RESUMO
The production of the sugars fructose and lactulose from lactose using the enzymes ß-galactosidase and glucose isomerase immobilized on bacterial cellulose (BC) membranes has been investigated. Lactose is hydrolyzed by ß-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.
Assuntos
Celulose , Enzimas Imobilizadas , Frutose , Lactose , Lactulose , beta-Galactosidase , Lactulose/química , Lactulose/metabolismo , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Lactose/química , Lactose/metabolismo , Celulose/química , Celulose/metabolismo , Frutose/química , Frutose/metabolismo , beta-Galactosidase/química , beta-Galactosidase/metabolismo , Isomerismo , Aldose-Cetose Isomerases/química , Aldose-Cetose Isomerases/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Biocatálise , Bactérias/enzimologia , Bactérias/química , Bactérias/metabolismoRESUMO
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 ß-galactosidase (ß-gal) of Lactobacillus delbruecki subsp. bulgaricus CRL450 (CRL450-ß-gal) from lactose, lactulose and lactitol. RESULTS: In the simulated gastrointestinal fluid method, no changes were observed. However, the oligosaccharides synthesized by CRL450-ß-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 ß (1â6) linked products showed higher resistance compared to ß (1â3) 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.
Assuntos
Digestão , Camundongos Endogâmicos C57BL , Oligossacarídeos , Prebióticos , beta-Galactosidase , Prebióticos/análise , Animais , beta-Galactosidase/metabolismo , beta-Galactosidase/química , Oligossacarídeos/metabolismo , Oligossacarídeos/química , Camundongos , Ratos , Suínos , Masculino , Lactulose/metabolismo , Lactulose/química , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Intestino Delgado/metabolismo , Intestino Delgado/enzimologia , Lactobacillus/metabolismo , Lactobacillus/enzimologia , Hidrólise , Lactose/metabolismo , Lactose/químicaRESUMO
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 α8/α9 loop exhibited longer half-lives at 65-75 °C as well as lower Km and higher kcat/Km 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.
Assuntos
Lactose , Lactulose , Lactulose/química , Lactose/química , Celobiose/química , Racemases e Epimerases/genética , Clostridiales , Desenho Assistido por ComputadorRESUMO
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.
Assuntos
Caseínas , Lactose , Caseínas/química , Lactose/metabolismo , Lactulose/química , Isomerismo , Reação de MaillardRESUMO
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 CsCE/Q371E had significantly enhanced substrate affinity (Km, 269.65 mM vs Km, 417.5 mM), reduced epimerization activity, and 3.3-fold increased isomerization activity over the original CsCE. 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.
Assuntos
Celobiose , Lactulose , Lactulose/química , Celobiose/metabolismo , Lactose/química , Isomerismo , Racemases e Epimerases/genética , Racemases e Epimerases/metabolismo , Especificidade por SubstratoRESUMO
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 ß-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.
Assuntos
Lactose , Lactulose , Catálise , Celobiose/química , Celobiose/metabolismo , Isomerismo , Lactose/metabolismo , Lactulose/química , Lactulose/metabolismo , Racemases e Epimerases/metabolismo , beta-Galactosidase/metabolismoRESUMO
The enzyme ß-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 ß-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. ß-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 KM and Vmax of ß-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 ß-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 ß-galactosidase from P. marcusii that can be used for the industrial production of prebiotic oligosaccharides.
Assuntos
Lactulose/metabolismo , Oligossacarídeos/biossíntese , Paracoccus/enzimologia , Prebióticos , beta-Galactosidase/metabolismo , Biocatálise , Configuração de Carboidratos , Cinética , Lactulose/química , Oligossacarídeos/químicaRESUMO
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.
Assuntos
Lacticaseibacillus casei/fisiologia , Polissacarídeos/química , Probióticos/química , Composição de Medicamentos , Estabilidade de Medicamentos , Humanos , Concentração de Íons de Hidrogênio , Lactulose/química , Tamanho da Partícula , Prebióticos , Amido Resistente , Sacarina/química , Trissacarídeos/químicaRESUMO
In this paper, 3 different biocatalysts of ß-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 ß-galactosidase in a kinetically controlled manner, and how medium composition influences this performance due to the kinetic properties of ß-galactosidase.
Assuntos
Enzimas Imobilizadas/química , Proteínas Fúngicas/química , Kluyveromyces/enzimologia , Lactulose , beta-Galactosidase/química , Biocatálise , Cinética , Lactulose/síntese química , Lactulose/químicaRESUMO
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.
