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.

Tolerance to acid and alkali by Streptococcus infantarius subsp. infantarius strain 25124 isolated from fermented nixtamal dough: Pozol. Studies in APT broth.

Pozol is a beverage prepared with maize dough made after boiling the kernels in limewater. This pretreatment could act as a selective force that shapes the starter microbiota, with microorganisms able to survive the fermentation. Since Streptococcus infantarius subsp. infantarius (Sii) dominates in pozol, we evaluated the effect of acid and alkali stresses on strain Sii-25124 in commercial APT broth as a first attempt to assess its adaptation capacity. Results suggest that Sii-25124 has adaptative advantages to pH changes that possibly contribute to its persistence even after the acidification of the dough. Its cardinal pH values were 4.0 and 11.0, with an optimum between 6.6 and 8.0. It showed alkali tolerance unlike other pozol Sii strains. Adaptation at pH 4.0, 10.0 and 11.0, compared with non-adapted cells, induced acid tolerance enhancing survival at pH 3.6 (P < 0.05); a 2 min heat shock at 62 °C induced alkali tolerance response enhancing survival at pH 10.5 (P < 0.05). The up-regulation of dnaK, groEL, ptsG and atpB was observed during 5 h of exposition at pH 3.6, 4.0 and 10.0, showing similar expression rates after induction by acid shock or alkaline stress. Changes of atpB were more evident having almost five-fold induction during long-term stress.

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.

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.

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.

Commercial probiotic bacteria and prebiotic carbohydrates: a fundamental study on prebiotics uptake, antimicrobials production and inhibition of pathogens.

BACKGROUND: Probiotics and prebiotics are among the most important functional food ingredients worldwide. The proven benefits of such ingredients to human health have encouraged the development of functional foods containing both probiotics and prebiotics. In this work, the production of antimicrobial compounds coupled to the uptake of commercial prebiotics by probiotic bacteria was investigated. RESULTS: The probiotic bacteria studied were able to take up commercial prebiotic carbohydrates to the same or higher extent than that observed for lactose (control carbohydrate). The growth of probiotic bacteria was coupled to the production of antimicrobials such as short-chain fatty acids (SCFA), H2 O2 and bacteriocins. A higher production of antimicrobial compounds was recorded with Oligomate 55® compared with Regulact® and Frutafit® (3-5 and 10-115 times higher SCFA and H2 O2 production, respectively). The probiotic bacteria grown with Oligomate 55® also produced bacteriocins and other non-identified antimicrobial compounds. The antimicrobials produced by the probiotic bacteria inhibited up to 50% the growth of model pathogens such as Escherichia coli, Listeria innocua and Micrococcus luteus compared with control cultures. CONCLUSIONS: The results here obtained are useful for the adequate selection of probiotic/prebiotics pairs and therefore in the development of efficient functional foods.

[The antihypertensive effect of fermented milks]. / El efecto antihipertensivo de las leches fermentadas.

There is a great variety of fermented milks containing lactic acid bacteria that present health-promoting properties. Milk proteins are hydrolyzed by the proteolytic system of these microorganisms producing peptides which may also perform other functions in vivo. These peptides are encrypted within the primary structure of proteins and can be released through food processing, either by milk fermentation or enzymatic hydrolysis during gastrointestinal transit. They perform different activities, since they act in the cardiovascular, digestive, endocrine, immune and nervous systems. Bioactive peptides that have an antihypertensive, antithrombotic, antioxidant and hypocholesterolemic effect on the cardiovascular system can reduce the risk factors for chronic disease manifestation and help improve human health. Most studied bioactive peptides are those which exert an antihypertensive effect by inhibiting the angiotensin-converting enzyme (ACE). Recently, the study of these peptides has focused on the implementation of tests to prove that they have an effect on health. This paper focuses on the production of ACEinhibitory antihypertensive peptides from fermented milks, its history, production and in vivo tests on rats and humans, on which its hypotensive effect has been shown.

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.

Impact of Heat-Killed Lactobacillus casei Strain IMAU60214 on the Immune Function of Macrophages in Malnourished Children.

Malnutrition is commonly associated with immunological deregulation, increasing the risk of infectious illness and death. The objective of this work was to determine the in vitro effects of heat-killed Lactobacillus casei IMAU60214 on monocyte-derived macrophages (MDMs) from well-nourished healthy children, well-nourished infected children and malnourished infected children, which was evaluated by an oxygen-dependent microbicidal mechanism assay of luminol-increase chemiluminescence and the secretion of tumor necrosis factor (TNF-α), interleukin (IL-1ß), IL-6 and IL-10, as well as phagocytosis using zymosan and as its antibacterial activity against Salmonella typhimurium, Escherichia coli and Staphylococcus aureus. We found that reactive oxygen species (ROS), secretion cytokines (TNFα, IL-1ß, IL-6 and IL-10 levels), phagocytosis and bactericidal capacity increased in all groups after pre-treatment with heat-killed L. casei IMAU60214 at a ratio of 500:1 (bacteria:MDM) over 24 h compared with MDM cells without pre-treatment. The results could indicate that heat-killed L. casei IMAU60214 is a potential candidate for regulating the immune function of macrophages.

