Prebiotic Oligosaccharides Research Articles

A-049 Effect of Supplementation With Prebiotics on Levels of 25-Hydroxy Vitamin D and on Biomarkers of Bone Remodelling-Results From the OSTEOME Clinical Trial

Abstract Background It is proven that intestinal microbes regulate bone metabolism and can affect the quality of the bone. Prebiotics contain no microorganisms, only substances stimulating their growth. Moreover, flavonoids and catechines are transformed by the microbes into bioactive compounds and polyphenols can modify the microflora composition. OSTEOME is a randomized double-blinded clinical trial to evaluate the efficacy of dietary supplements enriched with prebiotics, in advancing the role of gut microbiota in bone remodelling. We present the effect of supplementation with prebiotics on the serum levels of 25-hydroxy vitamin D (25OHD) and on levels bone markers Osteocalcin (OCN), and C-Terminal Telopeptide of Type-I Collagen (CTX). Methods We enrolled 120 postmenopausal women with osteopenia. Participants were randomized into three groups of 40-participants. Group-A received a supplement containing 600mg calcium citrate and 2400IUof vitamin D3 only, group-B received a supplement additionally containing flavonoids (120mg Naringin and 120mg Hesperidine) and group-C received additionally prebiotic oligosaccharides (2000mg fructooligosaccharides and acacia gum). Blood collections were performed at baseline and at 3, 6 and 9 months for the measurement of bone remodelling biomarkers. Levels of serum Calcium (sCa), 25OHD, as well as levels bone markers OCN, and CTX were measured at all timepoints of follow-up. Results The mean age of group-A, group-B and group-C participants was 60.72(+8.08), 62.37(+7.70) and 62.09(+7.09) (p=NS), and the mean BMIs were 25.35(+3.96), 25.89(+3.80) and 26.25(+4.56) (p=NS). All women were osteopenic as mean total hip T-score was -1.282(+0.617), -1.193(+0.659) and -1.273(+0.573) at baseline for all three groups respectively. The results of OCN, CTX and 25OHD are shown in the table. Conclusions Only patients in group-C showed significant changes in CTX and 25OHD at 9-months, and OCN at 6- and 9-months. Addition of prebiotics to supplements might improve the vitamin D absorption from the gut and give positive balance to biomarkers of bone metabolism.

Comparative Study of Prebiotics for Infants Using a Fecal Culture System: Insights into Responders and Non-Responders

Background: The gut microbiota of breast-fed infants is dominated by infant-type human-residential bifidobacteria (HRB) that contribute to infant health; thus, it is crucial to develop infant formulas that promote the establishment of a gut microbiota enriched with infant-type HRB, closely resembling that of breastfed infants. Methods: We compared various non-digestible prebiotic oligosaccharides and their combinations using a fecal culture system to explore which candidates could promote the growth of all infant-type HRB and rarely yield non-responders. The analysis included lactulose (LAC), raffinose (RAF), galactooligosaccharides (GOS), and short- and long-chain fructooligosaccharides. Fecal samples were collected from seven infants aged 1.5–10.2 months and cultured with each oligosaccharide individually or their combinations. Results: No single oligosaccharide effectively promoted the growth of all infant-type HRB, although GOS promoted the growth of HRB other than Bifidobacterium longum subsp. longum. Only the LAC/RAF/GOS group evenly and effectively promoted the growth of all infant-type HRB. Accordingly, acetate production was higher in fecal cultures supplemented with GOS or LAC/RAF/GOS than in the other cultures, suggesting that it is a superior combination for all infant-type HRB and rarely yields non-responders. Conclusions: This study can aid in developing infant formulas that help align the gut microbiota of formula-fed infants with that of breastfed infants.

In Vitro Utilization of Prebiotics by Listeria monocytogenes.

