 2.1 Å resolutions, respectively. These structures reveal details of the structural elements that define the catalytic activity and substrate binding of HsBglA, and provide a possible interpretation for its high catalytic potency for transgalactosylation reaction.     

Xylitol and Erythritol Decrease Adherence of Polysaccharide-Producing Oral Streptococci

Xylitol consumption decreases counts of mutans streptococci. However, the mechanism behind this decrease is not well understood. We studied not only type strains and clinical isolates of mutans streptococci, but also other polysaccharide-forming oral streptococci. Growth inhibition and adherence of cells to a smooth glass surface—reflecting synthesis of water-insoluble polysaccharides were studied in the presence of 2% (0.13 mol/l) and 4% (0.26 mol/l) xylitol. The effect of xylitol was compared to a novel polyol sweetener, erythritol. Except for Streptococcus mutans 10449 and S. sobrinus OMZ 176, the glass surface adhesion of most polysaccharide-forming streptococci was reduced by the presence of both 4% xylitol and erythritol. For the S. mutans and S. sobrinus type strains, the growth inhibition with 4% xylitol and erythritol was 36–77% and for the clinical S. mutans isolates 13–73%. Of the other oral streptococci, only S. sanguinis was inhibited with 4% xylitol (45–55%). For both polyols, the magnitude of the growth inhibition observed was not associated with the magnitude of the decrease in adherence (xylitol: r = −0.18; erythritol: r = 0.49). In conclusion, both xylitol and erythritol can decrease polysaccharide-mediated cell adherence contributing to plaque accumulation through a mechanism not dependent on growth inhibition.     

Design of β‐galactosidase/silica biocatalysts: Impact of the enzyme properties and immobilization pathways on their catalytic performance

The use of heterogeneous biocatalysis in industrial applications is advantageous and the enzyme stability improvement is a continuous challenge. Therefore, we designed β‐galactosidase heterogeneous biocatalysts by immobilization, involving the support synthesis and enzyme selection (from Bacillus circulans, Kluyveromyces lactis, and Aspergillus oryzae). The underivatized, tailored, macro‐mesoporous silica exhibited high surface area, offered high enzyme immobilization yields and activity. Its chemical activation with glyoxyl groups bound the enzyme covalently, which suppressed lixiviation and conferred higher pH and thermal stability (120‐fold than for the soluble enzyme), without observable reduction of activity/stability due to the presence of silica. The best balance between the immobilization yield (68%), activity (48%), and stability was achieved for Bacillus circulans β‐galactosidase immobilized on glyoxyl‐activated silica, without using stabilizing agents or modifying the enzyme. The enzyme stabilization after immobilization in glyoxyl‐activated silica was similar to that observed in macroporous agarose‐glyoxyl support, with the reported microbiological and mechanical advantages of inorganic supports. The whey lactolysis at pH 6.0 and 25°C by using this catalyst (1 mg ml^?1, 290 UI g^?1) was still 90%, even after 10 cycles of 10 min, in batch process but it could be also implemented on continuous processes at industrial level with similar results.