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1.
ACS Synth Biol ; 13(6): 1866-1878, 2024 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-38836566

RESUMO

3-Fucosyllactose (3-FL) is an important fucosylated human milk oligosaccharide (HMO) with biological functions such as promoting immunity and brain development. Therefore, the construction of microbial cell factories is a promising approach to synthesizing 3-FL from renewable feedstocks. In this study, a combinatorial engineering strategy was used to achieve efficient de novo 3-FL production in Escherichia coli. α-1,3-Fucosyltransferase (futM2) from Bacteroides gallinaceum was introduced into E. coli and optimized to create a 3-FL-producing chassis strain. Subsequently, the 3-FL titer increased to 5.2 g/L by improving the utilization of the precursor lactose and down-regulating the endogenous competitive pathways. Furthermore, a synthetic membraneless organelle system based on intrinsically disordered proteins was designed to spatially regulate the pathway enzymes, producing 7.3 g/L 3-FL. The supply of the cofactors NADPH and GTP was also enhanced, after which the 3-FL titer of engineered strain E26 was improved to 8.2 g/L in a shake flask and 10.8 g/L in a 3 L fermenter. In this study, we developed a valuable approach for constructing an efficient 3-FL-producing cell factory and provided a versatile workflow for other chassis cells and HMOs.


Assuntos
Escherichia coli , Fucosiltransferases , Engenharia Metabólica , Trissacarídeos , Escherichia coli/genética , Escherichia coli/metabolismo , Trissacarídeos/metabolismo , Trissacarídeos/biossíntese , Engenharia Metabólica/métodos , Fucosiltransferases/genética , Fucosiltransferases/metabolismo , Lactose/metabolismo , Bacteroides/genética , Bacteroides/metabolismo , Fermentação , Oligossacarídeos
2.
Food Res Int ; 189: 114554, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38876592

RESUMO

Listeria monocytogenes, a widespread food-borne pathogen, utilizes diverse growth substrates including mono- and di-saccharides via PEP-phosphotransferase (PTS) systems. We evaluated a collection of L. monocytogenes isolates of different origins for their ability to utilize lactose, a disaccharide composed of galactose and glucose and the main carbon source in milk and dairy products. Notably, the dairy-associated outbreak strain F2365 could not utilize lactose efficiently, conceivably due to a frameshift mutation (lacR887del) resulting in a truncated LacR. Transcriptional activator LacR is involved in the expression of two PTS systems, encoded by the lpo operon lmo1718-1720 in combination with lmo2708 and the lmo2683-2685 operon, and linked to lactose and/or cellobiose metabolism in L. monocytogenes. Via experimental evolution of the ancestral strain F2365, an evolved isolate F2365 EV was obtained which showed enhanced growth and metabolism of lactose. Using the lactose-positive model strain L. monocytogenes EGDe as a control, HPLC experiments showed that EGDe and F2365 EV could consume lactose and utilize the glucose moiety, while the galactose moiety was exported from the cells. Genome sequencing of F2365 EV found the original lacR887del mutation was still present but an additional point mutation lmo2766C415T had occurred, resulting in an amino acid substitution in the putative regulator Lmo2766. The lmo2766 gene is located next to operon lmo2761-2765 with putative PTS genes in the genome. Notably, comparative RNAseq analysis confirmed that the lmo2761-2765 operon was strongly upregulated in F2365 EV in the presence of lactose but not in EGDe and F2365. Conversely, the LacR-regulated lpo operon, lmo2708, and lmo2683-2685 operon were only upregulated in EGDe. Additional growth and HPLC experiments, using mutants constructed in lactose-positive L. monocytogenes EGDe, showed reduced growth of the EGDe lacR887del mutant with no utilization of lactose, while the double mutant EGDe lacR887dellmo2766C415T showed enhanced growth and lactose utilization. Hence, these results demonstrate that an amino acid substitution in the Lmo2766 regulator activates a previously silent lactose utilization pathway encoded by PTS operon lmo2761-2765, facilitating the growth and metabolism of L. monocytogenes with lactose as a substrate. This finding enhances our understanding of the metabolic capabilities and adaptability of L. monocytogenes, offering a broader view of the lactose utilization capacity of this pathogen.


Assuntos
Lactose , Listeria monocytogenes , Listeria monocytogenes/genética , Listeria monocytogenes/metabolismo , Listeria monocytogenes/crescimento & desenvolvimento , Lactose/metabolismo , Óperon , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Surtos de Doenças , Regulação Bacteriana da Expressão Gênica , Microbiologia de Alimentos , Leite/microbiologia , Animais , Laticínios/microbiologia
3.
Sci Rep ; 14(1): 13376, 2024 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-38862572

