No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
β-galactosidase covalent immobilization over large-pore mesoporous silica supports for the production of high galacto-oligosaccharides (GOS)
Abstract
Large-pore mesoporous silica supports with different structure and pore sizes distribution have been tested for glyoxyl multipoint covalent immobilization of Pectinex Ultra SP-L, a commercial preparation including the enzyme β-galactosidase from Aspergillus aculeatus. The prepared biocatalysts have been assayed in the production of galacto-oligosaccharides (GOS) from lactose as prebiotic functional food, aiming to optimize the yield to the GOS having the highest presumed prebiotic effect, tri- and tetra- GOS (high-GOS). Immobilization of β-galactosidase over glyoxyl-modified silica supports, with pores, large enough to accommodate the enzyme within their structure, led to an enhancement of the enzyme activity in terms of high-GOS production relative to the free enzyme under the same reaction conditions. Remarkably, such an improvement was achieved without previous purification of β-galactosidase from the commercial source. An enhancement of the transgalactosylation activity over the hydrolytic activity, due to the relatively hydrophobic nature of organically modified silica surface of the supports, led to an increase in the selectivity to high-GOS. The best biocatalyst in the series was that based in hexagonal ultra-large-pore SBA-15 (ULP-SBA-15), which presents the most adequate balance between confinement effect within its pore framework and transgalactosylation activity leading to a remarkable yield to high-GOS of 20.2% vs. 11.2% for the enzyme in free conditions. Furthermore, reusability of these silica-based biocatalysts in three consecutive 24 h reaction-cycles has been successfully performed at 50 °C, temperature that minimizes the thermal deactivation of the enzyme.
... GOSs are non-digestible oligosaccharides which resist most digestive enzymes and can stimulate the growth and/or activity of the bacteria being members of the microbial consortia in the colon, including bifidobacteria (Panesar et al., 2006;Roberfroid et al., 2010;O'Callaghan & van Sinderen et al., 2016;Hidalgo-Cantabrana et al., 2017;Zhang et al., 2023). Several studies revealed the beneficial effects of GOSs on health (Sanders et al., 2019;Sangwan et al., 2015) and focused on the access to products with high GOS content (Rodriguez-Colinas et al., 2014;González-Delgado et al., 2018;Urrutia et al., 2018). ...
... In addition, the enzymes can be easily removed from the milk at an optimal time point (e.g., by filtration); consequently, the products-finally reaching the consumers-are absent from the lactase enzymes which have been added previously for removal of LAC. Several solid supports, such as biopolymer-based beads (Bedade et al., 2019;Flores et al., 2019;Kurayama et al., 2020), silica gels (González-Delgado et al., 2018), hydrogels (Wolf et al., 2021), magnetic nanoparticles (Nguyen et al., 2019), and inorganic particles (Husain et al., 2011;Khan et al., 2019), have been developed and tested for enzyme immobilization so far. In addition, it has also been reviewed that not only solid supports but liposomes may also be good candidates to carry and deliver enzymes such as β-galactosidases (Mohammadi et al., 2021). ...
The lactose intolerance—as a limiting factor for dairy milk consumption—has a high prevalence worldwide. Dairy milk and milk-derived products are major sources of multiple inorganic compounds and nutrients and thus are considered to be functional foods. β-galactosidases are able to hydrolyze lactose and are therefore widely applied for the production of lactose-free products. In addition, they are capable of the synthesis of galacto-oligosaccharides (GOSs); thus, the dairy industry has a special interest in applying them for the enrichment of dairy products with prebiotic GOSs. In this work, we studied two commercially available β-galactosidase products: Saphera 2600L and Nola Fit 5500. Both enzyme solutions contain a recombinant β-galactosidase of Bifidobacterium bifidum and have already been authorized for food industrial application, but the information about their hydrolytic and/or synthetic activities is only limited. After immobilization on chitosan beads, the enzymes were used for lactose hydrolysis and simultaneous synthesis of GOSs, by performing the reactions in pasteurized milk (skim milk). Both immobilized β-galactosidase exhibited elevated lactose hydrolysis (vmax increased from ~ 1 to ~ 4 mM/min) and GOS synthesis as compared to the free enzymes. The enzyme-coated beads were efficiently re-used at least 15 cycles; the residual lactose concentration was < 2 mg/ml after each cycle. After treatment, GOSs were present in ≤ 9% of the total sugar content, indicating that the prepared low-lactose milks were enriched in prebiotic GOSs. The application of immobilized Saphera 2600L and Nola Fit 5500 β-galactosidases may be implemented for the large-scale production of GOS-enriched low-lactose milk.
Graphical Abstract
... Large-pore mesoporous silica was used to support β-galactosidase covalently. Yields of tri-and tetra-GOS were doubled to 20% by immobilized enzyme compared to 11% by free enzyme (González-Delgado et al., 2018). Dextranase can directly catalyze GOS production in orange juice to produce a functional healthy beverage. ...
Microbial enzymes catalyze various reactions inside and outside living cells. Among the widely studied enzymes, fungal enzymes have been used for some of the most diverse purposes, especially in bioremediation, biosynthesis, and many nature-inspired commercial applications. To improve their stability and catalytic ability, fungal enzymes are often immobilized on assorted materials, conventional as well as nanoscale. Recent advances in fungal enzyme immobilization provide effective and sustainable approaches to achieve improved environmental and commercial outcomes. This review aims to provide a comprehensive overview of commonly studied fungal enzymes and immobilization technologies. It also summarizes recent advances involving immobilized fungal enzymes for the degradation or assembly of compounds used in the manufacture of products, such as detergents, food additives, and fossil fuel alternatives. Furthermore, challenges and future directions are highlighted to offer new perspectives on improving existing technologies and addressing unexplored fields of applications.
... In industry, enzymatic processes are usually carried out using soluble enzymes (Choi et al., 2015;Zhu et al., 2017), which prevents the recovery of this enzyme from the reaction medium. Over the years, several enzymatic immobilization methods showed an alternative to the use of soluble enzymes (Aburto et al., 2018;González-Delgado et al., 2018;Katrolia et al., 2019;Urrutia et al., 2018). The main advantages of immobilized enzymes compared to soluble ones includes the easy separation from the product, which minimizes or eliminates the contamination of the final product with the enzyme (Grosová et al., 2008), its efficient reuse (Genari et al., 2003), allowing the use in continuous operation in bioreactors (Hernaiz and Crout, 2000;Krajewska, 2004), improvement of stability, both in conditions of storage, and under operational conditions (Mei et al., 2018;Rempel et al., 2018;Vaz et al., 2016), and increased stability to heat denaturation, organic solvents or pH extremes (Sheldon and van Pelt, 2013). ...
Milk whey (MW) and milk whey permeate (MWP) constitute the main part of co-products derived from milk processing. The use of the transgalactosylation activity of β-galactosidases is an excellent alternative to add value to these co-products since this is the route for the synthesis of galactooligosaccharides (GOS), a valuable prebiotic sugar. Hereby, we immobilized a β-galactosidase from Bacillus circulans onto glutaraldehyde-activated chitosan beads. The immobilized enzyme was characterized and the parameters affecting GOS synthesis (pH, temperature, and concentration of MW and MWP) were evaluated. The optimum pH and temperature of activity for the immobilized β-galactosidase were 50 °C and pH 6.0, respectively. The immobilization improves enzyme thermal stability by 2.9 times. In the batch process, the immobilized enzyme exhibited excellent operational stability at 40 °C, being able to hydrolyze 91% lactose after 17 cycles of reuse. Regarding GOS synthesis, the immobilized enzyme could be reused for 10 consecutive cycles retaining 74% of the initial activity at 50 °C. The maximum concentration of GOS achieved, using MW and MWP, was 159.4 g L⁻¹ and 168.8 g L⁻¹, respectively. Therefore, these results proved that it was possible to obtain, in high concentration, a useful and valuable prebiotic sugar from co-products of milk processing.
... These silica microparticles (average diameter 7.5 µm) have a randomly arranged, cage-like pore structure with an average pore diameter of 35 nm and a pore volume of 1.23 cm 3 /gm. 28,29 The combination of larger pore diameter and cage-like pore geometry not only enables the accommodation of larger diameter drug molecules such as vancomycin, but also translates to a higher drug loading and faster release rate of drug, 30,31 a desirable attribute to achieve bone concentrations well above the minimum inhibitory concentration (MIC) of the infecting organism. ...
The objective of this study was to develop a novel microcomposite implant to be used in the treatment of osteomyelitis following total joint arthroplasty, with the dual purpose of releasing high local concentrations of antibiotic to eradicate the infection while providing adequate mechanical strength to maintain the dynamic or static spacer. Vancomycin‐loaded microcomposite implants were fabricated by incorporating drug‐loaded microparticles comprised of mesoporous silica into commonly employed polymethylmethacrylate (PMMA) bone cement, to yield a final drug loading of 10% w/w. In vitro release kinetics at 37°C were monitored by reverse‐phase high‐performance liquid chromatography, and compared to the release kinetics of current therapy implants consisting of drug alone incorporated at 10% w/w directly into PMMA bone cement. Results demonstrated a sevenfold improvement in the elution profile of microcomposite systems over current therapy implants. In vivo delivery of vancomycin to bone from microcomposite implants (70% of payload) was significantly higher than that from current therapy implants (approx. 22% of payload) and maintained significantly higher bone concentrations for up to 2 weeks duration. The elastic modulus showed no statistical difference between microcomposite implants and current standard therapy implants before drug elution, and maintenance of acceptable strength of microcomposite implants postdrug elution. These results demonstrate that we have developed a novel microcomposite spacer that will release continuously high antibiotic concentrations over a prolonged period of time, offering the possibility to eliminate infection and avoid the emergence of new resistant bacterial strains, while maintaining the requisite mechanical properties for proper space maintenance and joint fixation.
