Galacto-oligosaccharides Research Articles

Galactooligosaccharide (GOS) Reduces Branched Short-Chain Fatty Acids, Ammonium, and pH in a Short-Term Colonic Fermentation Model

Prebiotics beneficially affect the gut microbiome. Bimuno®, a prebiotic supplement containing galactooligosaccharides (GOS), has multiple demonstrated prebiotic effects. Using short-term colonic incubations, the influence of GOS on the colonic microbiota of three healthy human adults was evaluated. Colonic reactors inoculated with fecal samples were untreated (blank) or supplemented with GOS. pH, gas pressure, short-chain fatty acids (SCFAs), lactic acid, branched SCFAs, ammonium, and microbial community composition were evaluated at 0 h, 6 h, 24 h, and 48 h. pH decreased and gas pressure increased (+29.01 kPa) with GOS treatment versus blank. Total SCFA (+22.4 mM), acetate (+14.1 mM), propionate (+5.5 mM), and butyrate (+5.8 mM) were higher for GOS than blank. Acetate and propionate production were highest earlier in the experiment, while butyrate production was highest between 24 h and 48 h. With GOS, lactic acid production increased between 0 h and 6 h (+14.4 mM) followed by apparent consumption. Levels of branched SCFAs and ammonium were low with GOS and reduced versus blank (respectively, −2.1 mM and −256.0 mg/L). GOS significantly increased the relative abundance of Bifidobacterium longum (LDA = 4; p = 0.006), and significantly increased the absolute abundance of Bifidobacteriaceae (p < 0.001), Lactobacillaceae (p < 0.05), Bifidobacterium adolescentis (LDA = 4.5; p < 0.001), and Bifidobacterium ruminantium (LDA= 3.2; p = 0.01). This in vitro model demonstrated the prebiotic potential of GOS as supplementation resulted in increased beneficial bacteria, SCFA, and lactic acid and decreased branched SCFA, pH, and ammonium.

Analysis of Akkermansia muciniphila in Mulberry Galacto-Oligosaccharide Medium via Comparative Transcriptomics.

Akkermansia muciniphila is a common member of the human gut microbiota and belongs to the phylum Verrucomicrobia. Decreased levels of A. muciniphila are associated with many diseases, so it is thought to be a beneficial resident of the intestinal mucosal layer. In this study, we found that different prebiotics promoted the proliferation of A. muciniphila, and mulberry galacto-oligosaccharide (MGO) had the greatest effect. We cultured A. muciniphila in a brian heart infusion (BHI) medium containing 5% galactooligosaccharides (GOS), mulberry polysaccharide solution (MPS), and MGO, and transcriptomic analyses were performed. The results revealed that, after 6 days of cultivation, the numbers of upregulated functional genes (based on Gene Ontology) were approximately 0.7 and 19% higher with MPS and MGO, respectively, than with GOS. Analysis using the Kyoto Encyclopedia of Genes and Genomes showed that, when A. muciniphila was cultured with MGO, genes that were upregulated were enriched in the carbohydrate metabolism, the metabolism of cofactors and vitamins, the energy metabolism, the amino acid metabolism, and the lipid metabolism. Upregulated genes included galM and pfkA in the galactose metabolism, and pgi, pfk, fbaA, tpiA, gapA, pgk, gpml, eno, pyk, and lpd in the glycolysis/gluconeogenesis pathway. Real-time quantitative PCR results were consistent with the RNA-Seq data. This work provides valuable knowledge which can be available for the functional application of A. muciniphila and MGO.

Sex difference in prebiotics on gut and blood-brain barrier dysfunction underlying stress-induced anxiety and depression.