RESUMO

In individuals with hearing loss, protection of residual hearing is essential following cochlear implantation to facilitate acoustic and electric hearing. Hearing preservation requires slow insertion, atraumatic electrode and delivery of the optimal quantity of a pharmacological agent. Several studies have reported variable hearing outcomes with osmotic pump-mediated steroid delivery. New drugs, such as sialyllactose (SL) which have anti-inflammatory effect in many body parts, can prevent tissue overgrowth. In the present study, the positive effects of the pharmacological agent SL against insults were evaluated in vitro using HEI-OC1 cells. An animal model to simulate the damage due to electrode insertion during cochlear implantation was used. SL was delivered using osmotic pumps to prevent loss of the residual hearing in this animal model. Hearing deterioration, tissue fibrosis and ossification were confirmed in this animal model. Increased gene expressions of inflammatory cytokines were identified in the cochleae following dummy electrode insertion. Following the administration of SL, insertion led to a decrease in hearing threshold shifts, tissue reactions, and inflammatory markers. These results emphasize the possible role of SL in hearing preservation and improve our understanding of the mechanism underlying hearing loss after cochlear implantation.


Assuntos
Implante Coclear , Perda Auditiva , Lactose , Animais , Lactose/análogos & derivados , Lactose/farmacologia , Perda Auditiva/prevenção & controle , Perda Auditiva/tratamento farmacológico , Audição/efeitos dos fármacos , Cóclea/efeitos dos fármacos , Cóclea/metabolismo , Camundongos , Modelos Animais de Doenças , Linhagem Celular , Citocinas/metabolismo , Masculino , Ácidos Siálicos
4.
AAPS PharmSciTech ; 25(6): 138, 2024 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-38890193

RESUMO

Unexpected cross-contamination by foreign components during the manufacturing and quality control of pharmaceutical products poses a serious threat to the stable supply of drugs and the safety of customers. In Japan, in 2020, a mix-up containing a sleeping drug went undetected by liquid chromatography during the final quality test because the test focused only on the main active pharmaceutical ingredient (API) and known impurities. In this study, we assessed the ability of a powder rheometer to analyze powder characteristics in detail to determine whether it can detect the influence of foreign APIs on powder flow. Aspirin, which was used as the host API, was combined with the guest APIs (acetaminophen from two manufacturers and albumin tannate) and subsequently subjected to shear and stability tests. The influence of known lubricants (magnesium stearate and leucine) on powder flow was also evaluated for standardized comparison. Using microscopic morphological analysis, the surface of the powder was observed to confirm physical interactions between the host and guest APIs. In most cases, the guest APIs were statistically detected due to characteristics such as their powder diameter, pre-milling, and cohesion properties. Furthermore, we evaluated the flowability of a formulation incorporating guest APIs for direct compression method along with additives such as microcrystalline cellulose, potato starch, and lactose. Even in the presence of several additives, the influence of the added guest APIs was successfully detected. In conclusion, powder rheometry is a promising method for ensuring stable product quality and reducing the risk of unforeseen cross-contamination by foreign APIs.


Assuntos
Contaminação de Medicamentos , Pós , Reologia , Pós/química , Reologia/métodos , Contaminação de Medicamentos/prevenção & controle , Excipientes/química , Acetaminofen/química , Celulose/química , Preparações Farmacêuticas/química , Controle de Qualidade , Aspirina/química , Química Farmacêutica/métodos , Lactose/química , Composição de Medicamentos/métodos , Lubrificantes/química , Princípios Ativos
5.
An Acad Bras Cienc ; 96(3): e20221063, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38865506

RESUMO

This study evaluated the influence of milk production, number of lactations, and days in milk (DIM) on the quality and composition of milk from dairy cows housed in a compost barn (CB) system. The study was carried out using a six-year database, counting 31,268 observations from 2,037 cows of European breeds. Multiparous cows showed higher fat and protein production. Lactose showed high levels for primiparous and the initial stage of lactation (4.65%) and was negatively influenced by somatic cell count (SCC). Milk urea nitrogen was higher (14.01%) from 106 to 205 days in milk, and the other components were higher at >305 days. Therefore, the solids content was higher in the first and second lactations due to the high contents of lactose, fat, and milk protein, but lactose was reduced over lactations. In contrast, high DIM increased SCC and concentrated solids due to lower milk production. The effect of milk production, stage, and lactation order on the composition and milk quality of herds housed in CB showed the same pattern as in other production systems.


Assuntos
Indústria de Laticínios , Lactação , Leite , Animais , Lactação/fisiologia , Leite/química , Leite/citologia , Feminino , Bovinos , Indústria de Laticínios/métodos , Compostagem , Lactose/análise , Fatores de Tempo , Proteínas do Leite/análise
6.
Int J Pharm ; 659: 124290, 2024 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-38821435

RESUMO

The influence of hydroxypropyl cellulose type (HPC-SSL SFP, HPC-SSL), concentration (2 %, 3.5 %, 5 %) and filler (lactose, calcium hydrogen phosphate (DCP)/microcrystalline cellulose (MCC)) on twin-screw wet granulation and subsequent tableting was studied. The aim was to identify the formulation of the highest tabletability which still fulfills the requirements of the disintegration. Lactose combined with 5 % binder enabled a higher tabletability and a faster disintegration than DCP/MCC. It was found that tabletability of lactose formulations can be increased by higher binder concentration and higher compression pressure while tabletability of DCP/MCC formulations can be only increased by higher compression pressure. It was observed that batches containing DCP/MCC failed the disintegration test, if the highest binder concentration and the highest compression pressure were used. To ensure a fast disintegration, the compression pressure or at least the binder concentration had to be low. Changing the disintegrant and its localization improved the DCP/MCC formulation, resulting in faster disintegration than lactose tablets. However, it also resulted in a lower tabletability. In this study best tablets were achieved with 3.5 % or 5 % binder and lactose as filler. These tablets presented the highest tabletability but still disintegrated in less than 500 s.