... β-GAL from Aspergillus aculeatus (Pectinex Ultra SP-L) was covalently immobilized by multipoint covalent attachment to organically modified silica, functionalized with glyoxyl groups. The prepared biocatalysts have been assayed in the production of GOS from lactose, as prebiotic functional food (Gonzalez-Delgado et al., 2018). ...
... As is discussed above, the pore size influences the catalytic activity of immobilized enzymes [60,61]. Fig. S1 (SI) shows that indeed silica contains larger pores (some of which can accommodate GOx with a hydrodynamic diameter of 7.6 nm [62]) than alumina which would be consistent with the biocatalyst activity, as larger pores were reported to enhance enzymatic catalysis [63,64]. However, after the iron oxide NP formation in the silica or alumina pores, the pore sizes decrease, while the catalytic activity increases, revealing that other factors could be also responsible for the differences in catalytic activities. ...
Immobilized enzymes are better suited for industrial applications than free enzymes due to their favorable properties such as ease of separation and reuse, and enhanced stability and storage life. β-Galactosidases are an important class of glycosidases with hydrolysis and transglycosylation activities, which are applied in industries for lactose hydrolysis and prebiotics synthesis worldwide. The recent innovations in immobilized β-galactosidases have improved the performance of the immobilized enzymes and broadened their applications in the fields of food, energy, and medicine. Innovations in β-galactosidase immobilization methods include rational adsorption based on enzyme features, layer by layer adsorption for strengthened ionic bonding, 3-D printing for rapid and elaborate entrapment, modifications of either materials or enzymes for ingenious covalent binding, nontoxic crosslinking, carrier-free immobilization, and oriented immobilization either through protein engineering or enzyme display on cells, membranes, and phages, along with innovations in carrier materials involving the introduction of graphene derivatives, polyaniline nanomaterials, nanofibers, nucleotide molecules, Langmuir−Blodgett films, and so on. These innovations have partially solved the problems associated with traditional methods, resulting in enzymes with highly retained activity, excellent stability, reduced microbial contamination, enzyme leakage, and reagent toxicity. The immobilized β-galactosidases with potential economic and environmental benefits have been extendedly used for hydrolysis of prodrugs for disease treatment, assembly of biosensors for lactose detection, synthesis of bioactive carbohydrates, and even production of food additives and industrial products, such as tagatose and bioethanol. This review describes the innovations in β-galactosidases immobilization and the applications of these immobilized enzymes. It not only enables the fully understanding of β-galactosidases, but also provides a valuable reference for the immobilization of other industrially-important enzymes.
The concern for better life quality has been encouraging the bioprocess industries to develop technological strategies to obtain new biomolecules. Galactooligosaccharides (GOS) are an important class of food-grade oligosaccharides, being classified as non-digestible, and which present prebiotic potential, promoting better conditions of health and well-being. The main benefits include the selective stimulation of beneficial microorganisms, the decrease in the formation of toxic compounds, the increase in the absorption of minerals, improvement of an immune response, and a reduction in the severity of obesity and diabetes. This review approaches the recent methodologies and strategies to obtain GOS, their health benefits, purification, and technological properties for industrial application. Improvements in the process are continuously being investigated, with the technique of enzyme immobilization representing a potentially promising strategy. Sustainable GOS productions have been reported by the use of agro-industrial residues, such as cheese whey. Despite these advances, the main concern of the process consists in the low yield, which implies high investments in the purification of the bioproducts. Technological and nutritional approaches to the GOS application in different industrial sectors are also reported.
Over the past decade, galactooligosaccharides (GOS) have been globally recognized as prebiotic substances and have fascinated various researchers worldwide. Owing to their various physiological properties as well as enormous health benefits, the demand for GOS among the growing population has substantially increased. GOS can be extracted from a variety of natural sources, but in small quantities, and there is a need to scale up GOS production to fulfill ever-increasing demand. Both chemical and enzymatic methods have been reported for production, yet in comparison to the former, the latter techniques have been most widely explored owing to the various techno-economic benefits. Various biotechnological approaches, such as whole cells, free, immobilized and recombinant enzymes, have been proposed by researchers globally to enhance the production of GOS, each having their merits and demerits. Despite this, there is still a need for a robust strategy that can not only augment the yield, but also curb the cost of production. This chapter provides detailed insights into the various production strategies of GOS, their role as potential prebiotics and the worldwide production and consumption of GOS.
The kinetic model of the hydrolysis of lactose by β-galactosidase from Aspergillus niger immobilized on a commercial ceramic monoliths was estimated in the attendance of lactose and its hydrolysis reaction products galactose and glucose. The aim of this work was to developing kinetic model of lactase hydrolysis by Aspergillus niger. The variables in this study are temperature, pH, enzyme concentration, substrate concentration and final product. The optimum temperature used to achieve the best hydrolysis performance in the kinetic model selection was 55 and 60 °C. The optimum pH used for enzyme activity was about3.5 to 4. Five kinetic models were proposed to confirm experimental data the enzymatic reaction of the lactose hydrolysis by the β-galactosidase. The kinetics of lactose hydrolysis by both Immobilized and soluble lactases were scrutinized in a batch reactor system in the lack of any mass conduction restriction. In both instance the galactose inhibition kinetic models predicted the experimental data. The model is capable to fit the experimental data correctly in the extensive experimental span studied.
Enzyme immobilization is a well-known method for the improvement of enzyme reusability and stability. To achieve very high effectiveness of the enzyme immobilization, not only does the method of attachment need to be optimized, but the appropriate support must be chosen. The essential necessities addressed to the support applied for enzyme immobilization can be focused on the material features as well as on the stability and resistances in certain conditions. Ceramic membranes and nanoparticles are the most widespread supports for enzyme immobilization. Hence, the immobilization of enzymes on ceramic membrane and nanoparticles are summarized and discussed. The important properties of the supports are particle size, pore structure, active surface area, volume to surface ratio, type and number of reactive available groups, as well as thermal, mechanical, and chemical stability. The modifiers and the crosslinkers are crucial to the enzyme loading amount, the chemical and physical stability, and the reusability and catalytical activity of the immobilized enzymes. Therefore, the chemical and physical methods of modification of ceramic materials are presented. The most popular and used modifiers (e.g. APTES, CPTES, VTES) as well as activating agents (GA, gelatin, EDC and/or NHS) applied to the grafting process are discussed. Moreover, functional groups of enzymes are presented and discussed since they play important roles in the enzyme immobilization via covalent bonding. The enhanced physical, chemical, and catalytical properties of immobilized enzymes are discussed revealing the positive balance between the effectiveness of the immobilization process, preservation of high enzyme activity, its good stability, and relatively low cost.
Silica-based materials are considered very versatile and flexible, easily optimized to various applications that can go from catalysis, biosensor support, enzyme/drug carriers, inorganic fillers to coatings/polymers and many others. With emphasis on colloidal silica, the multitude of synthesis pathways, mostly comprising the gaseous (fumed pyrogenic silicas) and liquid (wet) routes (Stöber, surfactant-templated, precipitated, and biomimetic silicas), are responsible for the development of different silica-based materials with varied surface chemistry, morphology, porosity, size, and surface area. Given this, their use as support matrix to develop silica-metal hybrids is a common and current trend with various potential applications. In this review, the most common synthesis pathways to develop colloidal silicas and silica-metal hybrids are discussed. A special emphasis regarding to their applications within the food sector is given, more specifically, as host/carriers for enzyme immobilization in food production and processing, as materials to produce sensor-like devices for food-related analyte detection in food safety monitoring, and as inorganic fillers in polymer composite formation. This last application is related to the added improvements (e.g., mechanical, gas barrier, antibacterial, etc.) in the polymeric matrix, posing as potential alternatives to be used in food packaging.
