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Background: The general assumption that prebiotics reach the colon without any alterations has been challenged. Some in vitro and in vivo studies have demonstrated that 'non-digestible' oligosaccharides are digested to different degrees depending on their structural composition. In the present study, we compared different methods aiming to assess the digestibility of oligosaccharides synthesized by β-galactosidase (β-gal) of Lactobacillus delbruecki subsp. bulgaricus CRL450 (CRL450-β-gal) from lactose, lactulose and lactitol., Results: In the simulated gastrointestinal fluid method, no changes were observed. However, the oligosaccharides synthesized by CRL450-β-gal were partially hydrolyzed in vitro, depending on their structure and composition, with rat small intestinal extract (RSIE) and small intestinal brush-border membrane vesicles (BBMV) from pig. Digestion of some oligosaccharides increased when mixtures were fed to C57BL/6 mice used as in vivo model; however, lactulose-oligosaccharides were the most resistant to the physiological conditions of mice. In general β (1→6) linked products showed higher resistance compared to β (1→3) oligosaccharides., Conclusion: In vitro digestion methods, without disaccharidases, may underestimate the importance of carbohydrates hydrolysis in the small intestine. Although BVMM and RSIE digestion assays are appropriate in vitro methods for these studies, in vivo studies remain the most reliable for understanding what actually happens in the digestion of oligosaccharides. © 2024 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry., (© 2024 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.)

The production of the sugars fructose and lactulose from lactose using the enzymes β-galactosidase and glucose isomerase immobilized on bacterial cellulose (BC) membranes has been investigated. Lactose is hydrolyzed by β-galactosidase at 30 °C to glucose and galactose at a high conversion rate, while at the same temperature, glucose isomerase is not effective in converting the produced glucose to fructose. The rate of the isomerization reaction of glucose to fructose at 70 °C has been studied. Two types of enzyme immobilization were investigated: immobilization in one stage and immobilization in two stages. The results showed that BC membrane increased three-fold the yield and the reaction rate of fructose and lactulose production from lactose. The noteworthy enhancement of BC membranes' impact on the isomerization reaction by immobilized enzymes grants permission for a novel research avenue within the context of white biotechnology development. Additionally, this effect amplifies the role of BC in sustainability and the circular economy., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Kanellaki Maria reports financial support was provided by Epanek2014–2020 Operational Programme., (Copyright © 2024. Published by Elsevier Ltd.)

Cellobiose 2-epimerase (CE) is ideally suited to synthesize lactulose from lactose, but the poor thermostability and catalytic efficiency restrict enzymatic application. Herein, a non-characterized CE originating from Caldicellulosiruptor morganii (CmCE) was discovered in the NCBI database. Then, a smart mutation library was constructed based on FoldX ΔΔG calculation and modeling structure analysis, from which a positive mutant D226G located within the α 8 /α 9 loop exhibited longer half-lives at 65-75 °C as well as lower K m and higher k cat /K m values compared with CmCE. Molecular modeling demonstrated that the improvement of D226G was largely attributed to the rigidification of the flexible loop, the compactness of the catalysis pocket and the increment of substrate-binding capability. Finally, the yield of synthesizing lactulose catalyzed by D226G reached 45.5%, higher than the 35.9% achieved with CmCE. The disclosed effect of the flexible loop on enzymatic stability and catalysis provides insight to redesign efficient CEs to biosynthesize lactulose., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Lactulose is an isomer of lactose, formed under thermal processing of milk. Alkaline conditions favor the isomerization of lactose. As reducing sugar, lactose and lactulose could participate in the Maillard reaction and cause protein glycation in milk products. In this study, the influence of lactose and lactulose on the functional and structural properties of glycated casein was investigated. The results demonstrated that compared with lactose, lactulose led to severer changes in molecular weight, more disordered spatial structure and decrease of tryptophan fluorescence intensity of casein. Besides, the glycation degree and advanced glycation end products (AGEs) results suggested that lactulose exhibited stronger glycation ability than lactose due to the higher proportion of open chain in solution. Furthermore, higher glycation degree induced by lactulose resulted in lower solubility, surface hydrophobicity, digestibility and emulsifying capacity of casein-glycoconjugates compared with lactose. The results of this study are essential for tracking the effects of harmful Maillard reaction products on the quality of milk and dairy products., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Enzymatic isomerization of lactose into lactulose via cellobiose 2-epimerase (CE) could provide an eco-friendly route for the industrial production of lactulose, a valuable food prebiotic. However, poor substrate affinity for lactose and preference for epimerization over isomerization hinder this application. Previous studies on CE improvement have focused on random mutagenesis or active site rational design; little is known about the relationship between substrate binding and enzyme efficacy, which was hence the subject of this study. First, residues 372W and 308W were identified as key for disaccharide recognition in CEs based on crystal structure alignment of the N -acetyl-glucosamine 2-epimerase superfamily and site-directed mutation. This binding domain was then reshaped through site saturation mutagenesis, resulting in seven mutants with enhanced isomerization activity. The optimal mutant Cs CE/Q371E had significantly enhanced substrate affinity ( K m , 269.65 mM vs K m , 417.5 mM), reduced epimerization activity, and 3.3-fold increased isomerization activity over the original Cs CE. Molecular dynamics simulation further revealed that substituting Gln-371 with Glu strengthened the hydrogen-bonding network and altered the active site-substrate interactions, increasing the substrate stability and shifting the catalytic direction. This study uncovered new information about the substrate binding region and its mechanisms and impact on CE catalytic performance, paving the way for potential commercial applications.

Lactulose, a semisynthetic nondigestive disaccharide with versatile applications in the food and pharmaceutical industries, has received increasing interest due to its significant health-promoting effects. Currently, industrial lactulose production is exclusively carried out by chemical isomerization of lactose via the Lobry de Bruyn-Alberda van Ekenstein (LA) rearrangement, and much work has been directed toward improving the conversion efficiency in terms of lactulose yield and purity by using new chemo-catalysts and integrated catalytic-purification systems. Lactulose can also be produced by an enzymatic route offering a potentially greener alternative to chemo-catalysis with fewer side products. Compared to the controlled trans-galactosylation by β-galactosidase, directed isomerization of lactose with high isomerization efficiency catalyzed by the most efficient lactulose-producing enzyme, cellobiose 2-epimerase (CE), has gained much attention in recent decades. To further facilitate the industrial translation of CE-based lactulose biotransformation, numerous studies have been reported on improving biocatalytic performance through enzyme mediated molecular modification. This review summarizes recent developments in the chemical and enzymatic production of lactulose. Related catalytic mechanisms are also highlighted and described in detail. Emerging techniques that aimed at advancing lactulose production, such as the boronate affinity-based technique and molecular biological techniques, are reviewed. Finally, perspectives on challenges and opportunities in lactulose production and purification are also discussed., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have influenced the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