Assuntos
Celulose , Composição de Medicamentos , Excipientes , Lactose , Comprimidos , Celulose/química , Celulose/análogos & derivados , Lactose/química , Excipientes/química , Composição de Medicamentos/métodos , Fosfatos de Cálcio/química , Química Farmacêutica/métodos , Pressão , Solubilidade
7.
Nutrition ; 124: 112446, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38703635

RESUMO

OBJECTIVE: The human gut microbiota is composed of many viruses, bacteria, and fungi. Escherichia coli representatives are facultative anaerobic bacteria in the colon that play a crucial role in the metabolism of lactose, vitamin synthesis, and immune system modulation. E. coli forms a biofilm on the epithelial cell surface of the intestine that can be modified by diet compounds, i.e., gluten, xylitol, lactose, and probiotics. METHODS: In the present study, the impact of probiotic-derived Lactobacillus rhamnosus GG strain on non-pathogenic E. coli biofilm was examined. The mono- and multispecies biofilm was also treated with gluten, xylitol, and lactose. We used 96-well plates to obtain biofilm growth. Biofilm was stained using crystal violet. To evaluate the type of interaction in mono- and multispecies biofilm, a new formula was introduced: biofilm interaction ratio index (BIRI). To describe the impact of nutrients on biofilm formation, the biofilm formation impact ratio (BFIR) was calculated. RESULTS: The biofilms formed by both examined species are stronger than in monocultures. All the BIRI values were above 3.0. It was found that the monospecies biofilm of L. rhamnosus is strongly inhibited by gluten (84.5%) and the monospecies biofilm of E. coli by xylitol (85.5%). The mixed biofilm is inhibited by lactose (78.8%) and gluten (90.6%). CONCLUSION: The relations between bacteria in the mixed biofilm led to changes in biofilm formation by E. coli and L. rhamnosus GG. Probiotics might be helpful in rebuilding the gut microbiota after broad spectrum antibiotic therapy, but only if gluten and lactose are excluded from diet.


Assuntos
Biofilmes , Escherichia coli , Microbioma Gastrointestinal , Glutens , Lacticaseibacillus rhamnosus , Lactose , Probióticos , Xilitol , Biofilmes/efeitos dos fármacos , Xilitol/farmacologia , Humanos , Lacticaseibacillus rhamnosus/fisiologia , Microbioma Gastrointestinal/fisiologia , Microbioma Gastrointestinal/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Probióticos/farmacologia
8.
Int J Biol Macromol ; 271(Pt 1): 132478, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38772465

RESUMO

Bioconversion of lactose to functional lactose derivatives attracts increasing attention. Lactulose is an important high-value lactose derivative, which has been widely used in pharmaceutical, nutraceutical, and food industries. Lactulose can be enzymatically produced from lactose by cellobiose 2-epimerase (CEase). Several studies have already focused on the food-grade expression of CEase, but they are all aimed at the biosynthesis of epilactose. Herein, we reported for the first time the biosynthesis of lactulose using the recombinant food-grade Bacillus subtilis. Lactulose biosynthesis was optimized by varying lactulose-producing CEases and expression vectors. Caldicellulosiruptor saccharolyticus CEase and pP43NMK were determined to be the optimal CEase and expression vector. Fine-tuning of CEase expression was investigated by screening a beneficial N-terminal coding sequence. After fed-batch cultivation, the highest fermentation isomerization activity reached 11.6 U/mL. Lactulose was successfully produced by the broth of the engineered B. subtilis with a yield of 52.1 %.


Assuntos
Bacillus subtilis , Lactose , Lactulose , Lactulose/metabolismo , Lactulose/biossíntese , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Lactose/metabolismo , Fermentação , Engenharia Metabólica/métodos , Engenharia Genética
9.
Parasit Vectors ; 17(1): 232, 2024 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-38769548

