Safety evaluation of 3'-siallylactose sodium salt supplementation on growth and clinical parameters in neonatal piglets.

Sialyllactose (SL) is an abundant oligosaccharide in human milk with health benefits that include intestinal maturation, gut microbiota modulation, and cognitive development. Recent technological advances support large scale production of different forms of sialyllactose, which will enable their use as a food ingredient. The objective of the study was to investigate the dose-dependent effects of novel enzymatically-synthesized 3'-sialyllactose (3'SL) sodium salt supplemented to swine milk replacer on growth, hematological parameters and tissue histology in a pre-clinical neonatal pig model. Forty-five two-day-old male and female pigs were provided one of four experimental diets for 21 days. Diets were formulated to contain 0 (CON), 140 (LOW), 200 (MOD) or 500 (HIGH) mg/L of 3'SL sodium salt. Samples were collected on days 8 and 22 of the study for hematological and histological analyses. The addition of 3'SL sodium salt to formula at all doses was well-tolerated by neonatal piglets and supported growth and development comparable to those observed in the CON group. In addition, serum chemistries as well as hematology and organ microscopic structure were unaffected by 3'SL (p > 0.05). These data provide supportive evidence for the safety of supplementation of this enzymatically-synthesized 3'SL sodium salt to human infant formula.

Toxicological evaluation of 6'-sialyllactose (6'-SL) sodium salt.

We performed a series of toxicity studies on the safety of 6'-sialyllactose (6'-SL) sodium salt as a food ingredient. 6'-SL sodium salt, up to a maximum dose of 5000 µg/plate, did not increase the number of revertant colonies in five strains of Salmonella typhimurium in the presence or absence of S9 metabolic activation. A chromosomal aberration assay (using Chinese hamster lung cells) found no clastogenic effects at any concentration of 6'-SL sodium salt in the presence or absence of S9 metabolic activation. An in vivo bone marrow micronucleus test in Kunming mice showed no clastogenic activities with 6'-SL sodium salt doses up to 2000 mg/kg body weight (bw). In an acute toxicity study, the mean lethal dose of 6'-SL sodium salt was greater than 20 g/kg bw in rats. In a 13-week subchronic toxicity investigation, no effects were found at doses up to 5.0 g/kg bw of 6'-SL sodium salt in food consumption, body weight, clinical signs, blood biochemistry and hematology, urinalysis, or ophthalmic and histological macroscopic examination of organs. The no-observed-adverse-effect level (NOAEL) was 5.0 g/kg bw/day in rats.

Erratum to: Synthesis of Curcumin Glycosides with Enhanced Anticancer Properties Using One-Pot Multienzyme Glycosylation Technique.

This erratum is being published to correct the author's contribution and name of above manuscript by Gurung et al. that was published in Journal of Microbiology and Biotechnology (2017, 27: 1639-1648). The first author (Rit Bahadur Gurung) and the second author (So Youn Gong) contributed equally to this article. And the seventh author (Tae Jin Oh) should appear as 'Tae-Jin Oh'.

Toxicological evaluation of 3'-sialyllactose sodium salt.

The safety of 3'-sialyllactose (3'-SL) sodium salt was evaluated by testing for gene mutations, in vivo and in vitro clastogenic activity, and animal toxicity in beagle dogs and rats. The results of all mutagenicity and genotoxicity tests were negative, indicating that 3'-SL does not have any mutagenic or clastogenic potential. The mean lethal dose (LD50) of 3'-SL sodium salt was well above 20 g/kg body weight (bw) in rats. A dose escalation acute toxicity study in Beagle dogs also indicated no treatment-related abnormalities. Subsequent 28-day and 90-day toxicity studies in Sprague- Dawley (SD) rats involved dietary exposure to 500, 1,000, and 2000 mg/kg bw of 3'-SL sodium salt and a water (vehicle) control. There were no treatment-related abnormalities on clinical observations, body weight, food consumption, behavior, hematology, clinical chemistry, organ weights, relative organ weights, urinalysis parameters, or necropsy and histopathological findings. The No Observed Adverse Effect Level (NOAEL) of 3'-SL sodium salt was determined to be higher than 2000 mg/kg bw/day in an oral subchronic toxicity study in rats, indicating that the substance is an ordinary carbohydrate with the lowest toxicity rating. Results confirm that 3'-SL sodium salt has a toxicity profile similar to other non-digestible carbohydrates and naturally occurring human milk oligosaccharides (HMOs) and support its safety for human consumption in foods.