Most of the previous studies have demonstrated the potential antidepressive and anxiolytic role of prebiotic supplement in male subjects, yet few have females enrolled. Herein, we explored whether prebiotics administration during chronic stress prevented depression-like and anxiety-like behavior in a sex-specific manner and the mechanism of behavioral differences caused by sex. Female and male C57 BL/J mice on normal diet were supplemented with or without a combination of fructo-oligosaccharides (FOS) and galacto-oligosaccharides (GOS) during 3- and 4-week chronic restraint stress (CRS) treatment, respectively. C57 BL/J mice on normal diet without CRS were used as controls. Behavior consequences, gut microbiota, dysfunction of gut and brain-blood barriers, and inflammatory profiles were measured. In the 3rd week, FOS + GOS administration attenuated stress-induced anxiety-like behavior in female, but not in male mice, and the anxiolytic effects in males were observed until the 4th week. However, protective effects of prebiotics on CRS-induced depression were not observed. Changes in the gene expression of tight junction proteins in the distal colon and hippocampus, and decreased number of colon goblet cells following CRS were restored by prebiotics only in females. In both female and male mice, prebiotics alleviated stress-induced BBB dysfunction and elevation in pro-inflammatory cytokines levels, and modulated gut microbiota caused by stress. Furthermore, correlation analysis revealed that anxiety-like behaviors were significantly correlated with levels of pro-inflammatory cytokines and gene expression of tight junction proteins in the hippocampus of female mice, and the abundance of specific gut microbes was also correlated with anxiety-like behaviors, pro-inflammatory cytokines, and gene expression of tight junction proteins in the hippocampus of female mice. Female mice were more vulnerable to stress and prebiotics than males. The gut microbiota, gut and blood-brain barrier, and inflammatory response may mediate the protective effects of prebiotics on anxiety-like behaviors in female mice.

Cyclic Production of Galacto-Oligosaccharides through Ultrafiltration-Assisted Enzyme Recovery

Galacto-oligosaccharides (GOS) are prebiotics manufactured enzymatically from lactose as substrate. The growing GOS market facilitates the valorization of dairy by-products which represent cheap and abundant sources of lactose. Large-scale GOS production typically employs soluble enzymes in batch reactors that are commonly associated with low enzyme usability and, therefore, high operational expenditures. In this study, we investigate the possibility of recovering enzymes by ultrafiltration (UF) and reusing them in repeated reaction steps. The proposed process scheme included 24 h batch reaction steps with Biolacta N5, a commercial enzyme preparation of Bacillus circulans origin. The reaction steps were followed by UF steps to separate the carbohydrate products from the enzymes by applying a volume concentration factor of 8.6. Then, the collected biocatalysts were reused for repeated cycles by adding fresh lactose. Enzyme losses were quantified with a direct method by analyzing the underlying relationship between reaction rates and enzyme dosage obtained from additional experiments conducted with known enzyme loads. Within five cycles, the enzyme activity declined gradually from 923 to 8307 U·kg−1, and the half-life was estimated as ca. 15.3 h. The outcomes of this study may serve as a basis for further optimization of the reported process scheme with enhanced enzyme usability.

Human milk oligosaccharides, antimicrobial drugs, and the gut microbiota of term neonates: observations from the KOALA birth cohort study

ABSTRACT The infant gut microbiota affects childhood health. This pioneer microbiota may be vulnerable to antibiotic exposures, but could be supported by prebiotic oligosaccharides found in breast milk and some infant formulas. We sought to characterize the effects of several exposures on the neonatal gut microbiota, including human milk oligosaccharides (HMOs), galacto-oligosaccharides (GOS), and infant/maternal antimicrobial exposures. We profiled the stool microbiota of 1023 one-month-old infants from the KOALA Birth Cohort using 16S rRNA gene amplicon sequencing. We quantified 15 HMOs in breast milk from the mothers of 220 infants, using high-performance liquid chromatography-mass spectrometry. Both breastfeeding and antibiotic exposure decreased gut microbial diversity, but each was associated with contrasting shifts in microbiota composition. Other factors associated with microbiota composition included C-section, homebirth, siblings, and exposure to animals. Neither infant exposure to oral antifungals nor maternal exposure to antibiotics during pregnancy were associated with infant microbiota composition. Four distinct groups of breast milk HMO compositions were evident, corresponding to maternal Secretor status and Lewis group combinations defined by the presence/absence of certain fucosylated HMOs. However, we found the strongest evidence for microbiota composition associations between two non-fucosylated HMOs: 6’-sialyllactose (6’-SL) and lacto-N-hexaose (LNH). Among 111 exclusively formula-fed infants, the GOS-supplemented formula was associated with a lower relative abundance of Clostridium perfringens. In conclusion, the gut microbiota is sensitive to some prebiotic and antibiotic exposures during early infancy and understanding their effects could inform future strategies for safeguarding a health-promoting infant gut microbiota.