RESUMO

BACKGROUND: Schistosoma japonicum eggs lodge in the liver and induce a fibrotic granulomatous immune response in the liver of host. Galectin 3 (Gal-3) is a protein implicated in fibrosis in multiple organs. However, the pathology and molecular mechanisms promoting hepatic granuloma formation remain poorly understood. METHODS: To investigate the effect of blocking galectin-receptor interactions by α-lactose on liver immunopathology in mice with S. japonicum infection, C57BL/6 mice were infected with S. japonicum and alpha (α)-lactose was intraperitoneally injected to block the interactions of galectins and their receptors. RESULTS: Compared with S. japonicum-infected mice, there were significantly decreased Gal-3 mRNA and protein expression levels, decreased intensity of Gal-3 fluorescence in the liver, decreased serum ALT and AST levels, decreased egg numbers of S. japonicum in the liver section, attenuated hepatic and spleen pathology, and alleviated liver fibrosis accompanied with decreased protein expression levels of fibrosis markers [α-smooth muscle actin (α-SMA), collagen I, and collagen IV] in the liver of S. japonicum-infected mice blocked galectin-receptor interactions with hematoxylin-eosin staining, Masson's trichrome staining, immunohistochemistry, or Western blot analysis. Compared with S. japonicum-infected mice, blocking galectin-receptor interactions led to increased eosinophil infiltration and higher eosinophil cationic protein (ECP) expression in the liver, accompanied by increased mRNA levels of eosinophil granule proteins [ECP and eosinophil peroxidase (EPO)], IL-5, CCL11, and CCR3 in the liver and decreased mRNA levels of Gal-3 and M2 macrophage cytokines (TGF-ß, IL-10, and IL-4) in the liver and spleen by using quantitative real-time reverse transcription-polymerase chain reaction. In addition, there were increased Beclin1 protein expression and protein expression ratio of LC3B-II/LC3B-I and decreased p62 protein expression and protein expression ratios of phospho-mTOR/mTOR and phospho-AKT/AKT by Western blot; increased double-labeled F4/80+/LC3B+ cells by immunofluorescence staining; increased M1 macrophage polarization in the liver of S. japonicum-infected mice blocked galectin-receptor interactions by flow cytometric analysis and immunofluorescence staining. CONCLUSIONS: Our data found that blockage of galectin-receptor interactions downregulated Gal-3, which in turn led to reduced liver functional damage, elevated liver eosinophil recruitment, promoted macrophage autophagy through the Akt/mTOR signaling pathway, and alleviated liver pathology and fibrosis. Therefore, Gal-3 plays a pivotal role during S. japonicum infection and could be a target of pharmacologic potential for liver fibrosis induced by S. japonicum infection.


Assuntos
Galectina 3 , Cirrose Hepática , Camundongos Endogâmicos C57BL , Schistosoma japonicum , Esquistossomose Japônica , Animais , Esquistossomose Japônica/parasitologia , Esquistossomose Japônica/complicações , Cirrose Hepática/parasitologia , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , Camundongos , Galectina 3/metabolismo , Galectina 3/genética , Fígado/parasitologia , Fígado/patologia , Fígado/metabolismo , Feminino , Lactose/farmacologia , Lactose/análogos & derivados , Galectinas/metabolismo , Galectinas/genética
10.
Int J Pharm ; 657: 124190, 2024 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-38701910

RESUMO

Lubricants are essential for most tablet formulations as they assist powder flow, prevent adhesion to tableting tools and facilitate tablet ejection. Magnesium stearate (MgSt) is an effective lubricant but may compromise tablet strength and disintegratability. In the design of orodispersible tablets, tablet strength and disintegratability are critical attributes of the dosage form. Hence, this study aimed to conduct an in-depth comparative study of MgSt with alternative lubricants, namely sodium lauryl sulphate (SLS), stearic acid (SA) and hydrogenated castor oil (HCO), for their effects on the tableting process as well as tablet properties. Powder blends were prepared with lactose, sodium starch glycolate or crospovidone as the disintegrant, and a lubricant at different concentrations. Angle of repose was determined for the mixtures. Comparative evaluation was carried out based on the ejection force, tensile strength, liquid penetration and disintegratability of the tablets produced. As the lubricant concentration increased, powder flow and tablet ejection improved. The lubrication efficiency generally decreased as follows: MgSt > HCO > SA > SLS. Despite its superior lubrication efficacy, MgSt is the only lubricant of four evaluated that reduced tablet tensile strength. Tablet disintegration time was strongly determined by tensile strength and liquid penetration, which were in turn affected by the lubricant type and concentration. All the above factors should be taken into consideration when deciding the type and concentration of lubricant for an orodispersible tablet formulation.


Assuntos
Excipientes , Lubrificantes , Ácidos Esteáricos , Comprimidos , Resistência à Tração , Lubrificantes/química , Ácidos Esteáricos/química , Excipientes/química , Composição de Medicamentos/métodos , Pós/química , Dodecilsulfato de Sódio/química , Óleo de Rícino/química , Povidona/química , Amido/química , Amido/análogos & derivados , Lactose/química , Administração Oral , Solubilidade , Química Farmacêutica/métodos
11.
Appl Microbiol Biotechnol ; 108(1): 349, 2024 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-38809317