The enzyme β-galactosidase can synthesise novel prebiotics such as oligosaccharides derived from lactulose (OsLu) which can be added as a supplement in infant food formula. In this study, the intracellular β-galactosidase produced by the alkaliphilic bacterium Paracoccus marcusii was extracted and purified to homogeneity using hydrophobic and metal affinity chromatography. The purification resulted in 18 U/mg specific activity, with a yield of 8.86% and an 18-fold increase in purity. The purified enzyme was a monomer with an 86 kDa molecular weight as determined by SDS PAGE and Q-TOF-LC/MS. β-Galactosidase was highly active at 50 °C and pH 6-8. The enzyme displayed an alkali tolerant nature by maintaining more than 90% of its initial activity over a pH range of 5-9 after 3 h of incubation. Furthermore, the enzyme activity was enhanced by 37% in the presence of 5 M NaCl and 3 M KCl, indicating its halophilic nature. The effects of metal ions, solvents, and other chemicals on enzyme activity were also studied. The kinetic parameters K M and V max of β-galactosidase were 1 mM and 8.56 μmoles/ml/min and 72.72 mM and 11.81 μmoles/ml/min on using oNPG and lactose as substrates. P. marcusii β-galactosidase efficiently catalysed the transgalactosylation reaction and synthesised 57 g/L OsLu from 300 g/L lactulose at 40 °C. Thus, in this study we identified a new β-galactosidase from P. marcusii that can be used for the industrial production of prebiotic oligosaccharides., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The probiotics are extremely sensitive to various environmental factors, which imposes limitation on their health and functional effectiveness. Thus, development of delivery system for protection of viable cells while passing through different stages of the human digestion system is key factor in application of probiotic products. In our study, the effects of several polysaccharides such as alginate, κ-carrageenan, locust bean gum, gellan gum, xanthan gum and their combination with various prebiotic components (resistant starch, lactulose, lactosucrose) on encapsulation of probiotic Lactobacillus casei 01 strain were studied. Both regular and unregular beads with size distributions from 2 mm up to 5 mm were obtained. The encapsulation efficiencies varied from 64.4% up to 79%. Based on the texture's profiles, the capsules can be grouped into 5 clusters with squared Euclidean distance 3.5. Meanwhile, the starch-alginate and the lactosucrose LS55L - alginate beads were found to be the most stable and to have massive textural properties, whereas the gellan gum - xanthan gum and the chitosan coated alginate beads emerged as the softest. Encapsulation significantly improved the degree of gastric tolerance of probiotic cells even in the presence of pepsin. The INFOGEST in vitro digestion protocol was adapted to investigate the protection effects of different capsules. The highest survival (with loss rate of lower than 1 log CFU/g) was observed in the case of the cells encapsulated in starch-alginate beads. Moreover, the alginate microcapsules combined with lactosucrose LS55L also provided very promising shield for probiotics from the low pH of gastric conditions. Our findings suggest that incorporation of prebiotics into alginate-base encapsulation would be good idea in development of micro delivery systems that helps the survival of probiotics and their delivery to the target sites of action in human body., (Copyright © 2021 Elsevier B.V. All rights reserved.)

In this paper, 3 different biocatalysts of β-galactosidase from Kluyveromyces lactis have been prepared by immobilization in chitosan activated with glutaraldehyde (Chi_Glu_Gal), glyoxyl agarose (Aga_Gly_Gal) and agarose coated with polyethylenimine (Aga_PEI_Gal). These biocatalysts have been used to catalyze the synthesis of lactulose from lactose and fructose. Aga-PEI-Gal only produces lactulose at 50 °C, and not at 25 or 37 °C, Aga_Gly_Gal was unable to produce lactulose at any of the assayed temperatures while Chi_Glu_Gal produced lactulose at all assayed temperatures, although a lower yield was obtained at 25 or 37 °C. The pre-incubation of this biocatalyst at 50 °C permitted to obtain similar yields at 25 or 37 °C than at 50 °C. The use of milk whey instead of pure lactose and fructose produced an improvement in the yields using Aga_PEI_Gal and a decrease using Chi_Glu_Gal. The operational stability also depends on the reaction medium and of biocatalyst. This study reveals how enzyme immobilization may greatly alter the performance of β-galactosidase in a kinetically controlled manner, and how medium composition influences this performance due to the kinetic properties of β-galactosidase., Competing Interests: Declaration of competing interest The authors declare no conflict of interest. The authors report no commercial or proprietary interest in any product or concept discussed in this article., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Parkinson's disease (PD) is a neurodegenerative disease characterized by selective dopaminergic (DAergic) neuronal degeneration in the substantia nigra (SN) and proteinaceous α-synuclein-positive Lewy bodies and Lewy neuritis. As a chemical chaperone to promote protein stability and an autophagy inducer to clear aggregate-prone proteins, a disaccharide trehalose has been reported to alleviate neurodegeneration in PD cells and mouse models. Its trehalase-indigestible analogs, lactulose and melibiose, also demonstrated potentials to reduce abnormal protein aggregation in spinocerebellar ataxia cell models. In this study, we showed the potential of lactulose and melibiose to inhibit α-synuclein aggregation using biochemical thioflavin T fluorescence, cryogenic transmission electron microscopy (cryo-TEM) and prokaryotic split Venus complementation assays. Lactulose and melibiose further reduced α-synuclein aggregation and associated oxidative stress, as well as protected cells against α-synuclein-induced neurotoxicity by up-regulating autophagy and nuclear factor, erythroid 2 like 2 (NRF2) pathway in DAergic neurons derived from SH-SY5Y cells over-expressing α-synuclein. Our findings strongly indicate the potential of lactulose and melibiose for mitigating PD neurodegeneration, offering new drug candidates for PD treatment.

β-Galactosidase was produced by the yeast Kluyveromyces lactis NRRL Y1564 in cheese whey supplemented with yeast extract under the optimal temperature of 30 °C, delivering an enzymatic activity of 4418.37 U/g cell after 12 h of process. In order to develop more stable biocatalysts, the enzyme produced by fermentation was immobilized on 2.0% w/v chitosan activated with glutaraldehyde, epichlorohydrin or glycidol, producing a highly active and stable biocatalyst capable of hydrolyzing lactose and producing lactulose simultaneously. The biocatalyst obtained by immobilization in chitosan-glutaraldehyde showed high storage stabilities (100% of its activity when stored at 4 °C 105 days). Regarding the milk lactose hydrolysis by both the soluble and the immobilized enzyme, the conversions obtained were 38.0% and 42.8%, respectively. In this study, by using a biocatalyst deriving from enzyme immobilization to chitosan support, a lactulose production of 17.32 g/L was also possible.

The influence of the amorphization technique on the physicochemical properties of amorphous lactulose was investigated. Four different amorphization techniques were used: quenching of the melt, milling, spray-drying, and freeze-drying, and amorphous samples were analyzed by differential scanning calorimetry, NMR spectroscopy, and powder X-ray diffraction analysis. Special attention was paid to the tautomeric composition and to the glass transition of amorphized materials. It was found that the tautomeric composition of the starting physical state (crystal, liquid, or solution) is preserved during the amorphization process and has a strong repercussion on the glass transition of the material. The correlation between these two properties as well as the plasticizing effect of the different tautomers was clarified by molecular dynamics simulations.