Dietary soy galactooligosaccharides affect the performance, intestinal function, and gut microbiota composition of growing chicks

The objective of this experiment was to investigate the effects of the dietary soy galactooligosaccharides (GOS), raffinose and stachyose, on performance, gastrointestinal health, and systemic stress in young broilers. Birds were fed a GOS-devoid diet based on soy protein isolate (SPI) or the SPI diet with 0.9, 1.8, 2.7, or 3.6% added stachyose and raffinose in a ratio of 4:1 at the expense of corn starch. These 5 treatments were administered to 10 replicate cages of 8 birds. Performance was measured weekly and excreta moisture, N retention, apparent metabolizeable energy, and complete blood cell counts were determined at 14 and 21 d. At 21 d, 2 birds per cage were orally gavaged with fluorescein isothiocyanate-dextran (FITC-d) and serum samples were analyzed for FITC-d as a marker of gut leakage. Additionally, intestinal morphology, crop presumptive lactic acid bacteria (LAB) counts, crop and cecal pH, and cecal microbiota via16S rRNA microbial sequencing were evaluated at 21 d. From 0 to 21 d, feed intake increased linearly (P < 0.01) as dietary GOS increased, whereas BWG increased (P < 0.05) quadratically. Feed conversion ratio increased (P < 0.01) linearly as GOS increased. There were linear increases (P < 0.05) in excreta moisture as dietary GOS increased at 14 and 21 d, as well as dose-dependent responses (P < 0.05) in N retention, AME, and AMEn. There was a quadratic increase (P < 0.05) in crop LAB recovery and a linear decrease (P < 0.01) in ceca pH as GOS increased. At 14 d, a linear increase (P < 0.05) in blood heterophil to lymphocyte ratio was observed as dietary GOS increased. Serum concentrations of FITC-d increased quadratically (P < 0.01) to dietary GOS. Increasing levels of GOS influenced alpha and beta diversities and composition of gut microbiota, including the abundance of Ruminococcus and Bifidobacterium. Results from this trial indicate that soy-derived GOS exert dose-dependent effects on nutrient utilization and intestinal health in young broilers.

Differential Effects of Oligosaccharides, Antioxidants, Amino Acids and PUFAs on Heat/Hypoxia-Induced Epithelial Injury in a Caco-2/HT-29 Co-Culture Model.

(1) Exposure of intestinal epithelial cells to heat and hypoxia causes a (heat) stress response, resulting in the breakdown of epithelial integrity. There are indications that several categories of nutritional components have beneficial effects on maintaining the intestinal epithelial integrity under stress conditions. This study evaluated the effect of nine nutritional components, including non-digestible oligosaccharides (galacto-oligosaccharides (GOS), fructo-oligosaccharides (FOS), chitosan oligosaccharides (COS)), antioxidants (α-lipoic acid (ALA), resveratrol (RES)), amino acids (l-glutamine (Glu), l-arginine (Arg)) and polyunsaturated fatty acids (PUFAs) (docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA)), on heat/hypoxia-induced epithelial injury. (2) Two human colonic cell lines, Caco-2 and HT-29, were co-cultured and pre-treated with the nutritional components for 48 h. After pre-treatment, the cells were exposed to heat/hypoxia (42 °C, 5% O2) for 2 h. Epithelial integrity was evaluated by measuring trans-epithelial electrical resistance (TEER), paracellular Lucifer Yellow (LY) permeability, and tight junction (TJ) protein expression. Heat stress and oxidative stress levels were evaluated by determining heat-shock protein-70 (HSP-70) expression and the concentration of the lipid peroxidation product malondialdehyde (MDA). (3) GOS, FOS, COS, ALA, RES, Arg, and EPA presented protective effects on epithelial damage in heat/hypoxia-exposed Caco-2/HT-29 cells by preventing the decrease in TEER, the increase in LY permeability, and/or decrease in TJ proteins zonula occludens-1 (ZO-1) and claudin-3 expression. COS, RES, and EPA demonstrated anti-oxidative stress effects by suppressing the heat/hypoxia-induced MDA production, while Arg further elevated the heat/hypoxia-induced increase in HSP-70 expression. (4) This study indicates that various nutritional components have the potential to counteract heat/hypoxia-induced intestinal injury and might be interesting candidates for future in vivo studies and clinical trials in gastrointestinal disorders related to heat stress and hypoxia.