RESUMO

Galacto-oligosaccharides (GOS) are prebiotic compounds that are mainly used in infant formula to mimic bifidogenic effects of mother's milk. They are synthesized by ß-galactosidase enzymes in a trans-glycosylation reaction with lactose. Many ß-galactosidase enzymes from different sources have been studied, resulting in varying GOS product compositions and yields. The in vivo role of these enzymes is in lactose hydrolysis. Therefore, the best GOS yields were achieved at high lactose concentrations up to 60%wt, which require a relatively high temperature to dissolve. Some thermostable ß-glucosidase enzymes from thermophilic bacteria are also capable of using lactose or para nitrophenyl-galactose as a substrate. Here, we describe the use of the ß-glucosidase BglA from Thermotoga maritima for synthesis of oligosaccharides derived from lactose and cellobiose and their detailed structural characterization. Also, the BglA enzyme kinetics and yields were determined, showing highest productivity at higher lactose and cellobiose concentrations. The BglA trans-glycosylation/hydrolysis ratio was higher with 57%wt lactose than with a nearly saturated cellobiose (20%wt) solution. The yield of GOS was very high, reaching 72.1%wt GOS from lactose. Structural elucidation of the products showed mainly ß(1 → 3) and ß(1 → 6) elongating activity, but also some ß(1 → 4) elongation was observed. The ß-glucosidase BglA from T. maritima was shown to be a very versatile enzyme, producing high yields of oligosaccharides, particularly GOS from lactose. KEY POINTS: • ß-Glucosidase of Thermotoga maritima synthesizes GOS from lactose at very high yield. • Thermotoga maritima ß-glucosidase has high activity and high thermostability. • Thermotoga maritima ß-glucosidase GOS contains mainly (ß1-3) and (ß1-6) linkages.


Assuntos
Celobiose , Lactose , Oligossacarídeos , Thermotoga maritima , beta-Glucosidase , Thermotoga maritima/enzimologia , Thermotoga maritima/genética , Lactose/metabolismo , Celobiose/metabolismo , beta-Glucosidase/metabolismo , beta-Glucosidase/genética , beta-Glucosidase/química , Cinética , Oligossacarídeos/metabolismo , Glicosilação , Hidrólise , Temperatura , Estabilidade Enzimática
12.
Food Res Int ; 183: 114175, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38760120

RESUMO

Lactose hydrolysed concentrated milk was prepared using ß-galactosidase enzyme (4.76U/mL) with a reaction period of 12 h at 4 °C. Addition of polysaccharides (5 % maltodextrin/ß-cyclodextrin) to concentrated milk either before or after lactose hydrolysis did not result in significant differences (p > 0.05) in degree of hydrolysis (% DH) of lactose and residual lactose content (%). Three different inlet temperatures (165 °C, 175 °C and 185 °C) were used for the preparation of powders which were later characterised based on physico-chemical and maillard browning characteristics. Moisture content, solubility and available lysine content of the powders decreased significantly, whereas, browning parameters i.e., browning index, 5-hydroxymethylfurfural, furosine content increased significantly (p < 0.05) with an increase in inlet air temperature. The powder was finally prepared with 5 % polysaccharide and an inlet air temperature of 185 °C which reduced maillard browning. Protein-polysaccharide interactions were identified using Fourier Transform infrared spectroscopy, fluorescence spectroscopy and determination of free amino groups in the powder samples. Maltodextrin and ß-cyclodextrin containing powder samples exhibited lower free amino groups and higher degree of graft value as compared to control sample which indicated protein-polysaccharide interactions. Results obtained from Fourier Transform infrared spectroscopy also confirmed strong protein-polysaccharide interactions, moreover a significant decrease in fluorescence intensity was also observed in the powder samples. These interactions between the proteins and polysaccharides reduced the maillard browning in powders.


Assuntos
Furaldeído , Lactose , Reação de Maillard , Leite , Polissacarídeos , Pós , Lactose/química , Polissacarídeos/química , Leite/química , Animais , Espectroscopia de Infravermelho com Transformada de Fourier , Furaldeído/análogos & derivados , Furaldeído/química , beta-Galactosidase/metabolismo , beta-Ciclodextrinas/química , Hidrólise , Secagem por Atomização , Temperatura , Lisina/química , Lisina/análogos & derivados , Solubilidade , Espectrometria de Fluorescência , Proteínas do Leite/química , Manipulação de Alimentos/métodos
13.
Bioorg Chem ; 147: 107395, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38705105

RESUMO

Fluorination of carbohydrate ligands of lectins is a useful approach to examine their binding profile, improve their metabolic stability and lipophilicity, and convert them into 19F NMR-active probes. However, monofluorination of monovalent carbohydrate ligands often leads to a decreased or completely lost affinity. By chemical glycosylation, we synthesized the full series of methyl ß-glycosides of N,N'-diacetylchitobiose (GlcNAcß(1-4)GlcNAcß1-OMe) and LacdiNAc (GalNAcß(1-4)GlcNAcß1-OMe) systematically monofluorinated at all hydroxyl positions. A competitive enzyme-linked lectin assay revealed that the fluorination at the 6'-position of chitobioside resulted in an unprecedented increase in affinity to wheat germ agglutinin (WGA) by one order of magnitude. For the first time, we have characterized the binding profile of a previously underexplored WGA ligand LacdiNAc. Surprisingly, 4'-fluoro-LacdiNAc bound WGA even stronger than unmodified LacdiNAc. These observations were interpreted using molecular dynamic calculations along with STD and transferred NOESY NMR techniques, which gave evidence for the strengthening of CH/π interactions after deoxyfluorination of the side chain of the non-reducing GlcNAc. These results highlight the potential of fluorinated glycomimetics as high-affinity ligands of lectins and 19F NMR-active probes.