Assuntos
Autofagia/efeitos dos fármacos , Lactulose/farmacologia , Melibiose/farmacologia , Neurônios/metabolismo , Agregados Proteicos , Regulação para Cima , alfa-Sinucleína/metabolismo , Sequência de Aminoácidos , Antioxidantes/farmacologia , Benzotiazóis/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Neurônios Dopaminérgicos/efeitos dos fármacos , Neurônios Dopaminérgicos/metabolismo , Fluorescência , Proteínas de Fluorescência Verde/metabolismo , Humanos , Lactulose/química , Melibiose/química , Crescimento Neuronal/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Agregados Proteicos/efeitos dos fármacos , Trealose/química , Trealose/farmacologia , Regulação para Cima/efeitos dos fármacos , alfa-Sinucleína/químicaRESUMO
ß-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/gcell 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.
Assuntos
Queijo , Proteínas Fúngicas/química , Kluyveromyces/enzimologia , Lactulose/síntese química , Soro do Leite/química , beta-Galactosidase/química , Lactose/química , Lactulose/químicaRESUMO
Morphological and structural properties of amorphous disaccharide lactulose (C12H22O11), 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.
Assuntos
Composição de Medicamentos/métodos , Lactulose/química , Varredura Diferencial de Calorimetria , Cristalização , Dessecação , Estabilidade de Medicamentos , Liofilização , Isomerismo , Microscopia Eletrônica de Varredura , Simulação de Dinâmica Molecular , Nêutrons , Tamanho da Partícula , Pós , Difração de Raios XRESUMO
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.
Assuntos
Dessecação/métodos , Lactulose/química , Ciência dos Materiais/métodos , Varredura Diferencial de Calorimetria , Difusão , Liofilização/métodos , Isomerismo , Lactulose/síntese química , Espectroscopia de Ressonância Magnética , Simulação de Dinâmica Molecular , Pós , Solubilidade , Vitrificação , Difração de Raios XRESUMO
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.
Assuntos
Bactérias/crescimento & desenvolvimento , Microbioma Gastrointestinal/efeitos dos fármacos , Lactulose/farmacologia , Animais , Bactérias/genética , Bacteroides/genética , Bacteroides/crescimento & desenvolvimento , Bacteroidetes/genética , Bacteroidetes/crescimento & desenvolvimento , Firmicutes/genética , Firmicutes/crescimento & desenvolvimento , Microbioma Gastrointestinal/genética , Helicobacteraceae/genética , Helicobacteraceae/crescimento & desenvolvimento , Sequenciamento de Nucleotídeos em Larga Escala , Lactobacillaceae/genética , Lactobacillaceae/crescimento & desenvolvimento , Lactulose/química , Camundongos , RNA Ribossômico 16S/genéticaRESUMO
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 5â¯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 (pHâ¯=â¯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.
Assuntos
Fracionamento Químico/métodos , Lactose/química , Lactulose/análise , Boratos/química , Catálise , Cromatografia Líquida de Alta Pressão , Temperatura Alta , Interações Hidrofóbicas e Hidrofílicas , Isomerismo , Lactose/análise , Lactulose/química , Hidróxido de Sódio/químicaRESUMO
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.
Assuntos
Lactulose/imunologia , Lactulose/farmacologia , Oligossacarídeos/imunologia , Oligossacarídeos/farmacologia , Animais , Suplementos Nutricionais , Feminino , Imunoglobulina A/sangue , Imunoglobulina A/imunologia , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Imunoglobulina M/sangue , Imunoglobulina M/imunologia , Lactulose/química , Camundongos , Oligossacarídeos/química , Baço/efeitos dos fármacos , Baço/imunologia , Coloração e Rotulagem , Timo/efeitos dos fármacos , Timo/imunologia , Aumento de Peso/efeitos dos fármacosRESUMO
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 (α2â3)-linked sialic acid from donor substrates to nonreducing terminal ß-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.
Assuntos
Glicoproteínas/química , Lactose/química , Lactulose/química , Ácido N-Acetilneuramínico/química , Neuraminidase/química , Oligossacarídeos/química , Proteínas de Protozoários/química , Trypanosoma cruzi/enzimologia , BiocatáliseRESUMO
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.
Assuntos
Comunicação Celular/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Lactulose/farmacologia , Leucócitos Mononucleares/efeitos dos fármacos , Oligodesoxirribonucleotídeos/farmacologia , Sinergismo Farmacológico , Células Epiteliais/citologia , Células Epiteliais/imunologia , Células HT29 , Humanos , Interferon gama/genética , Interferon gama/imunologia , Interleucina-10/genética , Interleucina-10/imunologia , Lactulose/química , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/imunologia , Oligodesoxirribonucleotídeos/químicaRESUMO
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.
Assuntos
Proteínas Fúngicas/metabolismo , Lactulose/química , Lectinas Tipo C/metabolismo , Oligossacarídeos/análise , Receptores Imunológicos/metabolismo , Saccharomyces cerevisiae/metabolismo , Prebióticos , ProteômicaRESUMO
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.