Development of Galactooligosaccharide (GOS) added gummies: sensory, characterization and shelf quality

Sugar loaded confectioneries have a rapidly growing market globally. Consumption of such products may lead to multiple health risks. Products like gummies can be substituted with healthy sugar alternatives such as Galactooligosaccharide (GOS), which can be consumed by all age groups.The aim of the study was to develop standard gummies and sugar substituted gummies, conduct sensory analysis, shelf life studies and characterize it for its physico chemical properties.The standard gummies were made using agar, sugar, citric acid, water, and FSSAI (Food Safety Standards Authority of India) certified natural colours and flavours, while GOS supplemented gummies were made by replacing sugar in varied amounts, upto 100%. A trained panel (n = 8) evaluated the gummies using a composite score card in triplicates for a variety of sensory attributes. GOS recovery analysis, physicochemical variables such as colour, moisture, pH, and texture were assessed. Shelf life Studies of 100 percent GOS supplemented gummies were carried out at accelerated temperatures (37 °C) over a period of 6 months.The results revealed that gummies with varying levels of GOS were acceptable to the panelists, with no significant differences in the keeping quality. However, F test revealed a significant improvement (p < 0.05) in the texture of the gummies with a slight reduction in colour and flavour at the end of 6 months. The moisture content and pH values were 24.8% and 3.37 respectively. HPLC analysis revealed a recovery of 95% GOS in the prepared gummies.Hence, sugar can be substituted with 100% GOS to fulfil the increasing demand for healthy confectioneries without any change in organoleptic qualities and shelf life for 6 months.Graphical

Physico-chemical analysis of newly prepared prebiotic chocolates by using Galacto Oligosaccharides (GOS)

In India, chocolate is very famous mood freshening sweet food product specially for children. But high consumption of chocolate may be harmful for human health, because most of the chocolates contain high fat. This research has been done to increase the health benefits of chocolate by using cocoa and Galacto Oligosaccharides (GOS). Many researchers have shown their research that GOS is very beneficial for human health such as it helps to lower down the cholesterol levels in the blood, to prevent colon cancer, and helps to improve mineral absorption in human body. Basically, GOS is a type of prebiotic e.g., food for probiotic bacteria. Prebiotics are non-digestible ingredients that benefit the host by boosting the growth and activity of one or a few bacterial species that are already present in the colon. It was also studied by many researchers that cocoa powder contains a lot of caffeine, flavanols, which are polyphenolic compounds. Flavanols, particularly epicatechin monomer and oligomer cocoa flavanols have been linked to a number of health advantages, including boosting nitric oxide synthase, enhancing blood flow and arterial flexibility, lowering blood pressure and platelet aggregation, and reducing inflammation. The main objectives of this research are to develop the formulation of prebiotic chocolate and to evaluate the physicochemical parameters of the newly prepared chocolate. After physico-chemical analysis of carbohydrate, fat, crude fibre and pH, it was found that both the newly prepared prebiotic enriched chocolates (T1 and T2) were significantly different (p≤0.01) from control (T0) and after analysis of protein content, it was also found that both the newly prepared prebiotic enriched chocolates (T1 and T2) were insignificantly different (p≤0.01) from control (T0).

In vitro prebiotic activity of rhLf and galactooligosaccharides on infant intestinal microbiota.

human lactoferrin (Lf) and human milk oligosaccharides possess a wide range of functions. So, the present study focusses on the role of Lf and/or galactooligosaccharides (GOS) in the modulation of gut microbiota composition. recombinant human lactoferrin (rhLf) was added to the first infant formula (0.10, 0.15, 0.20 %) alone or in combination with GOS (1 %) in vessels of a small-scale batch culture fermentation model. Short-chain fatty acids (SCFAs), microbial population groups, and pH were monitored through fermentation for 24 hours. insignificant changes were observed in pH values and acetic acid accumulated during fermentation. Propionic acid content has been insignificantly increased while butyric acid has been insignificantly decreased. Moreover, increments in all bacterial groups except for Bacteroides were observed through the fermentation process. Lactobacillus and Bifidobacterium showed an increase in relation to initial time over the fermentation process, demonstrating the prebiotic effect of lactoferrin and GOS. After 24 hours of fermentation, all tested ingredients showed significant similarities in Enterococcus for controls except for 0.20 % rhLf + 1 % GOS, which provoked a diminution of Enterococci growth. despite the importance of the batch culture fermentation technique in uncovering the prebiotic activity of food ingredients, it is not useful for detecting the prebiotic nature of Lf due to its nature as a protein. Thus, Lf maybe shows its prebiotic activity on the gut microbiota through other mechanisms.