Listeria monocytognes is an emerging pathogen responsible for the serious foodborne disease, listeriosis. The commensal gut microbiota is the first line of defense against pathogen internalization. The gut microbiome can be modified by prebiotic substrates, which are frequently added to food products and dietary supplements. Prebiotics should selectively support the growth of beneficial microbes and thus improve host health. Nevertheless, little is known about their effect on the growth of L. monocytogenes. The aim of this study was to evaluate the growth ability of four L. monocytogenes strains, representing the most common serotypes, on prebiotic oligosaccharides (beta-(1,3)-D-glucan, inulin, fructooligosaccharides, galactooligosaccharides, lactulose, raffinose, stachyose and 2'-fucosyllactose and a mixture of human milk oligosaccharides) as a sole carbon source. The results showed that only beta-(1,3)-D-glucan was metabolized by L. monocytogenes. These cell culture data suggest that beta-(1,3)-D-glucan may not be selectively utilized by healthy commensal bacteria, and its role in intestinal pathogen growth warrants further exploration in vivo.

Exploring Levansucrase Operon Regulating Levan-Type Fructooligosaccharides (L-FOSs) Production in Priestia koreensis HL12.

Levan biopolymer and levan-type fructooligosaccharides (L-FOSs) are β-2,6-linked fructans that have been used as non-digestible dietary fiber and prebiotic oligosaccharides in food and cosmeceutical applications. In this study, we explore the operon responsible for levan and L-FOSs production in Priestia koreensis HL12. Presented is the first genomic perspective on sucrose utilization and the levan biosynthesis pathway in this bacterium. Regarding sequence annotation, the putative levansucrase operon responsible for β-2,6-linked fructan was identified in the genome of strain HL12, and comprises sacB levansucrase gene belonging to GH68, located adjacent to levB endo-levanase gene, which belongs to GH32. Importantly, sugars related with the levan biosynthesis pathway are proposed to be transported via 3 types of transportation systems, including multiple ABCSugar and glucose/H+ transporters, as well as glucose- and fructose-specific PTS systems. Based on product profile analysis, the HL12 strain exhibited high efficiency in levan production from high sucrose concentration (300 g/l), achieving the highest yield of 127 g/l (equivalent to 55% conversion based on sucrose consumption), together with short-chain L-FOSs (DP3-5) and long-chain L-FOSs with respective size larger than DP6 after 48 h incubation. These findings highlight the potential of P. koreensis HL12 as a whole-cell biocatalyst for producing levan and L-FOSs, and underscore its novelty in converting sugars into high-value-added products for diverse commercial and industrial applications.

Selectivities of the β-galactosidase of Bacillus circulans in the production of galactooligosaccharides, lactulose and lactosucrose

There has been recent interest in using the β-galactosidase of Bacillus circulans to produce prebiotic oligosaccharides, including galactooligosaccharides (GOS), lactulose and lactosucrose. The types and amounts of the transgalactosylation products produced depend on the selectivities of the enzyme for the various transgalactosylation and hydrolysis reactions that occur. To date, the selectivities of the β-galactosidase of B. circulans for these reactions have not been adequately characterized. In the current work, we undertake four case studies using literature data for systems in which different product mixtures are produced (i) GOS; (ii) GOS and disaccharides; (iii) GOS and lactulose; and (iv) GOS and lactosucrose. We analyze this data to obtain quantitative estimates of the relevant selectivities. We show that, in the production of GOS, the isoform β-Gal-A is significantly less selective against hydrolysis reactions than the other β-galactosidase isoforms. In the production of lactulose, the β-galactosidase of B. circulans gives high lactulose yields, despite having a 19- to 33-fold preference for producing GOS over lactulose. In the production of lactosucrose, the β-galactosidase of B. circulans also prefers to produce GOS.