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.

Evaluation of Immunomodulatory Activities of the Heat-Killed Probiotic Strain Lactobacillus casei IMAU60214 on Macrophages In Vitro.

Most Lactobacillus species have beneficial immunological ("immunoprobiotic") effects in the host. However, it is unclear how probiotic bacteria regulate immune responses. The present study investigated the effects of heat-killed Lactobacillus casei IMAU60214 on the activity of human monocyte-derived macrophages (MDMs). Human MDMs were treated with heat-killed L. casei at a ratio (bacteria/MDM) of 50:1, 100:1, 250:1, and 500:1, and then evaluated for the following: NO production, by Griess reaction; phagocytosis of FITC-labeled Staphylococcus aureus particles; cytokine secretion profile (tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, IL-6, IL-12p70, IL-10, and transforming growth factor (TGF)-ß) by ELISA; and costimulatory molecule (CD80 and CD86) surface expression, by flow cytometry. Heat-killed L. casei IMAU60214 enhanced phagocytosis, NO production, cytokine release, and surface expression of CD80 and CD86 in a dose-dependent manner. All products were previously suppressed by pretreatment with a Toll-like receptor 2 (TLR2)-neutralizing antibody. Overall, our findings suggest that this probiotic strain promotes an M1-like pro-inflammatory phenotype through the TLR2 signaling pathway. These effects on macrophage phenotype help explain the probiotic efficacy of Lactobacillus and provide important information for the selection of therapeutic targets and treatments compatible with the immunological characteristics of this probiotic strain.

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.

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).

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.

Determination of the secondary structure of Kluyveromyces lactis beta-galactosidase by circular dichroism and its structure-activity relationship as a function of the pH.

The secondary structure of Kluyveromyces lactis beta-galactosidase was determined by circular dichroism. It is mainly a beta-type protein, having 22% beta-turns, 14% parallel beta-sheet, 25% antiparallel beta-sheet, 34% unordered structure, and only 5% alpha-helix. The structure-activity relationship as a function of the pH was also studied. The pH conditions leading to the highest secondary structure content (100% ellipticity) of the enzyme was found at pH 7.0; at pH 6.5-7.0, the percent ellipticity decreased slightly, suggesting little structural change, but the activity decreased significantly, probably because of variations in critical residues. On the other hand, at pH's above 7.0, a more noticeable change in ellipticity was observed due to structural changes; the CD analysis showed a small increase in the helical content toward higher pH, whereas the maximum activity was found at pH 7.5, meaning that the changes produced in the secondary structure at this pH favored the interaction between the enzyme and the substrate.

Activity of the enzymes involved in the synthesis of exopolysaccharide precursors in an overproducing mutant ropy strain of Streptococcus thermophilus.

The activities of some enzymes belonging to the Leloir pathway, phosphoglucomutase, UDP-glucose pyrophosphorylase, UDP-galactose 4-epimerase and galactose 1-P uridyl transferase, were studied in a wild ropy, a non-ropy and an overproducing mutant ropy strain of Streptococcus thermophilus. These activities were assayed over successive culture transfers along with exocellular polysaccharide (EPS) production. The overproducing mutant ropy strain showed increments in polysaccharide production over successive culture transfers, as opposed to reductions in production by the wild ropy strain. The observed variations among strains in the enzyme activities that were analysed in relation to EPS production suggest their involvement in the synthesis of sugar-nucleotide EPS precursors.

ß-lactoglobulin's conformational requirements for ligand binding at the calyx and the dimer interphase: a flexible docking study.