Candida antarctica lipase B (CALB) produced by recombinant Pichia pastoris was covalently immobilized to a carboxyl‐functionalized hollow mesoporous silica sphere (HMS) synthesized by sol‐gel precipitation. The HMS was characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), X‐ray diffractometer (XRD), and Brunauer‐Emmett‐Teller (BET). The immobilized CALB was characterized through Fourier transform infrared spectra (FT‐IR) and elemental analysis to verify the binding of enzyme. The free and immobilized CALB were tested in tributyrin hydrolysis. Results revealed that CALB loading capacity of HMS‐COOH was about 28.6 mg/g support and the corresponding enzyme activity was up to about 2700 U/g support at optimal pH and temperature, which was 6.5 and 30 °C respectively. Moreover, compared with free CALB, the immobilized CALB presented more resistant towards the reaction pH and temperature changes. Furthermore, the immobilized CALB retained 60 % residual activity after eight batches of recycling and exhibited better thermal stability and storage stability, which was promising in the potential industrial applications. An efficient method for convalent immobilization of CALB was developed by conjugation to functionalized HMS via amide bonds, with eco‐friendly, efficient, and easy operation conditions. Compared with free CALB, immobilized CALB presented more resistant towards the reaction pH and temperature changes. The immobilized CALB retained 60 % residual activity after eight batches of recycling and exhibited improved thermal stability and storage stability
Here we report the preparation of biomimetic fibrin/chitosan/keratin hybrid scaffolds with a synergistic combination of ferulic acid loaded silica microspheres for antimicrobial wound dressing applications. The infrared and X-ray powder diffraction studies confirm the homogenous nature of the prepared hybrid scaffolds without any major interactions between the constituents. The developed hybrid scaffolds show good thermal, porosity, compression and water uptake properties. Scanning electron microscopic analysis shows that the as-synthesized ferulic acid loaded silica microspheres exhibit an average size of 35 ± 10 μm and also exposes the smooth surface with interconnected porosity in the prepared hybrid scaffolds. The incorporated ferulic acid loaded silica microspheres hybrid scaffolds show effective antimicrobial activity against the common wound pathogens. In vitro NIH3T3 fibroblast cell culture and drug release studies reveal that the prepared hybrid scaffolds have enhanced cell proliferation and adhesion with a prolonged drug release for about 72 h. In vitro wound healing and actin cytoskeleton analysis reveal that the incorporated ferulic acid loaded silica microspheres in fibrin/chitosan/keratin hybrid scaffolds facilitates cell growth and migration to damaged area through cell-cell interactions. These results suggest that the prepared hybrid scaffolds with ferulic acid loaded silica microspheres have great potential for soft tissue engineering applications particularly for the treatment of chronic and infected wounds.
Enzyme-assisted catalysis have long been considered a risky venture for many industrial processes despite their unique advantages, specificity, and activity. The use of free enzymes often lack economic viability and commercial feasibility for industrial use since they are expensive, incapable to withstand harsh conditions during downstream processing, and are extremely difficult to recover. Developing novel interventions employing nanomaterials as an immobilization template has greatly improved in minimizing these obstacles, thereby promoting activity and recycling of enzymes. As a result, self-assembled enzymes over nanomaterials have demonstrated great success particularly in biomedical sector, using novel nanobiocatalysts (NBCs) for diagnostic and therapeutic purposes. However, the use of such hybrid system for the production of value-added biochemical products is yet another domain, which has not been much explored. This chapter thus provides some useful insights using enzyme-nanohybrids for bioprocessing applications, particularly in the field of carbohydrate hydrolysis, biotransformation, and biofuel production. Several nanocomposites were considered for enzyme immobilization owing to their extraordinary characteristics such as non-toxic, ease in modification, high porosity ,and extremely stable toward adverse conditions. All selected studies were critically evaluated in terms of enzyme used, immobilization strategies, process parameters (enzymes’ stability, activity, loading, and recycling), and product specifications (yield, productivity, and purity). Relevant challenges encountered using nanomaterials, especially for the production of industrially relevant biochemical products, were also discussed at the end, which needs to be addressed in near future.
Human milk oligosaccharides (HMOs) are a family of structurally distinct carbohydrate oligomers present in human milk. HMOs protect breastfed babies against infection and promote the development of infant health and cognition. In the gut, fucosylated HMOs in particular function as decoy receptors that intercept epithelial attachment of enteric pathogens and hence help reduce infection. Infant formulae made from bovine milk are essentially devoid of HMOs, which creates a large impetus for biosynthetic production of HMOs. Certain microbial α-L-fucosidases (EC 3.2.1.51, EC 3.2.1.111), specifically various retaining α-L-fucosidases of glycoside hydrolase family 29 (GH29), are capable of catalysing transfucosylation. The use of GH29 α-L-fucosidases to promote transfucosylation reactions thus represents a strategy for biocatalytic synthesis of fucosylated HMOs. The purpose of this review is to present the current knowledge on the use of such α-L-fucosidases for synthesis of fucosylated HMOs by enzymatic transfucosylation. We summarize the available data obtained for both wild type and engineered microbial α-L-fucosidases, discuss enzyme and substrate sources, and review factors governing transglycosylation performance, particularly the use of protein engineering. We describe the mechanistic reaction details of α-l-fucosidase transfucosylation, and examine details of enzyme mutation strategies promoting transfucosylation. Finally, we list recommendations for future reaction targets based on currently abundant substrate sources.
Heterogeneous catalysis plays a key role in promoting green chemistry through many routes. The functionalizable reactive silanols highlight silica as a beguiling support for the preparation of heterogeneous catalysts. Metal active sites anchored on functionalized silica (FS) usually demonstrate the better dispersion and stability due to their firm chemical interaction with FSs. Having certain functional groups in structure, FSs can act as the useful catalysts for few organic reactions even without the need of metal active sites which are termed as the covetous reusable organocatalysts. Magnetic FSs have laid the platform where the effortless recovery of catalysts is realized just using an external magnet, resulting in the simplified reaction procedure. Using FSs of multiple functional groups, we can envisage the shortened reaction pathway and, reduced chemical uses and chemical wastes. Unstable bio‐molecules like enzymes have been stabilized when they get chemically anchored on FSs. The resultant solid bio‐catalysts exhibited very good reusability in many catalytic reactions. Getting provoked from the green chemistry aspects and benefits of FS‐based catalysts, we confer the recent literature and progress focusing on the significance of FSs in heterogeneous catalysis. This review covers the preparative methods, types and catalytic applications of FSs. A special emphasis is given to the metal‐free FS catalysts, multiple FS‐based catalysts and magnetic FSs. Through this review, we presume that the contribution of FSs to green chemistry can be well understood. The future perspective of FSs and the improvements still required for implementing FS‐based catalysts in practical applications have been narrated at the end of this review.
Levulinic acid, being one the most important building block precursors, is of prime importance in a variety of industries. The esters of levulinic acid have significant applications in fields of flavor and fragrance. In this study, surface functionalized SBA-15 was used to immobilize Candida antarctica lipase B for the production of different alkyl levulinates. APTS (3-aminopropyl) triethoxysilane functionalized SBA-15 was used to immobilize lipase (Lip) cross-linked with GLU (glutaraldehyde) (named SBA-15-APTS-GLU-Lip). Various aspects of the immobilized catalyst were studied including surface area analysis, morphology, stability using XRD, SEM, TEM, and FTIR techniques. Process optimization of the model reaction of levulinic acid and isoamyl alcohol was done for the production of isoamyl levulinate. The highest conversion of 94% was achieved within 2 h at 50 °C. The kinetics was found to obey ternary dead end complex mechanism with isoamyl alcohol inhibition. The results indicate that the synthesized biocatalyst shows high operational stability and reusability up to 4 cycles.
This paper describes a new support that permits to efficient immobilization of L-asparaginase (L-ASNase). For this purpose, Fe3O4 magnetic nanoparticles were synthesized and coated by MCM-41. 3-chloropropyltrimethoxysilane (CPTMS) was used as a surface modifying agent for covalent immobilization of L-ASNase on the magnetic nanoparticles. The chemical structure; thermal, morphological, and magnetic properties; chemical composition; and zeta potential value of Fe3O4@MCM-41-Cl were characterized by Fourier transform infrared spectroscopy (FTIR), thermogravimetric analysis (TGA), differential thermal analysis (DTA), differential scanning calorimetry (DSC), vibrating sample magnetometer (VSM), scanning electron microscope (SEM), energy dispersive X-ray (EDX), X-ray diffraction patterns (XRD), and zeta-potential measurement. The immobilization efficiency onto Fe3O4@MCM-41-Cl was detected as 63%. The reusability, storage, pH, and thermal stabilities of the immobilized L-ASNase were investigated and compared to that of soluble one. The immobilized enzyme maintained 42.2% of its original activity after 18 cycles of reuse. Furthermore, it was more stable towards pH and temperature compared with soluble enzyme. The Michaelis–Menten kinetic properties of immobilized L-ASNase showed a lower Vmax and a similar Km compared to soluble L-ASNase. Immobilized enzyme had around 47 and 32.5% residual activity upon storage a period of 28 days at 4 and 25 °C, respectively. In conclusion, the Fe3O4@MCM-41-Cl@L-ASNase core–shell nanoparticles could successfully be used in industrial and medical applications.
Aerosol processing technologies represent a major route of innovation in the mushrooming field of heterogeneous catalysts preparation.
A magnetic silica/titania material with planned textural properties was obtained by sol-gel method through hydrolysis and condensation of tetraethyl orthosilicate and titanium isopropoxide in the presence of magnetite particles. The resultant material (SiTiMPs) was used as support for direct immobilization of tyrosinase (SiTiMPs-Ty) avoiding additional modification steps. This was possible due to the chemical reactivity of titania due to its surface acidity and also to the textural properties of the material like pore size near 20 nm and specific surface area of 158 m² g⁻¹. These characteristics combined to the presence of magnetite lead to possibility to prepare carbon paste electrodes with SiTiMPs-Ty material with high electroactive area when tested in the ferricyanide/ferrocyanide redox system. This electrode was applied as efficient electrochemical biosensor for catechol and catecholamines, such as dopamine and epinephrine. It can be observed great sensitivities for all analytes tested in wide linear range. The calculated detection limits were 0.23 μmol L⁻¹, 0.72 μmol L⁻¹ and 2.94 μmol L⁻¹ for catechol, dopamine and epinephrine, respectively. This electrode presented excellent response for simultaneous analyses of catechol and epinephrine, with high sensitivity and low detection limits. The present sensor showed good reproducibility for catechol detection with a relative standard deviation of 6.9% (n = 5).