Application of hollow fiber membranes for producing an ultrafiltration permeate from colostrum whey enriched in bioactive compounds

Bovine colostrum whey, despite being a source of bioactive peptides and prebiotic oligosaccharides with dietary and nutraceutical applications, presents significant processing challenges due to its high viscosity, making hollow fiber membranes, with their superior ability to control membrane fouling, well-suited for handling such fluids. The objective of this work was to determine optimal ultrafiltration conditions of a 10 kDa hollow fiber membrane to increase efficiency (i.e., permeate flux), selectively retain whey proteins, and facilitate the permeation of peptides (< 10,000 Da) and oligosaccharides (∼600 Da) from bovine colostrum whey. The impact of feed flow rate (3, 6, 9 L min-1) and transmembrane pressure (1, 2, 3 bar) were evaluated on permeate flux, protein retention, and non-protein nitrogen permeation. Higher permeate fluxes were observed at higher feed flows and moderate transmembrane pressure, demonstrating the ability of turbulence to disrupt the concentration polarization layer at the membrane surface and decrease fouling, as exhibited in the resistance analyses. Higher feed flow significantly increased the cumulative permeate flux during colostrum whey concentration (64.6 ± 0.1 L h-1m-2 at 9 L min-1 vs. 12.5 ± 5.9 L h-1m-2 at 3 L min-1, at continuous diafiltration mode and 2 bar), with continuous diafiltration experiments yielding similar permeate fluxes to the ones achieved in discontinuous mode. Processing scale at 12.7 L (2 bar, 9 L min-1, and continuous diafiltration) enabled 97% protein retention and 95% peptide and oligosaccharide permeation, producing a permeate with 1.3 g L-1 peptide and 0.42 g L-1 oligosaccharide. The results of this study highlight the impact of key ultrafiltration conditions of bovine colostrum whey using a hollow fiber membrane for the effective recovery of peptides and oligosaccharides for subsequent evaluation of their biological properties and commercial applications.

Combined effect of oligosaccharides combination on the growth of probiotics: synergistic or superposable?

SummarySynbiotics confer benefits to our health. Study on the promotion effects of prebiotics combinations on probiotics growth is necessary for synbiotics preparation. However, there exists few reports on how to combine different prebiotics. The aim of the study was to explore combined effect of prebiotics combinations in modulating probiotics growth for rational synbiotics development. Four prebiotic oligosaccharides were screened for their promotion effects on the growth of four representative probiotics. Then, SynergyFinder 3.0 was used for the first time to rationally analyse interaction of various oligosaccharides combinations. Results showed that there was no synergism between any of two oligosaccharides under the conditions studied, but rather a superposition effect. Finally, the optimal oligosaccharides combinations based on the maximum specific growth rate and the maximum cell yield were selected. This study advanced our knowledge of oligosaccharides utilisation by probiotics and provided guidance on the rational selection of oligosaccharides combinations for specific probiotics, which was important for the design of synergistic synbiotics.

Surface-Displayed Mannanolytic and Chitinolytic Enzymes Using Peptidoglycan Binding LysM Domains.

Using Lactiplantibacillus plantarum as a food-grade carrier to create non-GMO whole-cell biocatalysts is gaining popularity. This work evaluates the immobilization yield of a chitosanase (CsnA, 30 kDa) from Bacillus subtilis and a mannanase (ManB, 40 kDa) from B. licheniformis on the surface of L. plantarum WCFS1 using either a single LysM domain derived from the extracellular transglycosylase Lp_3014 or a double LysM domain derived from the muropeptidase Lp_2162. ManB and CsnA were fused with the LysM domains of Lp_3014 or Lp_2162, produced in Escherichia coli and anchored to the cell surface of L. plantarum. The localization of the recombinant proteins on the bacterial cell surface was successfully confirmed by Western blot and flow cytometry analysis. The highest immobilization yields (44-48%) and activities of mannanase and chitosanase on the displaying cell surface (812 and 508 U/g of dry cell weight, respectively) were obtained when using the double LysM domain of Lp_2162 as an anchor. The presence of manno-oligosaccharides or chito-oligosaccharides in the reaction mixtures containing appropriate substrates and ManB or CsnA-displaying cells was determined by high-performance anion exchange chromatography. This study indicated that non-GMO Lactiplantibacillus chitosanase- and mannanase-displaying cells could be used to produce potentially prebiotic oligosaccharides.

