Haemophilus influenzae carriage and antibiotic resistance profile in Belgian infants over a three-year period (2016-2018).

Background: Non-typeable Haemophilus influenzae has become increasingly important as a causative agent of invasive diseases following vaccination against H. influenzae type b. The emergence of antibiotic resistance underscores the necessity to investigate typeable non-b carriage and non-typeable H. influenzae (NTHi) in children. Methods: Nasopharyngeal swab samples were taken over a three-year period (2016-2018) from 336 children (6-30 months of age) attending daycare centers (DCCs) in Belgium, and from 218 children with acute otitis media (AOM). Biotype, serotype, and antibiotic resistance of H. influenzae strains were determined phenotypically. Mutations in the ftsI gene were explored in 129 strains that were resistant or had reduced susceptibility to beta-lactam antibiotics. Results were compared with data obtained during overlapping time periods from 94 children experiencing invasive disease. Results: Overall, NTHi was most frequently present in both carriage (DCC, AOM) and invasive group. This was followed by serotype "f" (2.2%) and "e" (1.4%) in carriage, and "b" (16.0%), "f" (11.7%), and "a" (4.3%) in invasive strains. Biotype II was most prevalent in all studied groups, followed by biotype III in carriage and I in invasive strains. Strains from both groups showed highest resistance to ampicillin (26.7% in carriage vs. 18.1% in invasive group). A higher frequency of ftsI mutations were found in the AOM group than the DCC group (21.6 vs. 14.9% - p = 0.056). Even more so, the proportion of biotype III strains that carried a ftsI mutation was higher in AOM compared to DCC (50.0 vs. 26.3% - p < 0.01) and invasive group. Conclusion: In both groups, NTHi was most frequently circulating, while specific encapsulated serotypes for carriage and invasive group were found. Biotypes I, II and III were more frequently present in the carriage and invasive group. The carriage group had a higher resistance-frequency to the analyzed antibiotics than the invasive group. Interestingly, a higher degree of ftsI mutations was found in children with AOM compared to DCC and invasive group. This data helps understanding the H. influenzae carriage in Belgian children, as such information is scarce.

Polyols Induce the Production of Antifungal Compounds by Lactobacillus plantarum.

Mycotoxins may be present in nuts, coffee, cereals, and grapes, among other products. Increasing concerns about human health and environmental protection have driven the application of biological control techniques that can inhibit fungal contaminants. In this study, the growth inhibition of the ochratoxigenic fungus Aspergillus carbonarius Ac 162 was evaluated using 5 lactic acid bacteria (LAB). The LAB studied were Lactobacillus plantarum MZ801739 (J), Lactobacillus plantarum MZ809351 (31) and Lactobacillus plantarum MZ809350 (34), isolated in the Ivory Coast, and Lactobacillus plantarum MN982928 (3) and Leuconostoc citreum MZ801735 (23), isolated in Mexico. J, 31, 34, 3 and 23 are the internal strain codes from our laboratory. LAB were cultivated in De Man, Rogosa and Sharpe (MRS) broth, and different polyols (glycerol, mannitol, sorbitol, and xylitol) were added to the culture broth to stimulate the production of antifungal compounds. The fungal inhibition studies were performed using the poisoned food technique. The highest inhibition of A. carbonarius growth was obtained by cultivating L. plantarum MZ809351 in the presence of xylitol and glycerol. Under these conditions, 1 L of the L. plantarum MZ809351 cultures were used to identify antifungal compounds. The compounds were concentrated by solid-phase extraction and then characterized by GC-MS. In addition to 9-octadecenoic acid, 3 diketopiperazines or cyclic dipeptides were identified, including cyclo (Leu-Leu), cyclo (Pro-Gly) and cyclo (Val-Phe), which were compounds related to microbial antifungal activities. Xylitol and glycerol induced the production of these antifungal compounds against A. carbonarius Ac 162. On the other hand, adding xylitol and glycerol to the MRS broth reduced the Ochratoxin A (OTA) content to 56.8 and 54.7%, respectively. This study shows the potential for using L. plantarum MZ809351 as a biocontrol agent to prevent the growth of A. carbonarius and reduce the production of OTA in foods.

Antimicrobial susceptibility of commensal Neisseria in a general population and men who have sex with men in Belgium.