Synthesis of Curcumin Glycosides with Enhanced Anticancer Properties Using One-Pot Multienzyme Glycosylation Technique.

Curcumin is a natural polyphenolic compound, widely acclaimed for its antioxidant, antiinflammatory, antibacterial, and anticancerous properties. However, its use has been limited due to its low-aqueous solubility and poor bioavailability, rapid clearance, and low cellular uptake. In order to assess the effect of glycosylation on the pharmacological properties of curcumin, one-pot multienzyme (OPME) chemoenzymatic glycosylation reactions with UDP- α-D-glucose or UDP-α-D-2-deoxyglucose as donor substrate were employed. The result indicated significant conversion of curcumin to its glycosylated derivatives: curcumin 4'-O-ß- glucoside, curcumin 4',4''-di-O-ß-glucoside, curcumin 4'-O-ß-2-deoxyglucoside, and curcumin 4',4''-di-O-ß-2-deoxyglucoside. The products were characterized by ultra-fast performance liquid chromatography, high-resolution quadruple-time-of-flight electrospray ionization-mass spectrometry, and NMR analyses. All the products showed improved water solubility and comparable antibacterial activities. Additionally, the curcumin 4'-O-ß-glucoside and curcumin 4'-O-ß-2-deoxyglucoside showed enhanced anticancer activities compared with the parent aglycone and diglycoside derivatives. This result indicates that glycosylation can be an effective approach for enhancing the pharmaceutical properties of different natural products, such as curcumin.

Donor specificity of YjiC glycosyltransferase determines the conjugation of cytosolic NDP-sugar in in vivo glycosylation reactions.

Escherichia coli BL21 (DE3) was engineered by blocking glucose-1-phosphate utilizing glucose phosphate isomerase (pgi), glucose-6-phosphate dehydrogenase (zwf) and uridylyltransferase (galU) genes to produce pool of four different rare dTDP-sugars. The cytosolic pool of dTDP-l-rhamnose, dTDP-d-viosamine, dTDP-4-amino 4,6-dideoxy-d-galactose, and dTDP-3-amino 3,6-dideoxy-d-galactose was generated by overexpressing respective dTDP-sugars biosynthesis genes from various microbial sources. A flexible glycosyltransferase YjiC, from Bacillus licheniformis DSM 13 was also overexpressed to transfer sugar moieties to 3-hydroxyl group of 3-hydroxyflavone, a core unit of flavonoids. Among four rare dTDP-sugars generated in cytosol of engineered strains, YjiC solely transferred l-rhamnose from dTDP-l-rhamnose and tuned to rhamnosyltransferase.

Enzymatic synthesis of lactosylated and sialylated derivatives of epothilone A.

Epothilone A is a derivative of 16-membered polyketide natural product, which has comparable chemotherapeutic effect like taxol. Introduction of sialic acids to these chemotherapeutic agents could generate interesting therapeutic glycoconjugates with significant effects in clinical studies. Since, most of the organisms biosynthesize sialic acids in their cell surface, they are key mediators in cellular events (cell-cell recognition, cell-matrix interactions). Interaction between such therapeutic sugar parts and cellular polysaccharides could generate interesting result in drugs like epothilone A. Based on this hypothesis, epothilone A glucoside (epothilone A 6-O-ß-D-glucoside) was further decorated by conjugating enzymatically galactose followed by sialic acids to generate epothilone A 7-O-ß-D-glucopyranosyl, 4'-O-α-D-galactoside i.e., lactosyl epothilone A (lac epoA) and two sialosides of epothilone A namely epothilone A 7-O-ß-D-glucopyranosyl, 4'-O-α-D-galactopyranosyl 3″-O-α-N-acetyl neuraminic acid and epothilone A 7-O-ß-D-glucopyranosyl, 4'-O-α-D-galactopyranosyl 6″-O-α-N-acetylneuraminic acid i.e., 3'sialyllactosyl epothilone A: 3'SL-epoA, and 6'sialyllactosyl epothilone A: 6'SL-epoA, respectively. These synthesized analogs were spectroscopically analyzed and elucidated, and biologically validated using HUVEC and HCT116 cancer cell lines.