Morphological and structural properties of amorphous disaccharide lactulose (C 12 H 22 O 11 ), obtained by four different amorphization methods (milling, quenching of the melt form, spray-drying, and freeze-drying), are investigated by scanning electron microscopy, polarized neutron scattering, and molecular dynamics simulations. While major differences on the morphology of the different amorphous samples are revealed by scanning electron microscopy images, only subtle structural differences have been found by polarized neutron scattering. Microstructure of the milled sample appears slightly different from the other amorphized materials with the presence of remaining crystalline germs which are not detected by X-ray diffraction. Quantitative phase analysis shows that these remaining crystallites are present in a ratio between 1 and 4%, and their size remains between 20 and 30 nm despite a long milling time of about 8 h. The impact of the change in tautomeric concentrations on the physical properties of lactulose in the amorphous state has been investigated from molecular dynamics simulations. It is suggested that chemical differences between lactulose tautomers could be at the origin of small structural differences detected by polarized neutron scattering.

Sialylated oligosaccharides are known to have beneficial effects, such as increasing the level of bifidobacteria, reducing the levels of blood endotoxin and blood ammonia, and enhancing the body's immune system. However, it is unknown whether sialylated lactuloses have modulatory effects on the intestinal microbiota. In this study, 60 healthy mice were randomly divided into six groups, namely, a normal control group, a lactulose group, a Kdn-α2,3-lactulose group, a Kdn-α2,6-lactulose group, a Neu5Ac-α2,3-lactulose group, and a Neu5Ac-α2,6-lactulose group. After 14 days of lactulose administration, the feces of three mice from each group were collected, and the intestinal microbiota were detected by Illumina MiSeq high-throughput sequencing targeting the V3-V4 region of the 16S rDNA gene. At the phylum level, the relative abundance of Firmicutes was increased in the sialylated lactulose groups, while the abundance of Bacteroidetes was decreased. At the family level, sialylated lactulose intervention decreased the relative abundance of Bacteroidales S24-7 group and Helicobacteraceae and enhanced the abundance of Lactobacillaceae, which reflects the modulatory effect of sialylated lactulose on intestinal microbiota. Diversity analysis indicated that the index of Chao was higher in the sialylated lactulose groups than in the normal control group, and the Shannon and Simpson diversity indices were higher in the Kdnα-2,6-lactulose group and the Neu5Ac-α2,3-lactulose group than in the normal control group. The results of the intestinal microbiota sample composition indicated that there were differences between the sialylated lactulose groups and the normal control group. Thus, sialylated lactulose could be used as a functional food component with potential therapeutic applications in manipulating intestinal microbiota to exert beneficial effects on the host's health.

An HPLC method suitable for rapid monitoring of lactulose production by isomerization from lactose was developed. The separation of lactose and lactulose under hydrophilic interaction liquid chromatography (HILIC) mode was achieved with resolution 1.5 within 5 min. Since isocratic elution was used, there is no extra time necessary for the column equilibration. Application of the method was illustrated on monitoring lactulose isomerization with catalysis of sodium hydroxide in the presence of sodium tetraborate at 70 °C (pH = 11). The conversion yield obtained for lactulose was 86%, and corresponding purity 76%. For the first time, a polyhydroxy stationary phase for separation of lactose and lactulose is reported., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The aim of this study was to investigate the immune modulatory influences of sialylated lactuloses in mice. The effects of the four sialylated lactuloses by gavage methods on the weight gain rate, organ, serum and spleen immunoglobulin of mice were investigated. Neu5Ac-α2,3-lactulose group and Kdn-α2,3-lactulose group had significantly higher weight gain rate than control group. The weight gain rate, thymus index and spleen index of Kdn-α2,3-lactulose group were significantly higher than control group and lactulose group. Liver and small intestine of Neu5Ac-α2,3-lactulose group, Neu5Ac-α2,6-lactulose group and Kdn-α2,6-lactulose group showed different degree of damage. IgG levels of serum and spleen in Neu5Ac-α2,6-lactulose group and Kdn-α2,6-lactulose group were significantly higher than control group and lactulose group. The contents of IgG in serum and spleen of Kdn-α2,3-lactulose group were significantly lower than that of control group, while the contents of IgA and IgM in serum were significantly higher than those of control group. The IgA level increased by 12.23% and 58.77% comparing with lactulose group and control group, respectively. The IgM level in serum of Kdn-α2,3-lactulose group mice increased by 43.88% and 8.05% comparing with control group and lactulose group, respectively. The IgA level and IgM level in spleen of Kdn-α2,3-lactulose group mice increased by 49.05% and 47.25% comparing with control group. In short, Kdn-α2,3-lactulose is relatively safe and superior to use as a food supplement or potential drug candidate. Our results also indicate that some other sialylated oligosaccharides are potentially harmful to organisms, they may cause some side effects., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Sialylated oligosaccharides contribute 12.6-21.9% of total free oligosaccharides in human milk ( hMOS). These acidic hMOS possess prebiotic properties and display antiadhesive effects against pathogenic bacteria. Only limited amounts of sialylated hMOS are currently available. The aim of our work is to enzymatically synthesize sialylated oligosaccharides mimicking hMOS functionality. In this study, we tested mixtures of glucosylated-lactose (GL34), galactosylated-lactulose (LGOS), and galacto-oligosaccharide (Vivinal GOS) molecules, as trans-sialylation acceptor substrates. The recombinant trans-sialidase enzyme from Trypanosoma cruzi (TcTS) was used for enzymatic decoration, transferring (α2→3)-linked sialic acid from donor substrates to nonreducing terminal β-galactopyranosyl units of these acceptor substrates. The GL34 F2 2-Glc-Lac compound with an accessible terminal galactosyl residue was sialylated efficiently (conversion degree of 47.6%). TcTS sialylated at least 5 LGOS structures and 11 Vivinal GOS DP3-4 compounds. The newly synthesized sialylated oligosaccharides are interesting as potential hMOS mimics for applications in biomedical and functional-food products.

In this article, we show that crystalline lactulose can be amorphized directly in the solid state by mechanical milling. Moreover, compared to similar materials, the amorphization kinetics of lactulose is found to be very rapid and the amorphous state thus obtained appears to be very stable against recrystallization on heating. These features make lactulose a model compound for this type of solid state transformation. The ease of crystalline lactulose to be amorphized on milling is explained by comparing elastic constants of lactulose with those of several other disaccharides. These constants have been determined by molecular dynamics simulations. The article also shows how isothermal dissolution calorimetry can be used effectively for the determination of amorphization kinetics during grinding when the usual characterization techniques (differential scanning calorimetry and powder X-ray diffraction) fail., (Copyright © 2019 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

Lactulose, a non-digestible oligosaccharide and functional food, promotes Bifidobacteria growth. Here we show that lactulose, beyond its prebiotic action, may have direct immunomodulatory effects as well. In synergy with CpG-ODN, a bacterial DNA mimetic, lactulose enhances basolateral concentrations of IFN-γ, IL-10, and galectin-9 in the co-culture model of epithelial and immune cells.