Two-domain GH30 xylanase from human gut microbiota as a tool for enzymatic production of xylooligosaccharides: Crystallographic structure and a synergy with GH11 xylosidase

Production of value-added compounds and sustainable materials from agro-industrial residues is essential for better waste management and building of circular economy. This includes valorization of hemicellulosic fraction of plant biomass, the second most abundant biopolymer from plant cell walls, aiming to produce prebiotic oligosaccharides, widely explored in food and feed industries. In this work, we conducted biochemical and biophysical characterization of a prokaryotic two-domain R. champanellensis xylanase from glycoside hydrolase (GH) family 30 (RcXyn30A), and evaluated its applicability for XOS production from glucuronoxylan in combination with two endo-xylanases from GH10 and GH11 families and a GH11 xylobiohydrolase. RcXyn30A liberates mainly long monoglucuronylated xylooligosaccharides and is inefficient in cleaving unbranched oligosaccharides. Crystallographic structure of RcXyn30A catalytic domain was solved and refined to 1.37 Å resolution. Structural analysis of the catalytic domain releveled that its high affinity for glucuronic acid substituted xylan is due to the coordination of the substrate decoration by several hydrogen bonds and ionic interactions in the subsite −2. Furthermore, the protein has a larger β5-α5 loop as compared to other GH30 xylanases, which might be crucial for creating an additional aglycone subsite (+3) of the catalytic site. Finally, RcXyn30A activity is synergic to that of GH11 xylobiohydrolase.

Effect of prebiotic oligosaccharides on bowel habit and the gut microbiota in children with functional constipation (Inside study): study protocol for a randomised, placebo-controlled, multi-centre trial

BackgroundFunctional constipation (FC) in children is a common gastrointestinal disorder with a worldwide-pooled prevalence of 9.5%. Complaints include infrequent bowel movements, painful defecation due to hard and/or large stools, faecal incontinence, and abdominal pain. Prebiotic oligosaccharides have been shown to relieve constipation symptoms in young adults and elderly. However, sufficient evidence is lacking linking additional prebiotic intake to improve symptoms in children with FC. We hypothesise that prebiotic oligosaccharides are able to relieve symptoms of constipation in young children as well.MethodsIn the present randomised, double-blind, placebo-controlled, multi-centre study, we will study the effects of two prebiotic oligosaccharides in comparison to placebo on constipation symptoms in children of 1–5 years (12 to 72 months) of age diagnosed with FC according to the Rome IV criteria for functional gastrointestinal disorders. The primary outcome measure will be change in stool consistency. Secondary outcomes include stool frequency and stool consistency in a number of cases (%). Tertiary outcomes include among others painful defecation, use of rescue medication, and quality of life. In addition, the impact on gut microbiome outcomes such as faecal microbiota composition and metabolites will be investigated. Participants start with a run-in period, after which they will receive supplements delivered in tins with scoops for 8 weeks, containing one of the two prebiotic oligosaccharides or placebo, followed by a 4-week wash-out period.DiscussionThis randomised double-blind, placebo-controlled multi-centre study will investigate the effectiveness of prebiotic oligosaccharides in children aged 1–5 years with FC.Trial registrationClinicalTrials.gov NCT04282551. Registered on 24 February 2020.

Enzymatic synthesis of Artemisia capillaris-derived prebiotic oligosaccharide using Leuconostoc mesenteroides MKSR dextransucrase and its potential health functional effects