Non-pathogenic Neisseria are a reservoir of antimicrobial resistance genes for pathogenic Neisseria meningitidis and Neisseria gonorrhoeae. Men who have sex with men (MSM) are at risk of co-colonization with resistant non-pathogenic and pathogenic Neisseria. We assessed if the antimicrobial susceptibility of non-pathogenic Neisseria among MSM differs from a general population and if antimicrobial exposure impacts susceptibility. We recruited 96 participants at our center in Belgium: 32 employees, 32 MSM who did not use antibiotics in the previous 6 months, and 32 MSM who did. Oropharyngeal Neisseria were cultured and identified with MALDI-TOF-MS. Minimum inhibitory concentrations for azithromycin, ceftriaxone and ciprofloxacin were determined using E-tests® and compared between groups with non-parametric tests. Non-pathogenic Neisseria from employees as well as MSM were remarkably resistant. Those from MSM were significantly less susceptible than employees to azithromycin and ciprofloxacin (p < 0.0001, p < 0.001), but not ceftriaxone (p = 0.3). Susceptibility did not differ significantly according to recent antimicrobial exposure in MSM. Surveilling antimicrobial susceptibility of non-pathogenic Neisseria may be a sensitive way to assess impact of antimicrobial exposure in a population. The high levels of antimicrobial resistance in this survey indicate that novel resistance determinants may be readily available for future transfer from non-pathogenic to pathogenic Neisseria.

Novel bioactive peptides sequences released by in vitro digestion of proteins isolated from Amaranthus hypochondriacus.

Amaranth is a protein-rich seed, highly consumed in Mexico and reported as a potential source of bio-peptides with physiological function. Therefore, the objective of this study was to evaluate and identify bioactive peptide sequences from amaranth protein fractions released by pepsin and pancreatin in vitro digestion. Multifunctional bioactivities of peptides were measured through antihypertensive, antithrombotic and antioxidant assays. Peptide sequences were identified by analysis with a Nano-LC-ESI-MS/MS. With these results, it was demonstrated that the in vitro enzymatic digestion mechanism allows the release of multifunctional bioactive peptide fractions from amaranth proteins.

Improvement of Fucosylated Oligosaccharides Synthesis by α-L-Fucosidase from Thermotoga maritima in Water-Organic Cosolvent Reaction System.

The effects of water activity (aw), pH, and temperature on transglycosylation activity of α-L-fucosidase from Thermotoga maritima in the synthesis of fucosylated oligosaccharides were evaluated using different water-organic cosolvent reaction systems. The optimum conditions of transglycosylation reaction were the pH range between 7 and 10 and temperature 90-95 °C. The addition of organic cosolvent decreased α-L-fucosidase transglycosylation activity in the following order: acetone > dimethyl sulfoxide (DMSO) > acetonitrile (0.51 > 0.42 > 0.18 mM/h). However, the presence of DMSO and acetone enhanced enzyme-catalyzed transglycosylation over hydrolysis as demonstrated by the obtained transglycosylation/hydrolysis rate (rT/H) values of 1.21 and 1.43, respectively. The lowest rT/H was calculated for acetonitrile (0.59), though all cosolvents tested improved the transglycosylation rate in comparison to a control assay (0.39). Overall, the study allowed the production of fucosylated oligosaccharides in water-organic cosolvent reaction media using α-L-fucosidase from T. maritima as biocatalyst.

Synthesis of fucosylated oligosaccharides with α-L-fucosidase from Thermotoga maritima immobilized on Eupergit® CM.

Fucosylated oligosaccharides present in human milk perform various biological functions that benefit infants' health. These compounds can be also obtained by enzymatic synthesis. In this work, the effect of the immobilization of α-L-fucosidase from Thermotoga maritima on the synthesis of fucosylated oligosaccharides was studied, using lactose and 4-nitrophenyl-α-L-fucopyranoside (pNP-Fuc) as acceptor and donor substrates, respectively, and Eupergit® CM as an immobilization support. The enzyme was immobilized with 90% efficiency at pH 8 and ionic strength of 1.5 M. Immobilization decreased enzyme affinity for the donor substrate as shown by a 1.5-times higher KM value and a 22-times decrease of the kcat/KM ratio in comparison to the unbound enzyme. In contrast, no effect was observed on the synthesis/hydrolysis ratio (rs/rh) when α-L-fucosidase was immobilized. Also, the effect of initial concentration of substrates was studied. An increase of the acceptor concentration improved the yields of fucosylated oligosaccharides regardless enzyme immobilization. The synthesis yields of 38.9 and 40.6% were obtained using Eupergit® CM-bound or unbound enzyme, respectively, and 3.5 mM pNP-Fuc and 146 mM lactose. In conclusion, α-L-fucosidase from Thermotoga maritima was efficiently immobilized on Eupergit® CM support without affecting the synthesis of fucosylated oligosaccharides.