Lactulose-derived oligosaccharides (OsLu) are prebiotic galactooligosaccharides (GOS) beneficial for human health including immunomodulatory properties; however, the molecular mechanism is unclear. OsLu produced by enzymatic synthesis can be purified with Saccharomyces cerevisiae (OsLu-Sc). We show that this purification introduces yeast-derived proteins reactive to Dectin-2, an innate immune receptor for fungal polysaccharides. Using a cell-based bioassay, we tested the binding of OsLu and GOS samples to Dectin-2. While OsLu purified with active charcoal and commercial GOS failed to bind to Dectin-2, we found OsLu-Sc bound to this receptor. The carbohydrate-binding incompetent mutant of Dectin-2 failed to bind to OsLu-Sc. These data suggest that OsLu-Sc introduced carbohydrate ligands for Dectin-2. In accordance with this, proteomic analysis revealed OsLu-Sc contained S. cerevisiae-derived mannoproteins. Therefore, our data highlight the importance of the purification method for OsLu, which may positively affect the bioactivity of OsLu. Data are available via ProteomeXchange with identifier PXD010495., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Background: Lactulose, a valuable lactose-originated 'bifidus factor' product, is exclusively produced by chemical-based isomerization commercially. A complexing agent of sodium aluminate exhibiting high conversion efficiency and strong recyclable stability is more practical for industrial applications. In this study, efficient purification of high-purity lactulose through recycling of sodium aluminate and further desalination by nanofiltration (NF) was implemented on a pilot scale., Results: Over 99.5% of the catalyst was prior recycled in the form of Al(OH) 3 precipitate by pH-induced precipitation and centrifugation; residual aluminum was further absorbed by ion exchange resin to an acceptable level (≤10 mg kg -1 ). Subsequently, impurities (monosaccharides and NaCl) were ideally separated from lactulose syrup by NF based on their significant retention differences (lactulose 94.8-97.2% > lactose 86.2-93.5% > monosaccharides 36.3-48.7% > NaCl 9.5-31.1%). High-purity (>95%) lactulose was obtained with >90% yield in both constant and variable volume diafiltration (CVD and VVD) modes when the volume dilution ratio (V c /V f ) was 4.0 and 2.5 respectively. Both experimental and predicted results showed that the VVD mode was more water-saving in practice., Conclusion: This is the first trial purification of lactulose syrup from chemical isomerization of lactose catalyzed by sodium aluminate, and the applied methodology is a promising industrial-scale purification strategy. © 2018 Society of Chemical Industry., (© 2018 Society of Chemical Industry.)

Boronate affinity materials have been widely used for enrichment of cis-diol molecules. In this work, phenylboronic acid functionalized adsorbents were prepared via a simple and efficient procedure grafting phenylboronic acid groups onto amino macroporous resins. Elemental analysis has confirmed the successful functionalization of AR-1M and AR-2M with approximately 2.17% and 0.73% weight percentage of boron. Comparatively, AR-1M possessed higher lactulose adsorption capacity ( q e-Lu , 84.78 ± 0.95 mg/g dry resin) under neutral conditions (pH = 7), while the introduced glutaraldehyde spacer arms on AR-2M resulted in excellent adsorption selectivity (α ≈ 23), high adsorption efficiency (π ≈ 22%), and fast adsorption/desorption rate. The purity of lactulose (Pu D Lu ) through pH-driven adsorption (pH 7-8) and desorption (pH 1.5) can be effectively improved depending on the ratio of lactulose to lactose. When lactulose/lactose ≥ 1:1, Pu D Lu ≈ 95% was achieved. No significant drop in q e-Lu (>90%) was observed after ten-consecutive repeats. Results demonstrated that the newly developed method may achieve satisfactory performance in lactulose purification.

Oligosaccharides derived from lactulose (β-D-Galp-(1 → 4)-D-Fru) are drawing more and more attention nowadays because of their strong resistance to gut digestion, and the interest to discover novel prebiotics. Compared to galactooligosaccharides, currently known structures of lactulose oligosaccharides are very limited. In this study, the wild-type β-galactosidase BgaD-D of Bacillus circulans ATCC 31382, as well as the derived mutant R484H, were used to synthesize oligosaccharides from lactulose. In total, 9 oligosaccharide structures were identified by MALDI-TOF-MS and NMR spectroscopy analysis. Trisaccharide β-D-Galp-(1 → 4)-β-D-Galp-(1 → 4)-D-Fru was the major structure produced by the wild-type enzyme, while the R484H mutant showed a preference for synthesis of β-D-Galp-(1 → 3)-β-D-Galp-(1 → 4)-D-Fru. Our study greatly enriched the structural information about oligosaccharides derived from lactulose., (Copyright © 2018. Published by Elsevier Ltd.)

High-efficiency lactulose-producing enzyme of Caldicellulosiruptor saccharolyticus cellobiose 2-epimerase (WT- CsCE) was immobilized in the form of cross-linked enzyme aggregates (CLEAs). Conditions for enzyme aggregation and cross-linking were optimized, and a sugar-assisted strategy with less damage to enzyme secondary structures was developed to improve the activity yield of CLEAs up to approximately 65%. The resulting CLEAs with multiple-layer network structures exhibited an enlarged optimal temperature range (70-80 °C) and maintained higher activity at 50-90 °C. Besides, CLEAs retained more than 95% of their initial activity after 10 successive batches at 60 °C, demonstrating superior reusability. Moreover, CLEAs displayed an equivalent or higher catalytic ability to free WT- CsCE in lactulose biosynthesis, and the final sugar ratios were similar, lactulose 58.8-61.7%, epilactose 9.3-10.2%, and lactose 27.8-30%, with a constant isomerization selectivity of 0.84-0.87 regardless of enzymes used and temperature applied. The proposed strategy is the first trial for enzymatic synthesis of lactulose catalyzed by CLEAs of WT- CsCE.

Background: Lactulose has various uses in the food and pharmaceutical fields. Thermostable enzymes have many advantages for industrial exploitation, including high substrate solubilities as well as reduced risk of process contamination., Results: Enzymatic synthesis of lactulose employing a transgalactosylation reaction by a recombinant thermostable glycoside hydrolase (GH1) from the hyperthermophilic archaeon Caldivirga maquilingensis IC-167 was investigated. The optimal pH for lactulose production was found to be 4.5, while the optimal temperature was 85 °C, before it dropped moderately to 83% at 90 °C. However, the relative activity for lactulose synthesis dropped sharply to 35% at 95 °C. At optimal reaction conditions of 70% (w/w) initial sugar substrates with molar ratio of lactose to fructose of 1:4, 15 U mL -1 enzyme concentration and 85 °C, the time course reaction produced a maximum lactulose concentration of 108 g L -1 at 4 h, corresponding to a lactulose yield of 14% and 27 g L -1  h -1 productivity with 84% lactose conversion. The transgalactosylation reaction for lactulose synthesis was greatly influenced by the ratio of galactose donor to acceptor., Conclusion: This novel GH1 may be useful for process applications owing to its high activity in very concentrated substrate reaction media and promising thermostability. © 2017 Society of Chemical Industry., (© 2017 Society of Chemical Industry.)