Assuntos
Dissacarídeos , Aglutininas do Germe de Trigo , Dissacarídeos/química , Dissacarídeos/síntese química , Aglutininas do Germe de Trigo/química , Aglutininas do Germe de Trigo/metabolismo , Halogenação , Estrutura Molecular , Acetilglucosamina/química , Acetilglucosamina/metabolismo , Lactose/análogos & derivados
14.
PLoS One ; 19(5): e0303872, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38771780

RESUMO

BACKGROUND: Antimicrobial resistance (AMR) is among the top public health concerns in the globe. Estimating the prevalence of multidrug resistance (MDR), MDR index (MDR-I) and extended-spectrum beta-lactamase (ESBL)-producing lactose fermenting Enterobacteriaceae (LFE) is important in designing strategies to combat AMR. Thus, this study was designed to determine the status of MDR, MDR-I and ESBL-producing LFE isolated from the human-dairy interface in the northwestern part of Ethiopia, where such information is lacking. METHODOLOGY: A cross-sectional study was conducted from June 2022 to August 2023 by analyzing 362 samples consisting of raw pooled milk (58), milk container swabs (58), milker's hand swabs (58), farm sewage (57), milker's stool (47), and cow's feces (84). The samples were analyzed using standard bacteriological methods. The antimicrobial susceptibility patterns and ESBL production ability of the LFE isolates were screened using the Kirby-Bauer disk diffusion method, and candidate isolates passing the screening criteria were phenotypically confirmed by using cefotaxime (30 µg) and cefotaxime /clavulanic acid (30 µg/10 µg) combined-disk diffusion test. The isolates were further characterized genotypically using multiplex polymerase chain reaction targeting the three ESBL-encoding- genes namely blaTEM, blaSHV, and blaCTX-M. RESULTS: A total of 375 bacterial isolates were identified and the proportion of MDR and ESBL-producing bacterial isolates were 70.7 and 21.3%, respectively. The MDR-I varied from 0.0 to 0.81 with an average of 0.30. The ESBL production was detected in all sample types. Genotypically, the majority of the isolates (97.5%), which were positive on the phenotypic test, were carrying one or more of the three genes. CONCLUSION: A high proportion of the bacterial isolates were MDR; had high MDR-I and were positive for ESBL production. The findings provide evidence that the human-dairy interface is one of the important reservoirs of AMR traits. Therefore, the implementation of AMR mitigation strategies is highly needed in the area.


Assuntos
Antibacterianos , Farmacorresistência Bacteriana Múltipla , Enterobacteriaceae , Lactose , beta-Lactamases , Humanos , Etiópia , beta-Lactamases/genética , beta-Lactamases/metabolismo , Enterobacteriaceae/genética , Enterobacteriaceae/efeitos dos fármacos , Enterobacteriaceae/isolamento & purificação , Enterobacteriaceae/enzimologia , Lactose/metabolismo , Farmacorresistência Bacteriana Múltipla/genética , Estudos Transversais , Antibacterianos/farmacologia , Animais , Testes de Sensibilidade Microbiana , Bovinos , Infecções por Enterobacteriaceae/microbiologia , Cefotaxima/farmacologia , Leite/microbiologia , Fermentação , Fezes/microbiologia
15.
Appl Microbiol Biotechnol ; 108(1): 338, 2024 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-38771321

RESUMO

Fucosyl-oligosaccharides (FUS) provide many health benefits to breastfed infants, but they are almost completely absent from bovine milk, which is the basis of infant formula. Therefore, there is a growing interest in the development of enzymatic transfucosylation strategies for the production of FUS. In this work, the α-L-fucosidases Fuc2358 and Fuc5372, previously isolated from the intestinal bacterial metagenome of breastfed infants, were used to synthesize fucosyllactose (FL) by transfucosylation reactions using p-nitrophenyl-α-L-fucopyranoside (pNP-Fuc) as donor and lactose as acceptor. Fuc2358 efficiently synthesized the major fucosylated human milk oligosaccharide (HMO) 2'-fucosyllactose (2'FL) with a 35% yield. Fuc2358 also produced the non-HMO FL isomer 3'-fucosyllactose (3'FL) and traces of non-reducing 1-fucosyllactose (1FL). Fuc5372 showed a lower transfucosylation activity compared to Fuc2358, producing several FL isomers, including 2'FL, 3'FL, and 1FL, with a higher proportion of 3'FL. Site-directed mutagenesis using rational design was performed to increase FUS yields in both α-L-fucosidases, based on structural models and sequence identity analysis. Mutants Fuc2358-F184H, Fuc2358-K286R, and Fuc5372-R230K showed a significantly higher ratio between 2'FL yields and hydrolyzed pNP-Fuc than their respective wild-type enzymes after 4 h of transfucosylation. The results with the Fuc2358-F184W and Fuc5372-W151F mutants showed that the residues F184 of Fuc2358 and W151 of Fuc5372 could have an effect on transfucosylation regioselectivity. Interestingly, phenylalanine increases the selectivity for α-1,2 linkages and tryptophan for α-1,3 linkages. These results give insight into the functionality of the active site amino acids in the transfucosylation activity of the GH29 α-L-fucosidases Fuc2358 and Fuc5372. KEY POINTS: Two α-L-fucosidases from infant gut bacterial microbiomes can fucosylate glycans Transfucosylation efficacy improved by tailored point-mutations in the active site F184 of Fuc2358 and W151 of Fuc5372 seem to steer transglycosylation regioselectivity.