Boosting the stability of β-galactosidase immobilized onto soy-protein isolate-glutaraldehyde-functionalized carrageenan beads

Uncontrolled enzyme-immobilizer interactions were evident after immobilizing β-galactosidase onto soy-protein isolate-glutaraldehyde-functionalized carrageenan beads. Such interactions triggered shortcomings in the immobilized β-galactosidase (iβGL) thermal and storage stabilities. The thermal stability of the iβGL was somewhat lesser than that of the free βGL. Moreover, the iβGL suffered an initial sharp fall-off in its activity after storing it. Thus, approaches were adopted to prevent the occurrence of such uncontrolled enzyme-immobilizer interactions, and accordingly, boost the stability of the iβGL. These approaches involved neutralizing the covalently reactive GA entities via glycine and also altering the functionalizing GA concentrations. Nonetheless, no improvement was recorded in the iβGL thermal stability and this indicated that the uncontrolled enzyme-immobilizer interactions were not mediated via GA. Another approach was then attempted which involved treating the iβGL with lactose. The lactose-treated iβGL (LT-iβGL) presented superior thermal stability as was verified from its smaller kd and bigger t1/2 and D-values. The LT-iβGL t1/2 values were 5.60 and 3.53 fold higher than those presented by the free βGL at 62 and 65 °C, respectively. Moreover, the LT- iβGL presented loftier ΔG than did the free βGL. The storage stability of the LT- iβGL was also superior as it offered 100.41% of its commencing activity on its 43rd storage day. Thus, it could be concluded that lactose prevented the uncontrolled enzyme-immobilizer interactions. Finally, advantageous galacto-oligosaccharides (GOS) were prepared via the iβGL. The GOS were then analyzed with mass spectrometry, and it was shown that their degree of polymerization reached up to 7.

The Milk Active Ingredient, 2'-Fucosyllactose, Inhibits Inflammation and Promotes MUC2 Secretion in LS174T Goblet Cells In Vitro.

In several mice inflammatory models, human milk oligosaccharides (HMOs) were shown to protect the intestinal barrier by promoting mucin secretion and suppressing inflammation. However, the functions of the individual HMOs in enhancing mucin expression in vivo have not been compared, and the related mechanisms are not yet to be clarified. In this study, we investigated the modulatory effects of 2′-fucosyllactose (2′-FL), 3′-sialyllactose (3′-SL), galacto-oligosaccharide (GOS) and lactose (Lac) on goblet cells’ functions in vitro. The appropriate dosage of the four chemicals was assessed in LS174T cells using the CCK-8 method. Then they were supplemented into a homeostasis and inflammatory environment to further investigate their effects under different conditions. Mucin secretion-related genes, including mucin 2 (MUC2), trefoil factor family 3 (TFF3), resistin-like β (RETNLB), carbohydrate sulfotransferase 5 (CHST5) and galactose-3-O-sulfotransferase 2 (GAL3ST2), in LS174T cells were detected using quantitative RT-qPCR. The results showed that 2′-FL (2.5 mg/mL, 72 h) was unable to increase MUC2 secretion in a steady-state condition. Comparatively, it exhibited a greater ability to improve mucin secretion under an inflammatory condition compared with GOS, demonstrated by a significant increase in TFF3 and CHST5 mRNA expression levels (p > 0.05). However, 3′-SL and Lac exhibited no effects on mucin secretion. To further investigate the underlying mechanism via which 2′-FL enhanced goblet cells’ secretion function, the NOD-like receptor family pyrin domain containing 6 (NLRP6) gene, which is closely related to MUC2 secretion, was silenced using the siRNA method. After silencing the NLRP6 gene, the mRNA expression levels of MUC2, TFF3 and CHST5 in the (2′-FL + tumor necrosis factor α (TNF-α) + NLRP6 siRNA) group were significantly decreased compared with the (2′-FL + TNF-α) group (p > 0.05), indicating that NLRP6 was essential for MUC2 expression in goblet cells. We further found that 2′-FL could significantly decrease toll-like receptor 4 (TLR4, p < 0.05), myeloid differential protein-88 (MyD88, p < 0.05) and nuclear factor kappa-B (NF-κB, p < 0.05) levels in LS174T inflammatory cells, even when the NLRP6 was silenced. Altogether, these results indicated that in goblet cells, 2′-FL exerts its function via multiple processes, i.e., by promoting mucin secretion through NLRP6 and suppressing inflammation by inhibiting the TLR4/MyD88/NF-κB pathway.

Food contributing to fermentable oligosaccharide, disaccharide, monosaccharide, and polyols intake in Korean adults.