This study focuses on the enzymatic synthesis of prebiotic oligosaccharide from Artemisia capillaris (AC) using Leuconostoc mesenteroides MKSR dextransucrase and explores their antidiabetic, antidementia, and antioxidant effects through in vitro experiments. A Box-Behnken design was used to optimize transglycosylation conditions to produce Artemisia capillaris-derived oligosaccharide (ACOD) containing isomaltooligosaccahrides (IMO), dextran, and A. capillaris-derived substances with high α-glucosidase inhibition (AGI). The optimized conditions were 3.62 brix of AC, 182.54 mM sucrose, and 11.304 mM maltose. ACOD completely inhibited α-glucosidase activity, with 1.52-fold increase compared to other samples, such as OD (IMO and dextran, transglycosylated by dextransucrase without AC) and AC + OD (a simple mixture of AC and OD). Additionally, ACOD showed noteworthy acetylcholinesterase (103.91%) and butyrylcholinesterase (103.30%) inhibitory activity and potent antioxidant properties in terms of DPPH radical scavenging activity (70.04%), reducing power (100%), and ferric reducing antioxidant power (FRAP). ACOD (3.60) showed a higher prebiotic index compared with AC (-23.11), OD (-2.27), and AC + OD (-9.12). In conclusion, ACOD offers enhanced multi-biological activities as a potential health-functional prebiotic. These findings hold promise for applications in functional foods and pharmaceuticals, expanding our understanding of prebiotic functionality.

Evaluation of Prebiotic Glycan Composition in Human Milk and Infant Formula: Profile of Galacto-Oligosaccharides and Absolute Quantification of Major Milk Oligosaccharides by UPLC-Cyclic IM-MS and UPLC-MS/MS.

Prebiotic oligosaccharides have attracted immense interest in the infant formula (IF) industry due to their unique health benefits for infants. There is a need for the reasonable supplementation of prebiotics in premium IF products. Herein, we characterized the profile of galacto-oligosaccharides (GOS) in human milk (HM) and IF using ultrahigh-performance liquid chromatography-cyclic ion mobility-mass spectrometry (UPLC-cIM-MS) technique. Additionally, we further performed a targeted quantitative analysis of five essential HM oligosaccharides (HMOs) in HM (n = 196), IF (n = 50), and raw milk of IF (n = 10) by the high-sensitivity UPLC-MS/MS method. HM exhibited a more abundant and variable HMO composition (1183.19 to 2892.91 mg/L) than IF (32.91 to 56.31 mg/L), whereas IF contained extra GOS species and non-negligible endogenous 3'-sialyllactose. This also facilitated the discovery of secretor features within the Chinese population. Our study illustrated the real disparity in the prebiotic glycome between HM and IF and provided crucial reference for formula improvement.

Sustainable production of cellulose and hemicellulose-derived oligosaccharides from pineapple leaves: Impact of hydrothermal pretreatment and controlled enzymatic hydrolysis

Globally, the demands for sustainably sourced functional foods like prebiotic oligosaccharides have been constantly increasing. This study assessed the potential of pineapple leaves (PL) as lignocellulosic feedstock for sustainable production of cellulose and hemicellulose-derived oligosaccharides through its hydrothermal pretreatment (HT) followed by controlled enzymatic hydrolysis. PL was subjected to HT at 160, 175, and 190 °C for 20, 30, 60, and 90 min without any catalyst for xylooligosaccharide (XOS) production, whereas, the resulting solid content after HT was subjected to controlled enzymatic hydrolysis by commercial cellulase using conduritol B epoxide (0.5–5 mM) for glucooligosaccharides (GOS) production. HT at 160 °C for 60 min resulted in maximum yield of XOS and GOS at 23.7 and 18.3 %, respectively, in the liquid phase. Controlled enzymatic hydrolysis of HT treated (160 °C) PL solids for 20 and 30 min yielded ∼ 174 mg cellobiose/g dry biomass within 24 h, indicating overall high oligosaccharide production.