ß-lactoglobulin (BLG) is an abundant milk protein relevant for industry and biotechnology, due significantly to its ability to bind a wide range of polar and apolar ligands. While hydrophobic ligand sites are known, sites for hydrophilic ligands such as the prevalent milk sugar, lactose, remain undetermined. Through the use of molecular docking we first, analyzed the known fatty acid binding sites in order to dissect their atomistic determinants and second, predicted the interaction sites for lactose with monomeric and dimeric BLG. We validated our approach against BLG structures co-crystallized with ligands and report a computational setup with a reduced number of flexible residues that is able to reproduce experimental results with high precision. Blind dockings with and without flexible side chains on BLG showed that: i) 13 experimentally-determined ligands fit the calyx requiring minimal movement of up to 7 residues out of the 23 that constitute this binding site. ii) Lactose does not bind the calyx despite conformational flexibility, but binds the dimer interface and an alternate Site C. iii) Results point to a probable lactolation site in the BLG dimer interface, at K141, consistent with previous biochemical findings. In contrast, no accessible lysines are found near Site C. iv) lactose forms hydrogen bonds with residues from both monomers stabilizing the dimer through a claw-like structure. Overall, these results improve our understanding of BLG's binding sites, importantly narrowing down the calyx residues that control ligand binding. Moreover, our results emphasize the importance of the dimer interface as an insufficiently explored, biologically relevant binding site of particular importance for hydrophilic ligands. Furthermore our analyses suggest that BLG is a robust scaffold for multiple ligand-binding, suitable for protein design, and advance our molecular understanding of its ligand sites to a point that allows manipulation to control binding.

Lactase production by solid-state cultivation of Kluyveromyces marxianus CDBBL 278 on an inert support: effect of inoculum, buffer, and nitrogen source.

A study was carried out to select the conditions for cultivation of Kluyveromyces marxianus CDBBL 278 in solid-state culture (SSC) using polyurethane foam (PUF) as an inert support. PUF was impregnated with culture media containing lactose (50 g/L) as the carbon and energy source. Evaluation of culture parameters during different growth phases was carried out by respirometry. The effect of inoculum level, buffer capacity of the medium, and nitrogen source upon the yield of biomass on lactose (Yx/s) and production of lactase and inulinase was investigated. The highest lactase titre was achieved with an inoculum level of 1 x 10(7) cells per gram of wet matter (gwm) and 20% of the total nitrogen source provided as urea. The best biomass yield (0.37) was obtained when less than 40% of the total nitrogen was provided as urea. Using potassium phosphate allowed 90% substrate consumption in 30 h. In the best conditions, intracellular lactase and extracellular inulinase activities of 1147.7 IU/gX and 241.6 IU/gX were obtained, respectively, with a lag phase of 13.8 h and a rate of respiratory activity (microCO2) of 0.23 +/- 0.01 h(-1). To our knowledge, this is the first report on lactase production by K. marxianus CDBBL 278 in SSC. This study gives basic information about biomass yield and enzyme production using lactose as the sole carbon source in SSC on an inert support.

Role of lysine epsilon-amino groups of beta-lactoglobulin on its activating effect of Kluyveromyces lactis beta-galactosidase.

Native beta-lactoglobulin binds and increases the activity of Kluyveromyces lactis beta-galactosidase. Construction of a three-dimensional (3D) model of beta-lactoglobulin showed that lysine residues 15, 47, 69, and 138 are the most exposed ones, thus the ones more likely to interact with beta-galactosidase. Molecular docking estimated the interaction energies of amino acid residues with either lactose or succinic anhydride, showing that Lys(138) is the most likely to react with both. Affinity chromatography demonstrated that succinylated beta-lactoglobulin diminished its ability to bind to the enzyme. Furthermore, when activity was measured in the presence of succinylated beta-lactoglobulin, its activating effect was lost. Since succinylation specifically blocks Lys epsilon-amino groups, their loss very likely causes the disappearance of the activating effect. Results show that the activating effect of beta-lactoglobulin on beta-galactosidase activity is due to the interaction between both proteins and that this interaction is very likely to occur through the Lys epsilon-amino groups of beta-lactoglobulin.

Influence of water activity in the synthesis of galactooligosaccharides produced by a hyperthermophilic beta-glycosidase in an organic medium.

The present study evaluated the influence of water activity and lactose concentration on the synthesis of galactooligosaccharides (GOS), by means of a hyperthermophilic beta-glycosidase in an organic system. The production of GOS gradually grew as water activity increased in the reaction system; later, their synthesis decreased as water activity increased. The authors used the response surface methodology to study how different water activities and different concentrations of lactose influenced the synthesis of GOS and their length. In every case, the variable that proved to have the greatest effect on GOS synthesis was water activity. Maximum GOS3 synthesis was reached at a water activity interval of 0.44-0.57, with lactose concentrations of 0.06%-0.1%, while GOS4 and GOS5 maxima were reached at water activity intervals of 0.47-0.57 and 0.49-0.60, respectively. The research showed that higher water activity was required to synthesize GOS of greater length. Synthesis of GOS would then depend on the flexibility of the enzyme, which in turn would depend on water activity of the reaction system. This hypothesis was supported by experiments in which the reaction temperature was modified in order to change the flexibility of the enzyme, thus leading to longer GOS.