Background: Current health concerns have driven consumers to request products with nutritional and physiological advantages, which can be achieved by using prebiotic ingredients. Lactulose is a prebiotic with excellent functional properties and can be easily incorporated into meat products through the addition of liquid whey. This study investigated the technological and sensorial quality of restructured cooked ham elaborated without liquid whey added (control) and with liquid whey containing different contents (0, 30, 60 and 100 g kg -1 ) of lactulose., Results: Liquid whey did not change any technological or sensorial characteristics of the product, but the general acceptability decreased due to addition of lactulose. Samples with higher lactulose concentrations had lower moisture content, pH and refreezing loss and increased carbohydrate content. Control and whey added samples had higher lightness and lower intense color than samples with lactulose. Liquid whey additions with higher lactulose content increased hardness and chewiness of the samples., Conclusion: Restructured cooked hams formulated with liquid whey and 30 g kg -1 of lactulose had minimal effects on the technological properties and sensory characteristics and, due to the possible benefits conferred by the prebiotic, is a potential alternative to provide meat products with prebiotic activity. © 2017 Society of Chemical Industry., (© 2017 Society of Chemical Industry.)

Background: Lactulose, a synthetic disaccharide, has received increasing interest due to its role as a prebiotic, specifically proliferating Bifidobacilli and Lactobacilli and enhancing absorption of calcium and magnesium. The use of cellobiose 2-epimerase (CE) is considered an interesting alternative for industrial production of lactulose. CE reversibly converts D-glucose residues into D-mannose residues at the reducing end of unmodified β-1,4-linked oligosaccharides, including β-1,4-mannobiose, cellobiose, and lactose. Recently, a few CE 3D structure were reported, revealing mechanistic details. Using this information, we redesigned the substrate binding site of CE to extend its activity from epimerization to isomerization., Results: Using superimposition with 3 known CE structure models, we identified 2 residues (Tyr114, Asn184) that appeared to play an important role in binding epilactose. We modified these residues, which interact with C2 of the mannose moiety, to prevent epimerization to epilactose. We found a Y114E mutation led to increased release of a by-product, lactulose, at 65 °C, while its activity was low at 37 °C. Notably, this phenomenon was observed only at high temperature and more reliably when the substrate was increased. Using Y114E, isomerization of lactose to lactulose was investigated under optimized conditions, resulting in 86.9 g/l of lactulose and 4.6 g/l of epilactose for 2 h when 200 g/l of lactose was used., Conclusion: These results showed that the Y114E mutation increased isomerization of lactose, while decreasing the epimerization of lactose. Thus, a subtle modification of the active site pocket could extend its native activity from epimerization to isomerization without significantly impairing substrate binding. While additional studies are required to scale this to an industrial process, we demonstrated the potential of engineering this enzyme based on structural analysis.

Background: One of the most promising uses of whey permeate (WP) is the synthesis of prebiotic oligosaccharides. Herein, commercial WP was submitted to chemical isomerization catalysed by sodium borate at an alkaline pH and subsequent purification using anion-exchange resins to remove boron. Subsequently, purified mixtures were used to synthesize prebiotic oligosaccharides using β-galactosidase from Bacillus circulans., Results: Isomerization of concentrated WP (200 g L -1 lactose) gave rise to levels of lactulose up to 155.5 g L -1 after 30 min of reaction (molar ratio of boron/lactose, 1/1; pH 12; 70 °C). Boron was removed from the isomerized WP (IWP) using the combination of a strong acid (IR-120, H + ) and a weak base (IRA-743) anion-exchange resins, reducing its level to <1 ppm, without loss of lactulose. During the transglycosylation reaction of purified IWP (lactose/lactulose ratio, 1/2.4) maximum content of prebiotic compounds was achieved, i.e. 690 g kg -1 WP after 3 h of reaction., Conclusion: This study shows that combined chemical-enzymatic reactions together with the purification of IWP results in an efficient synthesis of prebiotic oligosaccharides. © 2017 Society of Chemical Industry., (© 2017 Society of Chemical Industry.)

An efficient strategy for the synthesis of high-purity lactulose through chemical isomerization of lactose was developed using recyclable catalyst sodium aluminate. Maximum yield of 85.45±1.79% was obtained from 35% (w/v) of lactose using a 1:1M ratio of NaAlO 2 -lactose after reaction at 60°C and pH 12 for 50min, with a purity ratio (Pu) of approximately 95%. Al(OH) 3 precipitation from lactulose syrup through pH adjustment showing a superior decolorizing efficiency was implemented, which simplified the removal of catalyst and provided a convenient approach to achieve catalyst recovery. After dilution by 5 folds, nearly 98.5% of catalyst was removed through centrifugation and 85.50±1.81% of lactulose was recovered. Modified recycle use of catalyst without lactulose loss was carried out, after five consecutive recycles, total yield of lactulose (TY Lactulose ) and new yield of lactulose (NY Lactulose ) were of 80.44±1.05% and 73.87±0.50%, respectively, reflecting a high stability by using this recyclable methodology., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Background: Lactulose, a bioactive lactose derivative, has been widely used in food and pharmaceutical industries. Isomerisation of lactose to lactulose by cellobiose 2-epimerase (CE) has recently attracted increasing attention, since CE produces lactulose with high yield from lactose as a single substrate. In this study, a new lactulose-producing CE from Caldicellulosiruptor obsidiansis was extensively characterised., Results: The recombinant enzyme exhibited maximal activity at pH 7.5 and 70 °C. It displayed high thermostability with T m of 86.7 °C. The half-life was calculated to be 8.1, 2.8 and 0.6 h at 75, 80, and 85 °C, respectively. When lactose was used as substrate, epilactose was rapidly produced in a short period, and afterwards both epilactose and lactose were steadily isomerised to lactulose, with a final ratio of 35:11:54 for lactose:epilactose:lactulose. When the reverse reaction was investigated using lactulose as substrate, both lactose and epilactose appeared to be steadily produced from the start., Conclusion: The recombinant CE showed both epimerisation and isomerisation activities against lactose, making it an alternative promising biocatalyst candidate for lactulose production. © 2016 Society of Chemical Industry., (© 2016 Society of Chemical Industry.)