Assuntos
Microbioma Gastrointestinal , Metagenoma , Leite Humano , Trissacarídeos , alfa-L-Fucosidase , alfa-L-Fucosidase/genética , alfa-L-Fucosidase/metabolismo , Humanos , Trissacarídeos/metabolismo , Leite Humano/química , Lactose/metabolismo , Oligossacarídeos/metabolismo , Mutagênese Sítio-Dirigida , Lactente , Fucose/metabolismo
16.
Appl Microbiol Biotechnol ; 108(1): 354, 2024 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-38819482

RESUMO

Whey is a byproduct of dairy industries, the aqueous portion which separates from cheese during the coagulation of milk. It represents approximately 85-95% of milk's volume and retains much of its nutrients, including functional proteins and peptides, lipids, lactose, minerals, and vitamins. Due to its composition, mainly proteins and lactose, it can be considered a raw material for value-added products. Whey-derived products are often used to supplement food, as they have shown several physiological effects on the body. Whey protein hydrolysates are reported to have different activities, including antihypertensive, antioxidant, antithrombotic, opioid, antimicrobial, cytomodulatory, and immuno-modulatory. On the other hand, galactooligosaccharides obtained from lactose can be used as prebiotic for beneficial microorganisms for the human gastrointestinal tract. All these compounds can be obtained through physicochemical, microbial, or enzymatic treatments. Particularly, enzymatic processes have the advantage of being highly selective, more stable than chemical transformations, and less polluting, making that the global enzyme market grow at accelerated rates. The sources and different products associated with the most used enzymes are particularly highlighted in this review. Moreover, we discuss metagenomics as a tool to identify novel proteolytic enzymes, from both cultivable and uncultivable microorganisms, which are expected to have new interesting activities. Finally enzymes for the transformation of whey sugar are reviewed. In this sense, carbozymes with ß-galactosidase activity are capable of lactose hydrolysis, to obtain free monomers, and transgalactosylation for prebiotics production. KEY POINTS: • Whey can be used to obtain value-added products efficiently through enzymatic treatments • Proteases transform whey proteins into biopeptides with physiological activities • Lactose can be transformed into prebiotic compounds using ß-galactosidases.


Assuntos
Hidrolisados de Proteína , Proteínas do Soro do Leite , Proteínas do Soro do Leite/metabolismo , Hidrolisados de Proteína/metabolismo , Hidrolisados de Proteína/química , Prebióticos , Humanos , Soro do Leite/química , Soro do Leite/metabolismo , Lactose/metabolismo , beta-Galactosidase/metabolismo , beta-Galactosidase/genética
17.
Braz J Microbiol ; 55(2): 1735-1744, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38727922

RESUMO

AIMS: To develop and characterize a functional lactose-free ice cream with added ginger and honey, evaluate the survival of Lacticaseibacillus casei CSL3 under frozen storage and the simulated gastrointestinal tract (GIT), as well as antioxidant activity and product acceptability. METHODS AND RESULTS: The survival of Lacticaseibacillus casei CSL3 was evaluated for 180 days, under frozen storage, and GIT at 60 days. At 15 days of storage, proximal composition, antioxidant activity, color, pH, acidity, fusion, density, overrun, and sensory analysis were performed. Ice cream was an effective food matrix for maintaining the viability of CSL3, with concentrations > 7 log CFU g- 1 during storage and GIT. In addition, the analysis showed overrun and prebiotic characteristics through high values of antioxidant activity and phenolic compounds, good acceptability, and purchase intention. CONCLUSIONS: The product has satisfactory market potential (acceptance rate of 95.19% and purchase intention rate > 96%), and it could become another means of inserting probiotics in food.