Lactobacillus pentosus ABHEAU-05: An in vitro digestión resistant lactic acid bacterium isolated from a traditional fermented Mexican beverage / Lactobacillus pentosus ABHEAU-05: una bacteria ácido láctica resistente a la digestión in vitro aislada de una bebida fermentada tradicional mexicana

Abstract The health benefits attributed to probiotics generate interest in the search of competent strains adapted to several ecological niches, especially those related to traditional beverages and foods of each country. Pineapple tepache, a traditional Mexican fermented beverage, was used for the isolation of lactic acid bacteria with probiotic potential, one of which withstood the in vitro tests. The isolated strain AB-05, which exhibited the tested probiotic functional properties, was designated as Lactobacillus pentosus ABHEAU-05. The sequence was registered in GenBank under access code MK587617. This study is the first report of a lactic acid bacterium with in vitro digestion resistance isolated from pineapple tepache. The survival of L. pentosus ABHEAU-05 in a symbiotic medium was proven using fermented milk enriched with inulin. The in vitro digestion-resistant probiotic activity of lactobacilli was measured through analysis of pH and proteolysis. Results showed that L. pentosus grew properly in fermented milk; therefore, this microorganism could be used in the manufacture of this kind of products. The concentration of L. pentosus reached up to 8.5 logCFU/ml after 40 h of fermentation. In addition, the production of peptides and the decrease in pH indicated the vigorous and active metabolic state of the lactic acid bacterium tested. The activity and the concentration of this microorganism were maintained during refrigeration. The results of this research conclude that L. plantarum ABHEAU-05 is an in vitro digestion-resistant microorganism that can be used as a starter culture for the production of functional foods of dairy origin.

Resumen Los beneficios a la salud atribuidos a los probióticos generan interés en la búsqueda de cepas competentes adaptadas a varios nichos ecológicos, especialmente los relacionados con bebidas y alimentos tradicionales de cada país. En este estudio, se aisló del tepache de pina, una bebida fermentada tradicional mexicana, una bacteria láctica resistente a la digestión in vitro. Entre 5 bacterias aisladas, una de ellas soportó las pruebas simuladas de digestión gastrointestinal. Se analizó la resistencia a sales biliares, a condiciones ácidas y al ataque enz-imático con pepsina. La bacteria aislada, que exhibió las propiedades funcionales probióticas referidas, fue identificada como Lactobacillus pentosus y designada como L. pentosus ABHEAU-05. La secuencia fue depositada en GenBank (acceso MK587617). Se comprobó la supervivencia de L. pentosus ABHEAU-05 en una leche fermentada adicionada con inulina y su resistencia a la digestión in vitro mediante el análisis del pH y la proteólisis. Los resultados muestran que la leche fermentada es una matriz adecuada, donde L. pentosus ABHEAU-05 se desarrolla sin inconvenientes, alcanzando un título de 8,5 logufc/ml después de 40 h de fermentación. Además, la producción de péptidos y el descenso del pH indicaron el estado metabólico vigoroso y activo del microorganismo probiótico. Se concluye que L. pentosus ABHEAU-05 es un microorganismo resistente a la digestión in vitro, que puede servir como cultivo iniciador para la producción de alimentos de origen lácteo. Este es el primer informe acerca del aislamiento de una bacteria ácido láctica resistente a la digestión in vitro a partir del tepache de piña.

Spectroscopic studies and molecular modelling of the aflatoxin M1-bovine α-lactalbumin complex formation.