Three different fungal b-galactosidases, from three commercial sources, were studied for their capacity to produce galacto-oligosaccharides by trans-galactosylation from lactose. The enzymes were investigated in a soluble form for the GOS production at two concentrations of lactose (10 % and 30 % w/w). The maximum of GOS concentration (70.9 g · L-1) was obtained at 30 % of the initial lactose (pH 4.5, temperature 30 ℃) in the second hour of enzyme reaction. The GOS yield (24 % w/w) was not significantly affected by the process of immobilisation in comparison with the free enzyme preparation (22 % w/w), but time to obtain a maximal GOS concentration was prolonged. Galacto-oligosaccharides were effectively produced by immobilised β-galactosidase in the 15 repeated batch runs of 30 % (w/w) of lactose as the substrate.
The health benefits imparted by probiotics and prebiotics as well as synbiotics have been the subject of extensive research in the past few decades. These food supplements termed as functional foods have been demonstrated to alter, modify and reinstate the pre-existing intestinal flora. They also facilitate smooth functions of the intestinal environment. Most commonly used probiotic strains are: Bifidobacterium, Lactobacilli, S. boulardii, B. coagulans. Prebiotics like FOS, GOS, XOS, Inulin; fructans are the most commonly used fibers which when used together with probiotics are termed synbiotics and are able to improve the viability of the probiotics. Present review focuses on composition and roles of Probiotics, Prebiotics and Synbiotics in human health. Furthermore, additional health benefits like immune-modulation, cancer prevention, inflammatory bowel disease etc. are also discussed.
Graphical abstract
Pictorial summary of health benefits imparted by probiotics, prebiotics and synbiotics
Immobilization of Rhizomucor miehei lipase (RML) by two different methods is reported. In the first approach RML was directly immobilized on epoxy functionalized silica particles in a mild condition. In the other approach and to perform oriented immobilization, the epoxy functionalized support was partially modified by introducing iminodiacetic acid groups fallowed by addition of Cu2+. In this type of immobilization the covalent linkage takes place after initial adsorption of the enzyme on the support via metal chelate affinity interaction. The results showed higher thermal stability for the derivative obtained by this method. Co-solvent stability of the derivatives and free RML was also studied in presence of 10 and 20% of six polar organic solvents (DMSO, THF, acetonitrile, 1-propanol, 2-propanol and dioxane). The results showed improved stabilities for both derivatives, silica-epoxy-IDA-RML in particular. The ability of the immobilized preparations and the free enzyme to catalyze hydrolysis of fish oil was determined at different conditions. The results revealed that all the derivatives discriminate between cis-5,8,11,14,17-eicosapentaenoic acid (EPA) and cis-4,7,10,13,16,19-docosahexaenoic acid (DHA) in favor of EPA. The content of EPA and DHA increased with time and the amount of biocatalyst during the hydrolysis process. Reusability of the enzyme greatly improved after immobilization.
Prebiotics are short chain carbohydrates that are non-digestible by digestive enzymes in humans and selectively enhance the activity of some groups of beneficial bacteria. In the intestine, prebiotics are fermented by beneficial bacteria to produce short chain fatty acids. Prebiotics also render many other health benefits in the large intestine such as reduction of cancer risk and increase calcium and magnesium absorption. Prebiotics are found in several vegetables and fruits and are considered functional food components which present significant technological advantages. Their addition improves sensory characteristics such as taste and texture, and enhances the stability of foams, emulsions and mouthfeel in a large range of food applications like dairy products and bread. This contribution reviews bioactives from food sources with prebiotic properties. Additionally, food application of bioactive prebiotics, stimulation of the viability of probiotics, health benefits, epidemiological studies, and safety concerns of prebiotics are also reviewed.
Four ordered mesoporous silica supports with different pore structure characteristics were investigated for their drug loading and release abilities with regard to their structural variabilities as well as implications of surface modification. The (model) drug molecule in question was the poorly water soluble glucocorticoid Prednisolone, composed of a steroid skeleton with functional groups in the form of carbonyls and hydroxyls. Under non-aqueous conditions, such as those applied for drug loading, these functional groups are expected to interact with the surface silanols of silica, but this interaction could possibly also be enhanced by introducing amino groups to the silica surfaces. Thus, all four supports were further functionalized by surface hyperbranching of polyethylene imine, which was successfully incorporated to all supports in high amounts (>30wt%). However, the accessibility of the pore system after organic modification was dependent on the pore sizes and structures, highlighting the importance of using large-pore mesophases with adequate structures when aiming for applications involving (bulky) guest molecules. Additionally, after incorporation of large amounts of guest molecules (40wt%), full water accessibility was retained in that the loaded cargo could be rapidly released from the carrier matrixes, which is a crucial requirement when formulating poorly soluble substances. Results displayed that the release of Prednisolone from the silica supports occurred faster than the dissolution of the pure drug. All silica materials released in 5 hours more than 85% of the adsorbed drug, independently of the material. Thus, the confinement of Prednisolone inside the mesopores seems to be the main reason for the faster kinetic rate release. These constraints imply that Prednisolone becomes more mobile inside the pores, and therefore more soluble in water simulated body fluid. These results confirm the potential of silica supports as drug delivery carriers for drugs with limited water solubility such as steroids.
Pectinex Ultra SP-L, a commercial enzyme preparation obtained from Aspergillus aculeatus, containing β-galactosidase activity, was immobilized onto Eupergit C and was used for the production of galactooligosaccharides (GOS). Immobilization resulted in 100% binding yield and higher GOS yield (24%, w/v) with respect to soluble enzyme. Optimum conditions were not affected by immobilization, and optimum pH and temperature for free and immobilized enzyme were 4.0–5.0 and 55–60°C, respectively. Immobilized enzyme was more stable at high pH and temperatures. The amount of GOS produced from 30% (w/v) lactose solution using the free and immobilized enzyme was determined to be 12.8 and 15.8% (w/v) of the total sugar in the reaction mixture, respectively. The kinetic parameters for the free and immobilized enzyme were also determined. The immobilized Pectinex Ultra SP-L could be used for the production of galactooligosaccharides, since the immobilization yield is high (124%) and immobilized enzyme retains its activity for 20 days without any decrease.
The influence of synthesis time and temperature on the properties of SBA-15 is investigated for the samples prepared by using two different silica sources, tetraethylorthosilicate (TEOS) and sodium metasilicate. The samples studied were obtained via two-step synthesis, which involved an initial self-assembly of polymer and silica species at 40 °C for 2 hours followed by longer hydrothermal treatment at higher temperatures. The SBA-15 samples obtained from both silica sources were highly ordered as evidenced by SAXS spectra showing up to seven reflection peaks characteristic of the P6mm symmetry group. While for the samples studied the specific surface area decreased with increasing time and temperature of the hydrothermal treatment, their mesopore size distributions became narrower and their mesopore widths and mesopore volumes showed a tendency to increase. In contrast, a slight decrease in the mesopore wall thickness and micropore volume was observed with increasing time of this treatment. Also, the samples obtained from sodium metasilicate exhibited thicker mesopore walls than those prepared from TEOS. It was shown that the adsorption properties of SBA-15 can be tailored by adjusting the time and temperature of the hydrothermal treatment and simultaneously reducing the time of the initial self-assembly process from 24 h to 2 h.
Immobilization of enzymes may produce alterations in their observed activity, specificity or selectivity. Although in many cases an impoverishment of the enzyme properties is observed upon immobilization (caused by the distortion of the enzyme due to the interaction with the support) in some instances such properties may be enhanced by this immobilization. These alterations in enzyme properties are sometimes associated with changes in the enzyme structure. Occasionally, these variations will be positive. For example, they may be related to the stabilization of a hyperactivated form of the enzyme, like in the case of lipases immobilized on hydrophobic supports via interfacial activation. In some other instances, these improvements will be just a consequence of random modifications in the enzyme properties that in some reactions will be positive while in others may be negative. For this reason, the preparation of a library of biocatalysts as broad as possible may be a key turning point to find an immobilized biocatalyst with improved properties when compared to the free enzyme. Immobilized enzymes will be dispersed on the support surface and aggregation will no longer be possible, while the free enzyme may suffer aggregation, which greatly decreases enzyme activity. Moreover, enzyme rigidification may lead to preservation of the enzyme properties under drastic conditions in which the enzyme tends to become distorted thus decreasing its activity. Furthermore, immobilization of enzymes on a support, mainly on a porous support, may in many cases also have a positive impact on the observed enzyme behavior, not really related to structural changes. For example, the promotion of diffusional problems (e.g., pH gradients, substrate or product gradients), partition (towards or away from the enzyme environment, for substrate or products), or the blocking of some areas (e.g., reducing inhibitions) may greatly improve enzyme performance. Thus, in this tutorial review, we will try to list and explain some of the main reasons that may produce an improvement in enzyme activity, specificity or selectivity, either real or apparent, due to immobilization.