Assuntos
Mel , Sorvetes , Lacticaseibacillus casei , Probióticos , Zingiber officinale , Mel/análise , Zingiber officinale/química , Sorvetes/microbiologia , Sorvetes/análise , Lacticaseibacillus casei/química , Lacticaseibacillus casei/metabolismo , Probióticos/química , Humanos , Antioxidantes/química , Lactose/metabolismo , Trato Gastrointestinal/microbiologia , Armazenamento de Alimentos , Viabilidade Microbiana/efeitos dos fármacos
18.
Plant Foods Hum Nutr ; 79(2): 545-550, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38642193

RESUMO

Gluten-free foods (GF) availability on supermarket shelves is growing and it is expected to continue expanding in the years ahead. These foods have been linked to a lower content of fermentable oligosaccharides, disaccharides, monosaccharides and polyols (FODMAPs), molecules that trigger gastrointestinal symptoms in sensitive persons. In this study, the FODMAP content of 25 cereal-based GF foods in Spain (breakfast cereals, pasta, bread, biscuits, bakery products, and dough and puff pastry) and 25 gluten-containing equivalents (GC) available in the same supermarket were analysed and compared. Lactose, fructose, glucose, sorbitol, mannitol, raffinose, stachyose and fructans were quantified. In a like-by-like analysis, GF foods were found to generally contain fewer FODMAPs than their GC counterparts. The ingredients used in the manufacture of GF cereal-based foods may contribute to this fact. When the individually wrapped size was considered, the proportion of samples classified as high-FODMAPs in GC and GF foods showed a trend towards fewer samples in the GF. However, not all the GF samples were low-FODMAP. Altogether, our findings provide essential information for FODMAP content databases of GF products in Spain.


Assuntos
Dieta Livre de Glúten , Dissacarídeos , Grão Comestível , Glutens , Monossacarídeos , Oligossacarídeos , Polímeros , Grão Comestível/química , Espanha , Monossacarídeos/análise , Glutens/análise , Oligossacarídeos/análise , Dissacarídeos/análise , Polímeros/análise , Fermentação , Frutanos/análise , Lactose/análise , Pão/análise , Humanos , Rafinose/análise , Frutose/análise
19.
Int J Pharm ; 657: 124122, 2024 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-38621619

RESUMO

The aim of this study was to investigate how the propensity for aerosolisation in binary adhesive mixtures was affected by the drug load, and to determine whether these findings could be linked to different blend states. Binary blends of two different lactose carriers, each with varying size and morphology, were prepared together with budesonide. In vitro aerosolisation studies were conducted at four different pressure drops, ranging from 0.5 to 4 kPa, utilising a Next Generation Impactor. Several dispersion parameters were derived from the relationship between the quantity of dispersed API and the pressure drop. The evolution of the parameters with drug load was complex, especially at low drug loads. While similar responses were observed for both carriers, the range of drug load that could be used varied significantly. The choice of carrier not only influenced the capacity for drug loading but also affected the spatial distribution of the API within the mixture, which, in turn, affected its aerosolisation propensity. Thus, the drug dispersion process could be linked to different configurations of the lactose carrier and budesonide in the blends, i.e. blend states. In conclusion, the study suggests that the concept of blend states can provide an explanation for the complex dispersion process observed in adhesive blends.


Assuntos
Adesivos , Aerossóis , Budesonida , Portadores de Fármacos , Lactose , Budesonida/química , Budesonida/administração & dosagem , Lactose/química , Administração por Inalação , Adesivos/química , Portadores de Fármacos/química , Tamanho da Partícula , Química Farmacêutica/métodos
20.
Bioprocess Biosyst Eng ; 47(6): 919-929, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38644439

RESUMO

The growing need in the current market for innovative solutions to obtain lactose-free (L-F) milk is caused by the annual increase in the prevalence of lactose intolerance inside as well as the newborn, children, and adults. Various configurations of enzymes can yield two distinct L-F products: sweet (ß-galactosidase) and unsweet (ß-galactosidase and glucose oxidase) L-F milk. In addition, the reduction of sweetness through glucose decomposition should be performed in a one-pot mode with catalase to eliminate product inhibition caused by H2O2. Both L-F products enjoy popularity among a rapidly expanding group of consumers. Although enzyme immobilization techniques are well known in industrial processes, new carriers and economic strategies are still being searched. Polymeric carriers, due to the variety of functional groups and non-toxicity, are attractive propositions for individual and co-immobilization of food enzymes. In the presented work, two strategies (with free and immobilized enzymes; ß-galactosidase NOLA, glucose oxidase from Aspergillus niger, and catalase from Serratia sp.) for obtaining sweet and unsweet L-F milk under low-temperature conditions were proposed. For free enzymes, achieving the critical assumption, lactose hydrolysis and glucose decomposition occurred after 1 and 4.3 h, respectively. The tested catalytic membranes were created on regenerated cellulose and polyamide. In both cases, the time required for lactose and glucose bioconversion was extended compared to free enzymes. However, these preparations could be reused for up to five (ß-galactosidase) and ten cycles (glucose oxidase with catalase).


Assuntos
Enzimas Imobilizadas , Glucose Oxidase , Lactose , Leite , beta-Galactosidase , beta-Galactosidase/metabolismo , beta-Galactosidase/química , Leite/química , Lactose/metabolismo , Lactose/química , Glucose Oxidase/química , Glucose Oxidase/metabolismo , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Animais , Aspergillus niger/enzimologia , Glucose/metabolismo , Glucose/química , Catalase/metabolismo , Catalase/química , Membranas Artificiais
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