The dietary intake of foods with fermentable oligosaccharides, disaccharides, monosaccharides, and polyols (FODMAP) is known to adversely affect patients with irritable bowel syndrome (IBS). However, the effects of FODMAP have been studied predominantly among Western populations. This study aimed to identify foods high in FODMAP content which form a part of the Korean adult diet and obtain basic data for the preparation of IBS guidelines. An online survey of 1,000 adults from the general population in the age group of 20 to 40 years was performed. Data from 787 participants (men, 386; women, 401) were analyzed. The general characteristics of the participants, health status, IBS diagnosis using the Rome III diagnostic criteria, semi-quantitative food frequency questionnaire findings, and food items causing symptoms were analyzed. Overall, 169 participants (21.5%) had IBS. The contribution of the FODMAP nutrients in both IBS and healthy groups was as follows: fructan > lactose > excess fructose > sorbitol > mannitol > galacto-oligosaccharides (GOS). The fructan intake was 4.6 ± 2.2 g/day and 4.3 ± 2.5 g/day in the IBS and healthy groups (P = 0.014), respectively. In the IBS group, the ratio of the intake of fructan to the total FODMAP intake was 39.5%, 29.8%, and 5.8% through onions, garlic, and bananas, respectively. Fructan was the FODMAP nutrient most consumed by Korean adults. Therefore, given the difference in the dietary habits of each country's population, the dietary guidelines for IBS should be country specific.

Effect of Complex Prebiotics on the Intestinal Colonization Ability of Limosilactobacillus fermentum DALI02

Intestinal colonization is beneficial to the role of probiotics, and prebiotics can promote the adhesion and colonization of probiotics in the intestine. This study optimized the combination of complex prebiotics that could improve the growth ability and adhesion ability of Limosilactobacillus fermentum (L. fermentum) DALI02 to Caco-2 cells in vitro and determined the effect of its colonization quantity and colonization time in the immunocompromised rat model. The results showed that all five prebiotics (fructo-oligosaccharides (FOS), galacto-oligosaccharides (GOS), inulin, stachyose, and xylo-oligosaccharides (XOS)) significantly promoted the growth and adhesion of L. fermentum DALI02. It was found that 0.5% (w/w) inulin had the best growth promotion effect, and 0.5% FOS had the strongest adhesion promotion (the adhesion rate was increase by 1.75 times). In addition, 0.05% FOS, 0.20% GOS, 0.30% inulin, 0.20% stachyose, and 0.30% XOS could significantly improve the adhesion rate of L. fermentum DALI02 from 1.72% to 3.98%. After 1 w of intervention, the quantity of colonization in the fermented broth with prebiotics group was significantly higher than that in the fermented broth group. The intervention time was extended from 1 d to 4 w, and the amount of colonization of L. fermentum DALI02 in the fermented broth with prebiotics group increased significantly from 4.32 lgcopies/g to 5.12 lgcopies/g. After the intervention, the serum levels of lipopolysaccharide (LPS) and D-lactic acid in rats were significantly reduced, and the most significant was in the fermented broth with prebiotics group, with LPS and D-lactic acid levels of 74.11 pg/mL and 40.33 μmol/L, respectively. Complex prebiotics can promote the growth and adhesion of L. fermentum DALI02 and significantly increase the quantity of colonization and residence time of the strain in the intestine, which helps the restoration of intestinal barrier function and other probiotic effects.

Extensively Hydrolyzed Hypoallergenic Infant Formula with Retained T Cell Reactivity.

Immunoglobulin E (IgE)-mediated cow's milk allergy (CMA) can be life-threatening and affects up to 3% of children. Hypoallergenic infant formulas based on hydrolyzed cow's milk protein are increasingly considered for therapy and prevention of cow's milk allergy. The aim of this study was to investigate the allergenic activity and ability to induce T cell and cytokine responses of an infant formula based on extensively hydrolyzed cow's milk protein (whey) (eHF, extensively hydrolyzed formula) supplemented with Galactooligosaccharides (GOS) and Limosilactobacillus fermentum CECT5716 (LF) to determine its suitability for treatment and prevention of CMA. eHF and standard protein formula based on intact cow's milk proteins (iPF) with or without Galactooligosaccharide (GOS) and Limosilactobacillus fermentum CECT5716 (LF) were investigated with allergen-specific antibodies and tested for IgE reactivity and allergenic activity in basophil degranulation assays with sera from cow's milk (CM)-allergic infants/children. Their ability to stimulate T cell proliferation and cytokine secretion in cultured peripheral blood mononuclear cells (PBMC) from CM-allergic infants and children was studied with a FACS-based carboxyfluorescein diacetate succinimidyl ester (CFSE) dilution assay and xMAP Luminex fluorescent bead-based technology, respectively. An eHF supplemented with GOS and LF exhibiting almost no IgE reactivity and allergenic activity was identified. This eHF induced significantly lower inflammatory cytokine secretion as compared to an intact protein-based infant formula but retained T cell reactivity. Due to strongly reduced allergenic activity and induction of inflammatory cytokine secretion but retained T cell reactivity, the identified eHF may be used for treatment and prevention of CMA by induction of specific T cell tolerance.