Lactulose (4-O-β-d-galactopyranosyl-d-fructofuranose) is a prebiotic sugar derived from the milk sugar lactose (4-O-β-d-galactopyranosyl-d-glucopyranose). In our study we observed for the first time that known cellobiose 2-epimerases (CEs; EC 5.1.3.11) from mesophilic microorganisms were generally able to catalyze the isomerization reaction of lactose into lactulose. Commonly, CEs catalyze the C2-epimerization of d-glucose and d-mannose moieties at the reducing end of β-1,4-glycosidic-linked oligosaccharides. Thus, epilactose (4-O-β-d-galactopyranosyl-d-mannopyranose) is formed with lactose as substrate. So far, only four CEs, exclusively from thermophilic microorganisms, have been reported to additionally catalyze the isomerization reaction of lactose into lactulose. The specific isomerization activity of the seven CEs in this study ranged between 8.7 ± 0.1 and 1300 ± 37 pkat/mg. The results indicate that very likely all CEs are able to catalyze both the epimerization as well as the isomerization reaction, whereby the latter is performed at a comparatively much lower reaction rate.

Industrial application of Caldicellulosiruptor saccharolyticus cellobiose 2-epimerase (CsCE) for lactulose synthesis is limited by low enzyme activity and formation of epilactose as by-product. After four sequential rounds of random mutagenesis and screening, an optimal mutant G4-C5 was obtained. Compared with wild type (WT) enzyme, mutant G4-C5 demonstrated 2.8- and 3.0-fold increases in specific activity and kcat/Km for lactulose production, respectively, without compromising thermostability. DNA sequencing of mutant G4-C5 revealed five amino acid substitutions, namely, R5M, I52V, A12S, K328I and F231L, which were located on the protein surface, except for the mutation I52V. The yield of lactulose catalyzed by mutant G4-C5 increased to approximately 76% with no obvious epilactose detected, indicating that mutant G4-C5 was more suitable for lactulose production than the WT enzyme., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Lactose was isomerised to lactulose by microwave heating and purified by a methanolic procedure to a product with approximately 72% lactulose content. Afterwards, lactose and the lactulose-rich product (PLu) were conjugated with either whey protein isolate (WPI) or its antioxidant hydrolysate (WPH) through microwaving. Lactose had a higher Maillard reactivity than PLu, and WPH was more reactive than WPI. The browning intensity of WPI-sugar systems was however higher than that of WPH-sugar pairs. Atomic force microscopy showed larger (up to ≈103 nm) particles for WPI-PLu conjugates compared to WPH-PLu counterparts (up to ≈39 nm). The Maillard conjugation progressively increased the radical-scavenging activity of WPI/WPH-sugar pairs with increasing conjugation time and improved the foaming properties of WPI and WPH. The WPI/WPH-sugar conjugates showed higher solubility and emulsification index than unreacted counterpart pairs. For native WPI, β-lactoglobulin was not degraded by in vitro gastric digestion, whereas for WPH-PLu conjugates degraded completely., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Lactulose is considered as a prebiotic because it promotes the intestinal proliferation of Lactobacillus acidophilus which is added to various milk products. Moreover, lactulose is used in pharmaceuticals as a gentle laxative and to treat hyperammonemia. This study was aimed at the total synthesis of two Lactulose-derived oligosaccharides: one is 3-O-β-d-galactopyranosyl-d-fructose, d-fructose and β-d-galactose bounded together with β-1,3-glycosidic bound, the other is 1-O-β-d-galactopyranosyl-d-fructose, d-fructose and β-d-galactose bounded together with β-1,1-glycosidic bound, which were accomplished in seven steps from d-fructose and β-d-galactose and every step of yield above 75%. This synthetic route provided a practical and effective synthetic strategy for galactooligosaccharides, starting from commercially available monosaccharides. Then we evaluated on their prebiotic properties in the search for potential agents of regulating and improving the intestinal flora of human. The result showed that the prebiotic properties of Lactulose-derived oligosaccharides was much better than Lactulose. Among them, 3-O-β-d-galactopyranosyl-d-fructose displayed the most potent activity of proliferation of L. acidophilus., (Copyright © 2016 Elsevier Masson SAS. All rights reserved.)

Ammonium carbonate ((NH4)2CO3) was used as an alkaline catalyst of lactulose production from cheese whey. Maximum yield of 29.6% was obtained at reaction time of 28.44 min, (NH4)2CO3 of 0.76% at 97°C. During reaction, (NH4)2CO3 was fully decomposed to NH3 and CO2, and these gases were recovered. To boost up NH3 recovery, various methods such as heating, aeration, and pH adjustment were applied. The optimal condition for the purpose of NH3 retrieval was temperature of up to 60°C alongside aeration. Easy separation and recovery make (NH4)2CO3 a catalyst alternative to common alkaline chemicals especially for the weak alkaline reaction., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Cheese-whey, a major co-product of the dairy industry, has recently been the subject of many technological applications. We studied the bioconversion of whey into valuable bio-products such as a potential lactulose prebiotic and compounds with antioxidant properties. This paper examines efficiency, safety, and economics of electro-activation as an eco-friendly technology for a maximum valorization of whey. Thus, a bottom-up approach was therefore addressed. The effect of 4 experimental parameters--low working temperatures (0, 10, and 25 °C), current intensities (400, 600, and 800 mA), volume conditions (100, 200, and 300 mL), and feed concentrations [7, 14, and 28% (wt/vol)]--on lactose-whey isomerization to lactulose under electro-activation process were studied. Structural characteristics of whey proteins and antioxidant functionality were also investigated. The results showed a compromise to be reached between both parameters. Therefore, the maximum yield of 35% of lactulose was achieved after 40 min of reaction at the working temperature of 10 °C under 400 mA electric current field and 100-mL volume conditions with using feed solution at 7% (wt/vol). The isomerization of lactose to lactulose is accomplished by subsequent degradation to galactose, but only at a very small amount. Additionally, whey electro-activation showed significantly elevated antioxidant capacity compared with the untreated samples. The enhancement of antioxidant functionality of whey electro-activation resulted from the synergistic effect of its partial hydrolysis and the formation of antioxidant components that were able to scavenge free radicals. In conclusion, the findings of this study reveal that the whey treated by the safety electro-activation technology has both lactulose-prebiotic and antioxidant properties and could have a substantial application in the manufacture of pharmaceutical and functional foods., (Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Milk ultrafiltration permeate was heated at 97 °C in the presence of eggshell for 60 min. This decreased the ash content of permeate and converted ≈ 17% of lactose to lactulose. The isomerized permeate was subsequently purified to a lactulose-rich product (LRP; ≈ 70% lactulose content to total sugar) through crystallizing lactose out by methanol. The LRP and lactose were then conjugated with either whey protein isolate (WPI) or its antioxidant hydrolysate (WPH) through Maillard reaction at 90 °C. The amount of the Maillard reaction advanced products was higher for WPI-lactose system than WPH-lactose counterpart; whilst, the DPPH scavenging activities of WPH-sugar conjugates were significantly higher than those of WPI-sugar counterparts. Based on free amino groups content measurement, it was found that lactose is more reactive than LRP for Maillard conjugation with both WPI and WPH. Fourier transform infrared spectroscopy confirmed the bonding of the anomeric region of saccharide configuration of lactulose with WPH., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Trehalose, a chemical chaperone and mTOR-independent autophagy enhancer, has shown promise in models of Huntington's disease, Parkinson's disease and tauopathies. In this study, two trehalase analogs, lactulose and melibiose, were examined for their potentials in spinocerebellar ataxia treatment. Using a SCA3 ATXN3/Q75-GFP cell model, we found that the ATXN3/Q75 aggregation was significantly prohibited by lactulose and melibiose because of their abilities to up-regulate autophagy. Meanwhile, lactulose and melibiose reduced reactive oxygen species production in ATXN3/Q75 cells. Both of them further inhibited the ATXN3/Q75 aggregation in neuronally differentiated SH-SY5Y cells. These findings suggest the therapeutic applications of novel trehalose analogs in polyglutamine aggregation-associated neurodegenerative diseases.