Since the high incidence of aflatoxin M1 (AFM1) in milk and dairy products poses a serious risk to human health, this work aimed to investigate the complex formation between bovine α-lactalbumin (α-La) and AFM1 using different spectroscopic methods coupled with molecular docking studies. Fluorescence spectroscopy measurements demonstrated the AFM1 addition considerably reduced the α-La fluorescence intensity through a static quenching mechanism. The results indicated on the endothermic character of the reaction, and the hydrophobic interaction played a major role in the binding between AFM1 and α-La. The binding site stoichiometric value (n = 1.32) and a binding constant of 2.12 × 103 M-1 were calculated according to the Stern-Volmer equation. The thermodynamic parameters ΔH, ΔS and ΔGb were determined at 93.58 kJ mol-1, 0.378 kJ mol-1 K-1 and -19.17 ±â€¯0.96 kJ mol-1, respectively. In addition, far-UV circular dichroism studies revealed alterations in the α-La secondary structures when the α-La-AFM1 complex was formed. An increased content of the α-helix structures (from 35 to 40%) and the ß-sheets (from 16 to 19%) were observed. Furthermore, protein-ligand docking modelling demonstrated AFM1 could bind to the hydrophobic regions of α-La protein. Overall, the gathered results confirmed the α-La-AFM1 complex formation.

Development and Potential Usefulness of the COVID-19 Ag Respi-Strip Diagnostic Assay in a Pandemic Context.

Introduction: COVID-19 Ag Respi-Strip, an immunochromatographic (ICT) assay for the rapid detection of SARS-CoV-2 antigen on nasopharyngeal specimen, has been developed to identify positive COVID-19 patients allowing prompt clinical and quarantine decisions. In this original research article, we describe the conception, the analytical and clinical performances as well as the risk management of implementing the COVID-19 Ag Respi-Strip in a diagnostic decision algorithm. Materials and Methods: Development of the COVID-19 Ag Respi-Strip resulted in a ready-to-use ICT assay based on a membrane technology with colloidal gold nanoparticles using monoclonal antibodies directed against the SARS-CoV and SARS-CoV-2 highly conserved nucleoprotein antigen. Four hundred observations were recorded for the analytical performance study and thirty tests were analyzed for the cross-reactivity study. The clinical performance study was performed in a retrospective multi-centric evaluation on aliquots of 328 nasopharyngeal samples. COVID-19 Ag Respi-Strip results were compared with qRT-PCR as golden standard for COVID-19 diagnostics. Results: In the analytical performance study, the reproducibility showed a between-observer disagreement of 1.7%, a robustness of 98%, an overall satisfying user friendliness and no cross-reactivity with other virus-infected nasopharyngeal samples. In the clinical performance study performed in three different clinical laboratories during the ascendant phase of the epidemiological curve, we found an overall sensitivity and specificity of 57.6 and 99.5%, respectively with an accuracy of 82.6%. The cut-off of the ICT was found at CT <22. User-friendliness analysis and risk management assessment through Ishikawa diagram demonstrate that COVID-19 Ag Respi-Strip may be implemented in clinical laboratories according to biosafety recommendations. Conclusion: The COVID-19 Ag Respi-Strip represents a promising rapid SARS-CoV-2 antigen assay for the first-line diagnosis of COVID-19 in 15 min at the peak of the pandemic. Its role in the proposed diagnostic algorithm is complementary to the currently-used molecular techniques.

Lactobacillus pentosus ABHEAU-05: An in vitro digestion resistant lactic acid bacterium isolated from a traditional fermented Mexican beverage.

The health benefits attributed to probiotics generate interest in the search of competent strains adapted to several ecological niches, especially those related to traditional beverages and foods of each country. Pineapple tepache, a traditional Mexican fermented beverage, was used for the isolation of lactic acid bacteria with probiotic potential, one of which withstood the in vitro tests. The isolated strain AB-05, which exhibited the tested probiotic functional properties, was designated as Lactobacillus pentosus ABHEAU-05. The sequence was registered in GenBank under access code MK587617. This study is the first report of a lactic acid bacterium with in vitro digestion resistance isolated from pineapple tepache. The survival of L. pentosus ABHEAU-05 in a symbiotic medium was proven using fermented milk enriched with inulin. The in vitro digestion-resistant probiotic activity of lactobacilli was measured through analysis of pH and proteolysis. Results showed that L. pentosus grew properly in fermented milk; therefore, this microorganism could be used in the manufacture of this kind of products. The concentration of L. pentosus reached up to 8.5logCFU/ml after 40h of fermentation. In addition, the production of peptides and the decrease in pH indicated the vigorous and active metabolic state of the lactic acid bacterium tested. The activity and the concentration of this microorganism were maintained during refrigeration. The results of this research conclude that L. plantarum ABHEAU-05 is an in vitro digestion-resistant microorganism that can be used as a starter culture for the production of functional foods of dairy origin.

An Electronic Health Intervention for Latina Women Undergoing Breast Cancer Treatment (My Guide for Breast Cancer Treatment): Protocol for a Randomized Controlled Trial.