The effects of 2'-fucosyllactose and lacto-N-neotetraose, galacto-oligosaccharides, and maternal human milk oligosaccharide profile on iron absorption in Kenyan infants

BackgroundWhether prebiotic human milk oligosaccharides (HMO), such as 2'-fucosyllactose (2'-FL) and lacto-N-neotetraose (LNnT), enhance iron absorption in infants is unknown. Moreover, whether maternal HMO profile affects absorption of iron fortificants or the effects of prebiotic galacto-oligosaccharides (GOS) and/or HMO on iron absorption is uncertain. ObjectivesThe aim of this study was to test whether consumption of 3.0 g GOS or HMO enhances iron absorption from iron-fortified maize porridge in partially breastfed Kenyan infants and whether maternal HMO profile modulates these effects. MethodsIn a randomized, prospective crossover study, 55 infants (aged 8–12 mo) were fed test meals fortified with 1 of the following: 1) 5.0 mg iron as 54Fe-labeled ferrous fumarate (FeFum); 2) 5.0 mg iron as 58FeFum and 3.0 g GOS (FeFum+GOS); and 3) 5.0 mg iron as 57FeFum and 2.0 g 2'-FL and 1.0 g LNnT (FeFum+HMO). Fractional iron absorption (FIA) was assessed by erythrocyte incorporation of iron isotopes. HMO profiles were determined by capillary gel electrophoresis with laser-induced florescence detection. Data were analyzed with mixed-effect models, and iron dialyzability was measured in vitro. ResultsOf the 55 infants included, 49 were fed as instructed. FIA from the FeFum+GOS group [median (IQR) 22.2% (16.5%–25.9%)] was higher than that from the FeFum group [12.5% (9.5%–20.9%)] (P = 0.005). FIA from the FeFum+HMO group was 13.3% (7.1%–24.4%) and did not differ from the FeFum group (P = 0.923). Maternal HMO profile did not predict FIA or modulate the effects of GOS or HMO on FIA. Iron dialyzability ratios at pH 2 of FeFum+GOS to FeFum and FeFum+HMO to FeFum were 2.1 and 0.9 (P = 0.001 and P = 0.322), respectively. ConclusionsIn Kenyan infants consuming FeFum-fortified maize porridge, co-provision of 3.0 g GOS increased FIA by 78%, whereas co-provision of 3.0 g HMO did not affect FIA. Variations in maternal HMO profile, including secretor and Lewis phenotype, did not predict FIA. These data argue against a physiologic role for 2'-FL and LNnT in facilitating iron absorption in infancy.The study was registered at clinicaltrials.gov as NCT04163406 (https://clinicaltrials.gov/ct2/show/NCT04163406).

Diverse Galactooligosaccharides Differentially Reduce LPS-Induced Inflammation in Macrophages.

The effects of natural and synthetic galactooligosaccharides (GOS) on inflammation were explored by investigating the structure-activity relationship between the degree of GOS polymerization and in vitro anti-inflammatory activity, together with the potential underlying mechanism of their anti-inflammatory effects. The results demonstrated that GOS had strong anti-inflammatory effects in lipopolysaccharide (LPS)-induced RAW264.7 macrophages, including the inhibition of nitric oxide production and the reduced expression of pro-inflammatory mediators (interleukin-1β, interleukin-6, and tumor necrosis factor α), induced nitric oxide synthase (iNOS), cyclooxygenase 2 (COX-2), and proteins related to the Toll-like receptor 4 (TLR4)/nuclear factor (NF)-κB signaling pathway. GOS4, which has the highest degree of polymerization, exerted the strongest anti-inflammatory activity among the GOS examined. More importantly, our findings confirmed the anti-inflammatory effects of GOS on RAW264.7 macrophages via the TLR4/NF-κB pathway. Our experimental results could provide further support for the exploration of GOS in human nutrition and health.