In spite of their excellent catalytic properties, enzyme properties have to be usually improved before their implementation at industrial scale (where many cycles of high yield processes are desired). Generally, soluble enzymes have to be immobilized to be reused for long times in industrial reactors and, in addition to that, some other critical enzyme properties have to be improved like stability, activity, inhibition by reaction products, selectivity towards non-natural substrates. Some strategies to improve these enzyme properties during the performance of tailor-made enzyme immobilization protocols are here reviewed. In this way, immobilized enzymes may also exhibit much better functional properties than the corresponding soluble enzymes by very simple immobilization protocols. For example, multipoint and multisubunit covalent immobilization improve the stability of monomeric or multimeric enzymes. Moreover, enantioselectivity of different enzymes (e.g., lipases) may be also dramatically improved (from E = 1 to >100) by performing different immobilization protocols of the same enzyme. In all cases, enzyme engineering via immobilization techniques is perfectly compatible with other chemical or biological approaches to improve enzyme functions and the final success depend on the availability of a wide battery of immobilization protocols.
In the last decade, new trends for enzyme attachment to solid carriers have emerged in an attempt to rationalize the classical methods for enzyme immobilization. In silico analysis is becoming a powerful tool to predict the orientation of enzymes covalently attached to the carrier or the protein regions involved in adsorption to the support. Significantly, an array of algorithms has been established for the rational design of immobilized derivatives, which comprises both the protein size and the textural properties of the support. Ordered mesoporous materials open a challenging pathway to tailor immobilized enzymes with high volumetric activity and minimum lixiviation. In addition, fluorescence confocal microscopy is being successfully employed to understand the diffusional restrictions and the distribution of biomolecules within the support.
The enzyme β-galactosidase can be obtained from a wide variety of sources such as microorganisms, plants, and animals. The use of β-galactosidase for the hydrolysis of lactose in milk and whey is one of the promising enzymatic applications in food and dairy processing industries. The enzyme can be used in either soluble or immobilized forms but the soluble enzyme can be used only for batch processes and the immobilized form has the advantage of being used in batch wise as well as in continuous operation. Immobilization has been found to be convenient method to make enzyme thermostable and to prevent the loss of enzyme activity. This review has been focused on the different types of techniques used for the immobilization of β-galactosidase and its potential applications in food industry.
Use of amphiphilic triblock copolymers to direct the organization of polymerizing silica species has resulted in the preparation
of well-ordered hexagonal mesoporous silica structures (SBA-15) with uniform pore sizes up to approximately 300 angstroms.
The SBA-15 materials are synthesized in acidic media to produce highly ordered, two-dimensional hexagonal (space group p6mm) silica-block copolymer mesophases. Calcination at 500°C gives porous structures with unusually large interlattice d spacings of 74.5 to 320 angstroms between the (100) planes, pore sizes from 46 to 300 angstroms, pore volume fractions up
to 0.85, and silica wall thicknesses of 31 to 64 angstroms. SBA-15 can be readily prepared over a wide range of uniform pore
sizes and pore wall thicknesses at low temperature (35° to 80°C), using a variety of poly(alkylene oxide) triblock copolymers
and by the addition of cosolvent organic molecules. The block copolymer species can be recovered for reuse by solvent extraction
with ethanol or removed by heating at 140°C for 3 hours, in both cases, yielding a product that is thermally stable in boiling
water.
The production of galacto-oligosaccharides (GOS) from lactose by Aspergillus oryzae beta-galactosidase immobilized on cotton cloth was studied. A novel method of enzyme immobilization involving PEI-enzyme aggregate formation and growth of aggregates on individual fibrils of cotton cloth leading to multilayer immobilization of the enzyme was developed. A large amount of enzyme was immobilized (250 mg/g support) with about 90-95% efficiency. A maximum GOS production of 25-26% (w/w) was achieved at near 50% lactose conversion from 400 g/L of lactose at pH 4.5 and 40 degrees C. Tri- and tetrasaccharides were the major types of GOS formed, accounting for about 70% and 25% of the total GOS produced in the reactions, respectively. Temperature and pH affected not only the reaction rate but also GOS yield to some extend. A reaction pH of 6.0 increased GOS yield by as much as 10% compared with that of pH 4.5 while decreased the reaction rate of immobilized enzyme. The cotton cloth as the support matrix for enzyme immobilization did not affect the GOS formation characteristics of the enzyme under the same reaction conditions, suggesting diffusion limitation was negligible in the packed bed reactor and the enzyme carrier. Increase in the thermal stability of PEI-immobilized enzyme was also observed. The half-life for the immobilized enzyme on cotton cloth was close to 1 year at 40 degrees C and 21 days at 50 degrees C. Stable, continuous operation in a plug-flow reactor was demonstrated for about 3 days without any apparent problem. A maximum GOS production of 26% (w/w) of total sugars was attained at 50% lactose conversion with a feed containing 400 g/L of lactose at pH 4.5 and 40 degrees C. The corresponding reactor productivity was 6 kg/L/h, which is several-hundred-fold higher than those previously reported.
Highly ordered SBA-15 silicas with large cylindrical mesopores (approximately 15 nm) are successfully obtained with the help of NH4F by controlling the initial reaction temperatures in the presence of excess amounts of alkanes.
Ten strains of lactobacilli were assessed for their capacity to degrade inulin-type fructans, which are well-known prebiotics.
Both oligofructose and inulin were tested. The dairy isolate Lactobacillus acidophilus IBB 801 degraded only oligofructose. The human isolate Lactobacillus paracasei subsp. paracasei 8700:2 degraded oligofructose and long-chain inulin and grew rapidly on both energy sources. In both cases, fractions of
different degrees of polymerization were fermented. Moreover, large and short fractions of oligofructose were degraded simultaneously.
When L. paracasei subsp. paracasei 8700:2 grew on oligofructose-enriched inulin, oligofructose was preferentially metabolized. In all cases, lactic acid was
the main metabolic end product. Significant amounts of acetic acid, formic acid, and ethanol were produced when long-chain
inulin or oligofructose-enriched inulin was used as the sole energy source.
By finely tuning the TEOS/P123 molar ratio of the octane/water/P123/TEOS quadruple emulsion system and by controlling the synthesis conditions, an ultrafine emulsion system was isolated, under the confinement of which, nanoscale silica particles with ordered large mesopores (approximately 13 nm) have been successfully constructed; the obtained mesoporous silica particles have an unusual ultrafast enzyme adsorption speed and the amount of enzyme that can be immobilized is larger than that of conventional mesoporous silica, which has potential applications in the fast separation of biomolecules.
A rational optimization for the synthesis of galacto-oligosaccharides (GOS) from lactose catalysed by β-galactosidase from Aspergillus aculeatus, included in the commercial product Pectinex Ultra SP-L, has been performed by using experimental design and surface response methodology. This accurate tool optimized empirical production of the most desired high galacto-oligosaccharides (tri-GOS and tetra-GOS) up to 16.4% under the following reaction conditions: 59C, 4 U/mL free enzyme concentration, pH 6.5, 250 g/L initial lactose concentration and 20h of reaction. The statistical analysis revealed temperature and initial lactose concentration as critical parameters. The successful immobilization of the enzyme on a glyoxyl-functionalized porous silica support slightly increased the yield towards high-GOS (17.6%), especially tri-GOS yield (15.3%), under the optimized reaction conditions as compared to the free enzyme. Furthermore, the promotion of the transgalactosylation reaction towards tri-GOS production increased 1.5-fold the productivity of high-GOS as compared to the free enzyme.
Valorization of lactose is achieved by enzymatic conversion to lactose derivatives with nutraceutical properties. This review summarizes recent developments on composition and functional properties of lactose-derived oligosaccharides. Lactose conversion by β-galactosidase yields galacto-oligosaccharides (GOS); hetero-oligosaccharides with structural and/or functional similarity to human milk oligosaccharides are synthesized by transglycosylation with β-galactosidase, sialidase, glucansucrase, fructansucrase or α-fucosidase using lactose as donor or acceptor sugar. Application development for GOS was based on their prebiotic properties; intestinal fermentation of GOS to short-chain fatty acids confers health benefits. Several novel oligosaccharides exhibit additional potent biological activities that are based on direct interaction of oligosaccharides with glycan-binding domains of bacterial adhesins and toxins, and are highly specific to the oligosaccharide structure. Particularly the use of lactose-derived oligosaccharides to prevent binding of bacterial toxins or adhesins is supported by in vivo studies.
In this work, glutathione was grafted on mesoporous silica materials by silanization with (3-mercaptopropyl)trimethoxysilane, subsequent reaction with 2,2’-dipyridyldisulphide and a final treatment with glutathione. The obtained glutathione-functionalized materials have been characterized by powder X-ray diffraction, FT-IR, scanning electron microscopy, transmission electron microscopy, nitrogen gas sorption and X-ray fluorescence. In addition, the hybrid materials were used in the preparation of modified carbon paste electrodes MCPE by mixing quantities of glutathione-containing materials with paraffin oil and graphite for Cd(II) detection in aqueous media by square wave adsorptive stripping voltammetry. Two materials were used for the preparation of MCPE, (A) the first material was obtained by the grafting of glutathione on the pyridyldisulphide pending ligand of the mesoporous material, (B) the second one, was obtained by grafting a cadmium-glutathione complex on the pyridyldisulphide pending ligand of the mesoporous material. For the latter, elimination of cadmium ions was carried out by treatment with HCl 2M for 2 hours and wash with MilliQ water before preparing the MCPE. To achieve the most accurate and sensitive Cd(II) electrochemical measurements, optimization of the operating parameters in pre concentration and detection steps was performed. The responses are pH dependent, being the optimal conditions 120 s of electrolysis time in buffer solution (pH 6.4). The voltammetric responses increase linearly with the pre concentration time and with metal ion concentrations ranging from 1 to 100 ppb. The metal detection limits were 2 and 4 ppb for materials (A) and (B), respectively, after 5 min pre concentration time.