BACKGROUND: Among Latinas and Hispanics (henceforth referred to as Latinas), breast cancer is the most commonly diagnosed cancer and the leading cause of cancer-related deaths. However, few interventions have been developed to meet the needs of Latina women undergoing active treatment for breast cancer. OBJECTIVE: This paper aims to describe the procedures and methods of My Guide for Breast Cancer Treatment and the plans for conducting a multisite randomized controlled trial to investigate the feasibility and preliminary efficacy of this smartphone-based app for Latina women in active treatment for breast cancer. METHODS: Study participants will be randomized to the My Guide for Breast Cancer Treatment intervention or the enhanced usual care control condition for 12 weeks. Participants will have access to innovative features such as gamification via virtual awards to reinforce usage and an adaptive section that presents targeted material based on their self-reported concerns and needs. Using a stepped-care approach, intervention participants will also receive telecoaching to enhance their adherence to the app. Study outcomes and intervention targets will be measured at study enrollment (before randomization), 6 and 12 weeks after initial app use. General and disease-specific health-related quality of life (HRQoL) and symptom burden are the study's primary outcomes, whereas anxiety, depression, fear of cancer recurrence, physical activity, and dietary intake are secondary outcomes. RESULTS: Recruitment began in August 2019 and is expected to be completed by August 2020. We expect to submit study results for publication by fall 2020. CONCLUSIONS: My Guide for Breast Cancer Treatment has the potential to improve HRQoL and reduce symptom burden, and increase access to supportive care resources among Latina breast cancer patients. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): PRR1-10.2196/14339.

Sequence Identification of Bioactive Peptides from Amaranth Seed Proteins (Amaranthus hypochondriacus spp.).

Amaranthus hypochondriacus spp. is a commonly grown cereal in Latin America, known for its high protein content. The objective of this study was to separate and identify bioactive peptides found in amaranth seeds through enzymatically-assisted hydrolysis using alcalase and flavourzyme. Hydrolysis was carried out for each enzyme separately and compared to two-step continuous process where both enzymes were combined. The biological activity of the resulting three hydrolysates was analyzed, finding, in general, higher bioactive potential of the hydrolysate obtained in a continuous process (combined enzymes). Its fractions were separated by RP-HPLC, and their bioactivity was analyzed. In particular, two fractions showed the highest biological activity as ACE inhibitors with IC50 at 0.158 and 0.134, thrombin inhibitors with IC50 of 167 and 155, and antioxidants in ABTS assay with SC50 at 1.375 and 0.992 mg/L, respectively. Further sequence analysis of the bioactive peptides was carried out using MALDI-TOF, which identified amino acid chains that have not been reported as bioactive so far. Bibliographic survey allowed identification of similarities between peptides reported in amaranth and other proteins. In conclusion, amaranth proteins are a potential source of peptides with multifunctional activity.

Bigger and Better? Representativeness of the Influenza A Surveillance Using One Consolidated Clinical Microbiology Laboratory Data Set as Compared to the Belgian Sentinel Network of Laboratories.

Infectious diseases remain a serious public health concern globally, while the need for reliable and representative surveillance systems remains as acute as ever. The public health surveillance of infectious diseases uses reported positive results from sentinel clinical laboratories or laboratory networks, to survey the presence of specific microbial agents known to constitute a threat to public health in a given population. This monitoring activity is commonly based on a representative fraction of the microbiology laboratories nationally reporting to a single central reference point. However, in recent years a number of clinical microbiology laboratories (CML) have undergone a process of consolidation involving a shift toward laboratory amalgamation and closer real-time informational linkage. This report aims to investigate whether such merging activities might have a potential impact on infectious diseases surveillance. Influenza data was used from Belgian public health surveillance 2014-2017, to evaluate whether national infection trends could be estimated equally as effectively from only just one centralized CML serving the wider Brussels area (LHUB-ULB). The overall comparison reveals that there is a close correlation and representativeness of the LHUB-ULB data to the national and international data for the same time periods, both on epidemiological and molecular grounds. Notably, the effectiveness of the LHUB-ULB surveillance remains partially subject to local regional variations. A subset of the Influenza samples had their whole genome sequenced so that the observed epidemiological trends could be correlated to molecular observations from the same period, as an added-value proposition. These results illustrate that the real-time integration of high-throughput whole genome sequencing platforms available in consolidated CMLs into the public health surveillance system is not only credible but also advantageous to use for future surveillance and prediction purposes. This can be most effective when implemented for automatic detection systems that might include multiple layers of information and timely implementation of control strategies.