Cheese and Yogurt By-Products as Valuable Ingredients for the Production of Prebiotic Oligosaccharides

The growing global market of dairy products has led to the need for alternative approaches regarding whey valorization, which is the primary by-product of cheese and strained yogurt production. In this context, prebiotic galactooligosaccharides can be produced enzymatically from whey using commercially available β-galactosidases. A comparative study was conducted to assess the production of galactooligosaccharides from sweet and acid whey, thereby employing two commercial β-galactosidases from Aspergillus oryzae and Kluyveromyces lactis. The study considered the initial lactose content and enzyme load as variables. The maximum yields of galactooligosaccharides in concentrated sweet whey (15% w/v initial lactose) and raw acid whey (3.1% w/v initial lactose) reached 34.4 and 14.7% with lactase from Kluyveromyces lactis (0.13 U/mL), respectively. The corresponding galactooligosaccharide yields for lactase from Aspergillus oryzae were equal to 27.4 and 24.8% in the most concentrated sweet and acid whey, respectively, using enzyme loads of 2 U/mL in sweet whey and 1 U/mL in acid whey. Concerning the profile of the produced galactooligosaccharides, the Kluyveromyces lactis lactase hydrolyzed lactose more rapidly and resulted in higher levels of allolactose and lower levels of 6-galactosyl-lactose, compared to the lactase from Aspergillus oryzae, and achieved in both cases a polymerization degree of up to six.

Chemistry, Isolation, and Pharmaceutical Applications of Inulin

Abstract: Inulin (IN) is a prebiotic oligosaccharide reported in diverse sources of nature. The major sources encompass chicory, Jerusalem artichoke, onions, barley, garlic, rye, and wheat. The literature also reported its promising biological activities, e.g., antidiabetic, anticancer, antioxidant, immuneregulator and prebiotic for improving intestinal function, regulation of blood lipids, and so on. IN’s molecular flexibility, stabilization, and drug-targeting potential make it a unique polymer in pharmaceutical sciences and biomedical engineering. Further, its nutritional value and diagnostic application also widen its scope in food and medical sciences. The hydroxyl groups present in its structure offer chemical modifications, which could benefit advanced drug delivery such as controlled and sustained drug delivery, enhancement of bioavailability, cellular uptake, etc. This work reviews the isolation and purification of IN. The study also provides glimpses of the chemistry, chemical modification, and applications in pharmaceutical sciences and drug delivery.

Sensory improvement and antioxidant enhancement in silver carp hydrolysate using prebiotic oligosaccharides: insights from the Maillard reaction.

Our previous studies have highlighted the potential of silver carp hydrolysate (SCH) in managing chronic diseases. Unfortunately, its fishy smell and bitter taste limited consumer acceptance. Prebiotic oligosaccharides are often used as dietary supplements, ignoring their role as carbonyl ligands in the Maillard reaction to enhance food's sensory and antioxidant properties. This study aimed to improve SCH's sensory attributes and investigate its physicochemical properties and antioxidant activities using prebiotic oligosaccharides via the Maillard reaction. The results showed that xylo-oligosaccharide (XOS) had the highest reactivity among the oligosaccharides tested, and it greatly enhanced the taste and flavor of SCH, as well as its antioxidant activities (0.45 to 16.5 times). Specifically, XOS effectively reduced the fishy smell and bitter taste, imparting a caramel-like flavor and overall acceptability to SCH. The improved flavor profile was attributed to the increased presence of sulfur-containing and nitrogen oxide volatile flavor compounds, such as benzothiazole, methional, and furans, which also contributed to antioxidant effects. Sensory evaluation results indicated that SCH obtained from papain exhibited a stronger bitter taste than that obtained from alcalase. Additionally, XOS imparted a reddish-brown color to SCH due to the higher browning intensity. This study is the first to demonstrate that XOS in the Maillard reaction can effectively improve the undesirable flavor and taste of SCH while enhancing its antioxidant activities, providing a theoretical basis for developing SCH as a market-acceptable functional food ingredient.

Taste of common prebiotic oligosaccharides: impact of molecular structure.