Tyrosinase and Tyrosinase Inhibitors
From a commercial enzyme extract juice with purpose to stabilize it in the industry, has been able to extract an extract rich in beta-galactosidase activity. It has been designed to extract and purification protocol based on polyethyleneimine SUPPORTS agarose activated with next purification yields 80%.
SBA-15 silicas with cylindrical mesopores of diameter from 10 to 30 nm were synthesized within hours (2.5-7.5 hours) instead of one or more days required in hitherto reported syntheses of large-pore SBA-15 with (100) interplanar spacing above 13 nm. The synthesis was carried out using Pluronic P123 surfactant, 1,3,5-triisopropylbenzene swelling agent and silica hydrolysis/condensation catalyst, NH4F, under low-temperature initial synthesis conditions (12-17 °C) similar to those used in our earlier report on the synthesis of SBA-15 with a wide range of pore diameters (10-26 nm). Herein, it is shown that the low temperature step at which the ordered material forms can be shortened to one hour and the mesoporous material can be recovered at this stage. However, the attainment of a larger pore size and/or better structural ordering requires a hydrothermal treatment, which can be as short as 1-6 hours, if appropriately high temperature (150-170 °C) is selected. Additionally, improved synthesis conditions are reported for ultra-large-pore SBA-15 with (100) interplanar spacing of 25-30 nm. Standard SBA-15 synthesis without a swelling agent was also streamlined. Moreover, the synthesis of large-pore FDU-12 silica with face-centered cubic structure of spherical mesopores using Pluronic F127 surfactant and 1,3,5-trimethylbenzene swelling agent can be shortened to 9 hours. The streamlined synthesis procedures can be followed by surfactant removal via calcination with high ramping rate and short dwell time. The synthesis approach based on a short self-assembly step and short high-temperature hydrothermal treatment provides a convenient way to produce large-pore ordered mesoporous silicas suitable for numerous applications.
N-propylaniline functionalized 2D-hexagonal mesoporous SBA-15 material MFMS (monolayer N-propylaniline functionalized mesoporous SBA-15 type material) has been synthesized, where N-propylaniline group covalently grafted at the mesopore surface acts as a chromophoric fraction of the material for the sensing of metal ions from solutions. Powder XRD, HR TEM, FT IR, TGA, 13C/29Si solid state MAS-NMR, N2 sorption tools are employed to characterize the mesophase, framework-bonding and the surface properties of the material. Photoluminescence spectroscopy has been used to investigate the sensing behavior of this material towards various metal ions. This functionalized mesoporous material showed very high selectivity for sensing Hg2+ ions from its aqueous solution. With increasing concentration of Hg2+ ions from nanomolar to micromolar strength, strong fluorescence quenching is observed (upto 95.04%), while other main group and transition metal ions (Na+, K+, Ca2+, Mg2+, Cr3+, Mn2+, Fe3+, Co2+, Ni2+, Cu2+, Zn2+, Cd2+) caused insignificant change in the fluorescent intensity of the material, suggesting future potential of MFMS as a highly selective Hg(II)-sensor.
To test the digestibility of galactooligosaccharides (GOS) prepared from lactose through the transgalactosylation reaction of Sporobolomyces singularis cells and β-galactosidase from Kluyveromyces lactis, hydrogen gas in the exhalation of sixteen healthy subjects given GOS was analyzed. Hydrogen excretion following ingestion of GOS was substantially greater than that following ingestion of placebo. This fact shows that GOS was a mixture of non-digestible saccharides. Furthermore, to clarify non-digestible ingredients in GOS, we separated transgalactosylated di-, tri- and tetra-saccharides from GOS by Bio-Gel P-2 column, respectively. Then, resistance of these saccharides to the digestive enzymes was studied in vitro. The samples which contain di-, tri-, and tetra-saccharides were not digested in vitro by α-amylase of human saliva, the artificial gastric juice, and α-amylase of hog pancreas, but di-saccarides were partially digested by the enzymes of rat intestinal acetone powder. These results show that hydrogen excretion following ingestion of GOS was associated with bacterial degradation of tri-, and tetra-saccharides and some of di-saccarides in GOS after passing undigested through the small intestine into the large bowel.
Within the context of biomedicine and pharmaceutical sciences, the issue of (therapeutic) protein stabilization assumes particular relevance. Stabilization of protein and protein-like molecules translates into preservation of both structure and functionality during storage and/or targeting, and such stabilization is mostly attained through establishment of a thermodynamic equilibrium with the (micro)environment. The basic thermodynamic principles that govern protein structural transitions and the interactions of the protein molecule with its (micro)environment are, therefore, tackled in a systematic fashion. Highlights are given to the major classes of (bio)therapeutic molecules, viz. enzymes, recombinant proteins, (macro)peptides, (monoclonal) antibodies and bacteriophages. Modification of the microenvironment of the biomolecule via multipoint covalent attachment onto a solid surface followed by hydrophylic polymer co-immobilization, or physical containment within nanocarriers, are some of the (latest) strategies discussed aiming at full structural and functional stabilization of said biomolecules.
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.
A highly active and stable derivate of immobilized Bacillus circulans β-galactosidase was prepared for the synthesis of galacto-oligosaccharides (GOS) under repeated-batch operation. B. circulans β-galactosidase was immobilized on monofunctional glyoxyl agarose and three heterofunctional supports: amino-, carboxy-, and chelate-glyoxyl agarose. Glyoxyl agarose was the support with highest immobilization yield and stability being selected for the optimization of immobilization conditions and application in GOS synthesis. A central composite rotatable design was conducted to optimize contacted protein and immobilization time, using maximum catalytic potential as the objective function. Optimal conditions of immobilization were 28.9 mg/g and 36.4 h of contact, resulting in a biocatalyst with 595 IU/g and a half-life 89-fold higher than soluble enzyme. Immobilization process did not alter the synthetic capacity of β-galactosidase, obtaining the same GOS yield and product profile than the free enzyme. GOS yield and productivity remained unchanged along 10 repeated batches, with values of 39% (w/w) and 5.7 g GOS/g of biocatalyst·batch. Total product obtained after 10 batches of reaction was 56.5 g GOS/g of biocatalyst (1956 g GOS/g protein). Cumulative productivity in terms of mass of contacted protein was higher for the immobilized enzyme than for its soluble counterpart from the second batch of synthesis onwards.
A modification of the classical calcium alginate enzyme entrapment technique is described aiming to overcome some of the limitations of the former gel-based biocatalysts. Dried alginate entrapped enzymes (DALGEEs) were obtained dehydrating calcium alginate gel beads containing entrapped enzymes. A fructosyltransferase from Aspergillus aculeatus, present in Pectinex Ultra SP-L, was entrapped using this technique. The resulting DALGEEs were successfully tested both operating batchwise and in a continuous fixed-bed reactor for fructooligosaccharides (FOS) synthesis from sucrose. Interestingly, DALGEEs did not re-swell upon incubation in concentrated (600 g/L) sucrose solutions, probably due to the lowered water activity (aw) of such media. Confocal laser scanning microscopy of DALGEEs revealed that the enzyme molecules accumulated preferably in the shell of the particles. DALGEEs showed an approximately 30-fold higher volumetric activity (300 U/mL) compared with the calcium alginate gel beads. Moreover, a significant enhancement (40-fold) of the space-time-yield of fixed-bed bioreactors was observed when using DALGEEs as biocatalyst compared with gel beads (4030 g/day L of FOS vs. 103 g/day L). The operational stability of fixed-bed reactors packed with DALGEEs was extraordinary, providing a nearly constant FOS composition of the outlet during at least 700 h. It was also noticeable their resistance against microbial attack, even after long periods of storage at room temperature. The DALGEEs immobilisation strategy may also be useful for other biotransformations, in particular when they take place in low aw media.
Poly(styrenesulfonic acid)-functionalized materials based on poly(styrenesulfonic acid sodium salt) incorporated via aqueous atom transfer radical polymerization (ATRP) initiated from the surface of large-pore mesoporous SBA-15 silica support have been synthesized. The inorganic–organic nature of these hybrid materials makes them particularly desirable for acid-catalyzed reactions that require extended and hydrophobic surface areas with a narrow pore diameter distribution in the mesoporous range. Acidic hybrid materials were prepared by grafting the ATRP-initiator (3-(chlorodimethylsilyl)propyl bromoisobutyrate) on the silica surface, subsequent polymerization of the styrenesulfonic acid sodium salt monomer, and final sodium ion exchange by acid activation. Conventional and ultra-large-poreSBA-15 silica supports with nominal (BJH) pore diameter ranging from 8 to 32 nm were used for the incorporation of different polymer loadings at different polymerization times. The silylation of ATRP-initiator-functionalized SBA-15 supports has allowed a better control of the ATRP within the mesoporous structure. The use of ultra-large-poreSBA-15 supports provides a remarkable increase of the porosity which allowed us to properly allocate the polymer. The hybrid poly(styrenesulfonic acid)-modified materials showed good catalytic activities in the esterification of oleic acid with n-butanol, particularly in terms of intrinsic activity per acid site.