The scientific evidence on the bioactivity of oligosaccharides from lactulose has encouraged us to study their physicochemical modifications during the processing of milk and apple juice. The carbohydrate fraction with a degree of polymerization ⩾3 was stable in milk heated at temperatures up to 100°C for 30min and in apple juice heated up to 90°C for 15min. An assessment of the Maillard reaction in heated milk pointed out a higher formation of furosine in milk with oligosaccharides from lactulose as compared to its counterpart without this ingredient, due to a higher presence of galactose. The organoleptic properties of juice with oligosaccharides from lactulose were acceptable and similar to those of apple juice with commercial galactooligosaccharides. The results presented herein demonstrate that oligosaccharides from lactulose can be used as prebiotic ingredients in a wide range of functional foods, including those intended for diabetics and lactose intolerant individuals., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Lactulose synthesis was performed in a continuous stirred enzymatic membrane reactor. Each investigated operating condition (agitation, pH, feed molar ratio of lactose to fructose (mL/mF ratio), hydraulic residence time (HRT)) had an influence on reaction performances, in terms of lactulose concentration, productivity and selectivity. Lactulose concentration was maximum at an mL/mF ratio of 1/2. Higher than this ratio, synthesis of galactooligosaccharides was promoted rather than lactulose. At mL/mF ratios lower than 1/2, enzyme inhibition was pronounced to the detriment of lactulose production. At 7 or 9h HRT, higher lactulose concentrations were obtained than at shorter HRTs. Applying an mL/mF ratio of 1/2 and an HRT of 9h in a long-term operation, nearly constant lactulose concentration was reached after 23h and lasted up to 32h with a mean concentration of 14.51±0.07g/L and a reaction selectivity of 0.075-0.080mollactulose/molcons.lactose. After 7d, lactulose concentration reduced by 31%. A continuous synthesis of lactulose at lab-scale was shown to be amenable using a membrane reactor process. Moreover, for process evaluation, this study can bridge the gap between batch laboratory scale and continuous full-scale operation regarding lactulose synthesis., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The unique property of trehalose encourages its pharmaceutical application in aggregation-mediated neurodegenerative disorders, including Alzheimer's, Parkinson's, and many polyglutamine (polyQ)-mediated diseases. However, trehalose is digested into glucose by trehalase and which reduced its efficacy in the disease target tissues. Therefore, searching trehalase-indigestible analogs of trehalose is a potential strategy to enhance therapeutic effect. In this study, two trehalase-indigestible trehalose analogs, lactulose and melibiose, were selected through compound topology and functional group analyses. Hydrogen-bonding network analyses suggest that the elimination of the hydrogen bond between the linker ether and aspartate 321 (D321) of human trehalase is the key for lactulose and melibiose to avoid the hydrolyzation. Using polyQ-mediated spinocerebellar ataxia type 17 (SCA17) cell and slice cultures, we found the aggregation was significantly prohibited by trehalose, lactulose, and melibiose, which may through up-regulating of autophagy. These findings suggest the therapeutic applications of trehalase-indigestible trehalose analogs in aggregation-associated neurodegenerative diseases., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Sugar esters are considered as surfactants due to its amphiphilic balance that can lower the surface tension in oil/water mixtures. Enzymatic syntheses of these compounds are interesting both from economic and environmental considerations. A study was carried out to evaluate the effect of four solvents, temperature, substrate molar ratio, biocatalyst source, and immobilization methodology on the yield and specific productivity of lactulose palmitate monoester synthesis. Lipases from Pseudomonas stutzeri (PsL) and Alcaligenes sp. (AsL), immobilized in porous silica functionalized with octyl groups (adsorption immobilization, OS) and with glyoxyl-octyl groups (both adsorption and covalent immobilization, OGS), were used. The highest lactulose palmitate yields were obtained at 47 °C in acetone, for all biocatalysts, while the best lactulose:palmitic acid molar ratio differed according to the immobilization methodology, being 1:1 for AsL-OGS biocatalyst (20.7 ± 3%) and 1:3 for the others (30-50%).

An economical method of lactulose production from cheese whey was developed using sodium carbonate (Na2CO3). Three parameters such as temperature, reaction time, and Na2CO3 concentration were identified as experimental factors, and yield was selected as a response parameter. The experimental factors were optimised employing Response Surface Methodology (RSM). Maximum yield of 29.6% was obtained at reaction time of 20.41 min, Na2CO3 of 0.51% at 90 °C. To overcome this limited lactulose yield, due to the conversion of lactulose to galactose, fed batch system was applied using dried cheese whey as lactose source. By this system, limit was broken, and 15.8 g/L of lactulose is produced in hour., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

This study was aimed to statistically optimize CODES™ based Piroxicam (PXM) tablet for colon targeting. A 3(2) full factorial design was used for preparation of core tablet that was subsequently coated to get CODES™ based tablet. The experimental design of core tablets comprised of two independent variables: amount of lactulose and PEG 6000, each at three different levels and the dependent variable was %CDR at 12 h. The core tablets were evaluated for pharmacopoeial and non-pharmacopoeial test and coated with optimized levels of Eudragit E100 followed by HPMC K15 and finally with Eudragit S100. The in vitro drug release study of F1-F9 was carried out by change over media method (0.1 N HCl buffer, pH 1.2, phosphate buffer, pH 7.4 and phosphate buffer, pH 6.8 with enzyme β-galactosidase 120 IU) to select optimized formulation F9 that was subjected to in vivo roentgenography. Roentgenography study corroborated the in vitro performance, thus providing the proof of concept. The experimental design was validated by extra check point formulation and Diffuse Reflectance Spectroscopy revealed absence of any interaction between drug and formulation excipients. The shelf life of F9 was deduced as 12 months. Conclusively, colon targeted CODES™ technology based PXM tablets were successfully optimized and its potential of colon targeting was validated by roentgenography.