In vitro fermentation of fructooligosaccharide and galactooligosaccharide and their effects on gut microbiota and SCFAs in infants

• Gut microbes can degrade FOS and GOS to produce SCFAs and lactate. • The change of intestinal flora were the main factors affecting SCFAs accumulation. • The abundance of CAZymes was significantly higher in the GOS and FOS + GOS groups. • FOS and GOS may increase beneficial microbes and inhibit harmful microbes. Oligosaccharides are widely added to infant formula milk powders as prebiotics for intestinal health. Using in vitro fermentation conditions, this study investigated the fermentation characteristics of fructooligosaccharides (FOS) and galactooligosaccharides (GOS) with infant fecal inoculum. The changes in short-chain fatty acids (SCFAs) and lactate production were monitored by HPLC. The contents of SCFAs and lactate increased significantly in the GOS and FOS + GOS groups. Furthermore, the metagenomic analysis revealed that microbial communities were distinctively different. Most bacteria were significantly positively correlated with SCFAs production and negatively correlated with pH, particularly Bifidobacterium , Enterococcus , Weissella and Streptococcus . More importantly, the relative abundance of most carbohydrate-active enzymes (CAZymes, EC:2.3.1.54, EC:2.3.1.9 and EC:2.8.3.8, etc.) were significantly higher in the GOS and FOS + GOS groups. These results suggested that FOS and GOS had a significant regulatory effect on the composition and metabolism of the intestinal microbiota, further promoting the production of SCFAs and lactate.

In Vitro Production of Galactooligosaccharides by a Novel β-Galactosidase of Lactobacillus bulgaricus.

β-galactosidase is an enzyme with dual activity and important industrial application. As a hydrolase, the enzyme eliminates lactose in milk, while as a trans-galactosidase it produces prebiotic galactooligosaccharides (GOS) with various degrees of polymerization (DP). The aim of the present study is the molecular characterization of β-galactosidase from a Bulgarian isolate, Lactobacillus delbrueckii subsp. bulgaricus 43. The sequencing of the β-gal gene showed that it encodes a new enzyme with 21 amino acid replacements compared to all other β-galactosidases of this species. The molecular model revealed that the new β-galactosidase acts as a tetramer. The amino acids D207, H386, N464, E465, Y510, E532, H535, W562, N593, and W980 form the catalytic center and interact with Mg2+ ions and substrate. The β-gal gene was cloned into a vector allowing heterologous expression of E. coli BL21(DE3) with high efficiency, as the crude enzyme reached 3015 U/mL of the culture or 2011 U/mg of protein. The enzyme's temperature optimum at 55 °C, a pH optimum of 6.5, and a positive influence of Mg2+, Mn2+, and Ca2+ on its activity were observed. From lactose, β-Gal produced a large amount of GOS with DP3 containing β-(1→3) and β-(1→4) linkages, as the latter bond is particularly atypical for the L. bulgaricus enzymes. DP3-GOS formation was positively affected by high lactose concentrations. The process of lactose conversion was rapid, with a 34% yield of DP3-GOS in 6 h, and complete degradation of 200 g/L of lactose for 12 h. On the other hand, the enzyme was quite stable at 55 °C and retained about 20% of its activity after 24 h of incubation at this temperature. These properties expand our horizons as regards the use of β-galactosidases in industrial processes for the production of lactose-free milk and GOS-enriched foods.

Inhibition of Galactooligosaccharide (GOS) Degradation in High-Heat-Treated Goat’s Milk as a Raw Material for Functional Dairy Products

The aim of this study was to analyze the inhibition of galactooligosaccharide (GOS) degradation in heat-treated milk with permeate obtained by microfiltration and concentrated by ultrafiltration. An attempt was made to obtain raw material with a designed composition and stability in terms of GOS content for the production of fermented milk beverages. This study is important due to the versatile possibility of using milk and permeate with GOS in further processing and food production. During the heat treatment of goat’s milk, GOS degradation was approx. 16%. However, no changes in GOS content were observed in goat’s milk with permeate after 30 and 60 min of heating between 72 and 92 °C. Therefore, goat’s milk with permeate in terms of GOS content was stable for up to 60 min, regardless of the temperature. The addition of permeate effectively inhibits GOS degradation in milk. It has been shown that the produced raw material with a stable GOS content during heating can be further used for the production of kefir.