It is proposed herein that in order to obtain ultralarge-pore ordered mesoporous silicas using surfactant-templated synthesis with micelle expanders, one should select a micelle swelling agent with a moderate swelling ability to achieve an appreciable pore diameter enlargement while avoiding the formation of heterogeneous and/or poorly defined nanostructure. It is suggested to identify viable swelling agents based on the extent of solubilization of swelling agents in micellar solutions. On the basis of this reasoning, cyclohexane, 1,3,5-triethylbenzene and 1,3,5-triisopropylbenzene (TIPB) were selected for the synthesis of large-pore SBA-15 silicas with two-dimensional (2-D) hexagonal structures of cylindrical mesopores. SBA-15 with pore diameter tunable from 10 to 26 nm was obtained at initial synthesis temperature 12.25−20 °C using Pluronic P123 triblock copolymer as a micellar template and triisopropylbenzene as a micelle expander. Structures of the materials were characterized using small-angle X-ray scattering, TEM, and gas adsorption. The lowering of the initial synthesis temperature with adjustment of the amount of TIPB afforded pore diameters up to 26 nm (BJH pore diameters up to 34 nm) and (100) interplanar spacings up to 26 nm for 2-D hexagonal structure. As the initial synthesis temperature was lowered further, the pore diameter increased further (to 50 nm) with appreciable retention of cylindrical pore shape, but the pore structure became heterogeneous. The present approach makes silicas with 2D hexagonally ordered cylindrical pores of diameter up to 26 nm readily available and opens new opportunities in the synthesis of materials with other pore geometries and framework types.
Large-pore FDU-12 (LP-FDU-12) silicas with face-centered cubic structures of spherical mesopores of diameter 16–26nm were synthesized using Pluronic F127 triblock copolymer template. The materials were characterized using small-angle X-ray scattering and nitrogen adsorption. The initial temperature range suitable for LP-FDU-12 synthesis was shown to be much wider than that reported earlier (7–15°C vs. 14–15°C). However, the degree of structural ordering was lower and the pore size distribution was broader in case of temperatures of 7–9°C. The removal of the template by calcination resulted in a significant shrinkage (unit-cell parameter decrease by 10–16%) and afforded samples with low adsorption capacity, unless the synthesis involved a hydrothermal treatment in an HCl solution, which reduced the shrinkage and significantly increased the pore volume. The selection of time and temperature of the acid treatment allowed us to adjust the pore diameter of LP-FDU-12. However, the broadening of pore size distribution was observed for excessively long treatments at 130°C. The treatment at 130°C allowed us to tailor the pore entrance size in LP-FDU-12.
Galacto-oligosaccharides (GOS) have now been definitely established as prebiotic ingredients after in vitro and animal and human in vivo studies. Currently, GOS are produced by glycoside hydrolases (GH) using lactose as substrate. Converting lactose into GOS by GH results in mixtures containing GOS of different degrees of polymerization (DP), unreacted lactose, and monomeric sugars (glucose and galactose). Recent and future developments in the production of GOS aim at delivering purer and more efficient mixtures. To produce high-GOS-content mixtures, GH should not only have good ability to catalyze the transgalactosylation reaction relative to hydrolysis, but also have low affinity for the GOS formed relative to the affinity for lactose. In this article, several microbial GH, proposed for the synthesis of GOS, are hierarchized according to the referred performance indicators. In addition, strategies for process improvement are discussed. Besides the differences in purity of GOS mixtures, differences in the position of the glycosidic linkages occur, because different enzymes have different regiochemical selectivity. Depending on oligosaccharide composition, GOS products will vary in terms of prebiotic activity, as well as other physiological effects. This review focuses on GOS production from synthesis to purification processes. Physicochemical characteristics, physiological effects, and applications of these prebiotic ingredients are summarized. Regulatory aspects of GOS-containing food products are also highlighted with emphasis on the current process of health claims evaluation in Europe.
β-galactosidase from Kluyveromyces lactis was covalently immobilised on a Glyoxyl Sepharose (GS) support by multi-point attachment. The enzyme immobilisation process was very efficient; the supports immobilised almost all the protein responsible for the catalytic activity in a short period of time, retaining approximately 82% of the activity in the case of the optimal immobilised preparations. Stability of the GS derivatives varied as a function of enzyme-support incubation time. The optimal immobilised preparation was produced after 2 h of incubation with the support at alkaline pH. This derivative, obtained by multi-point covalent attachment, was 100-fold more stable at pH 7 and 50 °C than the cyanogen bromide Sepharose derivative obtained by a one-point covalent immobilisation method. Stabilisation was also observed under a wide range of experimental conditions. This method allowed the immobilisation of 9000 IU enzyme g−1 of support, resulting in highly active and stable derivatives suitable for industrial processes.
β-Galactosidase from Bacillus circulans was immobilized on hierarchical macro-mesoporous silica by multipoint covalent attachment by formation of Schiff bases between enzyme and support. The enzyme was effectively immobilized with high yields (around 60–80%) and expressed activity (around 50–80%) depending on the concentration of aldehyde groups in the carrier. Immobilization of β-galactosidase in chemically modified silica conferred excellent thermal stability to the biocatalyst and enzyme leaching was completely avoided. The effect of the concentration of functional groups in the silica surface was studied on the activity and thermal stability of the biocatalyst. The best hybrid catalyst was 370-fold more stable than the soluble enzyme at pH 6 and 55 °C.
The production of prebiotic galactooligosaccharides (GOS) by the β-galactosidase catalysed conversion of lactose has become commercially important. Yet it remains a challenge to sufficiently understand the structure and activity of β-galactosidase, to increase the efficiency of transgalactosylation and GOS production and to improve the quality of GOS products in a rational way. This review covers the broad but related aspects of GOS synthesis including: the structure and reaction mechanism of β-galactosidase, factors effecting yield and productivity of GOS synthesis systems, the structure of GOS products, models for the kinetics of GOS synthesis and reactor configurations for GOS synthesis. It aims to couple recent discoveries with established knowledge to enhance understanding of the complex biochemistry of GOS synthesis.
Glyoxyl agarose is constituted by quite thick agarose fibres containing a large number of very stable aldehyde groups attached to the support by very short spacer arms. Under alkaline conditions, these activated supports immobilize proteins, via, at least, a two-point reaction involving the region/s of the protein surface with the higher densities of amino groups. These bonds are weak Schiff's bases and the reversibility of the bonds has been used to convert this matrix into a chromatographic one. A more intense multipoint attachment between the immobilized protein and the activated support can be further promoted, with minimal loss of catalytic activity, by a long-term incubation of the protein–support conjugate under suitable conditions. The end-point of the preparation of agarose–protein conjugates is a very mild borohydride reduction. After that reduction, the enzyme remains attached to the support by means of very stable secondary amino bonds (with very similar physical properties to those of the former primary amino ones) and the remaining aldehyde groups on the support are converted into fully inert hydroxyl groups. Very active and highly stabilized derivatives of many enzymes and proteins have been prepared using these supports. The main features of these protein immobilization protocols are discussed here.
Galacto-oligosaccharides (GOS) are non-digestible carbohydrates, which are resistant to gastrointestinal digestive enzymes, but are fermented by specific colonic bacteria. After the discovery of bifidogenic activity of GOS and the development of an industrial production process by transgalactosidase activity of β-galactosidases, GOS have been extensively studied for their physiological roles on the intestinal flora and functions. GOS are selectively utilized by bifidobacteria in vitro, and show a bifidogenic effect at a daily intake of more than 2.5 g in human studies, whereas at least 5 g are needed to have effects on stool frequency. It is suggested that the products of fermentation of GOS in the colon, mainly short chain fatty acids, have a role in the improvement of the colonic environment, energy supply to the colonic epithelium, and calcium and magnesium absorption. These physiological effects in conjunction with the physicochemical characteristics of GOS enable GOS to be promising prebiotic food ingredients applicable to a variety of designed foods.
Aldehyde groups, moderately separated from support surfaces, are proposed as suitable active groups for developing strategies to insolubilize-stabilize enzymes by multipoint covalent attachment to activated preexistent supports. A method for preparation of glyoxyl-Sepharose CL gels, Ag-O-CH2-CHO, with very different surface densities of active groups, up to 17 aldehyde residues per 1000 Å2 of gel surface, is presented. These activated gels are very stable, even in moderately alkaline media, e.g., half-life time of these aldehyde groups at pH 10, 25°C, is 12 days. Experiments of insolubilization of the enzyme Penicillin G acylase on these supports indicate that one point amine-aldehyde attachments are fast but quite reversible. However, the binding of the enzyme on the support can be dramatically stabilized by two-point enzyme-support attachment. These qualities, as well as the absence of steric hindrance for the amine-aldehyde chemical reaction, make this activation method very suitable for designing intense, but not distorting, enzyme-support multiinteraction processes.
The unique properties of mesoporous silica (MPS) materials were utilized to immobilze enzymes: huge surface area, modifiable surface, and restricted pore nanospaces. The enzyme confinements in the nanochannels of MPS materials generate synergistic effects that enhance enzyme stability, improve product selectivity, and facilitate separation and reuse of enzymes. The physical chemistry of enzyme confinement, methods of immobilization, catalytic activity and advantages of protein confinements are discussed. In the end, we show that immobilized enzymes in the nanospaces of MPS can be applied as viable biocatalysts for chemical and pharmaceutical industries.