The two genes lacZ1 and lacZ2 from Klebsiella pneumoniae 285, encoding β-galactosidase isoenzymes II and III (KpBGase-II and -III), were each cloned downstream of a T7 promoter for expression in Escherichia coli BL21(DE3), and the resulting recombinant enzymes were characterized in detail. The optimum temperature and pH value of KpBGase-II were 40 °C and 7.5, and those of KpBGase-III were 50 °C and 8.0, respectively. KpBGase-III was more stable than KpBGase-II at higher temperature (>60°C). Both β-galactosidases were more active towards o-nitrophenyl-β- D-galactopyranoside as compared to lactose. The enzymatic synthesis of lactulose and 1-lactulose catalyzed by KpBGase-II and KpBGase-III was investigated. Using 400 g/L lactose and 200 g/L fructose as substrates, the resulting lactulose and 1-lactulose yields with KpBGase-II were 6.2 and 42.3 g/L, while those with KpBGase-III were 5.1 and 23.8 g/L, respectively. KpBGase-II has a potential for the production of 1-lactulose from lactose and fructose. Like other β-galactosidases, the two isozymes catalyze the transgalactosylation in the presence of fructose establishing the β-(1→1) linkage.

Intestinal microbiota modulation is becoming an interesting approach to manage inflammatory bowel disease and can be achieved by the administration of prebiotics. Previous studies showed the intestinal anti-inflammatory effects of the prebiotic lactulose. The aim of the present study was to test the preventative effects of oligosaccharides derived from lactulose with prebiotic properties (OsLu) in the trinitrobenzenesulfonic acid model of rat colitis and compare them with those of lactulose. Both treatments modified bacterial profile in intestinal contents, increasing the bifidobacteria and lactobacilli counts and up-regulating the production of short-chain fatty acids, although OsLu generated a larger amount. OsLu also inhibited to a greater extent different pro-inflammatory markers such as interleukins (IL) 1, 6, 12, and 23 and chemokines (MCP-1 and CINC-1). However, both prebiotics equally restored colonic epithelial integrity, evaluated both with a histological score (OsLu, 9.8 ± 2.2; and lactulose, 12.1 ± 2.1, vs colitic control, 27.3 ± 3.3) and by measuring several key proteins of the mucosal barrier (MUC-2, MUC-3, and TTF-3). OsLu effect was also associated with an inhibition of iNOS expression and a reduction of Th17 cell activity in the inflamed tissue that facilitated the intestinal mucosa barrier recovery. In conclusion, OsLu showed a better anti-inflammatory profile than lactulose in this model of experimental colitis.

Lipase-catalyzed synthesis of sugar esters, as lactulose palmitate, requires harsh conditions, making it necessary to immobilize the enzyme. Therefore, a study was conducted to evaluate the effect of different chemical surfaces of hierarchical meso-macroporous silica in the immobilization of two lipases from Pseudomonas stutzeri (PsL) and Alcaligenes sp. (AsL), which exhibit esterase activity. Porosity and chemical surface of silica supports, before and after functionalization and after immobilization, were characterized by gas adsorption and Fourier transform infrared (FTIR) spectroscopy. PsL and AsL were immobilized in octyl (OS), glyoxyl (GS), and octyl-glyoxyl silica (OGS). Hydrolytic activity, thermal and solvent stability, and sugar ester synthesis were evaluated with those catalysts. The best support in terms of expressed activity was OS in the case of PsL (100 IU g(-1)), while OS and OGS were the best for AsL with quite similar expressed activities (60 and 58 IU g(-1), respectively). At 60 °C in aqueous media the more stable biocatalysts were GS-PsL and OGS-AsL (half-lives of 566 and 248 h, respectively), showing the advantage of a heterofunctional support in the latter case. Lactulose palmitate synthesis was carried out in acetone medium (with 4% of equilibrium moisture) at 40 °C obtaining palmitic acid conversions higher than 20% for all biocatalysts, being the highest of those obtained with OGS-AsL and OS-PsL. Therefore, screening of different chemical surfaces on porous silica used as supports for lipase immobilization allowed obtaining active and stable biocatalyst to be employed in the novel synthesis of lactulose palmitate.

The aim of the present work was to study and develop an innovative, clean, and environmentally friendly process for lactulose synthesis by electroactivation of lactose. In this work, the electrode material (type 304 stainless steel, titanium, and copper), dimensionless interelectrode-membrane distance at the cathodic compartment (0.36, 0.68, and 1), and the membrane:electrode surface area ratio (0.23, 0.06, and 0.015) were considered to be the factors that could affect the kinetic conversion of lactose into lactulose. The reactions were conducted under an initial lactose concentration of 0.15mol/L at 10°C, Froude number (mixing speed) of 2.05×10(-2), and electric current intensity of 300mA for 30min. The highest lactulose formation yield of 32.50% (0.05mol/L) was obtained by using a copper electrode, interelectrode-membrane distance of 0.36, and membrane:electrode surface area ratio of 0.23. The 2-parameter Langmuir, Freundlich, and Temkin isotherm models were used for the prediction of the lactose isomerization kinetics as well as the 3-parameter Langmuir-Freundlich isotherm model. It was shown that the lactose isomerization kinetics into lactulose followed the Temkin and Langmuir-Freundlich models with coefficients of determination of 0.99 and 0.90 and a relative error of 1.42 to 1.56% and 4.27 to 4.37%, respectively., (Copyright © 2014 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Lactulose, a synthetic ketose disaccharide, has been widely used in food and pharmaceutical industries as prebiotic food additives and drugs against constipation and hepatic encephalopathy. Lactulose has, so far, been produced chemically using lactose on a commercial scale. The key problems associated with such chemical process are the cost of removal of the catalyst and colored by-products and the product safety. Enzymatic production of lactulose is safe, environment-friendly, and simpler in comparison to the chemical method. As a promising alternative to the chemical method, enzymatic conversion of lactose into lactulose by β-galactosidase or cellobiose 2-epimerase has recently gained a great deal of attention. This could be considered as a possible route for whey surplus because lactose is the major component of cheese whey. Herein, we summarize recent advances on the enzymatic synthesis of lactulose with emphasis on the selectivity of biocatalysts and their catalytic efficiency in free and immobilized forms. The production of 1-lactulose by enzymatic bioconversion of lactose has also been discussed. Furthermore, future research needs of β-galactosidase and cellobiose 2-epimerase for the enzymatic synthesis of lactulose and 1-lactulose are reviewed.

Batch synthesis of fructosyl-galacto-oligosaccharides from lactulose was performed with commercial β-galactosidase preparations from Aspergillus oryzae, Kluyveromyces lactis and Bacillus circulans. The enzyme from A. oryzae produced the highest yield and specific productivity of synthesis, being selected for further studies. Optimization of fructosyl-galacto-oligosaccharides synthesis was carried out using response surface methodology, considering temperature and initial sugar concentration as variables and yield and specific productivity as response parameters. Maximum yield of 0.41 g g(-1) fructosyl-galacto-oligosaccharides was obtained at 70°C and 60% w/w lactulose concentration, while maximum specific productivity of 1.2 g h(-1)mg(-1) was obtained at 70°C and 40% w/w lactulose concentration., (Copyright © 2012 Elsevier Ltd. All rights reserved.)