Synthesis of Fucosyl-Oligosaccharides Using α-l-Fucosidase from Lactobacillus rhamnosus GG.

Fucosyl-oligosaccharides are natural prebiotics that promote the growth of probiotics in human gut and stimulate the innate immune system. In this work, the release of α-lfucosidase by Lactobacillus rhamnosus GG, and the use of this enzyme for the synthesis of fucosyl-oligosaccharides were investigated. Since α-lfucosidase is a membrane-bound enzyme, its release from the cells was induced by addition of 4-nitrophenyl-α-l-fucopyranoside (pNP-Fuc). Enzyme activity associated with the cell was recovered at 78% of its total activity. Fucosyl-oligosaccharides where synthesized using α-l-fucosidase extract and pNP-Fuc as donor substrate, and D-lactose or D-lactulose as acceptor substrates, reaching a yield up to 25%. Fucosyllactose was obtained as a reaction product with D-lactose, and its composition was confirmed by mass spectrometry (MALDI-TOF MS). It is possible that the fucosyl-oligosaccharide synthesized in this study has biological functions similar to human milk oligosaccharides.

Bioactivity of Peptides Released During Lactic Fermentation of Amaranth Proteins with Potential Cardiovascular Protective Effect: An In Vitro Study.

Fermentation has shown to be an effective technique in bioactive peptides release. That is why in this study antihypertensive, antithrombotic, and antioxidant activity was evaluated during amaranth proteins fermentation with Lactobacillus casei Shirota and Streptococcus thermophilus 54102 in mono and combined culture. During fermentation an increase of free amine groups was observed, and no statistical differences among monocultures were shown, getting higher concentration in combined culture. This was related to antihypertensive and antioxidant activities, where the highest values were also found in the combined process (45% of angiotensin-converting enzyme inhibition, and 168 µmol Trolox equivalents per liter [TE/L] for 2,2-diphenyl-1-picrylhydrazyl, 268 µmol TE/L for 2,2'-azino-bis 3-ethylbenzothiazoline-6-sulfonic acid, and 381 µmol Fe2E/L for ferric reducing ability of plasma). On the contrary, antithrombotic activity was not related to free amine groups during fermentation, having the highest bioactivity in different moments in each experiment. L. casei Shirota and S. thermophilus 54102 are strains that are able to release bioactive peptides from amaranth protein, although amaranth is not a common matrix for the development of lactic acid bacteria. In addition, in this study it was observed for the first time that lactic acid strains are able to release bioactive peptides from amaranth protein. In addition, this methodology could be part for the development of fermented beverages, different from fermented milk, to diversify matrix to obtain a novel functional food.

Synthesis of Fucose-Containing Disaccharides by Glycosylhydrolases from Various Origins.

Glycosylhydrolases of various origins were used to produce fucose-containing disaccharides with prebiotic potential using different donor substrates and L-fucose as the acceptor substrate. Eight different disaccharides were synthesized as follows: three ß-D-galactosyl-L-fucosides with glycosidase CloneZyme Gly-001-02 using D-lactose as a donor substrate, two with a structure similar to prebiotics; one ß-D-galactosyl-L-fucose with ß-D-galactosidase from Aspergillus oryzae using D-lactose as a substrate donor; and four α-D-glucosyl-L-fucosides with α-D-glucosidase from Saccharomyces cerevisiae using D-maltose as a donor substrate. All disaccharides were purified and hydrolyzed. In all cases, an L-fucose moiety was present, and it was confirmed for ß-D-galactosyl-L-fucose by mass spectrometry. High concentrations of L-fucose as the acceptor substrate enhanced the synthesis of the oligosaccharides in all cases. The three enzymes were able to synthesize fucose-containing disaccharides when L-fucose was used as the acceptor substrate, and the highest yield was 20% using ß-D-galactosidase from Aspergillus oryzae.

Employment of fucosidases for the synthesis of fucosylated oligosaccharides with biological potential.