Enzyme interactions with material surfaces are of interest for industrial food and pharmaceutical transformations, biosensors, artificial cells, cell free reactions, drug and nutrition delivery technologies, and imaging. When in contact with a material surface, an enzyme may lose or appear to lose activity due to the nature of the enzyme, the nature of the material, and/or the nature of the interface between the enzyme, material, and substrate environment. The purpose of this review is to survey recent advances that have been made towards the preservation, optimization, and enhancement of enzyme activity on material surfaces within the context of well-known concepts that describe the loss of activity after immobilization. This review breaks down the immobilized enzyme system to look at the individual components of the system-namely the enzyme, the material, and the interface. For each piece, possible causes for the loss of enzyme activity are described as well as strategies that have been applied to limit the affect. At the conclusion we identify areas of future research needed to overcome limitations in the current state-of-the art for immobilized enzyme systems.
MCM-41 siliceous molecular sieves were used to test the applicability of the Kelvin equation for nitrogen adsorption in cylindrical pores of the size from 2 to 6.5 nm. It was shown that the Kelvin equation for the hemispherical meniscus, corrected for the statistical film thickness, is in quite good agreement with an experimental relation between the pore size and the capillary condensation pressure. The agreement can be made quantitative in the pore size range from ca. 2 to 6.5 nm, if a simple correction to the Kelvin equation is introduced. The required statistical film thickness curve (t-curve) was calculated using nitrogen adsorption data for large pore MCM-41 samples and the obtained results were extrapolated using an adsorption isotherm for a macroporous silica gel. Moreover, an accurate analytical representation of the t-curve was found. Since both the corrected Kelvin equation for cylindrical pores and the t-curve have simple analytical forms, they can conveniently be used in a variety of methods to evaluate porosity. It was shown that the BJH method with the corrected Kelvin equation accurately reproduces pore sizes of MCM-41 materials. A comparison was made between the specific surface areas for the MCM-41 samples calculated on the basis of the BET equation and those obtained using other independent methods. The results strongly suggest that when nitrogen adsorption data are used, the BET method overestimates the specific surface area of siliceous materials. The latter conclusion was supported by the examination of the obtained statistical film thickness curve.
β-Galactosidase or lactase is a very important enzyme in the food industry, being that from the yeast Kluyveromyces lactis the most widely used. Here we report its three-dimensional structure both in the free state and complexed with the product galactose. The monomer folds into five domains in a pattern conserved with the prokaryote enzymes of the GH2 family, although two long insertions in domains 2 and 3 are unique and related to oligomerization and specificity. The tetrameric enzyme is a dimer of dimers, with higher dissociation energy for the dimers than for its assembly. Two active centers are located at the interface within each dimer in a narrow channel. The insertion at domain 3 protrudes into this channel and makes putative links with the aglycone moiety of docked lactose. In spite of common structural features related to function, the determinants of the reaction mechanism proposed for Escherichia coli β-galactosidase are not found in the active site of the K. lactis enzyme. This is the first X-ray crystal structure for a β-galactosidase used in food processing.
Ultralarge-pore FDU-12 (ULP-FDU-12) silicas with face-centered cubic structures (Fm3m symmetry) of spherical mesopores were synthesized at low initial temperature (∼14 °C) using commercially available PEO-PPO-PEO triblock copolymer Pluronic F127 as a micellar template and xylene as a micelle expander. Xylene was selected on the basis of its predicted higher swelling ability for the Pluronic surfactant micelles in comparison to 1,3,5-trimethylbenzene that was used previously to obtain large-pore FDU-12. The optimization of the synthesis conditions afforded as-synthesized ULP-FDU-12 materials with unit-cell parameters up to 56 nm, which is comparable to the highest reported values for Fm3m structures templated by custom-made surfactants. Calcined silicas were obtained with unit-cell parameters up to 53 nm and pore diameters up to ∼36 nm (for N(2) adsorption at 77 K, the capillary condensation relative pressure was up to 0.938). The preferred silica source was tetraethylorthosilicate, but tetramethylorthosilicate was also found suitable. The pore diameter was dependent on the unit-cell size of the as-synthesized material, but was further tuned by adjusting the time and temperature of the treatment in the HCl solution. If the synthesis was performed at low temperature only, highly ordered closed-pore silicas were obtained at calcination temperatures as low as 450 °C. On the other hand, the hydrothermal treatments, including the acid treatment at 130 °C, afforded silicas with large pore entrance sizes. The present synthesis constitutes a major advancement in the synthesis of ordered silicas with very large open and closed spherical mesopores.
The thermodynamics of Zeolites, zeolite-related frame-works, and mesoporous silicas reflect a rich and closely balanced energy balanced energy landscape of competing energetic and entropic factors focuses on the energetics of two particular classes of generally metastable materials, the mesoporous and microporous framework structures represented by zeolites and their relatives. Thermal stability from a kinetic point of view is related to experimental conditions, where zeolites are synthesized under conditions in place of thermodynamic stability with respect to the starting material. The computer-assisted thermodynamic calculations offer additional insights into energetics of framework structures, facilitated by quantum mechanical calculations and molecular dynamic studies. Number of hydrated aluminosilicate zeolites can present a thermodynamic stability thereby indicating the exothermic enthalpy of hydration.
β-Galactosidase enzymes were extracted from pure cultures ofBifidobacterium angulatum, B. bifidum BB-12, B. adolescentis ANB-7, B. infantis DSM-20088, andB. pseudolongum DSM-20099 and used in glycosyl transfer reactions to synthesize oligosaccharides from lactose. At a lactose concentration
of 30% (wt/wt) oligosaccharide yields of 24.7 to 47.6% occurred within 7 h. Examination of the products by thin-layer chromatography
and methylation analysis revealed distinct product derived spectra from each enzyme. These were found to be different to that
of Oligomate 55, a commercial prebiotic galacto-oligosaccharide. Fermentation testing of the oligosaccharides showed an increase
in growth rate, compared to Oligomate 55, with products derived fromB. angulatum, B. bifidum, B. infantis, and B. pseudolongum. However B. adolescentis had a lower growth rates on its oligosaccharide compared with Oligomate 55. Mixed culture testing of the B. bifidum BS-4 oligosaccharide showed that the overall prebiotic effect was equivalent to that of Oligomate 55.
The design, implementation, and capabilities of an extensible visualization system, UCSF Chimera, are discussed. Chimera is segmented into a core that provides basic services and visualization, and extensions that provide most higher level functionality. This architecture ensures that the extension mechanism satisfies the demands of outside developers who wish to incorporate new features. Two unusual extensions are presented: Multiscale, which adds the ability to visualize large-scale molecular assemblies such as viral coats, and Collaboratory, which allows researchers to share a Chimera session interactively despite being at separate locales. Other extensions include Multalign Viewer, for showing multiple sequence alignments and associated structures; ViewDock, for screening docked ligand orientations; Movie, for replaying molecular dynamics trajectories; and Volume Viewer, for display and analysis of volumetric data. A discussion of the usage of Chimera in real-world situations is given, along with anticipated future directions. Chimera includes full user documentation, is free to academic and nonprofit users, and is available for Microsoft Windows, Linux, Apple Mac OS X, SGI IRIX, and HP Tru64 Unix from http://www.cgl.ucsf.edu/chimera/.
Mesoporous organic-inorganic hybrid materials, a new class of materials characterized by large specific surface areas and pore sizes between 2 and 15 nm, have been obtained through the coupling of inorganic and organic components by template synthesis. The incorporation of functionalities can be achieved in three ways: by subsequent attachment of organic components onto a pure silica matrix (grafting), by simultaneous reaction of condensable inorganic silica species and silylated organic compounds (co-condensation, one-pot synthesis), and by the use of bissilylated organic precursors that lead to periodic mesoporous organosilicas (PMOs). This Review gives an overview of the preparation, properties, and potential applications of these materials in the areas of catalysis, sorption, chromatography, and the construction of systems for controlled release of active compounds, as well as molecular switches, with the main focus being on PMOs.
A fructosyltransferase present in Pectinex Ultra SP-L, a commercial enzyme preparation from Aspergillus aculeatus, was purified to 107-fold and further characterised. The enzyme was a dimeric glycoprotein (20% (w/w) carbohydrate content) with a molecular mass of around 135 kDa for the dimer. Optimal activity/stability was found in the pH range 5.0-7.0 and at 60 degrees C. It was stable or slightly activated (upto 1.4-fold) in the presence of reducing agents, such as dithiothreitol and 2-mercaptoethanol, and detergents, such as sodium dodecylsulphate and Tween 80. The enzyme was able to transfer fructosyl groups from sucrose as donor producing the corresponding series of fructooligosaccharides: 1-kestose, nystose and fructosylnystose. Using sucrose as substrate, the k(cat) and K(m) values for transfructosylating activity were 1.62+/-0.09 x 10(4)s(-1) and 0.53+/-0.05 M, whereas for hydrolytic activity the corresponding values were 775+/-25s(-1) and 27+/-3 mM. At elevated sucrose concentrations, the fructosyltransferase from A. aculeatus showed a high transferase/hydrolase ratio that confers it a great potential for the industrial production of prebiotic fructooligosaccharides.