Efficient enzymatic systems for synthesis of novel α-mangostin glycosides exhibiting antibacterial activity against Gram-positive bacteria.

Two enzymatic systems were developed for the efficient synthesis of glycoside products of α-mangostin, a natural xanthonoid exhibiting anti-oxidant, antibacterial, anti-inflammatory, and anticancer activities. In these systems, one-pot reactions for the synthesis of UDP-α-D-glucose and UDP-α-D-2-deoxyglucose were modified and combined with a glycosyltransferase (GT) from Bacillus licheniformis DSM-13 to afford C-3 and C-6 position modified glucose and 2-deoxyglucose conjugated novel α-mangostin derivatives. α-Mangostin 3-O-ß-D-glucopyranoside, α-mangostin 6-O-ß-D-glucopyranoside, α-mangostin 3,6-di-O-ß-D-glucopyranoside, α-mangostin 3-O-ß-D-2-deoxyglucopyranoside, α-mangostin 6-O-ß-D-2-deoxyglucopyranoside, and α-mangostin 3,6-di-O-ß-D-2-deoxyglucopyranoside were successfully produced in practical quantities and characterized by high-resolution quadruple time-of-flight electrospray ionization-mass spectrometry (HR-QTOF ESI/MS), (1)H and (13)C NMR analyses. In excess of the substrate, the maximum productions of three α-mangostin glucopyranosides (4.8 mg/mL, 86.5 % overall conversion of α-mangostin) and three α-mangostin 2-deoxyglucopyronosides (4.0 mg/mL, 79 % overall conversion of α-mangostin) were achieved at 4-h incubation period. All the α-mangostin glycosides exhibited improved water solubility, and their antibacterial activity against three Gram-positive bacteria Micrococcus luteus, Bacillus subtilis, and Staphylococcus aureus was drastically enhanced by the glucosylation at C-3 position. In this study, diverse glycosylated α-mangostin were produced in significant quantities by using inexpensive starting materials and recycling co-factors within a reaction vessel without use of expensive NDP-sugars in the glycosylation reactions.

Assessing acceptor substrate promiscuity of YjiC-mediated glycosylation toward flavonoids.

The acceptor substrate promiscuity of YjiC, a UDP-glycosyltransferase from Bacillus licheniformis, was explored with seven different classes (flavonols, flavanols, flavones, flavanones, chalcone, stilbene, and isoflavonoids) of 23 flavonoid acceptors. For most of the polyphenols used in the reactions, the enzymatic bioconversion was significantly higher with the production of multiple glucosylated derivatives. This study highlights the highly flexible non-regiospecific glycosylation ability of YjiC toward polyphenolic compounds. The catalytic potential of YjiC could be useful to generate a library of natural product glucosides.

Enzymatic synthesis of apigenin glucosides by glucosyltransferase (YjiC) from Bacillus licheniformis DSM 13.

Apigenin, a member of the flavone subclass of flavonoids, has long been considered to have various biological activities. Its glucosides, in particular, have been reported to have higher water solubility, increased chemical stability, and enhanced biological activities. Here, the synthesis of apigenin glucosides by the in vitro glucosylation reaction was successfully performed using a UDP-glucosyltransferase YjiC, from Bacillus licheniformis DSM 13. The glucosylation has been confirmed at the phenolic groups of C-4' and C-7 positions ensuing apigenin 4'-O-glucoside, apigenin 7-O-glucoside and apigenin 4',7-O-diglucoside as the products leaving the C-5 position unglucosylated. The position of glucosylation and the chemical structures of glucosides were elucidated by liquid chromatography/mass spectroscopy and nuclear magnetic resonance spectroscopy. The parameters such as pH, UDP glucose concentration and time of incubation were also analyzed during this study.