Prebiotic oligosaccharides are naturally occurring nondigestible carbohydrates with demonstrated health benefits. They are also a chemically diverse class of nutrients, offering an opportunity to investigate the impact of molecular structure on oligosaccharide taste perception. Accordingly, a relevant question is whether these compounds are detected by the human gustatory system, and if so, whether they elicit sweet or "starchy" taste. Here, in 3 psychophysical experiments, we investigated the taste perception of 3 commercially popular prebiotics [fructooligosaccharides (FOS), galactooligosaccharides (GOS), xylooligosaccharides (XOS)] in highly pure form. Each of these classes of prebiotics differs in the type of glycosyl residue, and position and type of bond between those residues. In experiments I and II, participants were asked to discriminate a total of 9 stimuli [FOS, GOS, XOS; degree of polymerization (DP) of 2, 3, 4] prepared at 75mM in the presence and absence of lactisole, a sweet receptor antagonist. We found that all 9 compounds were detectable (P < 0.05). We also found that GOS and XOS DP 4 were discriminable even with lactisole, suggesting that their detection was not via the canonical sweet receptor. Accordingly, in experiment III, the taste of GOS and XOS DP 4 were directly compared with that of MOS (maltooligosaccharides) DP 4-6, which has been reported to elicit "starchy" taste. We found that GOS and MOS were perceived similarly although narrowly discriminable, while XOS was easily discriminable from both GOS and MOS. The current findings suggest that the molecular structure of oligosaccharides impacts their taste perception in humans.

Gram-scale chemical synthesis of galactosyllactoses and their impact on infant gut microbiota in vitro.

Galactooligosaccharides (GOS) are widely used as a supplement in infant nutrition to mimic the beneficial effects found in prebiotic human milk oligosaccharides (HMOs). However, the complexity of the GOS mixture makes it challenging to ascertain which of the GOS components contribute most to their health benefits. Galactosyllactoses (GLs) are lactose-based trisaccharides containing a β-galactopyranosyl residue at the 3'-position (3'galactosyllactose, 3'-GL), 4'-position (4'-galactosyllactose, 4'-GL), or the 6'-position (6'-galactosyllactose, 6'-GL). These GLs are of particular interest as they are present in both GOS mixtures and human milk at early stages of lactation. However, research on the potential health benefits of these individual GLs has been limited. Gram quantities are needed to assess their health benefits but these GLs are not readily available at this scale. In this study, we report the gram-scale chemical synthesis of 3'-GL, 4'-GL, and 6'-GL. All three galactosyllactoses were obtained on a gram scale in good purity from cheap and commercially available lactose. Furthermore, in vitro incubation of GLs with infant faecal microbiota demonstrates that the GLs were able to increase the abundance of Bifidobacterium and stimulate short chain fatty acid production.

Evaluation of transgalactosylation activity of commercial β-galactosidase from Bifidobacterium bifidum for synthesis of prebiotic oligosaccharides

Galacto-oligosaccharides (GOS) are products of transgalactosylation reaction of β-galactosidase when lactose is used as the substrate. These carbohydrates are considered prebiotics which stimulate beneficial effects to human health. In the present work, Saphera, a commercial preparation of β-galactosidase from Bifidobacterium bifidum, was biochemically characterised for production of GOS. Using o-nitrophenyl-β-D-galactopyranoside (oNPG) as the substrate, optimal activity for the enzyme was found to be at pH 6.0 and at 45°C. Ten (10) mM each of either Na+ or K+ enhanced enzyme activity by 10%, while Cu2+, Zn2+, Fe2+, and EDTA showed inhibitory effect on the enzyme activity. When incubated in 50 mM sodium phosphate and pH 6.5, the enzyme was found to have half-life time of 136 ± 6 and 2.1 ± 0.2 h at 30 and 50°C, respectively. The hydrolysis activity of the enzyme predominated when the initial lactose concentration used was 5% (w/v). When initial lactose concentration was increased to 20% (w/v), maximum GOS yield obtained was 10% (w/w) achieved at 86% lactose conversion. Analysis using high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) revealed that the major oligosaccharides produced by the enzyme were 3’-galactosylglucose, 3’-galactosyllactose, 3’-galactobiose, and allolactose, thus showing that this enzyme would prefer to form β-(13)- and β-(16)-linked GOS. Despite its predominantly hydrolytic activity, Saphera synthesised prebiotic GOS which could be interesting to dairy industry.