Fucosylated oligosaccharides play important physiological roles in humans, including in the immune response, transduction of signals, early embryogenesis and development, growth regulation, apoptosis, pathogen adhesion, and so on. Efforts have been made to synthesize fucosylated oligosaccharides, as it is difficult to purify them from their natural sources, such as human milk, epithelial tissue, blood, and so on. Within the strategies for its in vitro synthesis, it is remarkable the employment of fucosidases, enzymes that normally cleave the fucosyl residue from the non-reducing end of fucosylated compounds, as these enzymes are also capable of synthesizing them by means of a transfucosylation reaction. This review summarizes the progress in the use of fucosidases for the synthesis of compounds that have potential for industrial and commercial applications.

Improvement of the transfucosylation activity of α-L-fucosidase from Thermotoga maritima for the synthesis of fucosylated oligosaccharides in the presence of calcium and sodium.

The influence of CaCl2 and NaCl in the hydrolytic activity and the influence of CaCl2 in the synthesis of fucosylated oligosaccharides using α-L-fucosidase from Thermotoga maritima were evaluated. The hydrolytic activity of α-L-fucosidase from Thermotoga maritima displayed a maximum increase of 67% in the presence of 0.8 M NaCl with water activity (aw) of 0.9672 and of 138% in the presence of 1.1 M CaCl2 (aw 0.9581). In addition, the hydrolytic activity was higher when using CaCl2 compared to NaCl at aw of 0.8956, 0.9581 and 0.9672. On the other hand, the effect of CaCl2 in the synthesis of fucosylated oligosaccharides using 4-nitrophenyl-fucose as donor substrate and lactose as acceptor was studied. In these reactions, the presence of 1.1 M CaCl2 favored the rate of transfucosylation, and improved the yield of synthesis duplicating and triplicating it with lactose concentrations of 58 and 146 mM, respectively. CaCl2 did not significatively affect hydrolysis rate in these reactions. The combination of the activating effect of CaCl2, the decrement in aw and lactose concentration had a synergistic effect favoring the synthesis of fucosylated oligosaccharides.

Effect of thermoultrasound on aflatoxin M1 levels, physicochemical and microbiological properties of milk during storage.

The aim of this research was to determine the physicochemical properties, microbial counts and aflatoxin M1 (AFM1) levels of thermoultrasonicated, pasteurized and untreated milk (control) at days 1, 7 and 14 of storage. Thermoultrasound treatment was performed at a rate of 20 kHz for 10 or 15 min and 95% amplitude on homogenized and non-homogenized milk samples. Results showed that most physicochemical parameters were within the Mexican norms established for milk. Ultrasound treatment for 15 min reduced solids precipitation (p < 0.05) in unhomogenized milk during storage as compared to the pasteurized milk. All samples complied with aerobic mesophilic counts limits set by the Mexican norm except the control and the homogenized milk sample which was thermoultrasonicated for 10 min. Enterobacteriaceae counts of pasteurized and 15 min-thermoultrasound homogenized milks complied with the norm. The lowest levels of AFM1 were found in the 10 min-thermoultrasound unhomogenized milk (0.15 ±â€¯0.05 pg AFM⁠1E/mL) one day after storage. Thermoultrasound did not affect the color of samples but homogenized milk treated for 10 min exhibited less total color difference. A high phenolic content was found in thermoultrasound and pasteurized milks on day 1. Thermoultrasound could be an alternative to milk pasteurization that preserves the physicochemical and microbiological quality of milk while reducing AFM1 levels.

Synthesis of a Fucosylated Trisaccharide Via Transglycosylation by α-L-Fucosidase from Thermotoga maritima.

Fucosylated oligosaccharides, such as 2'-fucosyllactose in human milk, have important biological functions such as prebiotics and preventing infection. In this work, the effect of an acceptor substrate (lactose) and the donor substrate 4-nitrophenyl-α-L-fucopyranoside (pNP-Fuc) on the synthesis of a fucosylated trisaccharide was studied in a transglycosylation reaction using α-L-fucosidase from Thermotoga maritima. Conducting a matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), it was demonstrated that synthesized oligosaccharide corresponded to a fucosylated trisaccharide, and high-performance liquid chromatography (HPLC) of the hydrolyzed compound confirmed it was fucosyllactose. As the concentration of the acceptor substrate increased, the concentration and synthesis rate of the fucosylated trisaccharide also increased, and the highest concentration obtained was 0.883 mM (25.2% yield) when using the higher initial lactose concentration (584 mM). Furthermore, the lower donor/acceptor ratio had the highest synthesis, so at the molar ratio of 0.001, a concentration of 0.286 mM was obtained (32.5% yield).