Prebiotic Mannan Oligosaccharide Attenuates Cognitive and Behavioral Disorders in HIV-1 gp120 Transgenic Mouse Via the Microbiota-Gut-Brain Axis

Background The prevalence of HIV-associated neurocognitive disorders (HAND) remains high among HIV-1 infected patients, despite significant advances in antiretroviral therapy (HAART). HAND is characterized by clinical symptoms such as motor and attention deficits, impaired learning and memory, and in severe cases, dementia. The HIV-1 envelope protein gp120 is believed to have a crucial role in the development of these disorders HIV-1-related neurocognitive dysfunction is strongly linked to the host inflammatory regulator α7 nicotinic acetylcholine receptor (A7R) and the signaling of highly reactive oxygen species (ROS). A7R plays a crucial role in regulating calcium ion exchange in nerve cells, immune cells, and endothelial cells. In the brain, antioxidants are present to control and prevent the formation of harmful ROS, which further contribute to the severity of neurodegeneration. Assessing ROS can help predict oxidative tissue damage, including modifications in proteins, lipids, or DNA. ROS tend to accumulate in the small and large intestines, leading to oxidative stress and affecting cognitive function. The prebiotic mangan oligosaccharide (MOS) has been reported to reshape the gut microbiome and increase short-chain fatty acid (SCFAs) production. This, in turn, alters neuronal redox status through the gut-brain axis. There may be a critical role for HIV-1 gp120 (Gp120) protein in these disorders. The microbiota-gut-brain (MGB) axis is bidirectional, and gut microbiota influence brain disorders including HAND, which could be targeted by nutritional interventions. Methods: The HIV-1 gp120 transgenic mice (Gp120 transgenic mice), and wild-type (WT) mice were randomly divided into 4 groups (9 months old, n=6 per group): WT group, WT+MOS group, Gp120 transgenic group, Gp120 transgenic + MOS group. All MOS group mice were administered the prebiotic MOS (0.12% w/v in the drinking water).The drinking water added MOS or was replaced twice a week for 8 weeks and recorded water intake. In the in vitro cell experiment, the cortex of neonatal Sprague-Dawley (SD) rats (1-3 days) was dissected to prepare astrocytes. Cells were identified using Cy3-conjugated GFAP immunostaining. Cultured astrocytes were treated with 300 pM Gp120 protein for 24 hours, and then 0.2, 2, 20 or 200 μM MOS was applied for 24 hours, and different proteins level of expression were detected by Western blot. The ROS level were then analyzed on FACS Calibur-Tangerine flow cytometry in 502 nm. The alteration in mitochondrial membrane potential in astrocytes was assessed through the utilization of the JC-1 Assay Kit in mPMs. Results: As a result of treatment with MOS, the redox status of the brain and the neuroinflammatory responses were significantly balanced. Through correlation analysis, it was observed that a modified gut microbiome and increased butyrate production had a significant impact on behavioral changes and brain oxidative status. Our findings suggest that MOS could mitigate cognitive and behavioral disorders in the Gp120 transgenic mouse. MOS achieves this by suppressing Gp120-induced chronic inflammation and dysregulated production of reactive oxygen species (ROS), thereby improving HIV-1-related neurocognitive dysfunction. This is achieved by targeting A7R and intestinal ROS signaling. Additionally, our results indicate that SCFA supplementation in the brain of HAND mice alleviated behavioral disorders and balanced the HPA-axis and redox status. Conclusion: Overall, the present study demonstrated that MOS significantly reduced cognitive and mental deficits in Gp120 transgenic mice. As prebiotics, MOS could be translated into novel microbiota-targeted approaches to manage metabolic and neurodegenerative diseases. ( Acknowledgements: Natural Science Foundation of Guangdong Province, No. 208059478058 to H.C.; Corresponding author: Hong Cao, gzhcao@smu.edu.cn ) Keywords：HIV-1 gp120, mangan oligosaccharide, α7nAChR, reactive oxygen species